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[ [ "Novel substitution mutant receptors and their use in an ecdysone receptor-based inducible gene expression system", "This invention relates to the field of biotechnology or genetic engineering.", "Specifically, this invention relates to the field of gene expression.", "More specifically, this invention relates to novel substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system and methods of modulating the expression of a gene in a host cell for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic organisms." ], [ "1.A gene expression modulation system comprising a) a polynucleotide that encodes a first polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation, and b) a polynucleotide that encodes a second polypeptide comprising a nuclear receptor ligand binding domain that dimerizes with said Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "2.The gene expression modulation system of claim 1, said gene expression modulation system comprising: a) a first gene expression cassette comprising a polynucleotide that encodes a first polypeptide comprising: i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) a nuclear receptor ligand binding domain; and b) a second gene expression cassette comprising a polynucleotide that encodes a second polypeptide comprising: i) a transactivation domain; and ii) a nuclear receptor ligand binding domain wherein one of the nuclear receptor ligand binding domains is said Group B nuclear receptor ligand binding domain comprising a substitution mutation, and the other of said nuclear receptor ligand binding domains is a nuclear receptor ligand binding domain that dimerizes with said Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "3.The gene expression modulation system of claim 1, wherein the Group B nuclear receptor ligand binding domain is selected from the group consisting of a retinoid X receptor ligand binding domain, an H-2 region II binding protein (H-2RIIBP) ligand binding domain, a Nuclear Receptor co-regulator-1 (RCoR-1) ligand binding domain, an ultraspiracle protein ligand binding domain, a 2C1 nuclear receptor ligand binding domain, and a chorion factor 1 (CF-1) ligand binding domain.", "4.The gene expression modulation system of claim 3, wherein the Group B nuclear receptor ligand binding domain is encoded by a polynucleotide comprising a codon mutation that results in a substitution of an amino acid residue, wherein the amino acid residue is at a position equivalent to or analogous to a) 401 or 429 of SEQ ID NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 344, 355, 385, 431, 442, 462, 470, 472, 473, 495, 500, 511, 516, or 528 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, f) 455, 456, 457, and 458 of SEQ ID NO: 2, g) 470, 472, and 473 of SEQ ID NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2.5.The gene expression modulation system of claim 1, wherein the Group B nuclear receptor ligand binding domain comprising a substitution mutation is a retinoid X receptor ligand binding domain.", "6.The gene expression modulation system of claim 4, wherein the substitution mutation is selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, D344N, K355R, S385A, M431L, R442K, V462L, S470A, E471D, T473E, A495S, E500S, K511R, T516V, or A528S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451 V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.7.The gene expression modulation system of claim 2, wherein the DNA binding domain is selected from the group consisting of an ecdysone receptor DNA binding domain, a GAL4 DNA-binding domain, and a LexA DNA-binding domain.", "8.The gene expression modulation system of claim 2, wherein the transactivation domain is selected from the group consisting of an ecdysone receptor transactivation domain, a VP16 transactivation domain, a B42 acidic activator transactivation domain, and a p65 transactivation domain.", "9.A gene expression cassette comprising a polynucleotide that encodes a polypeptide selected from the group consisting of a) a polypeptide comprising a transactivation domain, a DNA-binding domain, and a Group B nuclear receptor ligand binding domain comprising a substitution mutation, b) a polypeptide comprising a DNA binding domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation, and c) a polypeptide comprising a transactivation domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "10.An isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the isolated polynucleotide comprises a codon mutation that results in a substitution of an amino acid residue at a position equivalent to or analogous to a) 401 or 429 of SEQ ID NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 344, 355, 385, 431, 442, 462, 470, 472, 473, 495, 500, 511, 516, or 528 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, f) 455, 456, 457, and 458 of SEQ ID NO: 2, g) 470, 472, and 473 of SEQ ID NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2.11.The isolated polynucleotide of claim 10, wherein the codon mutation results in a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, D344N, K355R, S385A, M431L, R442K, V462L, S470A, E471D, T473E, A495S, E500S, K511R, T516V, or A528S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451 V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.12.An expression vector comprising the isolated polynucleotide of claim 10 operatively linked to a transcription regulatory element.", "13.A host cell comprising the expression vector of claim 12, wherein the transcription regulatory element is operative in the host cell.", "14.An isolated polypeptide encoded by the isolated polynucleotide of claim 10.15.", "(canceled) 16.", "(canceled) 17.The isolated polypeptide of claim 14, wherein the substitution mutation is selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, D344N, K355R, S385A, M431L, R442K, V462L, S470A, E471D, T473E, A495S, E500S, K511R, T516V, or A528S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.18.A method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell the gene expression modulation system of claim 2; and b) introducing into the host cell a ligand; wherein the gene to be modulated is a component of a gene expression cassette comprising: i) a response element recognized by the DNA binding domain; ii) a promoter that is activated by the transactivation domain; and iii) a gene whose expression is to be modulated; whereby upon introduction of the ligand into the host cell, expression of the gene of b)iii) is modulated.", "19.The method of claim 18, wherein the ligand is a) a compound of the formula: wherein: E is a (C4-C6)alkyl containing a tertiary carbon or a cyano(C3-5)alkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2; R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, NH—CN, or joined with R3 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R3 is H, Et, or joined with R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R4, R5, and R6 are independently H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt; b) an ecdysone, 20-hydroxyecdysone, ponasterone A, or muristerone A; c) an oxysterol, a 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate, 7-ketocholesterol-3-sulfate, farnesol, a bile acid, a 1,1-biphosphonate ester, or a Juvenile hormone III; or d) a 9-cis-retinoic acid, 4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-napthyl)-ethenyl)benzoic acid (3-methyl-TTEB), ((E)-2)-2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-napthyl)propen-1-yl)-4-thiophenecarboxylic acid), 2-(5,6,7,8-tetra-hydro-3,5,5,8,8-tetramethyl-2-naphthyl)-2-(carboxyphenyl)-1,3-dioxolane, 4-(5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyl-dibenzo (b,e) (1,4)(diazepin-11-yl)-benzoic acid (HX600) or thiadiazepin analogs thereof, 3,7,11,15-tetramethyl hexadeconoic acid (phytanic acid), 6-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl)nicotinic acid, 2-(4-caroxyphenyl)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1,3-dithiane, or 4-(2-methyl)-1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl) propenyl)benzoic acid.", "20.The method of claim 18, wherein the method further comprises introducing into the host cell a second ligand, wherein the second ligand is 9-cis-retinoic acid or a synthetic analog of a retinoic acid.", "21.A host cell comprising the gene expression modulation system of claim 1.22.A non-human organism comprising the host cell of claim 21.23.The gene expression modulation system of claim 1, wherein in said second polypeptide comprising a nuclear receptor ligand binding domain that dimerizes with said Group B nuclear receptor ligand binding domain comprising a substitution mutation, said nuclear receptor ligand binding domain is selected from the group consisting of an ecdysone receptor ligand binding domain, a ubiquitous receptor ligand binding domain, an orphan receptor 1 ligand binding domain, a steroid hormone nuclear receptor 1 ligand binding domain, a retinoid X receptor interacting protein-15 ligand binding domain, a liver X receptor β ligand binding domain, a steroid hormone receptor like protein ligand binding domain, a liver X receptor ligand binding domain, a liver X receptor α ligand binding domain, a farnesoid X receptor ligand binding domain, a receptor interacting protein 14 ligand binding domain, and a farnesol receptor ligand binding domain.", "24.The gene expression modulation system of claim 23, wherein in said second polypeptide comprising a nuclear receptor ligand binding domain that dimerizes with said Group B nuclear receptor ligand binding domain comprising a substitution mutation, said nuclear receptor ligand binding domain is an ecdysone receptor ligand binding domain.", "25.The gene expression modulation system of claim 24, wherein said ecdysone receptor ligand binding domain is a Choristoneura fumiferana ecdysone receptor ligand binding domain.", "26.The gene expression modulation system of claim 3, wherein said Group B nuclear receptor ligand binding domain comprising a substitution mutation is a retinoid X receptor ligand binding domain.", "27.The gene expression modulation system of claim 26, wherein said retinoid X receptor ligand binding domain is a Homo sapiens retinoid X receptor ligand binding domain.", "28.A vector comprising the gene expression modulation system of claim 1.29.The vector of claim 28, wherein said vector is an expression vector.", "30.The vector of claim 28, wherein said vector is a viral vector.", "31.The vector of claim 28, wherein said vector is an adenoviral vector.", "32.The vector of claim 28, wherein said vector is a plasmid.", "33.A host cell comprising the gene expression modulation system of claim 1.34.The host cell of claim 33, wherein said host cell is selected from the group consisting of a bacterial cell, a fungal cell, a nematode cell, an insect cell, a fish cell, a plant cell, an avian cell, an animal cell, and a mammalian cell.", "35.The host cell of claim 33, wherein said host cell a mammalian cell.", "36.The host cell of claim 33, wherein said mammalian cell is a human cell." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.", "However, the citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.", "In the field of genetic engineering, precise control of gene expression is a valuable tool for studying, manipulating, and controlling development and other physiological processes.", "Gene expression is a complex biological process involving a number of specific protein-protein interactions.", "In order for gene expression to be triggered, such that it produces the RNA necessary as the first step in protein synthesis, a transcriptional activator must be brought into proximity of a promoter that controls gene transcription.", "Typically, the transcriptional activator itself is associated with a protein that has at least one DNA binding domain that binds to DNA binding sites present in the promoter regions of genes.", "Thus, for gene expression to occur, a protein comprising a DNA binding domain and a transactivation domain located at an appropriate distance from the DNA binding domain must be brought into the correct position in the promoter region of the gene.", "The traditional transgenic approach utilizes a cell-type specific promoter to drive the expression of a designed transgene.", "A DNA construct containing the transgene is first incorporated into a host genome.", "When triggered by a transcriptional activator, expression of the transgene occurs in a given cell type.", "Another means to regulate expression of foreign genes in cells is through inducible promoters.", "Examples of the use of such inducible promoters include the PR1-a promoter, prokaryotic repressor-operator systems, immunosuppressive-immunophilin systems, and higher eukaryotic transcription activation systems such as steroid hormone receptor systems and are described below.", "The PR1-a promoter from tobacco is induced during the systemic acquired resistance response following pathogen attack.", "The use of PR1-a may be limited because it often responds to endogenous materials and external factors such as pathogens, UV-B radiation, and pollutants.", "Gene regulation systems based on promoters induced by heat shock, interferon and heavy metals have been described (Wurn et al., 1986, Proc.", "Natl.", "Acad.", "Sci.", "USA 83: 5414-5418; Arnheiter et al., 1990, Cell 62: 51-61; Filmus et al., 1992 Nucleic Acids Research 20: 27550-27560).", "However, these systems have limitations due to their effect on expression of non-target genes.", "These systems are also leaky.", "Prokaryotic repressor-operator systems utilize bacterial repressor proteins and the unique operator DNA sequences to which they bind.", "Both the tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systems from the bacterium Escherichia coli have been used in plants and animals to control gene expression.", "In the Tet system, tetracycline binds to the TetR repressor protein, resulting in a conformational change that releases the repressor protein from the operator which as a result allows transcription to occur.", "In the Lac system, a lac operon is activated in response to the presence of lactose, or synthetic analogs such as isopropyl-b-D-thiogalactoside.", "Unfortunately, the use of such systems is restricted by unstable chemistry of the ligands, i.e.", "tetracycline and lactose, their toxicity, their natural presence, or the relatively high levels required for induction or repression.", "For similar reasons, utility of such systems in animals is limited.", "Immunosuppressive molecules such as FK506, rapamycin and cyclosporine A can bind to immunophilins FKBP12, cyclophilin, etc.", "Using this information, a general strategy has been devised to bring together any two proteins simply by placing FK506 on each of the two proteins or by placing FK506 on one and cyclosporine A on another one.", "A synthetic homodimer of FK506 (FK1012) or a compound resulted from fusion of FK506-cyclosporine (FKCsA) can then be used to induce dimerization of these molecules (Spencer et al., 1993, Science 262: 1019-24; Belshaw et al., 1996, Proc Natl Acad Sci USA 93: 4604-7).", "Gal4 DNA binding domain fused to FKBP12 and VP16 activator domain fused to cyclophilin, and FKCsA compound were used to show heterodimerization and activation of a reporter gene under the control of a promoter containing Gal4 binding sites.", "Unfortunately, this system includes immunosuppressants that can have unwanted side effects and therefore, limit its use for various mammalian gene switch applications.", "Higher eukaryotic transcription activation systems such as steroid hormone receptor systems have also been employed.", "Steroid hormone receptors are members of the nuclear receptor superfamily and are found in vertebrate and invertebrate cells.", "Unfortunately, use of steroidal compounds that activate the receptors for the regulation of gene expression, particularly in plants and mammals, is limited due to their involvement in many other natural biological pathways in such organisms.", "In order to overcome such difficulties, an alternative system has been developed using insect ecdysone receptors (EcR).", "Growth, molting, and development in insects are regulated by the ecdysone steroid hormone (molting hormone) and the juvenile hormones (Dhadialla, et al., 1998, Annu.", "Rev.", "Entomol.", "43: 545-569).", "The molecular target for ecdysone in insects consists of at least ecdysone receptor (EcR) and ultraspiracle protein (USP).", "EcR is a member of the nuclear steroid receptor super family that is characterized by signature DNA and ligand binding domains, and an activation domain (Koelle et al.", "1991, Cell, 67:59-77).", "EcR receptors are responsive to a number of steroidal compounds such as ponasterone A and muristerone A.", "Recently, non-steroidal compounds with ecdysteroid agonist activity have been described, including the commercially available insecticides tebufenozide and methoxyfenozide that are marketed world wide by Rohm and Haas Company (see International Patent Application No.", "PCT/EP96/00686 and U.S. Pat.", "No.", "5,530,028).", "Both analogs have exceptional safety profiles to other organisms.", "The insect ecdysone receptor (EcR) heterodimerizes with Ultraspiracle (USP), the insect homologue of the mammalian RXR, and binds ecdysteroids and ecdysone receptor response elements and activate transcription of ecdysone responsive genes (Riddiford et al., 2000).", "The EcR/USP/ligand complexes play important roles during insect development and reproduction.", "The EcR is a member of the steroid hormone receptor superfamily and has five modular domains, A/B (transactivation), C (DNA binding, heterodimerization)), D (Hinge, heterodimerization), E (ligand binding, heterodimerization and transactivation and F (transactivation) domains.", "Some of these domains such as A/B, C and E retain their function when they are fused to other proteins.", "Tightly regulated inducible gene expression systems or “gene switches” are useful for various applications such as gene therapy, large scale production of proteins in cells, cell based high throughput screening assays, functional genomics and regulation of traits in transgenic plants and animals.", "The first version of EcR-based gene switch used Drosophila melanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) and showed that these receptors in the presence of steroid, ponasteroneA, transactivate reporter genes in mammalian cell lines and transgenic mice (Christopherson et al., 1992; No et al., 1996).", "Later, Suhr et al., 1998 showed that non-steroidal ecdysone agonist, tebufenozide, induced high level of transactivation of reporter genes in mammalian cells through Bombyx mori EcR (BmEcR) in the absence of exogenous heterodimer partner.", "International Patent Applications No.", "PCT/US97/05330 (WO 97/38117) and PCT/US99/08381 (WO99/58155) disclose methods for modulating the expression of an exogenous gene in which a DNA construct comprising the exogenous gene and an ecdysone response element is activated by a second DNA construct comprising an ecdysone receptor that, in the presence of a ligand therefor, and optionally in the presence of a receptor capable of acting as a silent partner, binds to the ecdysone response element to induce gene expression.", "The ecdysone receptor of choice was isolated from Drosophila melanogaster .", "Typically, such systems require the presence of the silent partner, preferably retinoid X receptor (RXR), in order to provide optimum activation.", "In mammalian cells, insect ecdysone receptor (EcR) heterodimerizes with retinoid X receptor (RXR) and regulates expression of target genes in a ligand dependent manner.", "International Patent Application No.", "PCT/US98/14215 (WO 99/02683) discloses that the ecdysone receptor isolated from the silk moth Bombyx mori is functional in mammalian systems without the need for an exogenous dimer partner.", "U.S. Pat.", "No.", "6,265,173 B1 discloses that various members of the steroid/thyroid superfamily of receptors can combine with Drosophila melanogaster ultraspiracle receptor (USP) or fragments thereof comprising at least the dimerization domain of USP for use in a gene expression system.", "U.S. Pat.", "No.", "5,880,333 discloses a Drosophila melanogaster EcR and ultraspiracle (USP) heterodimer system used in plants in which the transactivation domain and the DNA binding domain are positioned on two different hybrid proteins.", "Unfortunately, these USP-based systems are constitutive in animal cells and therefore, are not effective for regulating reporter gene expression.", "In each of these cases, the transactivation domain and the DNA binding domain (either as native EcR as in International Patent Application No.", "PCT/US98/14215 or as modified EcR as in International Patent Application No.", "PCT/US97/05330) were incorporated into a single molecule and the other heterodimeric partners, either USP or RXR, were used in their native state.", "Drawbacks of the above described EcR-based gene regulation systems include a considerable background activity in the absence of ligands and non-applicability of these systems for use in both plants and animals (see U.S. Pat.", "Nos.", "5,880,333 and 6,265,173 B1).", "For most applications that rely on modulating gene expression, these EcR-based systems are undesirable.", "Therefore, a need exists in the art for improved systems to precisely modulate the expression of exogenous genes in both plants and animals.", "Such improved systems would be useful for applications such as gene therapy, large-scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic animals.", "Improved systems that are simple, compact, and dependent on ligands that are relatively inexpensive, readily available, and of low toxicity to the host would prove useful for regulating biological systems.", "Recently, Applicants have shown that an ecdysone receptor-based inducible gene expression system in which the transactivation and DNA binding domains are separated from each other by placing them on two different proteins results in greatly reduced background activity in the absence of a ligand and significantly increased activity over background in the presence of a ligand (pending application PCT/US01/09050, incorporated herein in its entirety by reference).", "This two-hybrid system is a significantly improved inducible gene expression modulation system compared to the systems disclosed in applications PCT/US97/05330 and PCT/US98/14215.The two-hybrid system exploits the ability of a pair of interacting proteins to bring the transcription activation domain into a more favorable position relative to the DNA binding domain such that when the DNA binding domain binds to the DNA binding site on the gene, the transactivation domain more effectively activates the promoter (see, for example, U.S. Pat.", "No.", "5,283,173).", "Briefly, the two-hybrid gene expression system comprises two gene expression cassettes; the first encoding a DNA binding domain fused to a nuclear receptor polypeptide, and the second encoding a transactivation domain fused to a different nuclear receptor polypeptide.", "In the presence of ligand, the interaction of the first polypeptide with the second polypeptide effectively tethers the DNA binding domain to the transactivation domain.", "Since the DNA binding and transactivation domains reside on two different molecules, the background activity in the absence of ligand is greatly reduced.", "A two-hybrid system also provides improved sensitivity to non-steroidal ligands for example, diarylhydrazines, when compared to steroidal ligands for example, ponasterone A (“PonA”) or muristerone A (“MurA”).", "That is, when compared to steroids, the non-steroidal ligands provide higher activity at a lower concentration.", "In addition, since transactivation based on EcR gene switches is often cell-line dependent, it is easier to tailor switching systems to obtain maximum transactivation capability for each application.", "Furthermore, the two-hybrid system avoids some side effects due to overexpression of RXR that often occur when unmodified RXR is used as a switching partner.", "In a preferred two-hybrid system, native DNA binding and transactivation domains of EcR or RXR are eliminated and as a result, these hybrid molecules have less chance of interacting with other steroid hormone receptors present in the cell resulting in reduced side effects.", "Applicants have recently made the surprising discovery that an invertebrate RXR can function similar to or better than a vertebrate RXR in an ecdysone receptor-based inducible gene expression system (see pending U.S. application 60/294,814, incorporated herein by reference in its entirety).", "RXR is a member of the nuclear receptor superfamily and classified into subfamily 2, Group B (referred to herein as “Group B nuclear receptors”).", "The members of each group share 40-60% amino acid identity in the E (ligand binding) domain (Laudet et al., A Unified Nomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97:161-163).", "In addition to the retinoid X receptor, other members of this nuclear receptor subfamily 2, Group B include: H-2 region II binding protein (H-2RIIBP), nuclear receptor co-regulator-1 (RCoR-1), ultraspiracle (USP), 2C1 nuclear receptor, and chorion factor 1 (CF-1).", "In an effort to provide improved nuclear receptor ligand binding domains, Applicants have now identified amino acid residues within Group B nuclear receptors that affect the ligand sensitivity and magnitude of induction in a nuclear receptor-based inducible gene expression system.", "Applicants describe herein the construction of Group B nuclear receptors that comprise substitution mutations (referred to herein as “substitution mutants”) at these critical residues and the demonstration that these substitution mutant nuclear receptors are useful in methods of modulating gene expression.", "As presented herein, Applicants' novel substitution mutant nuclear receptors and their use in a nuclear receptor-based inducible gene expression system provide an improved inducible gene expression system in both prokaryotic and eukaryotic host cells in which ligand sensitivity and magnitude of transactivation may be selected as desired, depending upon the application." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 : Reporter gene transactivation of VP16/MmRXRα-EF, VP16/LmUSP-EF, or VP16/MmRXRα-EF mutants E265D (RXRmutE/D) or G293S (RXRmutG/S) transfected into NIH3T3 cells along with GAL4/CfEcR-DEF and pFRLuc.", "The cells were grown in the presence of 0, 0.2, 1 and 10 μM GS™-E for 48 hours and the reporter activity was assayed.", "The numbers on the top of the bars show the maximum fold induction.", "FIG.", "2 : Reporter gene transactivation of VP16/MmRXRα-EF (RXR-E), VP16/LmUSP-EF (LmUSP-E), VP16/chimeric vertebrate RXR/invertebrate RXR (Chimera), or VP16/MmRXRα-EF mutants E401D (MutE265D), G429S (MutG293S) or three independent clones of double mutant E401D+G429S (DM1, DM2, and DM4) transfected into NIH3T3 cells along with GAL4/CfEcR-DEF and pFRLuc.", "The cells were grown in the presence of 0, 0.2, 1 and 10 μM GS™-E for 48 hours and the reporter activity was assayed.", "The numbers on the top of the bars show the maximum fold induction.", "FIG.", "3 : Reporter gene transactivation of GAL4CfEcRDEF, pFRLUC and VP16HsRXREFβ or its mutant version DNAs transfected into NIH3T3 cells.", "The transfected cells were grown in the presence of medium containing DMSO or 0.04, 0.2, 1, 5, or 25 μM GS™-E in DMSO.", "Reporter activity was assayed at 48 hours after adding ligands.", "The numbers on the top of the bars show the maximum fold induction.", "FIG.", "4 : Reporter gene transactivation of GAL4CfEcRDEF, pFRLUC and VP16HsRXREFβ or its mutant version DNAs transfected into NIH3T3 cells.", "The transfected cells were grown in the presence of medium containing DMSO or 0.04, 0.2, 1, 5, or 25 μM GS™-E in DMSO.", "Reporter activity was assayed at 48 hours after adding ligands.", "The numbers on the top of the bars show the maximum fold induction.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION This invention relates to the field of biotechnology or genetic engineering.", "Specifically, this invention relates to the field of gene expression.", "More specifically, this invention relates to novel nuclear receptors comprising a substitution mutation and their use in a nuclear receptor-based inducible gene expression system and methods of modulating the expression of a gene within a host cell using this inducible gene expression system.", "BACKGROUND OF THE INVENTION Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.", "However, the citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.", "In the field of genetic engineering, precise control of gene expression is a valuable tool for studying, manipulating, and controlling development and other physiological processes.", "Gene expression is a complex biological process involving a number of specific protein-protein interactions.", "In order for gene expression to be triggered, such that it produces the RNA necessary as the first step in protein synthesis, a transcriptional activator must be brought into proximity of a promoter that controls gene transcription.", "Typically, the transcriptional activator itself is associated with a protein that has at least one DNA binding domain that binds to DNA binding sites present in the promoter regions of genes.", "Thus, for gene expression to occur, a protein comprising a DNA binding domain and a transactivation domain located at an appropriate distance from the DNA binding domain must be brought into the correct position in the promoter region of the gene.", "The traditional transgenic approach utilizes a cell-type specific promoter to drive the expression of a designed transgene.", "A DNA construct containing the transgene is first incorporated into a host genome.", "When triggered by a transcriptional activator, expression of the transgene occurs in a given cell type.", "Another means to regulate expression of foreign genes in cells is through inducible promoters.", "Examples of the use of such inducible promoters include the PR1-a promoter, prokaryotic repressor-operator systems, immunosuppressive-immunophilin systems, and higher eukaryotic transcription activation systems such as steroid hormone receptor systems and are described below.", "The PR1-a promoter from tobacco is induced during the systemic acquired resistance response following pathogen attack.", "The use of PR1-a may be limited because it often responds to endogenous materials and external factors such as pathogens, UV-B radiation, and pollutants.", "Gene regulation systems based on promoters induced by heat shock, interferon and heavy metals have been described (Wurn et al., 1986, Proc.", "Natl.", "Acad.", "Sci.", "USA 83: 5414-5418; Arnheiter et al., 1990, Cell 62: 51-61; Filmus et al., 1992 Nucleic Acids Research 20: 27550-27560).", "However, these systems have limitations due to their effect on expression of non-target genes.", "These systems are also leaky.", "Prokaryotic repressor-operator systems utilize bacterial repressor proteins and the unique operator DNA sequences to which they bind.", "Both the tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systems from the bacterium Escherichia coli have been used in plants and animals to control gene expression.", "In the Tet system, tetracycline binds to the TetR repressor protein, resulting in a conformational change that releases the repressor protein from the operator which as a result allows transcription to occur.", "In the Lac system, a lac operon is activated in response to the presence of lactose, or synthetic analogs such as isopropyl-b-D-thiogalactoside.", "Unfortunately, the use of such systems is restricted by unstable chemistry of the ligands, i.e.", "tetracycline and lactose, their toxicity, their natural presence, or the relatively high levels required for induction or repression.", "For similar reasons, utility of such systems in animals is limited.", "Immunosuppressive molecules such as FK506, rapamycin and cyclosporine A can bind to immunophilins FKBP12, cyclophilin, etc.", "Using this information, a general strategy has been devised to bring together any two proteins simply by placing FK506 on each of the two proteins or by placing FK506 on one and cyclosporine A on another one.", "A synthetic homodimer of FK506 (FK1012) or a compound resulted from fusion of FK506-cyclosporine (FKCsA) can then be used to induce dimerization of these molecules (Spencer et al., 1993, Science 262: 1019-24; Belshaw et al., 1996, Proc Natl Acad Sci USA 93: 4604-7).", "Gal4 DNA binding domain fused to FKBP12 and VP16 activator domain fused to cyclophilin, and FKCsA compound were used to show heterodimerization and activation of a reporter gene under the control of a promoter containing Gal4 binding sites.", "Unfortunately, this system includes immunosuppressants that can have unwanted side effects and therefore, limit its use for various mammalian gene switch applications.", "Higher eukaryotic transcription activation systems such as steroid hormone receptor systems have also been employed.", "Steroid hormone receptors are members of the nuclear receptor superfamily and are found in vertebrate and invertebrate cells.", "Unfortunately, use of steroidal compounds that activate the receptors for the regulation of gene expression, particularly in plants and mammals, is limited due to their involvement in many other natural biological pathways in such organisms.", "In order to overcome such difficulties, an alternative system has been developed using insect ecdysone receptors (EcR).", "Growth, molting, and development in insects are regulated by the ecdysone steroid hormone (molting hormone) and the juvenile hormones (Dhadialla, et al., 1998, Annu.", "Rev.", "Entomol.", "43: 545-569).", "The molecular target for ecdysone in insects consists of at least ecdysone receptor (EcR) and ultraspiracle protein (USP).", "EcR is a member of the nuclear steroid receptor super family that is characterized by signature DNA and ligand binding domains, and an activation domain (Koelle et al.", "1991, Cell, 67:59-77).", "EcR receptors are responsive to a number of steroidal compounds such as ponasterone A and muristerone A.", "Recently, non-steroidal compounds with ecdysteroid agonist activity have been described, including the commercially available insecticides tebufenozide and methoxyfenozide that are marketed world wide by Rohm and Haas Company (see International Patent Application No.", "PCT/EP96/00686 and U.S. Pat.", "No.", "5,530,028).", "Both analogs have exceptional safety profiles to other organisms.", "The insect ecdysone receptor (EcR) heterodimerizes with Ultraspiracle (USP), the insect homologue of the mammalian RXR, and binds ecdysteroids and ecdysone receptor response elements and activate transcription of ecdysone responsive genes (Riddiford et al., 2000).", "The EcR/USP/ligand complexes play important roles during insect development and reproduction.", "The EcR is a member of the steroid hormone receptor superfamily and has five modular domains, A/B (transactivation), C (DNA binding, heterodimerization)), D (Hinge, heterodimerization), E (ligand binding, heterodimerization and transactivation and F (transactivation) domains.", "Some of these domains such as A/B, C and E retain their function when they are fused to other proteins.", "Tightly regulated inducible gene expression systems or “gene switches” are useful for various applications such as gene therapy, large scale production of proteins in cells, cell based high throughput screening assays, functional genomics and regulation of traits in transgenic plants and animals.", "The first version of EcR-based gene switch used Drosophila melanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) and showed that these receptors in the presence of steroid, ponasteroneA, transactivate reporter genes in mammalian cell lines and transgenic mice (Christopherson et al., 1992; No et al., 1996).", "Later, Suhr et al., 1998 showed that non-steroidal ecdysone agonist, tebufenozide, induced high level of transactivation of reporter genes in mammalian cells through Bombyx mori EcR (BmEcR) in the absence of exogenous heterodimer partner.", "International Patent Applications No.", "PCT/US97/05330 (WO 97/38117) and PCT/US99/08381 (WO99/58155) disclose methods for modulating the expression of an exogenous gene in which a DNA construct comprising the exogenous gene and an ecdysone response element is activated by a second DNA construct comprising an ecdysone receptor that, in the presence of a ligand therefor, and optionally in the presence of a receptor capable of acting as a silent partner, binds to the ecdysone response element to induce gene expression.", "The ecdysone receptor of choice was isolated from Drosophila melanogaster.", "Typically, such systems require the presence of the silent partner, preferably retinoid X receptor (RXR), in order to provide optimum activation.", "In mammalian cells, insect ecdysone receptor (EcR) heterodimerizes with retinoid X receptor (RXR) and regulates expression of target genes in a ligand dependent manner.", "International Patent Application No.", "PCT/US98/14215 (WO 99/02683) discloses that the ecdysone receptor isolated from the silk moth Bombyx mori is functional in mammalian systems without the need for an exogenous dimer partner.", "U.S. Pat.", "No.", "6,265,173 B1 discloses that various members of the steroid/thyroid superfamily of receptors can combine with Drosophila melanogaster ultraspiracle receptor (USP) or fragments thereof comprising at least the dimerization domain of USP for use in a gene expression system.", "U.S. Pat.", "No.", "5,880,333 discloses a Drosophila melanogaster EcR and ultraspiracle (USP) heterodimer system used in plants in which the transactivation domain and the DNA binding domain are positioned on two different hybrid proteins.", "Unfortunately, these USP-based systems are constitutive in animal cells and therefore, are not effective for regulating reporter gene expression.", "In each of these cases, the transactivation domain and the DNA binding domain (either as native EcR as in International Patent Application No.", "PCT/US98/14215 or as modified EcR as in International Patent Application No.", "PCT/US97/05330) were incorporated into a single molecule and the other heterodimeric partners, either USP or RXR, were used in their native state.", "Drawbacks of the above described EcR-based gene regulation systems include a considerable background activity in the absence of ligands and non-applicability of these systems for use in both plants and animals (see U.S. Pat.", "Nos.", "5,880,333 and 6,265,173 B1).", "For most applications that rely on modulating gene expression, these EcR-based systems are undesirable.", "Therefore, a need exists in the art for improved systems to precisely modulate the expression of exogenous genes in both plants and animals.", "Such improved systems would be useful for applications such as gene therapy, large-scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic animals.", "Improved systems that are simple, compact, and dependent on ligands that are relatively inexpensive, readily available, and of low toxicity to the host would prove useful for regulating biological systems.", "Recently, Applicants have shown that an ecdysone receptor-based inducible gene expression system in which the transactivation and DNA binding domains are separated from each other by placing them on two different proteins results in greatly reduced background activity in the absence of a ligand and significantly increased activity over background in the presence of a ligand (pending application PCT/US01/09050, incorporated herein in its entirety by reference).", "This two-hybrid system is a significantly improved inducible gene expression modulation system compared to the systems disclosed in applications PCT/US97/05330 and PCT/US98/14215.The two-hybrid system exploits the ability of a pair of interacting proteins to bring the transcription activation domain into a more favorable position relative to the DNA binding domain such that when the DNA binding domain binds to the DNA binding site on the gene, the transactivation domain more effectively activates the promoter (see, for example, U.S. Pat.", "No.", "5,283,173).", "Briefly, the two-hybrid gene expression system comprises two gene expression cassettes; the first encoding a DNA binding domain fused to a nuclear receptor polypeptide, and the second encoding a transactivation domain fused to a different nuclear receptor polypeptide.", "In the presence of ligand, the interaction of the first polypeptide with the second polypeptide effectively tethers the DNA binding domain to the transactivation domain.", "Since the DNA binding and transactivation domains reside on two different molecules, the background activity in the absence of ligand is greatly reduced.", "A two-hybrid system also provides improved sensitivity to non-steroidal ligands for example, diarylhydrazines, when compared to steroidal ligands for example, ponasterone A (“PonA”) or muristerone A (“MurA”).", "That is, when compared to steroids, the non-steroidal ligands provide higher activity at a lower concentration.", "In addition, since transactivation based on EcR gene switches is often cell-line dependent, it is easier to tailor switching systems to obtain maximum transactivation capability for each application.", "Furthermore, the two-hybrid system avoids some side effects due to overexpression of RXR that often occur when unmodified RXR is used as a switching partner.", "In a preferred two-hybrid system, native DNA binding and transactivation domains of EcR or RXR are eliminated and as a result, these hybrid molecules have less chance of interacting with other steroid hormone receptors present in the cell resulting in reduced side effects.", "Applicants have recently made the surprising discovery that an invertebrate RXR can function similar to or better than a vertebrate RXR in an ecdysone receptor-based inducible gene expression system (see pending U.S. application 60/294,814, incorporated herein by reference in its entirety).", "RXR is a member of the nuclear receptor superfamily and classified into subfamily 2, Group B (referred to herein as “Group B nuclear receptors”).", "The members of each group share 40-60% amino acid identity in the E (ligand binding) domain (Laudet et al., A Unified Nomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97:161-163).", "In addition to the retinoid X receptor, other members of this nuclear receptor subfamily 2, Group B include: H-2 region II binding protein (H-2RIIBP), nuclear receptor co-regulator-1 (RCoR-1), ultraspiracle (USP), 2C1 nuclear receptor, and chorion factor 1 (CF-1).", "In an effort to provide improved nuclear receptor ligand binding domains, Applicants have now identified amino acid residues within Group B nuclear receptors that affect the ligand sensitivity and magnitude of induction in a nuclear receptor-based inducible gene expression system.", "Applicants describe herein the construction of Group B nuclear receptors that comprise substitution mutations (referred to herein as “substitution mutants”) at these critical residues and the demonstration that these substitution mutant nuclear receptors are useful in methods of modulating gene expression.", "As presented herein, Applicants' novel substitution mutant nuclear receptors and their use in a nuclear receptor-based inducible gene expression system provide an improved inducible gene expression system in both prokaryotic and eukaryotic host cells in which ligand sensitivity and magnitude of transactivation may be selected as desired, depending upon the application.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1: Reporter gene transactivation of VP16/MmRXRα-EF, VP16/LmUSP-EF, or VP16/MmRXRα-EF mutants E265D (RXRmutE/D) or G293S (RXRmutG/S) transfected into NIH3T3 cells along with GAL4/CfEcR-DEF and pFRLuc.", "The cells were grown in the presence of 0, 0.2, 1 and 10 μM GS™-E for 48 hours and the reporter activity was assayed.", "The numbers on the top of the bars show the maximum fold induction.", "FIG.", "2: Reporter gene transactivation of VP16/MmRXRα-EF (RXR-E), VP16/LmUSP-EF (LmUSP-E), VP16/chimeric vertebrate RXR/invertebrate RXR (Chimera), or VP16/MmRXRα-EF mutants E401D (MutE265D), G429S (MutG293S) or three independent clones of double mutant E401D+G429S (DM1, DM2, and DM4) transfected into NIH3T3 cells along with GAL4/CfEcR-DEF and pFRLuc.", "The cells were grown in the presence of 0, 0.2, 1 and 10 μM GS™-E for 48 hours and the reporter activity was assayed.", "The numbers on the top of the bars show the maximum fold induction.", "FIG.", "3: Reporter gene transactivation of GAL4CfEcRDEF, pFRLUC and VP16HsRXREFβ or its mutant version DNAs transfected into NIH3T3 cells.", "The transfected cells were grown in the presence of medium containing DMSO or 0.04, 0.2, 1, 5, or 25 μM GS™-E in DMSO.", "Reporter activity was assayed at 48 hours after adding ligands.", "The numbers on the top of the bars show the maximum fold induction.", "FIG.", "4: Reporter gene transactivation of GAL4CfEcRDEF, pFRLUC and VP16HsRXREFβ or its mutant version DNAs transfected into NIH3T3 cells.", "The transfected cells were grown in the presence of medium containing DMSO or 0.04, 0.2, 1, 5, or 25 μM GS™-E in DMSO.", "Reporter activity was assayed at 48 hours after adding ligands.", "The numbers on the top of the bars show the maximum fold induction.", "DETAILED DESCRIPTION OF THE INVENTION Applicants have developed a novel nuclear receptor-based inducible gene expression system comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "Applicants have shown that the effect of such a substitution mutation may increase ligand binding activity or ligand sensitivity and may be steroid or non-steroid specific.", "Thus, Applicants' invention provides a Group B nuclear receptor-based inducible gene expression system useful for modulating expression of a gene of interest in a host cell.", "Applicants' novel inducible gene expression system and its use in methods of modulating gene expression in a host cell overcome the limitations of currently available inducible expression systems and provide the skilled artisan with an effective means to control gene expression.", "The present invention is useful for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, orthogonal ligand screening assays, functional genomics, proteomics, metabolomics, and regulation of traits in transgenic organisms, where control of gene expression levels is desirable.", "An advantage of Applicants' invention is that it provides a means to regulate gene expression and to tailor expression levels to suit the user's requirements.", "DEFINITIONS In this disclosure, a number of terms and abbreviations are used.", "The following definitions are provided and should be helpful in understanding the scope and practice of the present invention.", "In a specific embodiment, the term “about” or “approximately” means within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value or range.", "The term “substantially free” means that a composition comprising “A” (where “A” is a single protein, DNA molecule, vector, recombinant host cell, etc.)", "is substantially free of “B” (where “B” comprises one or more contaminating proteins, DNA molecules, vectors, etc.)", "when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is “A”.", "Preferably, “A” comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight.", "It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.", "The term “isolated” for the purposes of the present invention designates a biological material (nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present).", "For example, a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered “isolated”.", "The term “purified” does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds.", "It is rather a relative definition.", "A polynucleotide is in the “purified” state after purification of the starting material or of the natural material by at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.", "A “nucleic acid” is a polymeric compound comprised of covalently linked subunits called nucleotides.", "Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded.", "DNA includes but is not limited to cDNA, genomic DNA, plasmids DNA, synthetic DNA, and semi-synthetic DNA.", "DNA may be linear, circular, or supercoiled.", "A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.", "Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.", "The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.", "Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.", "In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).", "A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.", "The term “fragment” will be understood to mean a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence identical to the reference nucleic acid.", "Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.", "Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a nucleic acid according to the invention.", "As used herein, an “isolated nucleic acid fragment” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.", "An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.", "A “gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.", "“Gene” also refers to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence.", "“Native gene” refers to a gene as found in nature with its own regulatory sequences.", "“Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and/or coding sequences that are not found together in nature.", "Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.", "A chimeric gene may comprise coding sequences derived from different sources and/or regulatory sequences derived from different sources.", "“Endogenous gene” refers to a native gene in its natural location in the genome of an organism.", "A “foreign” gene or “heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer.", "Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.", "A “transgene” is a gene that has been introduced into the genome by a transformation procedure.", "“Heterologous” DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.", "Preferably, the heterologous DNA includes a gene foreign to the cell.", "The term “genome” includes chromosomal as well as mitochondrial, chloroplast and viral DNA or RNA.", "A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., 1989 infra).", "Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference).", "The conditions of temperature and ionic strength determine the “stringency” of the hybridization.", "Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms.", "For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS).", "Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5× or 6×SCC.", "High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5× or 6×SCC.", "Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.", "The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another.", "For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.", "Accordingly, the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as disclosed or used herein as well as those substantially similar nucleic acid sequences.", "In a specific embodiment of the invention, polynucleotides are detected by employing hybridization conditions comprising a hybridization step at Tm of 55° C., and utilizing conditions as set forth above.", "In a preferred embodiment, the Tm is 60° C.; in a more preferred embodiment, the Tm is 63° C.; in an even more preferred embodiment, the Tm is 65° C. Post-hybridization washes also determine stringency conditions.", "One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 minutes (min), then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 minutes, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 minutes.", "A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. Hybridization requires that the two nucleic acids comprise complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.", "The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art.", "The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences.", "The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.", "For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-0.51).", "For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8).", "In a specific embodiment of the invention, polynucleotides are detected by employing hybridization conditions comprising a hybridization step in less than 500 mM salt and at least 37 degrees Celsius, and a washing step in 2×SSPE at least 63 degrees Celsius.", "In a preferred embodiment, the hybridization conditions comprise less than 200 mM salt and at least 37 degrees Celsius for the hybridization step.", "In a more preferred embodiment, the hybridization conditions comprise 2×SSPE and 63 degrees Celsius for both the hybridization and washing steps.", "In one embodiment, the length for a hybridizable nucleic acid is at least about 10 nucleotides.", "Preferable a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least 30 nucleotides.", "Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.", "The term “probe” refers to a single-stranded nucleic acid molecule that can base pair with a complementary single stranded target nucleic acid to form a double-stranded molecule.", "As used herein, the term “oligonucleotide” refers to a nucleic acid, generally of at least 18 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule.", "Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.", "A labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid.", "Oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of a nucleic acid, or to detect the presence of a nucleic acid.", "An oligonucleotide can also be used to form a triple helix with a DNA molecule.", "Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer.", "Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.", "A “primer” is an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions.", "Such primers may be used in a polymerase chain reaction.", "“Polymerase chain reaction” is abbreviated PCR and means an in vitro method for enzymatically amplifying specific nucleic acid sequences.", "PCR involves a repetitive series of temperature cycles with each cycle comprising three stages: denaturation of the template nucleic acid to separate the strands of the target molecule, annealing a single stranded PCR oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase.", "PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.", "“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCR and means an in vitro method for enzymatically producing a target cDNA molecule or molecules from an RNA molecule or molecules, followed by enzymatic amplification of a specific nucleic acid sequence or sequences within the target cDNA molecule or molecules as described above.", "RT-PCR also provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.", "A DNA “coding sequence” is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.", "“Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence.", "Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.", "The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus.", "A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences.", "If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.", "“Open reading frame” is abbreviated ORF and means a length of nucleic acid sequence, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.", "The term “head-to-head” is used herein to describe the orientation of two polynucleotide sequences in relation to each other.", "Two polynucleotides are positioned in a head-to-head orientation when the 5′ end of the coding strand of one polynucleotide is adjacent to the 5′ end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds away from the 5′ end of the other polynucleotide.", "The term “head-to-head” may be abbreviated (5′)-to-(5′) and may also be indicated by the symbols (←→) or (3′←5′5′→3′).", "The term “tail-to-tail” is used herein to describe the orientation of two polynucleotide sequences in relation to each other.", "Two polynucleotides are positioned in a tail-to-tail orientation when the 3′ end of the coding strand of one polynucleotide is adjacent to the 3′ end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds toward the other polynucleotide.", "The term “tail-to-tail” may be abbreviated (3′)-to-(3′) and may also be indicated by the symbols (←→) or (5→3′3′←5′).", "The term “head-to-tail” is used herein to describe the orientation of two polynucleotide sequences in relation to each other.", "Two polynucleotides are positioned in a head-to-tail orientation when the 5′ end of the coding strand of one polynucleotide is adjacent to the 3′ end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds in the same direction as that of the other polynucleotide.", "The term “head-to-tail” may be abbreviated (5′)-to-(3′) and may also be indicated by the symbols (→→) or (5′→3′5′→3′).", "The term “downstream” refers to a nucleotide sequence that is located 3′ to reference nucleotide sequence.", "In particular, downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription.", "For example, the translation initiation codon of a gene is located downstream of the start site of transcription.", "The term “upstream” refers to a nucleotide sequence that is located 5′ to reference nucleotide sequence.", "In particular, upstream nucleotide sequences generally relate to sequences that are located on the 5′ side of a coding sequence or starting point of transcription.", "For example, most promoters are located upstream of the start site of transcription.", "The terms “restriction endonuclease” and “restriction enzyme” refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.", "“Homologous recombination” refers to the insertion of a foreign DNA sequence into another DNA molecule, e.g., insertion of a vector in a chromosome.", "Preferably, the vector targets a specific chromosomal site for homologous recombination.", "For specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome.", "Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.", "Several methods known in the art may be used to propagate a polynucleotide according to the invention.", "Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity.", "As described herein, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.", "A “vector” is any means for the cloning of and/or transfer of a nucleic acid into a host cell.", "A vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment.", "A “replicon” is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.", "The term “vector” includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.", "A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc.", "Possible vectors include, for example, plasmids or modified viruses including, for example bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives, or the Bluescript vector.", "For example, the insertion of the DNA fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate DNA fragments into a chosen vector that has complementary cohesive termini.", "Alternatively, the ends of the DNA molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) into the DNA termini.", "Such vectors may be engineered to contain selectable marker genes that provide for the selection of cells that have incorporated the marker into the cellular genome.", "Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.", "Viral vectors, and particularly retroviral vectors, have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects.", "Viral vectors that can be used include but are not limited to retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors.", "Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.", "In addition to a nucleic acid, a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).", "The term “plasmid” refers to an extra chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules.", "Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.", "A “cloning vector” is a “replicon”, which is a unit length of a nucleic acid, preferably DNA, that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment.", "Cloning vectors may be capable of replication in one cell type and expression in another (“shuttle vector”).", "Vectors may be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol.", "Chem.", "267:963-967; Wu and Wu, 1988, J. Biol.", "Chem.", "263:14621-14624; and Hartmut et al., Canadian Patent Application No.", "2,012,311, filed Mar.", "15, 1990).", "A polynucleotide according to the invention can also be introduced in vivo by lipofection.", "For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro.", "Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., 1987.Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 84:7413; Mackey, et al., 1988.Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 85:8027-8031; and Ulmer et al., 1993.Science 259:1745-1748).", "The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold, 1989.Science 337:387-388).", "Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U.S. Pat.", "No.", "5,459,127.The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages.", "Molecular targeting of liposomes to specific cells represents one area of benefit.", "It is clear that directing transfection to particular cell types would be particularly preferred in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain.", "Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey, et al., 1988, supra).", "Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.", "Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO95/21931).", "It is also possible to introduce a vector in vivo as a naked DNA plasmid (see U.S. Pat.", "Nos.", "5,693,622, 5,589,466 and 5,580,859).", "Receptor-mediated DNA delivery approaches can also be used (Curiel et al., 1992.Hum.", "Gene Ther.", "3:147-154; and Wu and Wu, 1987.J.", "Biol.", "Chem.", "262: 4429-4432).", "The term “transfection” means the uptake of exogenous or heterologous RNA or DNA by a cell.", "A cell has been “transfected” by exogenous or heterologous RNA or DNA when such RNA or DNA has been introduced inside the cell.", "A cell has been “transformed” by exogenous or heterologous RNA or DNA when the transfected RNA or DNA effects a phenotypic change.", "The transforming RNA or DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.", "“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance.", "Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.", "The term “genetic region” will refer to a region of a nucleic acid molecule or a nucleotide sequence that comprises a gene encoding a polypeptide.", "In addition, the recombinant vector comprising a polynucleotide according to the invention may include one or more origins for replication in the cellular hosts in which their amplification or their expression is sought, markers or selectable markers.", "The term “selectable marker” means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest.", "Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like.", "The term “reporter gene” means a nucleic acid encoding an identifying factor that is able to be identified based upon the reporter gene's effect, wherein the effect is used to track the inheritance of a nucleic acid of interest, to identify a cell or organism that has inherited the nucleic acid of interest, and/or to measure gene expression induction or transcription.", "Examples of reporter genes known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like.", "Selectable marker genes may also be considered reporter genes.", "“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.", "In general, a coding sequence is located 3′ to a promoter sequence.", "Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.", "It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions.", "Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.", "Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissue-specific promoters”.", "Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters”.", "Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters”.", "It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.", "A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence.", "For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.", "Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.", "A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.", "“Transcriptional and translational control sequences” are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.", "In eukaryotic cells, polyadenylation signals are control sequences.", "The term “response element” means one or more cis-acting DNA elements which confer responsiveness on a promoter mediated through interaction with the DNA-binding domains of the first chimeric gene.", "This DNA element may be either palindrome (perfect or imperfect) in its sequence or composed of sequence motifs or half sites separated by a variable number of nucleotides.", "The half sites can be similar or identical and arranged as either direct or inverted repeats or as a single half site or multimers of adjacent half sites in tandem.", "The response element may comprise a minimal promoter isolated from different organisms depending upon the nature of the cell or organism into which the response element will be incorporated.", "The DNA binding domain of the first hybrid protein binds, in the presence absence of a ligand, to the DNA sequence of a response element to initiate or suppress transcription of downstream gene(s) under the regulation of this response element.", "Examples of DNA sequences for response elements of the natural ecdysone receptor include: RRGG/TTCANTGAC/ACYY (see Cherbas L., et.", "al., (1991), Genes Dev.", "5, 120-131); AGGTCAN(n)AGGTCA, where N(n) can be one or more spacer nucleotides (see D'Avino PP., et.", "al., (1995), Mol.", "Cell.", "Endocrinol, 113, 1-9); and GGGTTGAATGAATTT (see Antoniewski C., et.", "al., (1994).", "Mol.", "Cell.", "Biol.", "14, 4465-4474).", "The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so, that the function of one is affected by the other.", "For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).", "Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.", "The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid or polynucleotide.", "Expression may also refer to translation of mRNA into a protein or polypeptide.", "The terms “cassette”, “expression cassette” and “gene expression cassette” refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination.", "The segment of DNA comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.", "“Transformation cassette” refers to a specific vector comprising a polynucleotide that encodes a polypeptide of interest and having elements in addition to the polynucleotide that facilitate transformation of a particular host cell.", "Cassettes, expression cassettes, gene expression cassettes and transformation cassettes of the invention may also comprise elements that allow for enhanced expression of a polynucleotide encoding a polypeptide of interest in a host cell.", "These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.", "For purposes of this invention, the term “gene switch” refers to the combination of a response element associated with a promoter, and an EcR based system which, in the presence of one or more ligands, modulates the expression of a gene into which the response element and promoter are incorporated.", "The terms “modulate” and “modulates” mean to induce, reduce or inhibit nucleic acid or gene expression, resulting in the respective induction, reduction or inhibition of protein or polypeptide production.", "The plasmids or vectors according to the invention may further comprise at least one promoter suitable for driving expression of a gene in a host cell.", "The term “expression vector” means a vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence following transformation into the host.", "The cloned gene, i.e., the inserted nucleic acid sequence, is usually placed under the control of control elements such as a promoter, a minimal promoter, an enhancer, or the like.", "Initiation control regions or promoters, which are useful to drive expression of a nucleic acid in the desired host cell are numerous and familiar to those skilled in the art.", "Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to: viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue specific promoter, developmental specific promoters, inducible promoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase promoters (useful for expression in Saccharomyces); AOX1 promoter (useful for expression in Pichia); β-lactamase, lac, ara, tet, trp, lPL, lPR, T7, tac, and trc promoters (useful for expression in Escherichia coli); light regulated-, seed specific-, pollen specific-, ovary specific-, pathogenesis or disease related-, cauliflower mosaic virus 35S, CMV 35S minimal, cassaya vein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose 1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase, stress inducible, rice tungro bacilliform virus, plant super-promoter, potato leucine aminopeptidase, nitrate reductase, mannopine synthase, nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters (useful for expression in plant cells); animal and mammalian promoters known in the art include, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3′ long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, an elongation factor 1 alpha (EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), pathogenesis or disease related-promoters, and promoters that exhibit tissue specificity and have been utilized in transgenic animals, such as the elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region active in pancreatic beta cells, immunoglobulin gene control region active in lymphoid cells, mouse mammary tumor virus control region active in testicular, breast, lymphoid and mast cells; albumin gene, Apo AI and Apo AII control regions active in liver, alpha-fetoprotein gene control region active in liver, alpha 1-antitrypsin gene control region active in the liver, beta-globin gene control region active in myeloid cells, myelin basic protein gene control region active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region active in skeletal muscle, and gonadotropic releasing hormone gene control region active in the hypothalamus, pyruvate kinase promoter, villin promoter, promoter of the fatty acid binding intestinal protein, promoter of the smooth muscle cell α-actin, and the like.", "In addition, these expression sequences may be modified by addition of enhancer or regulatory sequences and the like.", "Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor 1 (EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.", "Termination control regions, i.e., terminator or polyadenylation sequences, may also be derived from various genes native to the preferred hosts.", "Optionally, a termination site may be unnecessary, however, it is most preferred if included.", "In a preferred embodiment of the invention, the termination control region may be comprise or be derived from a synthetic sequence, synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, or the like.", "The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)” refer to DNA sequences located downstream (3′) of a coding sequence and may comprise polyadenylation [poly(A)] recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.", "The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor.", "“Regulatory region” means a nucleic acid sequence that regulates the expression of a second nucleic acid sequence.", "A regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin that are responsible for expressing different proteins or even synthetic proteins (a heterologous region).", "In particular, the sequences can be sequences of prokaryotic, eukaryotic, or viral genes or derived sequences that stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner.", "Regulatory regions include origins of replication, RNA splice sites, promoters, enhancers, transcriptional termination sequences, and signal sequences which direct the polypeptide into the secretory pathways of the target cell.", "A regulatory region from a “heterologous source” is a regulatory region that is not naturally associated with the expressed nucleic acid.", "Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences which do not occur in nature, but which are designed by one having ordinary skill in the art.", "“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence.", "When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA.", "“Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into protein by the cell.", "“cDNA” refers to a double-stranded DNA that is complementary to and derived from mRNA.", "“Sense” RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell.", "“Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene.", "The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or the coding sequence.", "“Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet has an effect on cellular processes.", "A “polypeptide” is a polymeric compound comprised of covalently linked amino acid residues.", "Amino acids have the following general structure: Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.", "A polypeptide of the invention preferably comprises at least about 14 amino acids.", "A “protein” is a polypeptide that performs a structural or functional role in a living cell.", "An “isolated polypeptide” or “isolated protein” is a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids).", "“Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.", "A “substitution mutant polypeptide” or a “substitution mutant” will be understood to mean a mutant polypeptide comprising a substitution of at least one (1) wild-type or naturally occurring amino acid with a different amino acid relative to the wild-type or naturally occurring polypeptide.", "A substitution mutant polypeptide may comprise only one (1) wild-type or naturally occurring amino acid substitution and may be referred to as a “point mutant” or a “single point mutant” polypeptide.", "Alternatively, a substitution mutant polypeptide may comprise a substitution of two (2) or more wild-type or naturally occurring amino acids with 2 or more amino acids relative to the wild-type or naturally occurring polypeptide.", "According to the invention, a Group B nuclear receptor ligand binding domain polypeptide comprising a substitution mutation comprises a substitution of at least one (1) wild-type or naturally occurring amino acid with a different amino acid relative to the wild-type or naturally occurring Group B nuclear receptor ligand binding domain polypeptide.", "Wherein the substitution mutant polypeptide comprises a substitution of two (2) or more wild-type or naturally occurring amino acids, this substitution may comprise either an equivalent number of wild-type or naturally occurring amino acids deleted for the substitution, i.e., 2 wild-type or naturally occurring amino acids replaced with 2 non-wild-type or non-naturally occurring amino acids, or a non-equivalent number of wild-type amino acids deleted for the substitution, i.e., 2 wild-type amino acids replaced with 1 non-wild-type amino acid (a substitution+deletion mutation), or 2 wild-type amino acids replaced with 3 non-wild-type amino acids (a substitution+insertion mutation).", "Substitution mutants may be described using an abbreviated nomenclature system to indicate the amino acid residue and number replaced within the reference polypeptide sequence and the new substituted amino acid residue.", "For example, a substitution mutant in which the twentieth (20th) amino acid residue of a polypeptide is substituted may be abbreviated as “x20z”, wherein “x” is the amino acid to be replaced, “20” is the amino acid residue position or number within the polypeptide, and “z” is the new substituted amino acid.", "Therefore, a substitution mutant abbreviated interchangeably as “E20A” or “Glu20Ala” indicates that the mutant comprises an alanine residue (commonly abbreviated in the art as “A” or “Ala”) in place of the glutamic acid (commonly abbreviated in the art as “E” or “Glu”) at position 20 of the polypeptide.", "A substitution mutation may be made by any technique for mutagenesis known in the art, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol.", "Chem.", "253:6551; Zoller and Smith, 1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 83:710), use of TAB® linkers (Pharmacia), restriction endonuclease digestion/fragment deletion and substitution, PCR-mediated/oligonucleotide-directed mutagenesis, and the like.", "PCR-based techniques are preferred for site-directed mutagenesis (see Higuchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp.", "61-70).", "“Fragment” of a polypeptide according to the invention will be understood to mean a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence.", "Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part.", "Such fragments of a polypeptide according to the invention may have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200, 240, or 300 amino acids.", "A “variant” of a polypeptide or protein is any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein.", "Different variants of the polypeptide or protein may exist in nature.", "These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification.", "The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements.", "These variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) variants in which one or more of the amino acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin.", "The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.", "), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.", "A variant polypeptide preferably comprises at least about 14 amino acids.", "A “heterologous protein” refers to a protein not naturally produced in the cell.", "A “mature protein” refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.", "“Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present.", "Pre- and propeptides may be but are not limited to intracellular localization signals.", "The term “signal peptide” refers to an amino terminal polypeptide preceding the secreted mature protein.", "The signal peptide is cleaved from and is therefore not present in the mature protein.", "Signal peptides have the function of directing and translocating secreted proteins across cell membranes.", "Signal peptide is also referred to as signal protein.", "A “signal sequence” is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell.", "This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide.", "The term “translocation signal sequence” is used herein to refer to this sort of signal sequence.", "Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.", "The term “homology” refers to the percent of identity between two polynucleotide or two polypeptide moieties.", "The correspondence between the sequence from one moiety to another can be determined by techniques known to the art.", "For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs.", "Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s) and size determination of the digested fragments.", "As used herein, the term “homologous” in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a “common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.)", "(Reeck et al., 1987, Cell 50:667.).", "Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity.", "However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and not a common evolutionary origin.", "Accordingly, the term “sequence similarity” in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., 1987, Cell 50:667).", "In a specific embodiment, two DNA sequences are “substantially homologous” or “substantially similar” when at least about 50% (preferably at least about 75%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences.", "Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.", "Defining appropriate hybridization conditions is within the skill of the art.", "See, e.g., Sambrook et al., 1989, supra.", "As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence.", "“Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology.", "“Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript.", "It is therefore understood that the invention encompasses more than the specific exemplary sequences.", "Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.", "Moreover, the skilled artisan recognizes that substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), with the sequences exemplified herein.", "Substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 70% identical to the DNA sequence of the nucleic acid fragments reported herein.", "Preferred substantially nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein.", "More preferred nucleic acid fragments are at least 90% identical to the DNA sequence of the nucleic acid fragments reported herein.", "Even more preferred are nucleic acid fragments that are at least 95% identical to the DNA sequence of the nucleic acid fragments reported herein.", "Two amino acid sequences are “substantially homologous” or “substantially similar” when greater than about 40% of the amino acids are identical, or greater than 60% are similar (functionally identical).", "Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program.", "The term “corresponding to” is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.", "A nucleic acid or amino acid sequence alignment may include spaces.", "Thus, the term “corresponding to” refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.", "A “substantial portion” of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol.", "Biol.", "215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/).", "In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.", "Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).", "In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.", "Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.", "The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.", "In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.", "“Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.)", "Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)", "Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.)", "Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.)", "Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.)", "Stockton Press, New York (1991).", "Preferred methods to determine identity are designed to give the best match between the sequences tested.", "Methods to determine identity and similarity are codified in publicly available computer programs.", "Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).", "Multiple alignment of the sequences may be performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS.", "5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).", "Default parameters for pairwise alignments using the Clustal method may be selected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences.", "“Sequence analysis software” may be commercially available or independently developed.", "Typical sequence analysis software will include but is not limited to the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol.", "Biol.", "215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA).", "Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified.", "As used herein “default values” will mean any set of values or parameters which originally load with the software when first initialized.", "“Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art.", "These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene.", "“Chemically synthesized”, as related to a sequence of DNA, means that the component nucleotides were assembled in vitro.", "Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines.", "Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell.", "The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host.", "Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.", "As used herein, two or more individually operable gene regulation systems are said to be “orthogonal” when; a) modulation of each of the given systems by its respective ligand, at a chosen concentration, results in a measurable change in the magnitude of expression of the gene of that system, and b) the change is statistically significantly different than the change in expression of all other systems simultaneously operable in the cell, tissue, or organism, regardless of the simultaneity or sequentially of the actual modulation.", "Preferably, modulation of each individually operable gene regulation system effects a change in gene expression at least 2-fold greater than all other operable systems in the cell, tissue, or organism.", "More preferably, the change is at least 5-fold greater.", "Even more preferably, the change is at least 10-fold greater.", "Still more preferably, the change is at least 100 fold greater.", "Even still more preferably, the change is at least 500-fold greater.", "Ideally, modulation of each of the given systems by its respective ligand at a chosen concentration results in a measurable change in the magnitude of expression of the gene of that system and no measurable change in expression of all other systems operable in the cell, tissue, or organism.", "In such cases the multiple inducible gene regulation system is said to be “fully orthogonal”.", "The present invention is useful to search for orthogonal ligands and orthogonal receptor-based gene expression systems such as those described in co-pending U.S. application 60/237,446, which is incorporated herein by reference in its entirety.", "Gene Expression Modulation System of the Invention Thus, Applicants have identified herein amino acid residues that affect the ligand sensitivity and magnitude of induction in a Group B-based inducible gene expression system.", "Applicants describe herein the construction of Group B nuclear receptors that comprise substitution mutations (referred to herein as “substitution mutants”) at these critical residues and the demonstration that these substitution mutant nuclear receptors are useful in methods of modulating gene expression.", "As presented herein, Applicants' novel substitution mutant nuclear receptors and their use in a nuclear receptor-based inducible gene expression system provides an improved inducible gene expression system in both prokaryotic and eukaryotic host cells in which ligand sensitivity and magnitude of transactivation may be selected as desired, depending upon the application.", "Thus, the present invention relates to novel substitution mutant Group B nuclear receptor polynucleotides and polypeptides, a nuclear receptor-based inducible gene expression system comprising such mutated Group B nuclear receptor polynucleotides and polypeptides, and methods of modulating the expression of a gene within a host cell using such a nuclear receptor-based inducible gene expression system.", "In particular, the present invention relates to a gene expression modulation system comprising at least one gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "Preferably, the Group B nuclear receptor ligand binding domain comprising a substitution mutation is from a retinoid X receptor α, retinoid X receptor β, retinoid X receptor γ, H-2 region II binding protein (H-2RIIBP), Nuclear Receptor co-regulator-1 (RCoR-1), ultraspiracle protein, 2C1 nuclear receptor, and chorion factor 1 (CF-1).", "More preferably, the Group B nuclear receptor ligand binding domain comprising a substitution mutation is from a vertebrate retinoid X receptor α, vertebrate retinoid X receptor β, vertebrate retinoid X receptor γ, or an invertebrate retinoid X receptor.", "In a specific embodiment, the gene expression modulation system comprises a) a first gene expression cassette comprising a polynucleotide that encodes a first polypeptide comprising a transactivation domain, a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and a Group B nuclear receptor ligand binding domain comprising a substitution mutation, and b) a second gene expression cassette comprising a polynucleotide that encodes a second polypeptide comprising a nuclear receptor ligand binding domain.", "The gene expression modulation system may further comprise a third gene expression cassette comprising: i) a response element recognized by the DNA-binding domain of the first polypeptide; ii) a promoter that is activated by the transactivation domain of the first polypeptide; and iii) a gene whose expression is to be modulated.", "In a preferred embodiment, the second polypeptide comprises a Group H nuclear receptor ligand binding domain selected from the group consisting of an ecdysone receptor (EcR), a ubiquitous receptor (UR), an orphan receptor 1 (OR-1), an NER-1, a receptor-interacting protein 15 (RIP-15), a liver X receptor β (LXRβ), a steroid hormone receptor-like protein (RLD-1), a liver X receptor (LXR), a liver X receptor (LXRα), a framesoid X receptor (FXR), a receptor-interacting protein 14 (RIP-14), and a farnesol receptor (HRR-1) ligand binding domains.", "In another specific embodiment, the gene expression modulation system comprises a first gene expression cassette comprising a polynucleotide that encodes a first polypeptide comprising a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated and a first nuclear receptor ligand binding domain, and a second gene expression cassette comprising a polynucleotide that encodes a second polypeptide comprising a transactivation domain and a second nuclear receptor ligand binding domain, wherein one of the nuclear receptor ligand binding domains is a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "In a preferred embodiment, the first polypeptide is substantially free of a transactivation domain and the second polypeptide is substantially free of a DNA binding domain.", "For purposes of the invention, “substantially free” means that the protein in question does not contain a sufficient sequence of the domain in question to provide activation or binding activity.", "The gene expression modulation system may further comprise a third gene expression cassette comprising: i) a response element recognized by the DNA-binding domain of the first polypeptide of the first gene expression cassette; ii) a promoter that is activated by the transactivation domain of the second polypeptide of the second gene expression cassette; and iii) a gene whose expression is to be modulated.", "In a preferred embodiment, when only one nuclear receptor ligand binding domain is a Group B ligand binding domain comprising a substitution mutation, the other nuclear receptor ligand binding domain may be from any other nuclear receptor that forms a dimer with the Group B ligand binding domain comprising the substitution mutation.", "In a specific embodiment, the Group B nuclear receptor ligand binding domain comprising a substitution mutation is a retinoid X receptor ligand binding domain comprising a substitution mutation, and the other nuclear receptor ligand binding domain (“partner”) is from a Group H nuclear receptor.", "In a preferred embodiment, the Group H nuclear receptor ligand binding domain is selected from the group consisting of an ecdysone receptor (EcR), a ubiquitous receptor (UR), an orphan receptor 1 (OR-1), an NER-1, a receptor-interacting protein 15 (RIP-15), a liver X receptor β (LXRβ), a steroid hormone receptor-like protein (RLD-1), a liver X receptor (LXR), a liver X receptor (LXRα), a farnesoid X receptor (FXR), a receptor-interacting protein 14 (RIP-14), and a farnesol receptor (HRR-1) ligand binding domains.", "The ecdysone receptor (EcR) ligand binding domain (LBD) may be from an invertebrate EcR, preferably selected from the class of Arthropod EcR.", "Preferably, the EcR is selected from the group consisting of a Lepidopteran EcR, a Dipteran EcR an Orthopteran EcR, a Homopteran EcR and a Hemipteran EcR.", "More preferably, the EcR ligand binding domain for use in the present invention is from a spruce budworm Choristoneura fumiferana EcR (“CfEcR”), a beetle Tenebrio molitor EcR (“TmEcR”), a Manduca sexta EcR (“MsEcR”), a Heliothies virescens EcR (“HvEcR”), a midge Chironomus tentans EcR (“CtEcR”), a silk moth Bombyx mori EcR (“BmEcR”), a squinting bush brown Bicyclus anynana EcR (“BanEcR”), a buckeye Junonia coenia EcR (“JcEcR”), a fruit fly Drosophila melanogaster EcR (“DmEcR”), a mosquito Aedes aegypti EcR (“AaEcR”), a blowfly Lucilia capitata (“LcEcR”), a blowfly Lucilia cuprina EcR (“LucEcR”), a blowfly Calliphora vicinia EcR (“CvEcR”), a Mediterranean fruit fly Ceratitis capitata EcR (“CcEcR”), a locust Locusta migratoria EcR (“LmEcR”), an aphid Myzus persicae EcR (“MpEcR”), a fiddler crab Celuca pugilator EcR (“CpEcR”), a whitefly Bamecia argentifoli EcR (“BaEcR”, SEQ ID NO: 74), a leafhopper Nephotetix cincticeps EcR (“NcEcR”, SEQ ID NO: 75) or an ixodid tick Amblyomma americanum EcR (“AmaEcR”).", "More preferably, the LBD is from a CfEcR, a DmEcR, or an AmaEcR.", "The “partner” nuclear receptor ligand binding domain may further comprise a truncation mutation, a deletion mutation, a second substitution mutation, or another modification.", "In a specific embodiment, the LBD is from a truncated EcR polypeptide.", "The EcR polypeptide truncation results in a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids.", "Preferably, the EcR polypeptide truncation results in a deletion of at least a partial polypeptide domain.", "More preferably, the EcR polypeptide truncation results in a deletion of at least an entire polypeptide domain.", "In a specific embodiment, the EcR polypeptide truncation results in a deletion of at least an A/B-domain, a C-domain, a D-domain, an F-domain, an A/B/C-domains, an A/B/1/2-C-domains, an A/B/C/D-domains, an A/B/C/D/F-domains, an A/B/F-domains, an A/B/C/F-domains, a partial E domain, or a partial F domain.", "A combination of several complete and/or partial domain deletions may also be performed.", "In a specific embodiment, the gene whose expression is to be modulated is a homologous gene with respect to the host cell.", "In another specific embodiment, the gene whose expression is to be modulated is a heterologous gene with respect to the host cell.", "The ligands for use in the present invention as described below, when combined with the ligand binding domain of the nuclear receptor(s), which in turn are bound to the response element linked to a gene, provide the means for external temporal regulation of expression of the gene.", "The binding mechanism or the order in which the various components of this invention bind to each other, that is, for example, ligand to ligand binding domain, DNA-binding domain to response element, transactivation domain to promoter, etc., is not critical.", "In a specific example, binding of the ligand to the ligand binding domain of a Group B nuclear receptor and optionally, its nuclear receptor ligand binding domain partner enables expression or suppression of the gene.", "This mechanism does not exclude the potential for ligand binding to the Group B nuclear receptor (GBNR) or its partner, and the resulting formation of active homodimer complexes (e.g.", "GBNR+GBNR or partner+partner).", "Preferably, one or more of the receptor domains is varied producing a hybrid gene switch.", "Typically, one or more of the three domains, a DNA binding domain (DBD), a ligand binding domain (LBD), and a transactivation domain (AD), may be chosen from a source different than the source of the other domains so that the hybrid genes and the resulting hybrid proteins are optimized in the chosen host cell or organism for transactivating activity, complementary binding of the ligand, and recognition of a specific response element.", "In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein from yeast (see Sadowski, et al.", "(1988) Nature, 335:563-564) or LexA protein from Escherichia coli (see Brent and Ptashne (1985), Cell, 43:729-736), or synthetic response elements specific for targeted interactions with proteins designed, modified, and selected for such specific interactions (see, for example, Kim, et al.", "(1997), Proc.", "Natl.", "Acad.", "Sci., USA, 94: 3616-3620) to accommodate hybrid receptors.", "Another advantage of the two-hybrid systems is that they allow choice of a promoter used to drive the gene expression according to a desired end result.", "Such double control can be particularly important in areas of gene therapy, especially when cytotoxic proteins are produced, because both the timing of expression as well as the cells wherein expression occurs can be controlled.", "When genes, operably linked to a suitable promoter, are introduced into the cells of the subject, expression of the exogenous genes is controlled by the presence of the system of this invention.", "Promoters may be constitutively or inducibly regulated or may be tissue-specific (that is, expressed only in a particular type of cell) or specific to certain developmental stages of the organism.", "The retinoid X receptor is a member of the nuclear receptor superfamily and classified into subfamily 2, Group B (referred to herein as “Group B nuclear receptors”).", "The members of each group share 40-60% amino acid identity in the E (ligand binding) domain (Laudet et al., A Unified Nomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97: 161-163).", "In addition to the retinoid X receptor, other members of this nuclear receptor subfamily 2, Group B include: H-2 region II binding protein (H-2RIIBP), Nuclear Receptor co-regulator-1 (RCoR-1), ultraspiracle protein (USP), 2C1 nuclear receptor, and chorion factor 1 (CF-1).", "Applicants previously demonstrated that a vertebrate RXR in partnership with an ecdysone receptor-based gene expression system provides an inducible gene expression system in yeast and mammalian cells that is characterized by increased ligand sensitivity and magnitude of transactivation (see pending application PCT/US01/09050).", "Recently, Applicants have shown that an invertebrate RXR can function as well as or better than a vertebrate RXR in an ecdysone receptor-based gene expression system by increasing gene transactivation and ligand sensitivity of the gene expression system (see pending application U.S. 60/294,814).", "As described herein, Applicants have now identified critical amino acid residues within the ligand binding domain of RXRs that affect transactivation and ligand sensitivity of a nuclear receptor-based expression system.", "In Examples 2-4 infra, Applicants have identified amino acids from invertebrate retinoid X receptors (see Examples 2 and 3) and invertebrate ultraspiracle proteins (see Example 4) ligand binding domains that differ from amino acids of vertebrate RXRs.", "Applicants made substitution mutants within vertebrate RXR ligand binding domains by replacing the wild type vertebrate RXR amino acid with that of an invertebrate RXR or USP at their analogous position and tested these substitution mutants for their ability to transactivate gene expression in a nuclear receptor-based inducible gene expression system.", "As presented in the Examples herein, Applicants have now identified several novel retinoid X receptor ligand binding domain substitution mutants that exhibit unexpected and surprising levels of transactivation activity and/or ligand sensitivity.", "Given the close relatedness of retinoid X receptor to other Group B nuclear receptors, Applicants' identified retinoid X receptor ligand binding domain substitution mutations are also expected to work when introduced into the analogous position of the ligand binding domains of other Group B nuclear receptors to modify ligand binding or ligand sensitivity in a Group B nuclear receptor-based gene expression system.", "Applicants' novel substitution mutated Group B nuclear receptor ligand binding domain polynucleotides and polypeptides are useful in a nuclear receptor-based inducible gene modulation system for various applications including gene therapy, expression of proteins of interest in host cells, production of transgenic organisms, and cell-based assays.", "In particular, Applicants describe herein a novel gene expression modulation system comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "This gene expression system may be a “single switch”-based gene expression system in which the transactivation domain, DNA-binding domain and ligand binding domain are on one encoded polypeptide.", "Alternatively, the gene expression modulation system may be a “dual switch”- or “two-hybrid”-based gene expression modulation system in which the transactivation domain and DNA-binding domain are located on two different encoded polypeptides.", "Applicants have demonstrated for the first time that a substitution mutated nuclear receptor can be used as a component of a nuclear receptor-based inducible gene expression system to modify ligand binding activity and/or ligand specificity in both prokaryotic and eukaryotic cells.", "As discussed herein, Applicants' findings are both unexpected and surprising.", "Preferably the Group B nuclear receptor-based gene expression modulation system of the present invention may be either heterodimeric and homodimeric.", "In a preferred embodiment, the Group B nuclear receptor ligand binding domain heterodimerizes with an ecdysone receptor ligand binding domain to form a function EcR complex.", "A functional EcR complex generally refers to a heterodimeric protein complex consisting of two members of the steroid receptor family, an ecdysone receptor protein obtained from various insects, and an ultraspiracle (USP) protein or the vertebrate homolog of USP, retinoid X receptor protein (see Yao, et al.", "(1993) Nature 366, 476-479; Yao, et al., (1992) Cell 71, 63-72).", "However, the complex may also be a homodimer as detailed below.", "The functional ecdysteroid receptor complex may also include additional protein(s) such as immunophilins.", "Additional members of the steroid receptor family of proteins, known as transcriptional factors (such as DHR38 or betaFTZ-1), may also be ligand dependent or independent partners for EcR, USP, and/or RXR.", "Additionally, other cofactors may be required such as proteins generally known as coactivators (also termed adapters or mediators).", "These proteins do not bind sequence-specifically to DNA and are not involved in basal transcription.", "They may exert their effect on transcription activation through various mechanisms, including stimulation of DNA-binding of activators, by affecting chromatin structure, or by mediating activator-initiation complex interactions.", "Examples of such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the promiscuous coactivator C response element B binding protein, CBP/p300 (for review see Glass et al., Curr.", "Opin.", "Cell Biol.", "9: 222-232, 1997).", "Also, protein cofactors generally known as corepressors (also known as repressors, silencers, or silencing mediators) may be required to effectively inhibit transcriptional activation in the absence of ligand.", "These corepressors may interact with the unliganded ecdysone receptor to silence the activity at the response element.", "Current evidence suggests that the binding of ligand changes the conformation of the receptor, which results in release of the corepressor and recruitment of the above described coactivators, thereby abolishing their silencing activity.", "Examples of corepressors include N-CoR and SMRT (for review, see Horwitz et al.", "Mol.", "Endocrinol.", "10 1167-1177, 1996).", "These cofactors may either be endogenous within the cell or organism, or may be added exogenously as transgenes to be expressed in either a regulated or unregulated fashion.", "Homodimer complexes of the ecdysone receptor protein, USP, or RXR may also be functional under some circumstances.", "The ecdysone receptor complex typically includes proteins that are members of the nuclear receptor superfamily wherein all members are generally characterized by the presence of an amino-terminal transactivation domain, a DNA binding domain (“DBD”), and a ligand binding domain (“LBD”) separated from the DBD by a hinge region.", "As used herein, the term “DNA binding domain” comprises a minimal polypeptide sequence of a DNA binding protein, up to the entire length of a DNA binding protein, so long as the DNA binding domain functions to associate with a particular response element.", "Members of the nuclear receptor superfamily are also characterized by the presence of four or five domains: AB, C, D, E, and in some members F (see U.S. Pat.", "No.", "4,981,784 and Evans, Science 240:889-895 (1988)).", "The “A/B” domain corresponds to the transactivation domain, “C” corresponds to the DNA binding domain, “D” corresponds to the hinge region, and “E” corresponds to the ligand binding domain.", "Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD corresponding to “F”.", "The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for ecdysone response elements.", "These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins.", "The EcR receptor, like a subset of the steroid receptor family, also possesses less well-defined regions responsible for heterodimerization properties.", "Because the domains of nuclear receptors are modular in nature, the LBD, DBD, and transactivation domains may be interchanged.", "Gene switch systems are known that incorporate components from the ecdysone receptor complex.", "However, in these known systems, whenever EcR is used it is associated with native or modified DNA binding domains and transactivation domains on the same molecule.", "USP or RXR are typically used as silent partners.", "Applicants have previously shown that when DNA binding domains and transactivation domains are on the same molecule the background activity in the absence of ligand is high and that such activity is dramatically reduced when DNA binding domains and transactivation domains are on different molecules, that is, on each of two partners of a heterodimeric or homodimeric complex (see PCT/US01/09050).", "Gene Expression Cassettes of the Invention The novel nuclear receptor-based inducible gene expression system of the invention comprises at least one gene expression cassette that is capable of being expressed in a host cell, wherein the gene expression cassette comprises a polynucleotide that encodes a polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "Thus, Applicants' invention also provides novel gene expression cassettes for use in the gene expression system of the invention.", "In a specific embodiment, the gene expression cassette that is capable of being expressed in a host cell comprises a polynucleotide that encodes a polypeptide selected from the group consisting of a) a polypeptide comprising a transactivation domain, a DNA-binding domain, and a Group B nuclear receptor ligand binding domain comprising a substitution mutation; b) a polypeptide comprising a DNA-binding domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation; and c) a polypeptide comprising a transactivation domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "In another specific embodiment, the present invention provides a gene expression cassette that is capable of being expressed in a host cell, wherein the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide selected from the group consisting of a) a hybrid polypeptide comprising a transactivation domain, a DNA-binding domain, and a Group B nuclear receptor ligand binding domain comprising a substitution mutation; b) a hybrid polypeptide comprising a DNA-binding domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation; and c) a hybrid polypeptide comprising a transactivation domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "A hybrid polypeptide according to the invention comprises at least two polypeptide fragments, wherein each polypeptide fragment is from a different source, i.e., a different nuclear receptor, a different species, etc.", "The hybrid polypeptide according to the invention may comprise at least two polypeptide domains, wherein each polypeptide domain is from a different source.", "In a specific embodiment, the Group B nuclear receptor ligand binding domain comprising a substitution mutation is from an retinoid X receptor α (RXRα), retinoid X receptor β (RXRβ), retinoid X receptor γ (RXRγ), H-2 region II binding protein (H-2RIIBP), nuclear receptor co-regulator-1 (RCoR-1), ultraspiracle protein (USP), 2C1 nuclear receptor, or chorion factor 1 (CF-1).", "In a preferred embodiment, the Group B nuclear receptor ligand binding domain is from a vertebrate retinoid X receptor α, vertebrate retinoid X receptor β, vertebrate retinoid X receptor γ, or an invertebrate retinoid X receptor.", "Thus, the present invention also provides a gene expression cassette comprising a polynucleotide that encodes a polypeptide selected from the group consisting of a) a polypeptide comprising a transactivation domain, a DNA-binding domain, and a retinoid X receptor ligand binding domain comprising a substitution mutation; b) a polypeptide comprising a DNA-binding domain and a retinoid X receptor ligand binding domain comprising a substitution mutation; and c) a polypeptide comprising a transactivation domain and a retinoid X receptor ligand binding domain comprising a substitution mutation.", "Preferably, the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide selected from the group consisting of a) a hybrid polypeptide comprising a transactivation domain, a DNA-binding domain, and a retinoid X receptor ligand binding domain comprising a substitution mutation; b) a hybrid polypeptide comprising a DNA-binding domain and a retinoid X receptor ligand binding domain comprising a substitution mutation; and c) a hybrid polypeptide comprising a transactivation domain and a retinoid X receptor ligand binding domain comprising a substitution mutation; wherein the encoded hybrid polypeptide comprises at least two polypeptide fragments, wherein each polypeptide fragment is from a different source.", "The retinoid X receptor (RXR) ligand binding domain (LBD) may be from an invertebrate or vertebrate RXR.", "Preferably, the RXR ligand binding domain for use in the present invention is from a human Homo sapiens (HsRXR), a mouse Mus musculus (MmRXR), a rat Rattus norvegicus (RnRXR), a chicken Gallus gallus (GgRXR), a domestic pig Sus scrofa domestica (SsRXR), a frog Xenopus laevis (XIRXR), a zebra fish Danio rerio (DrRXR), a beetle Tenebrio molitor RXR homolog (“TmRXR”), a locust Locusta migratoria USP/RXR homolog (referred to as either “LmUSP” or “LmRXR”), an aphid Myzus persicae RXR homolog (“MpRXR”), a honeybee Apis mellifera RXR homolog (“AmRXR”), a fiddler crab Celuca pugilator RXR homolog (“CpRXR”), an ixodid tick Amblyomma americanum RXR homolog 1 (“AmaRXR1”), an ixodid tick Amblyomma americanum RXR homolog 2 (“AmaRXR2”), a tunicate Polyandrocarpa misakiensis (PmRXR), or a jellyfish Tripedalia cystophora RXR.", "(TcRXR).", "More preferably, the LBD is from a MmRXR or a HsRXR.", "In a specific embodiment, the Group B nuclear receptor ligand binding domain is encoded by a polynucleotide comprising a codon mutation that results in a substitution of an amino acid residue, wherein the amino acid residue is at a position equivalent or analogous to an amino acid residue selected from the group consisting of a) 401 or 429 of SEQ ID NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 344, 355, 385, 431, 442, 462, 470, 472, 473, 495, 500, 511, 516, or 528 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, f) 455, 456, 457, and 458 of SEQ ID NO: 2, g) 470, 472, and 473 of SEQ ID NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2.In a preferred embodiment, the Group B nuclear receptor ligand binding domain is from a retinoid X receptor.", "In another specific embodiment, the Group B nuclear receptor ligand binding domain is encoded by a polynucleotide comprising a codon mutation that results in a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, and a serine residue at a position equivalent to or analogous to amino acid of amino acid residue 429 of SEQ ID NO: 1, d) a serine residue at a position equivalent or analogous to amino acid residues 337, 495, 500, or 528 of SEQ ID NO: 2, e) an asparagine residue at a position equivalent or analogous to amino acid residue 344 of SEQ ID NO: 2, f) an arginine residue at a position equivalent to or analogous to amino acid of amino acid residues 355 or 511 of SEQ ID NO: 2, g) an alanine residue at a position equivalent or analogous to amino acid residues 385 or 470 of SEQ ID NO: 2, h) a leucine residue at a position equivalent or analogous to amino acid residue 431 or 462 of SEQ ID NO: 2, i) a lysine residue at a position equivalent to or analogous to amino acid of amino acid residue 442 of SEQ ID NO: 2, j) an aspartic acid residue at a position equivalent or analogous to amino acid residue 472 of SEQ ID NO: 2, k) a glutamic acid at a position equivalent to or analogous to amino acid of amino acid residue 473 of SEQ ID NO: 2,1) a valine residue at a position equivalent to or analogous to amino acid of amino acid residue 516 of SEQ ID NO: 2, m) a leucine residue at a position equivalent to or analogous to amino acid of amino acid residue 321 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid of amino acid residue 322 of SEQ ID NO: 2, and a valine, residue at a position equivalent to or analogous to amino acid of amino acid residue 323 of SEQ ID NO: 2, n) a glutamic acid residue at a position equivalent to or analogous to amino acid of amino acid residue 450 of SEQ ID NO: 2, a valine residue at a position equivalent to or analogous to amino acid of amino acid residue 451 of SEQ ID NO: 2, and an arginine residue at a position equivalent to or analogous to amino acid of amino acid residue 452 of SEQ ID NO: 2, o) a lysine residue at a position equivalent to or analogous to amino acid of amino acid residue 455 of SEQ ID NO: 2, a serine residue at a position equivalent to or analogous to amino acid of amino acid residue 456 of SEQ ID NO: 2, an alanine residue at a position equivalent to or analogous to amino acid of amino acid residue 457 of SEQ ID NO: 2, and a glutamine residue at a position equivalent to or analogous to amino acid of amino acid residue 458 of SEQ ID NO: 2, p) an alanine residue at a position equivalent to or analogous to amino acid of amino acid residue 470 of SEQ ID NO: 2, an aspartic acid residue at a position equivalent to or analogous to amino acid of amino acid residue 472 of SEQ ID NO: 2, and a tyrosine residue at a position equivalent to or analogous to amino acid of amino acid residue 473 of SEQ ID NO: 2, q) a threonine residue at a position equivalent to or analogous to amino acid of amino acid residues 475, 477, and 478 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid of amino acid residue 476 of SEQ ID NO: 2, and a histidine residue at a position equivalent to or analogous to amino acid of amino acid residue 479 of SEQ ID NO: 2, or r) an aspartic acid residue at a position equivalent to or analogous to amino acid of amino acid residue 481 of SEQ ID NO: 2, a glutamic acid residue at a position equivalent to or analogous to amino acid of amino acid residue 482 of SEQ ID NO: 2, and a proline residue at a position equivalent to or analogous to amino acid of amino acid residue 483 of SEQ ID NO: 2.In a preferred embodiment, the Group B nuclear receptor ligand binding domain is from a retinoid X receptor.", "In another specific embodiment, the Group B nuclear receptor ligand binding domain comprising a substitution mutation is a retinoid X receptor ligand binding domain comprising a substitution mutation encoded by a polynucleotide comprising a codon mutation or codon mutations that results in a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, D344N, K355R, S385A, M431L, R442K, V462L, S470A, E471D, T473E, A495S, E500S, K511R, T516V, or A528S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.In another specific embodiment, the Group B nuclear receptor ligand binding domain comprises a substitution mutation at a position equivalent or analogous to amino acid residue a) 401 or 429 of SEQ ID NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 344, 355, 385, 431, 442, 462, 470, 472, 473, 495, 500, 511, 516, or 528 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, f) 455, 456, 457, and 458 of SEQ ID NO: 2, g) 470, 472, and 473 of SEQ ID NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2.In a preferred embodiment, the Group B nuclear receptor ligand binding domain is from a retinoid X receptor.", "Preferably, the Group B nuclear receptor ligand binding domain comprises a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, and a serine residue at a position equivalent to or analogous to amino acid of amino acid residue 429 of SEQ ID NO: 1, d) a serine residue at a position equivalent or analogous to amino acid residues 337, 495, 500, or 528 of SEQ ID NO: 2, e) an asparagine residue at a position equivalent or analogous to amino acid residue 344 of SEQ ID NO: 2, f) an arginine residue at a position equivalent to or analogous to amino acid of amino acid residues 355 or 511 of SEQ ID NO: 2, g) an alanine residue at a position equivalent or analogous to amino acid residues 385 or 470 of SEQ ID NO: 2, h) a leucine residue at a position equivalent or analogous to amino acid residue 431 or 462 of SEQ ID NO: 2, i) a lysine residue at a position equivalent to or analogous to amino acid of amino acid residue 442 of SEQ ID NO: 2, j) an aspartic acid residue at a position equivalent or analogous to amino acid residue 472 of SEQ ID NO: 2, k) a glutamic acid at a position equivalent to or analogous to amino acid of amino acid residue 473 of SEQ ID NO: 2, 1) a valine residue at a position equivalent to or analogous to amino acid of amino acid residue 516 of SEQ ID NO: 2, m) a leucine residue at a position equivalent to or analogous to amino acid of amino acid residue 321 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid of amino acid residue 322 of SEQ ID NO: 2, and a valine residue at a position equivalent to or analogous to amino acid of amino acid residue 323 of SEQ ID NO: 2, n) a glutamic acid residue at a position equivalent to or analogous to amino acid of amino acid residue 450 of SEQ ID NO: 2, a valine residue at a position equivalent to or analogous to amino acid of amino acid residue 451 of SEQ ID NO: 2, and an arginine residue at a position equivalent to or analogous to amino acid of amino acid residue 452 of SEQ ID NO: 2, o) a lysine residue at a position equivalent to or analogous to amino acid of amino acid residue 455 of SEQ ID NO: 2, a serine residue at a position equivalent to or analogous to amino acid of amino acid residue 456 of SEQ ID NO: 2, an alanine residue at a position equivalent to or analogous to amino acid of amino acid residue 457 of SEQ ID NO: 2, and a glutamine residue at a position equivalent to or analogous to amino acid of amino acid residue 458 of SEQ ID NO: 2, p) an alanine residue at a position equivalent to or analogous to amino acid of amino acid residue 470 of SEQ ID NO: 2, an aspartic acid residue at a position equivalent to or analogous to amino acid of amino acid residue 472 of SEQ ID NO: 2, and a tyrosine residue at a position equivalent to or analogous to amino acid of amino acid residue 473 of SEQ ID NO: 2, q) a threonine residue at a position equivalent to or analogous to amino acid of amino acid residues 475, 477, and 478 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid of amino acid residue 476 of SEQ ID NO: 2, and a histidine residue at a position equivalent to or analogous to amino acid of amino acid residue 479 of SEQ ID NO: 2, or r) an aspartic acid residue at a position equivalent to or analogous to amino acid of amino acid residue 481 of SEQ ID NO: 2, a glutamic acid residue at a position equivalent to or analogous to amino acid of amino acid residue 482 of SEQ ID NO: 2, and a proline residue at a position equivalent to or analogous to amino acid of amino acid residue 483 of SEQ ID NO: 2.In a preferred embodiment, the Group B nuclear receptor ligand binding domain is from a retinoid X receptor.", "In another specific embodiment, the Group B nuclear receptor ligand binding domain comprising a substitution mutation is a retinoid X receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation is selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, D344N, K355R, S385A, M431L, R442K, V462L, S470A, E471D, T473E, A495S, E500S, K511R, T516V, or A528S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.The DNA binding domain (DBD) can be any DNA binding domain with a known response element, including synthetic and chimeric DNA binding domains, or analogs, combinations, or modifications thereof.", "Preferably, the DBD is a GAL4 DBD, a LexA DBD, a transcription factor DBD, a Group B nuclear receptor member DBD, a steroid/thyroid hormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD, or a DBD.", "More preferably, the DBD is an ecdysone receptor (EcR) DBD [SEQ ID NO: 3 (polynucleotide) or SEQ ID NO: 4 (polypeptide)], a GAL4 DBD [SEQ ID NO: 5 (polynucleotide) or SEQ ID NO: 6 (polypeptide)], or a LexA DBD [(SEQ ID NO: 7 (polynucleotide) or SEQ ID NO: 8 (polypeptide)].", "The transactivation domain (abbreviated “AD” or “TA”) may be any Group B nuclear receptor member AD, steroid/thyroid hormone nuclear receptor AD, synthetic or chimeric AD, polyglutamine AD, basic or acidic amino acid AD, a VP16 AD, a GAL4 AD, an NF-κB AD, a BP64 AD, a B42 acidic activation domain (B42AD), a P65 activation domain (P65AD), or an analog, combination, or modification thereof.", "In a specific embodiment, the AD is a synthetic or chimeric AD, or is obtained from an ecdysone receptor, a glucocorticoid receptor, VP16, GAL4, NF-κB, or B42 acidic activation domain AD.", "Preferably, the AD is an EcR AD [SEQ ID NO: 9 (polynucleotide) or SEQ ID NO: 10 (polypeptide)], a VP16 AD [SEQ ID NO: 11 (polynucleotide) or SEQ ID NO: 12 (polypeptide)], a B42 AD [SEQ ID NO: 13 (polynucleotide) or SEQ ID NO: 14 (polypeptide)], or a p65 AD [SEQ ID NO: 15 (polynucleotide) or SEQ ID NO: 16 (polypeptide)].", "In a specific embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a) a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15; and a Group B nuclear receptor ligand binding domain comprising a substitution mutation encoded by a polynucleotide according to the invention; b) a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, and a Group B nuclear receptor ligand binding domain comprising a substitution mutation encoded by a polynucleotide according to the invention, or c) a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15; and a Group B nuclear receptor ligand binding domain comprising a substitution mutation encoded by a polynucleotide according to the invention.", "Preferably, the Group B nuclear receptor ligand binding domain comprising a substitution mutation is an ecdysone receptor ligand binding domain comprising a substitution mutation encoded by a polynucleotide according to the invention.", "In another specific embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a) a DNA-binding domain comprising an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; a transactivation domain comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention; b) a DNA-binding domain comprising an amino acid sequence acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention, or c) a transactivation domain comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention.", "Preferably, the Group B nuclear receptor ligand binding domain comprising a substitution mutation is an ecdysone receptor ligand binding domain comprising a substitution mutation according to the invention.", "The response element (“RE”) may be any response element with a-known DNA binding domain, or an analog, combination, or modification thereof.", "A single RE may be employed or multiple REs, either multiple copies of the same RE or two or more different REs, may be used in the present invention.", "In a specific embodiment, the RE is an RE from GAL4 (“GAL4RE”), LexA, a Group B nuclear receptor RE, a steroid/thyroid hormone nuclear receptor RE, or a synthetic RE that recognizes a synthetic DNA binding domain.", "Preferably, the RE is an ecdysone RE (EcRE) comprising a polynucleotide sequence of SEQ ID NO: 17, a GALORE comprising a polynucleotide sequence of SEQ ID NO: 18, or a LexA RE (operon, “op”) comprising a polynucleotide sequence of SEQ ID NO: 19 (“2XLexAopRE”).", "A steroid/thyroid hormone nuclear receptor DNA binding domain, activation domain or response element according to the invention may be obtained from a steroid/thyroid hormone nuclear receptor selected from the group consisting of thyroid hormone receptor α (TRα), thyroid receptor 1 (c-erbA-1), thyroid hormone receptor β (TRβ), retinoic acid receptor α (RARα), retinoic acid receptor β (RARβ, HAP), retinoic acid receptor γ (RARγ), retinoic acid receptor gamma-like (RARD), peroxisome proliferator-activated receptor α (PPARα), peroxisome proliferator-activated receptor β (PPARβ), peroxisome proliferator-activated receptor δ (PPARδ, NUC-1), peroxisome proliferator-activator related receptor (FFAR), peroxisome proliferator-activated receptor γ (PPARγ), orphan receptor encoded by non-encoding strand of thyroid hormone receptor α (REVERBα), v-erb A related receptor (EAR-1), v-erb related receptor (EAR-1A), γ), orphan receptor encoded by non-encoding strand of thyroid hormone receptor β (REVERBβ), v-erb related receptor (EAR-1β), orphan nuclear receptor BD73 (BD73), rev-erbA-related receptor (RVR), zinc finger protein 126 (HZF2), ecdysone-inducible protein E75 (E75), ecdysone-inducible protein E78 (E78), Drosophila receptor 78 (DR-78), retinoid-related orphan receptor α (RORα), retinoid Z receptor α (RZRα), retinoid related orphan receptor β (RORβ), retinoid Z receptor β (RZRβ), retinoid-related orphan receptor γ (RORγ), retinoid Z receptor γ (RZRγ), retinoid-related orphan receptor (TOR), hormone receptor 3 (HR-3), Drosophila hormone receptor 3 (DHR-3), manduca hormone receptor (MHR-3), Galleria hormone receptor 3 (GHR-3), C. elegans nuclear receptor 3 (CNR-3), Choristoneura hormone receptor 3 (CHR-3), C. elegans nuclear receptor 14 (CNR-14), ecdysone receptor (ECR), ubiquitous receptor (UR), orphan nuclear receptor (OR-1), NER-1, receptor-interacting protein 15 (RIP-15), liver X receptor β (LXRβ), steroid hormone receptor like protein (RLD-1), liver X receptor (LXR), liver X receptor α (LXRα), farnesoid X receptor (FXR), receptor-interacting protein 14 (RIP-14), HRR-1, vitamin D receptor (VDR), orphan nuclear receptor (ONR-1), pregnane X receptor (PXR), steroid and xenobiotic receptor (SXR), benzoate X receptor (BXR), nuclear receptor (MB-67), constitutive androstane receptor 1 (CAR-1), constitutive androstane receptor α (CARα), constitutive androstane receptor 2 (CAR-2), constitutive androstane receptor β (CARβ), Drosophila hormone receptor 96 (DHR-96), nuclear hormone receptor 1 (NHR-1), hepatocyte nuclear factor 4 (HNF-4), hepatocyte nuclear factor 4G (HNF-4G), hepatocyte nuclear factor 4B (HNF-4B), hepatocyte nuclear factor 4D (HNF-4D, DHNF-4), retinoid X receptor α (RXRα), retinoid X receptor β (RXRβ), H-2 region II binding protein (H-2RIIBP), nuclear receptor co-regulator-1 (RCoR-1), retinoid X receptor γ (RXRγ), Ultraspiracle (USP), 2C1 nuclear receptor, chorion factor 1 (CF-1), testicular receptor 2 (TR2), testicular receptor 2-11 (TR2-11), testicular receptor 4 (TR4), TAK-1, Drosophila hormone receptor (DHR78), Tailless (TLL), tailless homolog (TLX), XTLL, chicken ovalbumin upstream promoter transcription factor I (COUP-TFI), chicken ovalbumin upstream promoter transcription factor A (COUP-TFA), EAR-3, SVP-44, chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), chicken ovalbumin upstream promoter transcription factor B (COUP-TFB), ARP-1, SVP-40, SVP, chicken ovalbumin upstream promoter transcription factor III (COUP-TFIII), chicken ovalbumin upstream promoter transcription factor G (COUP-TFG), SVP-46, EAR-2, estrogen receptor α (ERα), estrogen receptor β (ERβ), estrogen related receptor β (ERRβ), estrogen related receptor α (ERRα), estrogen related receptor 2 (ERR2), estrogen related receptor β (ERRβ), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), progesterone receptor (PR), androgen receptor (AR), nerve growth factor induced gene B (NGFI-B), nuclear receptor similar to Nur-77 (TRS), N10, Orphan receptor (NUR-77), Human early response gene (NAK-1), Nurr related factor 1 (NURR-1), a human immediate-early response gene (NOT), regenerating liver nuclear receptor 1 (RNR-1), hematopoietic zinc finger 3 (HZF-3), Nur rekated protein-1 (TINOR), Nuclear orphan receptor 1 (NOR-1), NOR1 related receptor (MINOR), Drosophila hormone receptor 38 (DHR-38), C. elegans nuclear receptor 8 (CNR-8), C48D5, steroidogenic factor 1 (SF1), endozepine-like peptide (ELP), fushi tarazu factor 1 (FTZ-F1), adrenal 4 binding protein (AD4BP), liver receptor homolog (LRH-1), Ftz-F1-related orphan receptor A (xFFrA), Ftz-F1-related orphan receptor B (xFFrB), nuclear receptor related to LRH-1 (FFLR), nuclear receptor related to LRH-1 (PHR), fetoprotein transcription factor (FTF), germ cell nuclear factor (GCNFM), retinoid receptor-related testis-associated receptor (RTR), knirps (KNI), knirps related (KNRL), Embryonic gonad (EGON), Drosophila gene for ligand dependent nuclear receptor (EAGLE), nuclear receptor similar to trithorax (ODR7), Trithorax, dosage sensitive sex reversal adrenal hypoplasia congenita critical region chromosome X gene (DAX-1), adrenal hypoplasia congenita and hypogonadotropic hypogonadism (AHCH), and short heterodimer partner (SHP).", "Thus, the present invention also provides a gene expression cassette comprising: i) a response element comprising a domain recognized by a polypeptide comprising a DNA binding domain; a promoter that is activated by a polypeptide comprising a transactivation domain; and a gene whose expression is to be modulated.", "Genes of interest for use in Applicants' gene expression cassettes may be endogenous genes or heterologous genes.", "Nucleic acid or amino acid sequence information for a desired gene or protein can be located in one of many public access databases, for example, GENBANK, EMBL, Swiss-Prot, and PIR, or in many biology related journal publications.", "Thus, those skilled in the art have access to nucleic acid sequence information for virtually all known genes.", "Such information can then be used to construct the desired constructs for the insertion of the gene of interest within the gene expression cassettes used in Applicants' methods described herein.", "Examples of genes of interest for use in Applicants' gene expression cassettes include, but are not limited to: genes encoding therapeutically desirable polypeptides or products that may be used to treat a condition, a disease, a disorder, a dysfunction, a genetic defect, such as monoclonal antibodies, enzymes, proteases, cytokines, interferons, insulin, erythropoietin, clotting factors, other blood factors or components, viral vectors for gene therapy, virus for vaccines, targets for drug discovery, functional genomics, and proteomics analyses and applications, and the like.", "For purposes of this invention, nuclear receptors and Group B nuclear receptors also include synthetic and chimeric nuclear receptors and Group B nuclear receptors and their homologs.", "Polynucleotides of the Invention The novel nuclear receptor-based inducible gene expression system of the invention comprises at least one gene expression cassette comprising a polynucleotide that encodes a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "These gene expression cassettes, the polynucleotides they comprise, and the polypeptides they encode are useful as components of a nuclear receptor-based gene expression system to modulate the expression of a gene within a host cell.", "Thus, the present invention also provides an isolated polynucleotide that encodes a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "In a specific embodiment, the Group B nuclear receptor ligand binding domain is encoded by a polynucleotide comprising a codon mutation that results in a substitution of an amino acid residue, wherein the amino acid residue is at a position equivalent or analogous to an amino acid residue selected from the group consisting of a) 401 or 429 of SEQ ID NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 344, 355, 385, 431, 442, 462, 470, 472, 473, 495, 500, 511, 516, or 528 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, f) 455, 456, 457, and 458 of SEQ ID NO: 2, g) 470, 472, and 473 of SEQ ID NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2.In a preferred embodiment, the Group B nuclear receptor ligand binding domain is from a retinoid X receptor.", "In another specific embodiment, the Group B nuclear receptor ligand binding domain is encoded by a polynucleotide comprising a codon mutation that results in a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, and a serine residue at a position equivalent to or analogous to amino acid of amino acid residue 429 of SEQ ID NO: 1, d) a serine residue at a position equivalent or analogous to amino acid residues 337, 495, 500, or 528 of SEQ ID NO: 2, e) an asparagine residue at a position equivalent or analogous to amino acid residue 344 of SEQ ID NO: 2, f) an arginine residue at a position equivalent to or analogous to amino acid of amino acid residues 355 or 511 of SEQ ID NO: 2, g) an alanine residue at a position equivalent or analogous to amino acid residues 385 or 470 of SEQ ID NO: 2, h) a leucine residue at a position equivalent or analogous to amino acid residue 431 or 462 of SEQ ID NO: 2, i) a lysine residue at a position equivalent to or analogous to amino acid residue 442 of SEQ ID NO: 2, j) an aspartic acid residue at a position equivalent or analogous to amino acid residue 472 of SEQ ID NO: 2, k) a glutamic acid at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, 1) a valine residue at a position equivalent to or analogous to amino acid residue 516 of SEQ ID NO: 2, m) a leucine residue at a position equivalent to or analogous to amino acid residue 321 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid residue 322 of SEQ ID NO: 2, and a valine residue at a position equivalent to or analogous to amino acid residue 323 of SEQ ID NO: 2, n) a glutamic acid residue at a position equivalent to or analogous to amino acid residue 450 of SEQ ID NO: 2, a valine residue at a position equivalent to or analogous to amino acid residue 451 of SEQ ID NO: 2, and an arginine residue at a position equivalent to or analogous to amino acid residue 452 of SEQ ID NO: 2, o) a lysine residue at a position equivalent to or analogous to amino acid residue 455 of SEQ ID NO: 2, a serine residue at a position equivalent to or analogous to amino acid residue 456 of SEQ ID NO: 2, an alanine residue at a position equivalent to or analogous to amino acid residue 457 of SEQ ID NO: 2, and a glutamine residue at a position equivalent to or analogous to amino acid residue 458 of SEQ ID NO: 2, p) an alanine residue at a position equivalent to or analogous to amino acid residue 470 of SEQ ID NO: 2, an aspartic acid residue at a position equivalent to or analogous to amino acid residue 472 of SEQ ID NO: 2, and a tyrosine residue at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, q) a threonine residue at a position equivalent to or analogous to amino acid residues 475, 477, and 478 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid residue 476 of SEQ ID NO: 2, and a histidine residue at a position equivalent to or analogous to amino acid residue 479 of SEQ ID NO: 2, or r) an aspartic acid residue at a position equivalent to or analogous to amino acid residue 481 of SEQ ID NO: 2, a glutamic acid residue at a position equivalent to or analogous to amino acid of amino acid residue 482 of SEQ ID NO: 2, and a proline residue at a position equivalent to or analogous to amino acid residue 483 of SEQ ID NO: 2.In a preferred embodiment, the Group B nuclear receptor ligand binding domain is from a retinoid X receptor.", "In another specific embodiment, the Group B nuclear receptor ligand binding domain comprising a substitution mutation is a retinoid X receptor ligand binding domain comprising a substitution mutation encoded by a polynucleotide comprising a codon mutation or codon mutations that results in a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, D344N, K355R, S385A, M431L, R442K, V462L, S470A, E471D, T473E, A495S, E500S, K511R, T516V, or A528S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.The present invention also provides an isolated polynucleotide that encodes a polypeptide selected from the group consisting of a) a polypeptide comprising a transactivation domain, a DNA-binding domain, and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention; b) a polypeptide comprising a DNA-binding domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention; and c) a polypeptide comprising a transactivation domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention.", "In a specific embodiment, the isolated polynucleotide encodes a hybrid polypeptide selected from the group consisting of a) a hybrid polypeptide comprising a transactivation domain, a DNA-binding domain, and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention; b) a hybrid polypeptide comprising a DNA-binding domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention; and c) a hybrid polypeptide comprising a transactivation domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention.", "The present invention also relates to an isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation affects ligand binding activity or ligand sensitivity of the Group B nuclear receptor ligand binding domain.", "In particular, the present invention relates to an isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation reduces ligand binding activity or ligand sensitivity of the Group B nuclear receptor ligand binding domain.", "In a specific embodiment, the present invention relates to an isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation reduces steroid binding activity or steroid sensitivity of the Group B nuclear receptor ligand binding domain.", "Preferably, the isolated polynucleotide comprises a codon mutation that results in a substitution of an amino acid residue, wherein the amino acid residue is at a position equivalent or analogous to a) amino acid residue 401 or 429 of SEQ ID NO: 1, orb) amino acid residue 344, 431, 442, 495, 511, or 528 of SEQ ID NO: 2.More preferably, the isolated polynucleotide comprises a codon mutation that results in a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an asparagine residue at a position equivalent or analogous to amino acid residue 344 of SEQ ID NO: 2, d) a leucine residue at a position equivalent or analogous to amino acid residue 431 of SEQ ID NO: 2, e) a lysine residue at a position equivalent or analogous to amino acid residue 442 of SEQ ID NO: 2, f) a serine residue at a position equivalent or analogous to amino acid residue 495 or 528 of SEQ ID NO: 2, or g) an arginine residue at a position equivalent or analogous to amino acid residue 511 of SEQ ID NO: 2.Even more preferably, the isolated polynucleotide comprises a codon mutation that results in a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, and b) D344N, M431L, R442K, A495S, K511R, or A528S of SEQ ID NO: 2.In a specific embodiment, the present invention relates to an isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation reduces non-steroid ligand binding activity or ligand sensitivity of the Group B nuclear receptor ligand binding domain.", "Preferably, the isolated polynucleotide comprises a codon mutation that results in a substitution of an amino acid residue, wherein the amino acid residue is at a position equivalent or analogous to a) amino acid residue 401 or 429 of SEQ ID NO: 1, or b) amino acid residue 344, 431, 442, 495, 511, or 528 of SEQ ID NO: 2.More preferably, the isolated polynucleotide comprises a codon mutation that results in a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an asparagine residue at a position equivalent or analogous to amino acid residue 344 of SEQ ID NO: 2, d) a leucine residue at a position equivalent or analogous to amino acid residue 431 of SEQ ID NO: 2, e) a lysine residue at a position equivalent or analogous to amino acid residue 442 of SEQ ID NO: 2, f) a serine residue at a position equivalent or analogous to amino acid residue 495 or 528 of SEQ ID NO: 2, or g) an arginine residue at a position equivalent or analogous to amino acid residue 511 of SEQ ID NO: 2.Even more preferably, the isolated polynucleotide comprises a codon mutation that results in a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, and b) D344N, M431L, R442K, A495S, K511R, or A528S of SEQ ID NO: 2.In addition, the present invention also relates to an isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation enhances ligand binding activity or ligand sensitivity of the Group B nuclear receptor ligand binding domain.", "In a specific embodiment, the present invention relates to an isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation enhances steroid binding activity or steroid sensitivity of the Group B nuclear receptor ligand binding domain.", "Preferably, the isolated polynucleotide comprises a codon mutation that results in a substitution of an amino acid residue at a position equivalent or analogous to amino acid residue selected from the group consisting of a) 401 or 429 of SEQ ID NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 355, 385, 462, 470, 472, 473, or 500 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, f) 455, 456, 457, and 458 of SEQ ID NO: 2, g) 470, 472, and 473 of SEQ ID NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2.More preferably, the isolated polynucleotide comprises a codon mutation that results in a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, and a serine residue at a position equivalent to or analogous to amino acid residue 429 of SEQ ID NO: 1, d) a serine residue at a position equivalent or analogous to amino acid residue 337 or 500 of SEQ ID NO: 2, e) an arginine residue at a position equivalent to or analogous to amino acid residue 355 of SEQ ID NO: 2, f) an alanine residue at a position equivalent or analogous to amino acid residue 385 or 470 of SEQ ID NO: 2, g) a leucine residue at a position equivalent or analogous to amino acid residue 462 of SEQ ID NO: 2, h) an aspartic acid residue at a position equivalent or analogous to amino acid residue 472 of SEQ ID NO: 2, i) a glutamic acid at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, j) a leucine residue at a position equivalent or analogous to amino acid residue 321 of SEQ ID NO: 2, an arginine residue at a position equivalent or analogous to amino acid residue 322 of SEQ ID NO: 2, and a valine residue at a position equivalent or analogous to amino acid residue 323 of SEQ ID NO: 2, k) a glutamic acid residue at a position equivalent to or analogous to amino acid residue 450 of SEQ ID NO: 2, a valine residue at a position equivalent to or analogous to amino acid residue 451 of SEQ ID NO: 2, and an arginine residue at a position equivalent to or analogous to amino acid residue 452 of SEQ ID NO: 2, 1) a lysine residue at a position equivalent to or analogous to amino acid residue 455 of SEQ ID NO: 2, a serine residue at a position equivalent to or analogous to amino acid residue 456 of SEQ ID NO: 2, an alanine residue at a position equivalent to or analogous to amino acid residue 457 of SEQ ID NO: 2, and a glutamine residue at a position equivalent to or analogous to amino acid residue 458 of SEQ ID NO: 2, m) an alanine residue at a position equivalent to or analogous to amino acid residue 470 of SEQ ID NO: 2, an aspartic acid residue at a position equivalent to or analogous to amino acid residue 472 of SEQ ID NO: 2, and a tyrosine residue at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, n) a threonine residue at a position equivalent to or analogous to amino acid residues 475, 477, and 478 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid residue 476 of SEQ ID NO: 2, and a histidine residue at a position equivalent to or analogous to amino acid residue 479 of SEQ ID NO: 2, or o) an aspartic acid residue at a position equivalent to or analogous to amino acid residue 481 of SEQ ID NO: 2, a glutamic acid residue at a position equivalent to or analogous to amino acid residue 482 of SEQ ID NO: 2, and a proline residue at a position equivalent to or analogous to amino acid residue 483 of SEQ ID NO: 2.Even more preferably, the isolated polynucleotide comprises a codon mutation or codon mutations that results in a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, K355R, S385A, V462L, S470A, E471D, T473E, or E500S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.In another specific embodiment, the present invention relates to an isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation enhances non-steroid binding activity or non-steroid sensitivity of the Group B nuclear receptor ligand binding domain.", "Preferably, the isolated polynucleotide comprises a codon mutation that results in a substitution of an amino acid residue at a position equivalent or analogous to amino acid residue selected from the group consisting of a) 401 or 429 of SEQ ID NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 355, 385, 462, 470, 472, 473, or 500 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, f) 455, 456, 457, and 458 of SEQ ID NO: 2, g) 470, 472, and 473 of SEQ ID NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2.More preferably, the isolated polynucleotide comprises a codon mutation that results in a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, and a serine residue at a position equivalent to or analogous to amino acid residue 429 of SEQ ID NO: 1, d) a serine residue at a position equivalent or analogous to amino acid residue 337 or 500 of SEQ ID NO: 2, e) an arginine residue at a position equivalent to or analogous to amino acid residue 355 of SEQ ID NO: 2, f) an alanine residue at a position equivalent or analogous to amino acid residue 385 or 470 of SEQ ID NO: 2, g) a leucine residue at a position equivalent or analogous to amino acid residue 462 of SEQ ID NO: 2, h) an aspartic acid residue at a position equivalent or analogous to amino acid residue 472 of SEQ ID NO: 2, i) a glutamic acid at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, j) a leucine residue at a position equivalent or analogous to amino acid residue 321 of SEQ ID NO: 2, an arginine residue at a position equivalent or analogous to amino acid residue 322 of SEQ ID NO: 2, and a valine residue at a position equivalent or analogous to amino acid residue 323 of SEQ ID NO: 2, k) a glutamic acid residue at a position equivalent to or analogous to amino acid residue 450 of SEQ ID NO: 2, a valine residue at a position equivalent to or analogous to amino acid residue 451 of SEQ ID NO: 2, and an arginine residue at a position equivalent to or analogous to amino acid residue 452 of SEQ ID NO: 2, 1) a lysine residue at a position equivalent to or analogous to amino acid residue 455 of SEQ ID NO: 2, a serine residue at a position equivalent to or analogous to amino acid residue 456 of SEQ ID NO: 2, an alanine residue at a position equivalent to or analogous to amino acid residue 457 of SEQ ID NO: 2, and a glutamine residue at a position equivalent to or analogous to amino acid residue 458 of SEQ ID NO: 2, m) an alanine residue at a position equivalent to or analogous to amino acid residue 470 of SEQ ID NO: 2, an aspartic acid residue at a position equivalent to or analogous to amino acid residue 472 of SEQ ID NO: 2, and a tyrosine residue at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, n) a threonine residue at a position equivalent to or analogous to amino acid residues 475, 477, and 478 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid residue 476 of SEQ ID NO: 2, and a histidine residue at a position equivalent to or analogous to amino acid residue 479 of SEQ ID NO: 2, or o) an aspartic acid residue at a position equivalent to or analogous to amino acid residue 481 of SEQ ID NO: 2, a glutamic acid residue at a position equivalent to or analogous to amino acid residue 482 of SEQ ID NO: 2, and a proline residue at a position equivalent to or analogous to amino acid residue 483 of SEQ ID NO: 2.Even more preferably, the isolated polynucleotide comprises a codon mutation or codon mutations that results in a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, K355R, S385A, V462L, S470A, E471D, T473E, or E500S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.In another specific embodiment, the present invention relates to an isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation enhances both steroid binding activity or steroid sensitivity and non-steroid binding activity or non-steroid sensitivity of the Group B ligand binding domain.", "In another specific embodiment, the present invention also relates to an isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation increases ligand sensitivity of a heterodimer comprising the Group B nuclear receptor ligand binding domain comprising a substitution mutation and a dimerization partner.", "In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide.", "In another specific embodiment, the dimerization partner is a truncated EcR polypeptide.", "In addition, the present invention relates to an expression vector comprising a polynucleotide according the invention, operatively linked to a transcription regulatory element.", "Preferably, the polynucleotide encoding a nuclear receptor ligand binding domain comprising a substitution mutation is operatively linked with an expression control sequence permitting expression of the nuclear receptor ligand binding domain in an expression competent host cell.", "The expression control sequence may comprise a promoter that is functional in the host cell in which expression is desired.", "The vector may be a plasmid DNA molecule or a viral vector.", "Preferred viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, and vaccinia virus.", "The invention further relates to a replication defective recombinant virus comprising in its genome, the polynucleotide encoding a nuclear receptor ligand binding domain comprising a substitution mutation as described above.", "Thus, the present invention also relates to an isolated host cell comprising such an expression vector, wherein the transcription regulatory element is operative in the host cell.", "The present invention also relates to an isolated polypeptide encoded by a polynucleotide according to the invention.", "Polypeptides of the Invention The novel nuclear receptor-based inducible gene expression system of the invention comprises at least one gene expression cassette comprising a polynucleotide that encodes a polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "Thus, the present invention also provides an isolated polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention.", "In another specific embodiment, the Group B nuclear receptor ligand binding domain comprises a substitution mutation at a position equivalent or analogous to amino acid residue a) 401 or 429 of SEQ ID NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 344, 355, 385, 431, 442, 462, 470, 472, 473, 495, 500, 511, 516, or 528 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, 1) 455, 456, 457, and 458 of SEQ ID NO: 2, g) 470, 472, and 473 of SEQ ID NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2.Preferably, the Group B nuclear receptor ligand binding domain comprises a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, and a serine residue at a position equivalent to or analogous to amino acid residue 429 of SEQ ID NO: 1, d) a serine residue at a position equivalent or analogous to amino acid residue 337, 495, 500, or 528 of SEQ ID NO: 2, e) an asparagine residue at a position equivalent or analogous to amino acid residue 344 of SEQ ID NO: 2, f) an arginine residue at a position equivalent to or analogous to amino acid residue 355 or 511 of SEQ ID NO: 2, g) an alanine residue at a position equivalent or analogous to amino acid residue 385 or 470 of SEQ ID NO: 2, h) a leucine residue at a position equivalent or analogous to amino acid residue 431 or 462 of SEQ ID NO: 2, i) a lysine residue at a position equivalent to or analogous to amino acid residue 442 of SEQ ID NO: 2, j) an aspartic acid residue at a position equivalent or analogous to amino acid residue 472 of SEQ ID NO: 2, k) a glutamic acid at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, 1) a valine residue at a position equivalent to or analogous to amino acid residue 516 of SEQ ID NO: 2, m) a leucine residue at a position equivalent to or analogous to amino acid residue 321 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid residue 322 of SEQ ID NO: 2, and a valine residue at a position equivalent to or analogous to amino acid residue 323 of SEQ ID NO: 2, n) a glutamic acid residue at a position equivalent to or analogous to amino acid residue 450 of SEQ ID NO: 2, a valine residue at a position equivalent to or analogous to amino acid residue 451 of SEQ ID NO: 2, and an arginine residue at a position equivalent to or analogous to amino acid residue 452 of SEQ ID NO: 2, o) a lysine residue at a position equivalent to or analogous to amino acid residue 455 of SEQ ID NO: 2, a serine residue at a position equivalent to or analogous to amino acid residue 456 of SEQ ID NO: 2, an alanine residue at a position equivalent to or analogous to amino acid residue 457 of SEQ ID NO: 2, and a glutamine residue at a position equivalent to or analogous to amino acid residue 458 of SEQ ID NO: 2, p) an alanine residue at a position equivalent to or analogous to amino acid residue 470 of SEQ ID NO: 2, an aspartic acid residue at a position equivalent to or analogous to amino acid residue 472 of SEQ ID NO: 2, and a tyrosine residue at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, q) a threonine residue at a position equivalent to or analogous to amino acid residues 475, 477, and 478 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid residue 476 of SEQ ID NO: 2, and a histidine residue at a position equivalent to or analogous to amino acid residue 479 of SEQ ID NO: 2, or r) an aspartic acid residue at a position equivalent to or analogous to amino acid residue 481 of SEQ ID NO: 2, a glutamic acid residue at a position equivalent to or analogous to amino acid residue 482 of SEQ ID NO: 2, and a proline residue at a position equivalent to or analogous to amino acid residue 483 of SEQ ID NO: 2.In a preferred embodiment, the Group B nuclear receptor ligand binding domain is from a retinoid X receptor.", "In another specific embodiment, the Group B nuclear receptor ligand binding domain comprising a substitution mutation is a retinoid X receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation is selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, D344N, K355R, S385A, M431L, R442K, V462L, S470A, E471D, T473E, A495S, E500S, K511R, T516V, or A528S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.The present invention also provides an isolated polypeptide selected from the group consisting of a) an isolated polypeptide comprising a transactivation domain, a DNA-binding domain, and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention; b) an isolated polypeptide comprising a DNA-binding domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention; and c) an isolated polypeptide comprising a transactivation domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention.", "In a preferred embodiment, the Group B nuclear receptor ligand binding domain is from a retinoid X receptor.", "The present invention also provides an isolated hybrid polypeptide selected from the group consisting of a) an isolated hybrid polypeptide comprising a transactivation domain, a DNA-binding domain, and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention; b) an isolated hybrid polypeptide comprising a DNA-binding domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention; and c) an isolated hybrid polypeptide comprising a transactivation domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation according to the invention.", "In a preferred embodiment, the Group B nuclear receptor ligand binding domain is from a retinoid X receptor.", "The present invention also provides an isolated polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation that affects ligand binding activity or ligand sensitivity of the Group B nuclear receptor ligand binding domain.", "In particular, the present invention relates to an isolated Group B nuclear receptor polypeptide comprising a ligand binding domain comprising a substitution mutation that reduces ligand binding activity or ligand sensitivity of the Group B nuclear receptor ligand binding domain.", "In a specific embodiment, the present invention relates to an isolated polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation reduces steroid binding activity or steroid sensitivity of the Group B nuclear receptor ligand binding domain.", "Preferably, the isolated polypeptide comprises a substitution of an amino acid residue at a position equivalent or analogous to a) amino acid residue 401 or 429 of SEQ ID NO: 1, orb) amino acid residue 344, 431, 442, 495, 511, or 528 of SEQ ID NO: 2.More preferably, the isolated polypeptide comprises a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an asparagine residue at a position equivalent or analogous to amino acid residue 344 of SEQ ID NO: 2, d) a leucine residue at a position equivalent or analogous to amino acid residue 431 of SEQ ID NO: 2, e) a lysine residue at a position equivalent or analogous to amino acid residue 442 of SEQ ID NO: 2, f) a serine residue at a position equivalent or analogous to amino acid residue 495 or 528 of SEQ ID NO: 2, or g) an arginine residue at a position equivalent or analogous to amino acid residue 511 of SEQ ID NO: 2.Even more preferably, the isolated polypeptide comprises a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, and b) D344N, M431L, R442K, A495S, K511R, or A528S of SEQ ID NO: 2.In a specific embodiment, the present invention relates to an isolated polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation reduces non-steroid ligand binding activity or ligand sensitivity of the Group B nuclear receptor ligand binding domain.", "Preferably, the isolated polypeptide comprises a substitution of an amino acid residue at a position equivalent or analogous to a) amino acid residue 401 or 429 of SEQ ID NO: 1, orb) amino acid residue 344, 431, 442, 495, 511, or 528 of SEQ ID NO: 2.More preferably, the isolated polypeptide comprises a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an asparagine residue at a position equivalent or analogous to amino acid residue 344 of SEQ ID NO: 2, d) a leucine residue at a position equivalent or analogous to amino acid residue 431 of SEQ ID NO: 2, e) a lysine residue at a position equivalent or analogous to amino acid residue 442 of SEQ ID NO: 2, 1) a serine residue at a position equivalent or analogous to amino acid residue 495 or 528 of SEQ ID NO: 2, or g) an arginine residue at a position equivalent or analogous to amino acid residue 511 of SEQ ID NO: 2.Even more preferably, the isolated polypeptide comprises a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, and b) D344N, M431L, R442K, A495S, K511R, or A528S of SEQ ID NO: 2.In addition, the present invention relates to an isolated polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation that enhances ligand binding activity or ligand sensitivity of the Group B nuclear receptor ligand binding domain.", "In a specific embodiment, the present invention relates to an isolated polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation that enhances steroid binding activity or steroid sensitivity of the Group B nuclear receptor ligand binding domain.", "Preferably, the isolated polypeptide comprises a substitution of an amino acid residue at a position equivalent or analogous to amino acid residue selected from the group consisting of a) 401 or 429 of SEQ ID NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 355, 385, 462, 470, 472, 473, or 500 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, f) 455, 456, 457, and 458 of SEQ ID NO: 2, g) 470, 472, and 473 of SEQ ID NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2.More preferably, the isolated polypeptide comprises a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, and a serine residue at a position equivalent to or analogous to amino acid residue 429 of SEQ ID NO: 1, d) a serine residue at a position equivalent or analogous to amino acid residue 337 or 500 of SEQ ID NO: 2, c) an arginine residue at a position equivalent to or analogous to amino acid residue 355 of SEQ ID NO: 2, f) an alanine residue at a position equivalent or analogous to amino acid residue 385 or 470 of SEQ ID NO: 2, g) a leucine residue at a position equivalent or analogous to amino acid residue 462 of SEQ ID NO: 2, h) an aspartic acid residue at a position equivalent or analogous to amino acid residue 472 of SEQ ID NO: 2, i) a glutamic acid at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, j) a leucine residue at a position equivalent or analogous to amino acid residue 321 of SEQ ID NO: 2, an arginine residue at a position equivalent or analogous to amino acid residue 322 of SEQ ID NO: 2, and a valine residue at a position equivalent or analogous to amino acid residue 323 of SEQ ID NO: 2, k) a glutamic acid residue at a position equivalent to or analogous to amino acid residue 450 of SEQ ID NO: 2, a valine residue at a position equivalent to or analogous to amino acid residue 451 of SEQ ID NO: 2, and an arginine residue at a position equivalent to or analogous to amino acid residue 452 of SEQ ID NO: 2,1) a lysine residue at a position equivalent to or analogous to amino acid residue 455 of SEQ ID NO: 2, a serine residue at a position equivalent to or analogous to amino acid residue 456 of SEQ ID NO: 2, an alanine residue at a position equivalent to or analogous to amino acid residue 457 of SEQ ID NO: 2, and a glutamine residue at a position equivalent to or analogous to amino acid residue 458 of SEQ ID NO: 2, in) an alanine residue at a position equivalent to or analogous to amino acid residue 470 of SEQ ID NO: 2, an aspartic acid residue at a position equivalent to or analogous to amino acid residue 472 of SEQ ID NO: 2, and a tyrosine residue at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, n) a threonine residue at a position equivalent to or analogous to amino acid residues 475, 477, and 478 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid residue 476 of SEQ ID NO: 2, and a histidine residue at a position equivalent to or analogous to amino acid residue 479 of SEQ ID NO: 2, or o) an aspartic acid residue at a position equivalent to or analogous to amino acid residue 481 of SEQ ID NO: 2, a glutamic acid residue at a position equivalent to or analogous to amino acid residue 482 of SEQ ID NO: 2, and a proline residue at a position equivalent to or analogous to amino acid residue 483 of SEQ ID NO: 2.Even more preferably, the isolated polypeptide comprises a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, K355R, S385A, V462L, S470A, E471D, T473E, or E500S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.In another specific embodiment, the present invention relates to an isolated polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation that enhances non-steroid binding activity or non-steroid sensitivity of the Group B nuclear receptor ligand binding domain.", "Preferably, the isolated polypeptide comprises a substitution of an amino acid residue at a position equivalent or analogous to amino acid residue selected from the group consisting of a) 401 or 429 of SEQ ID NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 355, 385, 462, 470, 472, 473, or 500 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, f) 455, 456, 457, and 458 of SEQ ID NO: 2, g) 470, 472, and 473 of SEQ ID NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2.More preferably, the isolated polypeptide comprises a substitution of a) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, b) a serine residue at a position equivalent or analogous to amino acid residue 429 of SEQ ID NO: 1, c) an aspartic acid residue at a position equivalent or analogous to amino acid residue 401 of SEQ ID NO: 1, and a serine residue at a position equivalent to or analogous to amino acid residue 429 of SEQ ID NO: 1, d) a serine residue at a position equivalent or analogous to amino acid residue 337 or 500 of SEQ ID NO: 2, e) an arginine residue at a position equivalent to or analogous to amino acid residue 355 of SEQ ID NO: 2, f) an alanine residue at a position equivalent or analogous to amino acid residue 385 or 470 of SEQ ID NO: 2, g) a leucine residue at a position equivalent or analogous to amino acid residue 462 of SEQ ID NO: 2, h) an aspartic acid residue at a position equivalent or analogous to amino acid residue 472 of SEQ ID NO: 2, i) a glutamic acid at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, j) a leucine residue at a position equivalent or analogous to amino acid residue 321 of SEQ ID NO: 2, an arginine residue at a position equivalent or analogous to amino acid residue 322 of SEQ ID NO: 2, and a valine residue at a position equivalent or analogous to amino acid residue 323 of SEQ ID NO: 2, k) a glutamic acid residue at a position equivalent to or analogous to amino acid residue 450 of SEQ ID NO: 2, a valine residue at a position equivalent to or analogous to amino acid residue 451 of SEQ ID NO: 2, and an arginine residue at a position equivalent to or analogous to amino acid residue 452 of SEQ ID NO: 2,1) a lysine residue at a position equivalent to or analogous to amino acid residue 455 of SEQ ID NO: 2, a serine residue at a position equivalent to or analogous to amino acid residue 456 of SEQ ID NO: 2, an alanine residue at a position equivalent to or analogous to amino acid residue 457 of SEQ ID NO: 2, and a glutamine residue at a position equivalent to or analogous to amino acid residue 458 of SEQ ID NO: 2, m) an alanine residue at a position equivalent to or analogous to amino acid residue 470 of SEQ ID NO: 2, an aspartic acid residue at a position equivalent to or analogous to amino acid residue 472 of SEQ ID NO: 2, and a tyrosine residue at a position equivalent to or analogous to amino acid residue 473 of SEQ ID NO: 2, n) a threonine residue at a position equivalent to or analogous to amino acid residues 475, 477, and 478 of SEQ ID NO: 2, an arginine residue at a position equivalent to or analogous to amino acid residue 476 of SEQ ID NO: 2, and a histidine residue at a position equivalent to or analogous to amino acid residue 479 of SEQ ID NO: 2, or o) an aspartic acid residue at a position equivalent to or analogous to amino acid residue 481 of SEQ ID NO: 2, a glutamic acid residue at a position equivalent to or analogous to amino acid residue 482 of SEQ ID NO: 2, and a proline residue at a position equivalent to or analogous to amino acid residue 483 of SEQ ID NO: 2.Even more preferably, the isolated polypeptide comprises a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, K355R, S385A, V462L, S470A, E471D, T473E, or E500S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID NO: 2, e) D450E, A451V, and K452R of SEQ ID NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ID NO: 2, and i) E481D, Q482E, and Q483P of SEQ ID NO: 2.In another specific embodiment, the present invention relates to an isolated polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation enhances both steroid binding activity or steroid sensitivity and non-steroid binding activity or non-steroid sensitivity of the Group B ligand binding domain.", "In another specific embodiment, the present invention also relates to an isolated polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation increases ligand sensitivity of a heterodimer comprising the Group B nuclear receptor ligand binding domain comprising a substitution mutation and a dimerization partner.", "In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide.", "In another specific embodiment, the dimerization partner is a truncated EcR polypeptide.", "The present invention also relates to compositions comprising an isolated polypeptide according to the invention.", "Method of Modulating Gene Expression of the Invention Applicants' invention also relates to methods of modulating gene expression in a host cell using a gene expression modulation system according to the invention.", "Specifically, Applicants' invention provides a method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; and b) introducing into the host cell a ligand; wherein the gene to be modulated is a component of a gene expression cassette comprising: i) a response element comprising a domain recognized by the DNA binding domain of the gene expression system; ii) a promoter that is activated by the transactivation domain of the gene expression system; and iii) a gene whose expression is to be modulated, whereby upon introduction of the ligand into the host cell, expression of the gene is modulated.", "The invention also provides a method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; b) introducing into the host cell a gene expression cassette according to the invention, wherein the gene expression cassette comprises i) a response element comprising a domain recognized by the DNA binding domain from the gene expression system; ii) a promoter that is activated by the transactivation domain of the gene expression system; and iii) a gene whose expression is to be modulated; and c) introducing into the host cell a ligand; whereby upon introduction of the ligand into the host cell, expression of the gene is modulated.", "Applicants' invention also provides a method of modulating the expression of a gene in a host cell comprising a gene expression cassette comprising a response element comprising a domain to which the DNA binding domain from the first hybrid polypeptide of the gene expression modulation system binds; a promoter that is activated by the transactivation domain of the second hybrid polypeptide of the gene expression modulation system; and a gene whose expression is to be modulated; wherein the method comprises the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; and b) introducing into the host cell a ligand; whereby upon introduction of the ligand into the host, expression of the gene is modulated.", "Genes of interest for expression in a host cell using Applicants' methods may be endogenous genes or heterologous genes.", "Nucleic acid or amino acid sequence information for a desired gene or protein can be located in one of many public access databases, for example, GENBANK, EMBL, Swiss-Prot, and PIR, or in many biology related journal publications.", "Thus, those skilled in the art have access to nucleic acid sequence information for virtually all known genes.", "Such information can then be used to construct the desired constructs for the insertion of the gene of interest within the gene expression cassettes used in Applicants' methods described herein.", "Examples of genes of interest for expression in a host cell using Applicants' methods include, but are not limited to: antigens produced in plants as vaccines, enzymes like alpha-amylase, phytase, glucanes, xylase and xylanase, genes for resistance against insects, nematodes, fungi, bacteria, viruses, and abiotic stresses, nutraceuticals, antigens, pharmaceuticals, vitamins, genes for modifying amino acid content, herbicide resistance, cold, drought, and heat tolerance, industrial products, oils, protein, carbohydrates, antioxidants, male sterile plants, flowers, fuels, other output traits, genes encoding therapeutically desirable polypeptides or products that may be used to treat a condition, a disease, a disorder, a dysfunction, a genetic defect, such as monoclonal antibodies, enzymes, proteases, cytokines, interferons, insulin, erythropoietin, clotting factors, other blood factors or components, viral vectors for gene therapy, virus for vaccines, targets for drug discovery, functional genomics, and proteomics analyses and applications, and the like.", "Acceptable ligands are any that modulate expression of the gene when binding of the DNA binding domain of the gene expression system according to the invention to the response element in the presence of the ligand results in activation or suppression of expression of the genes.", "Preferred ligands include an ecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, and the like, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N′-diarylhydrazines such as those disclosed in U.S. Pat.", "Nos.", "6,013,836; 5,117,057; 5,530,028; and 5,378,726; dibenzoylalkyl cyanohydrazines such as those disclosed in European Application No.", "461,809; N-alkyl-N,N′-diaroylhydrazines such as those disclosed in U.S. Pat.", "No.", "5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those disclosed in European Application No.", "234,994; N-aroyl-N-alkyl-N′-aroylhydrazines such as those described in U.S. Pat.", "No.", "4,985,461; each of which is incorporated herein by reference and other similar materials including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, farnesol, bile acids, 1,1-biphosphonate esters, Juvenile hormone III, a 9-cis-retinoic acid, 4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-napthyl)-ethenyl)benzoic acid (3-methyl-TTEB), ((E)-2)-2-(5,6,7,8-tetrat-hydro-3,5,5,8,8-pentamethyl-2-napthyl)propen-1-yl)-4-thiophenecarboxylic acid), 2-(5,6,7,8-tetra-hydro-3,5,5,8,8-tetramethyl-2-naphthyl)-2-(carboxyphenyl)-1,3-dioxolane, 4-(5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyl-dibenzo (b,e) (1,4)diazepin-11-yl)-benzoic acid (HX600) or thiadiazepin analogs thereof, 3,7,11,15-tetramethyl hexadeconoic acid (phytanic acid), 6-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl)nicotinic acid, 2-(4-caroxyphenyl)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1,3-dithiane, or 4-(2-methyl)-1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl) propenyl)benzoic acid, and the like.", "In a preferred embodiment, the ligand for use in Applicants' method of modulating expression of gene is a compound of the formula: wherein: E is a (C4-C6)alkyl containing a tertiary carbon or a cyano(C3-C5)alkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C°CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2; R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C′CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, NH—CN, or joined with R3 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R3 is H, Et, or joined with R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R4, R5, and R6 are independently H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, C°CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.", "In another preferred embodiment, the ligand for use in Applicants' method of modulating expression of gene is an ecdysone, 20-hydroxyecdysone, ponasterone A, or muristerone A.", "In another preferred embodiment, the ligand for use in Applicants' method of modulating expression of gene is a 9-cis-retinoic acid, 4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-napthyl)-ethenyl)benzoic acid (3-methyl-TTEB), ((E)-2)-2-(5,6,7,8-tetrat-hydro-3,5,5,8,8-pentamethyl-2-napthyl)propen-1-yl)-4-thiophenecarboxylic acid), 2-(5,6,7,8-tetra-hydro-3,5,5,8,8-tetramethyl-2-naphthyl)-2-(carboxyphenyl)-1,3-dioxolane, 4-(5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyl-dibenzo (b,e) (1,4)diazepin-11-yl)-benzoic acid (HX600) or thiadiazepin analogs thereof, 3,7,11,15-tetramethyl hexadeconoic acid (phytanic acid), 6-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl)nicotinic acid, 2-(4-caroxyphenyl)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1,3-dithiane, or 4-(2-methyl)-1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl) propenyl)benzoic acid.", "In another preferred embodiment, a second ligand may be used in addition to the first ligand discussed above in Applicants' method of modulating expression of a gene.", "Preferably, this second ligand is 9-cis-retinoic acid or a synthetic analog of retinoic acid.", "Host Cells and Non-Human Organisms of the Invention As described above, the gene expression modulation system of the present invention may be used to modulate gene expression in a host cell.", "Expression in transgenic host cells may be useful for the expression of various genes of interest.", "Applicants' invention provides for modulation of gene expression in prokaryotic and eukaryotic host cells.", "Expression in transgenic host cells is useful for the expression of various polypeptides of interest including but not limited to antigens produced in plants as vaccines, enzymes like alpha-amylase, phytase, glucanes, xylase and xylanase, genes for resistance against insects, nematodes, fungi, bacteria, viruses, and abiotic stresses, nutraceuticals, antigens, pharmaceuticals, vitamins, genes for modifying amino acid content, herbicide resistance, cold, drought, and heat tolerance, industrial products, oils, protein, carbohydrates, antioxidants, male sterile plants, flowers, fuels, other output traits, therapeutic polypeptides, pathway intermediates; for the modulation of pathways already existing in the host for the synthesis of new products heretofore not possible using the host; cell based assays; functional genomics assays, biotherapeutic protein production, proteomics assays, and the like.", "Additionally the gene products may be useful for conferring higher growth yields of the host or for enabling an alternative growth mode to be utilized.", "Thus, Applicants' invention provides an isolated host cell comprising a gene expression system according to the invention.", "The present invention also provides an isolated host cell comprising a gene expression cassette according to the invention.", "Applicants' invention also provides an isolated host cell comprising a polynucleotide or a polypeptide according to the invention.", "In another embodiment, the invention relates to a host cell transfected with an expression vector according to the invention.", "The host cell may be a bacterial cell, a fungal cell, a nematode cell, an insect cell, a fish cell, a plant cell, an avian cell, an animal cell, or a mammalian cell.", "In still another embodiment, the invention relates to a method for producing a nuclear receptor ligand binding domain comprising a substitution mutation, wherein the method comprises culturing the host cell as described above in culture medium under conditions permitting expression of a polynucleotide encoding the nuclear receptor ligand binding domain comprising a substitution mutation, and isolating the nuclear receptor ligand binding domain comprising a substitution mutation from the culture.", "In a specific embodiment, the isolated host cell is a prokaryotic host cell or a eukaryotic host cell.", "In another specific embodiment, the isolated host cell is an invertebrate host cell or a vertebrate host cell.", "Preferably, the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a nematode cell, an insect cell, a fish cell, a plant cell, an avian cell, an animal cell, and a mammalian cell.", "More preferably, the host cell is a yeast cell, a nematode cell, an insect cell, a plant cell, a zebrafish cell, a chicken cell, a hamster cell, a mouse cell, a rat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell, a cow cell, a pig cell, a horse cell, a sheep cell, a simian cell, a monkey cell, a chimpanzee cell, or a human cell.", "Examples of preferred host cells include, but are not limited to, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterial species such as those in the genera Synechocystis, Synechococcus, Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella, plant species selected from the group consisting of an apple, Arabidopsis, bajra, banana, barley, beans, beet, blackgram, chickpea, chili, cucumber, eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat, animal, and mammalian host cells.", "In a specific embodiment, the host cell is a yeast cell selected from the group consisting of a Saccharomyces, a Pichia, and a Candida host cell.", "In another specific embodiment, the host cell is a Caenorhabdus elegans nematode cell.", "In another specific embodiment, the host cell is an insect cell.", "In another specific embodiment, the host cell is a plant cell selected from the group consisting of an apple, Arabidopsis, bajra, banana, barley, beans, beet, blackgram, chickpea, chili, cucumber, eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat cell.", "In another specific embodiment, the host cell is a zebrafish cell.", "In another specific embodiment, the host cell is a chicken cell.", "In another specific embodiment, the host cell is a mammalian cell selected from the group consisting of a hamster cell, a mouse cell, a rat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell, a cow cell, a pig cell, a horse cell, a sheep cell, a monkey cell, a chimpanzee cell and a human cell.", "Host cell transformation is well known in the art and may be achieved by a variety of methods including but not limited to electroporation, viral infection, plasmid/vector transfection, non-viral vector mediated transfection, Agrobacterium-mediated transformation particle bombardment, and the like.", "Expression of desired gene products involves culturing the transformed host cells under suitable conditions and inducing expression of the transformed gene.", "Culture conditions and gene expression protocols in prokaryotic and eukaryotic cells are well known in the art (see General Methods section of Examples).", "Cells may be harvested and the gene products isolated according to protocols specific for the gene product.", "In addition, a host cell may be chosen which modulates the expression of the inserted polynucleotide, or modifies and processes the polypeptide product in the specific fashion desired.", "Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification [e.g., glycosylation, cleavage (e.g., of signal sequence)] of proteins.", "Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.", "For example, expression in a bacterial system can be used to produce a non-glycosylated core protein product.", "However, a polypeptide expressed in bacteria may not be properly folded.", "Expression in yeast can produce a glycosylated product.", "Expression in eukaryotic cells can increase the likelihood of “native” glycosylation and folding of a heterologous protein.", "Moreover, expression in mammalian cells can provide a tool for reconstituting, or constituting, the polypeptide's activity.", "Furthermore, different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent.", "Applicants' invention also relates to a non-human organism comprising an isolated host cell according to the invention.", "In a specific embodiment, the non-human organism is a prokaryotic organism or a eukaryotic organism.", "In another specific embodiment, the non-human organism is an invertebrate organism or a vertebrate organism.", "Preferably, the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, a nematode, an insect, a fish, a plant, a bird, an animal, and a mammal.", "More preferably, the non-human organism is a yeast, a nematode, an insect, a plant, a zebrafish, a chicken, a hamster, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a cow, a pig, a horse, a sheep, a simian, a monkey, or a chimpanzee.", "In a specific embodiment, the non-human organism is a yeast selected from the group consisting of Saccharomyces, Pichia, and Candida.", "In another specific embodiment, the non-human organism is a Caenorhabdus elegans nematode.", "In another specific embodiment, the non-human organism is a plant selected from the group consisting of an apple, Arabidopsis, bajra, banana, barley, beans, beet, blackgram, chickpea, chili, cucumber, eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat.", "In another specific embodiment, the non-human organism is a Mus musculus mouse.", "Measuring Gene Expression/Transcription One useful measurement of Applicants' methods of the invention is that of the transcriptional state of the cell including the identities and abundances of RNA, preferably mRNA species.", "Such measurements are conveniently conducted by measuring cDNA abundances by any of several existing gene expression technologies.", "Nucleic acid array technology is a useful technique for determining differential mRNA expression.", "Such technology includes, for example, oligonucleotide chips and DNA microarrays.", "These techniques rely on DNA fragments or oligonucleotides which correspond to different genes or cDNAs which are immobilized on a solid support and hybridized to probes prepared from total mRNA pools extracted from cells, tissues, or whole organisms and converted to cDNA.", "Oligonucleotide chips are arrays of oligonucleotides synthesized on a substrate using photolithographic techniques.", "Chips have been produced which can analyze for up to 1700 genes.", "DNA microarrays are arrays of DNA samples, typically PCR products, that are robotically printed onto a microscope slide.", "Each gene is analyzed by a full or partial-length target DNA sequence.", "Microarrays with up to 10,000 genes are now routinely prepared commercially.", "The primary difference between these two techniques is that oligonucleotide chips typically utilize 25-mer oligonucleotides which allow fractionation of short DNA molecules whereas the larger DNA targets of microarrays, approximately 1000 base pairs, may provide more sensitivity in fractionating complex DNA mixtures.", "Another useful measurement of Applicants' methods of the invention is that of determining the translation state of the cell by measuring the abundances of the constituent protein species present in the cell using processes well known in the art.", "Where identification of genes associated with various physiological functions is desired, an assay may be employed in which changes in such functions as cell growth, apoptosis, senescence, differentiation, adhesion, binding to a specific molecules, binding to another cell, cellular organization, organogenesis, intracellular transport, transport facilitation, energy conversion, metabolism, myogenesis, neurogenesis, and/or hematopoiesis is measured.", "In addition, selectable marker or reporter gene expression may be used to measure gene expression modulation using Applicants' invention.", "Other methods to detect the products of gene expression are well known in the art and include Southern blots (DNA detection), dot or slot blots (DNA, RNA), northern blots (RNA), RT-PCR (RNA), western blots (polypeptide detection), and ELISA (polypeptide) analyses.", "Although less preferred, labeled proteins can be used to detect a particular nucleic acid sequence to which it hybridizes.", "In some cases it is necessary to amplify the amount of a nucleic acid sequence.", "This may be carried out using one or more of a number of suitable methods including, for example, polymerase chain reaction (“PCR”), ligase chain reaction (“LCR”), strand displacement amplification (“SDA”), transcription-based amplification, and the like.", "PCR is carried out in accordance with known techniques in which, for example, a nucleic acid sample is treated in the presence of a heat stable DNA polymerase, under hybridizing conditions, with one pair of oligonucleotide primers, with one primer hybridizing to one strand (template) of the specific sequence to be detected.", "The primers are sufficiently complementary to each template strand of the specific sequence to hybridize therewith.", "An extension product of each primer is synthesized and is complementary to the nucleic acid template strand to which it hybridized.", "The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers.", "Following a sufficient number of rounds of synthesis of extension products, the sample may be analyzed as described above to assess whether the sequence or sequences to be detected are present.", "Ligand Screening Assays The present invention also relates to methods of screening for a compound that induces or represses transactivation of a nuclear receptor ligand binding domain comprising a substitution mutation in a cell by contacting a nuclear receptor ligand binding domain with a candidate molecule and detecting reporter gene activity in the presence of the ligand.", "Candidate compounds may be either agonists or antagonists of the nuclear receptor ligand binding domain.", "In a preferred embodiment, the nuclear receptor ligand binding domain is expressed from a polynucleotide in the cell and the transactivation activity (i.e., expression or repression of a reporter gene) or compound binding activity is measured.", "Accordingly, in addition to rational design of agonists and antagonists based on the structure of a nuclear receptor ligand binding domain, the present invention contemplates an alternative method for identifying specific ligands of a nuclear receptor ligand binding domain using various screening assays known in the art.", "Any screening technique known in the art can be used to screen for Group B nuclear receptor ligand binding domain agonists or antagonists.", "For example, a suitable cell line comprising a nuclear receptor-based gene expression system according to the invention can be transfected with a gene expression cassette encoding a marker gene operatively linked to an inducible or repressible promoter.", "The transfected cells are then exposed to a test solution comprising a candidate agonist or antagonist compound, and then assayed for marker gene expression or repression.", "The presence of more marker gene expression relative to control cells not exposed to the test solution is an indication of the presence of an agonist compound in the test solution.", "Conversely, the presence of less marker gene expression relative to control cells not exposed to the test solution is an indication of the presence of an antagonist compound in the test solution.", "The present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and agonize or antagonize a Group B nuclear receptor ligand binding domain according to the invention in vivo.", "For example, natural products libraries can be screened using assays of the invention for molecules that agonize or antagonize nuclear receptor-based gene expression system activity.", "Identification and screening of antagonists is further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination.", "These techniques provide for the rational design or identification of agonists and antagonists.", "Another approach uses recombinant bacteriophage to produce large libraries.", "Using the “phage method” [Scott and Smith, 1990, Science 249:386-390 (1990); Cwirla, et al., Proc.", "Natl.", "Acad.", "Sci., 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)], very large libraries can be constructed (106-108 chemical entities).", "A second approach uses primarily chemical methods, of which the Geysen method [Geysen et al., Molecular Immunology 23:709-715 (1986); Geysen et al.", "J. Immunologic Method 102:259-274 (1987)] and the method of Fodor et al.", "[Science 251:767-773 (1991)] are examples.", "Furka et al.", "[14th International Congress of Biochemistry, Volume 5, Abstract FR:013 (1988); Furka, Int.", "J. Peptide Protein Res.", "37:487-493 (1991)], Houghton [U.S. Pat.", "No.", "4,631,211, issued December 1986] and Rutter et al.", "[U.S. Pat.", "No.", "5,010,175, issued Apr.", "23, 1991] describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.", "In another aspect, synthetic libraries [Needels et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 90:10700-4 (1993); Ohlmeyer et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 90:10922-10926 (1993); Lam et al., International Patent Publication.", "No.", "WO 92/00252; Kocis et al., International Patent Publication No.", "WO 9428028, each of which is incorporated herein by reference in its entirety], and the like can be used to screen for candidate ligands according to the present invention.", "The screening can be performed with recombinant cells that express a nuclear receptor ligand binding domain according to the invention, or alternatively, using purified protein, e.g.", "; produced recombinantly, as described above.", "For example, labeled, soluble nuclear receptor ligand binding domains can be used to screen libraries, as described in the foregoing references.", "In one embodiment, a Group B nuclear receptor ligand binding domain according to the invention may be directly labeled.", "In another embodiment, a labeled secondary reagent may be used to detect binding of a nuclear receptor ligand binding domain of the invention to a molecule of interest, e.g., a molecule attached to a solid phase support.", "Binding may be detected by in situ formation of a chromophore by an enzyme label.", "Suitable enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase.", "In a further embodiment, a two-color assay, using two chromogenic substrates with two enzyme labels on different acceptor molecules of interest, may be used.", "Cross-reactive and singly-reactive ligands may be identified with a two-color assay.", "Other labels for use in the invention include colored latex beads, magnetic beads, fluorescent labels (e.g., fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially Eu3+, to name a few fluorophores), chemiluminescent molecules, radio-isotopes, or magnetic resonance imaging labels.", "Two color assays may be performed with two or more colored latex beads, or fluorophores that emit at different wavelengths.", "Labeled molecules or cells may be detected visually or by mechanical/optical means.", "Mechanical/optical means include fluorescence activated sorting, i.e., analogous to FACS, and micromanipulator removal means.", "The present invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention.", "EXAMPLES Applicants have developed a novel nuclear receptor-based inducible gene expression system comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation.", "Applicants have shown that the effect of such a substitution mutation may increase ligand binding activity or ligand sensitivity and may be steroid or non-steroid specific.", "Thus, Applicants' invention provides a Group B nuclear receptor-based inducible gene expression system useful for modulating expression of a gene of interest in a host cell.", "Applicants' novel inducible gene expression system and its use in methods of modulating gene expression in a host cell overcome the limitations of currently available inducible expression systems and provide the skilled artisan with an effective means to control gene expression.", "Applicants' novel substitution mutated nuclear receptor polynucleotides and polypeptides are useful in a nuclear receptor-based inducible gene modulation system for various applications including but not limited to gene therapy, expression of proteins of interest in host cells, production of transgenic organisms, and cell-based assays.", "General Methods Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) and by T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc.", "and Wiley-Interscience (1987).", "Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art.", "Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A.", "Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass.", "(1989).", "All reagents, restriction enzymes and materials used for the growth and maintenance of host cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.", "), or Sigma Chemical Company (St. Louis, Mo.)", "unless otherwise specified.", "Manipulations of genetic sequences may be accomplished using the suite of programs available from the Genetics Computer Group Inc. (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).", "Where the GCG program “Pileup” is used the gap creation default value of 12, and the gap extension default value of 4 may be used.", "Where the CGC “Gap” or “Bestfit” program is used the default gap creation penalty of 50 and the default gap extension penalty of 3 may be used.", "In any case where GCG program parameters are not prompted for, in these or any other GCG program, default values may be used.", "The meaning of abbreviations is as follows: “h” means hour(s), “min” means minute(s), “sec” means second(s), “d” means day(s), “μl” means microliter(s), “ml” means milliliter(s), “L” means liter(s), “μM” means micromolar, “mM” means millimolar, “μg” means microgram(s), “mg” means milligram(s), “A” means adenine or adenosine, “T” means thymine or thymidine, “G” means guanine or guanosine, “C” means cytidine or cytosine, “xg” means times gravity, “nt” means nucleotide(s), “aa” means amino acid(s), “bp” means base pair(s), “kb” means kilobase(s), “k” means kilo, “μ” means micro, and “C” means degrees Celsius.", "Example 1 This Example describes the construction of several gene expression cassettes comprising novel substitution mutated Group B nuclear receptor polynucleotides and polypeptides of the invention for use in a nuclear receptor-based inducible gene expression system.", "Applicants constructed several gene expression cassettes based on the spruce budworm Choristoneura fumiferana EcR (“CfEcR”), mouse Mus musculus retinoid X receptor α (“MmRXRα”), human Homo sapiens retinoid X receptor β (“HsRXRβ”), locust Locusta migratoria ultraspiracle protein (referred to herein interchangeably as “LmUSP” or “LmRXR”), which is an invertebrate RXR homolog of vertebrate RXR, and C. fumiferana USP (“CfUSP”).", "The prepared receptor constructs comprise a ligand binding domain of either an EcR, an invertebrate USP, a vertebrate RXR, or an invertebrate RXR; and a GAL4 DNA binding domain (DBD) or a VP16 transactivation domain (AD).", "The reporter constructs include a reporter gene, luciferase or LacZ (β-galactosidase), operably linked to a synthetic promoter construct that comprises a GAL4 response element to which the Ga14 DBD binds.", "Various combinations of these receptor and reporter constructs were cotransfected into mammalian cells as described in Examples 2-4 infra.", "Gene Expression Cassettes: The gene expression cassettes (switches) were constructed as followed, using standard cloning methods available in the art.", "The following is a brief description of preparation and composition of each switch used in the Examples described herein.", "1.1—GAL4CfEcR-DEF/VP16LmUSP-EF: The wild-type D, E, and F domains from spruce budworm Choristoneura fumiferana EcR (“CfEcR-DEF”; SEQ ID NO: 20) were fused to a GAL4 DNA binding domain (“Gal4DNABD” or “Gal4 DBD”; SEQ ID NO: 5) and placed under the control of an SV40e promoter (SEQ ID NO: 21).", "The E and F domains from locust Locusta migratoria ultraspiracle protein (“LmUSP-EF”; SEQ ID NO: 22) were fused to the transactivation domain from VP16 (“VP16AD”; SEQ ID NO: 11) and placed under the control of an SV40e promoter (SEQ ID NO: 21).", "Five consensus GAL4 response element binding sites (“5XGAL4RE”; comprising 5 copies of a GAL4RE comprising SEQ ID NO: 18) were fused to a synthetic E1b minimal promoter (SEQ ID NO: 23) and placed upstream of the luciferase gene (SEQ ID NO: 24).", "1.2—GAL4CfEcR-DEF/VP16MmRXRα-EF: This construct was prepared in the same way as in switch 1.1 above except LmUSP-EF was replaced with the E and F domains of MmRXRα (“MmRXRα-EF”; SEQ ID NO: 25).", "1.3—GAL4CfEcR-DEF/VP16mutantMmRXRα-EF: This construct was prepared in the same way as in switch 1.2 above except wild-type MmRXRα-EF was replaced with mutant MmRXRα-EF comprising a ligand binding domain comprising a substitution mutation selected from Table 1 below.", "TABLE 1 Substitution Mutants of Mus musculus Retinoid X Receptor α (“MmRXRα”) Ligand Binding Domain (LBD).", "Corresponding amino MmRXRα Resulting “WT to acid in full length LBD Mutant” Amino MmRXRα Mutation Acid Substitution (SEQ ID NO: 1) E401D Glutamic Acid (E) to 401 Aspartic Acid (D) G429S Glycine (G) to Serine (S) 429 E401D/ Glutamic Acid (E) to 401 and 429 G429S Aspartic Acid (D) and respectively Glycine (G) to Serine (S) 1.4—GAL4CfEcR-DEF/VP16MmRXRαH1-7-LmUSP-H8-12chimera: This construct was prepared in the same way as in switch 1.1 above except LmUSP-EF was replaced with a chimeric ligand binding domain comprising helices (H) 1-7 of MmRXRα-EF and helices 8-12 of LmUSP-EF (SEQ ID NO: 26).", "1.5—GAL4CfEcR-DEF/VP16HsRXRβ-EF: This construct was prepared in the same way as in switch 1.1 above except LmUSP-EF was replaced with the E and F domains of HsRXRβ (“HsRXRβ-EF”; SEQ ID NO: 27).", "1.6—GAL4CfEcR-DEF/VP16mutantHsRXRβ-EF: This construct was prepared in the same way as in switch 1.5 above except wild-type HsRXRβ-EF was replaced with mutant HsRXRβ-EF comprising a ligand binding domain comprising a substitution mutation selected from Table 2 below.", "TABLE 2 Substitution Mutants of Homo sapiens Retinoid X Receptor β (“HsRXRβ”) Ligand Binding Domain (LBD).", "Corresponding HsRXRβ amino acid in full LBD Resulting “WT to Mutant” length HsRXRβ Mutation Amino Acid Substitution (SEQ ID NO: 2) G321L/ Glycine (G) to Leucine (L), 321, 322, and 323 P322R/ Proline (P) to Arginine (R), and respectively G323V Glycine (G) to Valine (V) T337S Threonine (T) to Serine (S) 337 D344N Aspartic Acid (D) to Asparagine (N) 344 K355R Lysine (K) to Arginine (R) 355 S385A Serine (S) to Alanine (A) 385 M431L Methionine (M) to Leucine (L) 431 R442K Arginine (R) to Lysine (K) 442 D450E/ Aspartic Acid (D) to Glutamic Acid (E), 450, 451, and 452 A451V/ Alanine (A) to Valine (V), and respectively K452R Lysine (K) to Arginine (R) S455K/ Serine (S) to Lysine (K), 455, 456, 457, and N456S/ Asparagine (N) to Serine (S), 458 respectively P457A/ Proline (P) to Alanine (A), and S458Q Serine (S) to Glutamine (Q) V462L Valine (V) to Leucine (L) 462 S470A Serine (S) to Alanine (A) 470 E472D Glutamic Acid (E) to Aspartic Acid (D) 472 T473E Threonine (T) to Glutamic Acid (E) 473 S470A/ Serine (S) to Alanine (A), 470, 472, and 473 E472D/ Glutamic Acid (E) to Aspartic Acid (D), respectively T473Y and Threonine (T) to Tyrosine (Y) C475T/ Cysteine (C) to Threonine (T), 475, 476, 477, 478, K476R/ Lysine (K) to Arginine (R), and 479 respectively Q477T/ Glutamine (Q) to Threonine (T), K478T/ Lysine (K) to Threonine (T), and Y479H Tyrosine (Y) to Histidine (H) E481D/ Glutamic Acid (E) to Aspartic Acid (D), 481, 482, and 483 Q482E/ Glutamine (Q) to Glutamic Acid (E), respectively Q483P and Glutamine (Q) to Proline (P) A495S Alanine (A) to Serine (S) 495 G500S Glycine (G) to Serine (S) 500 K511R Lysine (K) to Arginine (R) 511 T516V Threonine (T) to Valine (V) 516 A528S Alanine (A) to Serine (S) 528 Construction of Retinoid X Receptor Ligand Binding Domains Comprising a Substitution Mutation: In an effort to modify RXR transactivation activity, residues within the RXR ligand binding domains that were predicted to be important based upon sequence comparisons were mutated in RXRs from two different organisms.", "Tables 1 and 2 indicate the amino acid residues within the ligand binding domain of MmRxRα and HsRXRβ, respectively that were mutated and examined for modification of transactivation ability.", "Each one of the amino acid substitution mutations listed in Tables 1 and 2 was constructed in an RXR cDNA by PCR mediated site-directed mutagenesis.", "One double point mutant RXR LBD (see Table 1), four different triple point mutant RXR LBDs (see Table 2), one quadruple point mutant RXR LBD (see Table 2), and one quintuple point mutant RXR LBD (see Table 2) were also constructed.", "PCR site-directed mutagenesis was performed using the Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) using the reaction conditions and cycling parameters as follows.", "PCR site-directed mutagenesis was performed using 1× reaction buffer (supplied by manufacturer), 50 ng of dsDNA template, 125 ng of forward primer (FP), 125 ng of reverse complementary primer (RCP), and 1 μl of dNTP mix (supplied by manufacturer) in a final reaction volume of 50 μL.", "The forward primer and reverse complementary primer pairs used to produce each RXR substitution mutation are presented in Tables 3 and 4.The cycling parameters used consisted of one cycle of denaturing at 95° C. for 30 seconds, followed by 16 cycles of denaturing at 95° C. for 30 seconds, annealing at 55° C. for 1 minute, and extending at 68° C. for 22 minutes.", "TABLE 3 PCR Primers for Substitution Mutant MmRXRα Ligand Binding Domain Construction PRIMER PRIMER NUCLEOTIDE SEQUENCE MUTANT (SEQ ID NO:) (5′ TO 3′) E401D FP GTGTATGCGTCACTAGATGCGTACTGCAAACAC (SEQ ID NO: 28) E401D RCP GTGTTTGCAGTACGCATCTAGTGACGCATACAC (SEQ ID NO: 29) G429S FP GCACTGCGTTCCATCAGCCTCAAGTGCCTGGAG (SEQ ID NO: 30) G429S RCP CTCCAGGCACTTGAGGCTGATGGAACGCAGTGC (SEQ ID NO: 31) TABLE 4 PCR Primers for Substitution Mutant HsRXRβ Ligand Binding Domain Construction PRIMER PRIMER NUCLEOTIDE SEQUENCE MUTANT (SEQ ID NO:) (5′ TO 3′) G321L/ FP GACCAGGGCGTTGAGCGTCGTGTGGGAACCGGGGGT P322R/ (SEQ ID NO: 32) AGC G323V G321L/ RCP GCTACCCCCGGTTCCCACACGACGCTCAACGCCCTGG P322R/ (SEQ ID NO: 33) TC G323V T337S FP CCAAATGACCCTGTGTCTAACATCTGTCAGGC (SEQ ID NO: 34) T337S RCP GCCTGACAGATGTTAGACACAGGGTCATTTGG (SEQ ID NO: 35) D344N FP ATCTGTCAGGCAGCTAACAAACAGCTATTCACG (SEQ ID NO: 36) D344N RCP CGTGAATAGCTGTTTGTTAGCTGCCTGACAGAT (SEQ ID NO: 37) K355R FP CTTGTTGAGTGGGCGAGGAGGATCCCACACTTTTC (SEQ ID NO: 38) K355R RCP GAAAAGTGTGGGATCCTCCTCGCCCACTCAACAAG (SEQ ID NO: 39) S385A FP CTCATTGCCTCCTTTGCACACCGATCCATTGATG (SEQ ID NO: 40) S385A RCP CATCAATGGATCGGTGTGCAAAGGAGGCAATGAG (SEQ ID NO: 41) M43IL FP TCCAAAATGCGTGACCTGAGGATGGACAAGAC (SEQ ID NO: 42) M431L RCP GTCTTGTCCATCCTCAGGTCACGCATTTTGGA (SEQ ID NO: 43) R442K FP GAGCTTGGCTGCCTGAAGGCAATCATTCTGTTTAATC (SEQ ID NO: 44) R442K RCP GATTAAACAGAATGATTGCCTTCAGGCAGCCAAGCTC (SEQ ID NO: 45) D450E/ FP CATTCTGTTTAATCCAGAGGTCAGGGGCCTCTCCAAC A451V/ (SEQ ID NO: 46) CC K452R D450E/ RCP GGGTTGGAGAGGCCCCTGACCTCTGGATTAAACAGAA A451V/ (SEQ ID NO: 47) TG K452R S455K/ FP GATGCCAAGGGCCTCAAGTCCGCGCAGGAGGTGGAG N456S/ (SEQ ID NO: 48) GTCCTG P457A/ S458Q S455K/ RCP CAGGACCTCCACCTCCTGCGCGGACTTGAGGCCCTTG N456S/ (SEQ ID NO: 49) GCATC P457A/ S458Q V462L FP CCTAGTGAGGTGGAGCTCCTGCGGGAGAAAGTGTATG (SEQ ID NO: 50) V462L RCP CATACACTTTCTCCCGCAGGAGCTCCACCTCACTAGG (SEQ ID NO: 51) S470A FP GAGAAAGTGTATGCAGCACTGGAGACCTACTGC (SEQ ID NO: 52) S470A RCP GCAGTAGGTCTCCAGTGCTGCATACACTTTCTC (SEQ ID NO: 53) E472D FP GTGTATGCATCACTGGATACCTACTGCAAACAG (SEQ ID NO: 54) E472D RCP CTGTTTGCAGTAGGTATCCAGTGATGCATACAC (SEQ ID NO: 55) T473E FP GTATGCATCACTGGAGGAGTACTGCAAACAGAAG (SEQ ID NO: 56) T473E RCP CTTCTGTTTGCAGTACTCCTCCAGTGATGCATAC (SEQ ID NO: 57) S470A/ FP GAGAAAGTGTATGCAGCACTGGATGAGTACTGCAAAC E472D/ (SEQ ID NO: 58) AGAAG T473Y S470A/ RCP CTTCTGTTTGCAGTACTCATCCAGTGCTGCATACACTT E472D/ (SEQ ID NO: 59) TCTC T473Y C475T/ FP CATCACTGGAGACCTACACCAGAACGACGCACCCTGA K476R/ (SEQ ID NO: 60) GCAGCAGGGAC Q477T/ K478T/ Y479H C475T/ RCP GTCCCTGCTGCTCAGGGTGCGTCGTTCTGGTGTAGGT K476R/ (SEQ ID NO: 61) CTCCAGTGATG Q477T/ K478T/ Y479H E481D/ FP CAAACAGAAGTACCCTGACGAGCCGGGACGGTTTGCC Q482E/ (SEQ ID NO: 62) AAG Q483P E481D/ RCP CTTGGCAAACCGTCCCGGCTCGTCAGGGTACTTCTGT Q482F/ (SEQ ID NO: 63) TTG Q482E/ Q483P A495S FP CTGCTACGTCTTCCTTCCCTCCGGTCCATTGGC (SEQ ID NO: 64) A495S RCP GCCAATGGACCGGAGGGAAGGAAGACGTAGCAG (SEQ ID NO: 65) G500S FP GCCCTCCGGTCCATTAGCCTTAAGTGTCTAGAG (SEQ ID NO: 66) G500S RCP CTCTAGACACTTAAGGCTAATGGACCGGAGGGC (SEQ ID NO: 67) K511R FP CATCTGTTTTTCTTCAGGCTCATTGGTGACACC (SEQ ID NO: 68) K511R RCP GGTGTCACCAATGAGCCTGAAGAAAAACAGATG (SEQ ID NO: 69) T516V FP AGCTCATTGGTGACGTCCCCATCGACACCTTCC (SEQ ID NO: 70) T516V RCP GGAAGGTGTCGATGGGGACGTCACCAATGAGCT (SEQ ID NO: 71) A528S FP ATGGAGATGCTTGAGTCTCCCCATCAACTGGCC (SEQ ID NO: 72) A528S RCP GGCCAGTTGATGGGGAGACTCAAGCATCTCCAT (SEQ ID NO: 73) The resulting PCR nucleic acid products encoding the mutant RXR ligand binding domains were then each fused to a VP16 transactivation domain as described in Examples 1.3 and 1.6 above.", "The VP16/mutant RXR receptor constructs were tested for activity by transfecting them into NIH3T3 cells along with GAL4/CfEcR-DEF and pFRLuc in the presence of non-steroid ligand.", "Ligand: The non-steroidal ligand N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine (GS™-E) is a synthetic stable ecdysteroid ligand synthesized at Rohm and Haas Company.", "The ligand was dissolved in DMSO and the final concentration of DMSO was maintained at 0.1% in both controls and treatments.", "Transfections: DNAs corresponding to the various switch constructs outlined in Example 1.1-1.6 were transfected into mouse NIH3T3 cells (ATCC) as follows.", "Standard methods for culture and maintenance of the cells were followed.", "Cells were harvested when they reached 50% confluency and plated in 6-, 12- or 24-well plates at 125,000, 50,000, or 25,000 cells, respectively, in 2.5, 1.0, or 0.5 ml of growth medium containing 10% fetal bovine serum (FBS), respectively.", "The next day, the cells were rinsed with growth medium and transfected for four hours.", "Superfect™ (Qiagen Inc.) was used as the transfection reagent.", "For 12-well plates, 4 μl of Superfect™ was mixed with 100 μl growth medium.", "One μg of reporter construct and 0.25 μg of each receptor construct of the receptor pair to be analyzed were added to the transfection mix.", "A second reporter construct was added [pTKRL (Promega), 0.1 μg/transfection mix] that comprises a Renilla luciferase gene operably linked and placed under the control of a thymidine kinase (TK) constitutive promoter and was used for normalization.", "The contents of the transfection mix were mixed in a vortex mixer and let stand at room temperature for 30 minutes.", "At the end of incubation, the transfection mix was added to the cells maintained in 400 μl growth medium.", "The cells were maintained at 37° C. and 5% CO2 for four hours.", "At the end of incubation, 500 μl of growth medium containing 20% FBS and either dimethylsulfoxide (DMSO; control) or a DMSO solution of non-steroidal ligand was added and the cells were maintained at 37° C. and 5% CO2 for 48 hours.", "The cells were harvested and reporter activity was assayed.", "The same procedure was followed for 6 and 24 well plates as well except all the reagents were doubled for 6 well plates and reduced to half for 24-well plates.", "Reporter Assays Cells were harvested 40 hours after adding ligand.", "125 μl of passive lysis buffer (part of Dual-luciferase™ reporter assay system from Promega Corporation) were added to each well of the 24-well plate.", "The plates were placed on a rotary shaker for 15 minutes.", "Twenty μl of lysate were assayed.", "Luciferase activity was measured using Dual-luciferase™ reporter assay system from Promega Corporation following the manufacturer's instructions.", "β-Galactosidase was measured using Galacto-Star™ assay kit from TROPIX following the manufacturer's instructions.", "All luciferase and β-galactosidase activities were normalized using Renilla luciferase as a standard.", "Fold activities were calculated by dividing normalized relative light units (“RLU”) in ligand treated cells with normalized RLU in DMSO treated cells (untreated control).", "Example 2 In mammalian cells, the insect (Choristoneura fumiferana) ecdysone receptor (CfEcR) heterodimerizes with ultraspiracle (LISP) or its homolog retinoid X receptor (RXR) and transactivates genes that are placed under the control of cognate response elements.", "Ligand inducibility of this EcR-based transactivation depends on its heterodimerizing partner.", "As previously shown by Applicants, transactivation through CfEcR in partnership with insect USPs is ligand independent, whereas transactivation through CfEcR in partnership with invertebrate or vertebrate RXRs is ligand dependent (see pending U.S. patent application 60/294,814, incorporated herein by reference in its entirety).", "Applicants have now discovered that the sequence of RXR can be modified by substitution mutation of the ligand binding domain to influence the magnitude of transactivation as well as ligand sensitivity of an EcR-based inducible gene expression system.", "This Example describes the identification of three MmRXRα ligand binding domain substitution mutants that exhibit improved ligand sensitivity in response to non-steroidal ligand.", "Briefly, Applicants constructed three mouse RXR isoform cc ligand binding domain substitution mutants and created VP16/mutantMmRXRα-EF cDNA gene expression cassettes as described in Example 1 above using the Quikchange PCR-mediated site-directed mutagenesis kit (Stratagene, La Jolla, Calif.).", "The mutated cDNAs were tested along with GAL4/CfEcR-DEF in GAL4-driven luciferase reporter assays and the results were compared to wild type VP16/MmRXRα-EF and VP16/MmRXRα-LmUSPchimera-EF switches in mouse NIH3T3 cells.", "Transfections: DNAs corresponding to the various switch constructs outlined in Example 1, specifically switches 1.1-1.4, were transfected into mouse NIH3T3 cells (ATCC) as follows.", "Cells were harvested when they reached 50% confluency and plated in 24 well plates at 12,500 cells/well in 0.5 ml of growth medium containing 10% fetal bovine serum (FBS).", "The next day, the cells were rinsed with growth medium and transfected for four hours.", "Superfect™ (Qiagen Inc.) was found to be the best transfection reagent for 3T3 cells.", "Two μl of Superfect™ was mixed with 100 μl of growth medium and 50 ng of GAL4/CfEcR-DEF cassette, 50 ng of: VP16/LmUSP-EF, VP16/wild-typeMmRXRα-EF, VP16/MmRXRα-LmUSPchimera-EF, or VP16/mutantMmRXRα-EF, and 200 ng of pFRLuc were added to the transfection mix.", "A second reporter construct was added [pTKRL (Promega), 0.05 μg/transfection mix] that comprises a Renilla luciferase gene operably linked and placed under the control of a thymidine kinase (TK) constitutive promoter and was used for normalization.", "The contents of the transfection mix were mixed in a vortex mixer and let stand at room temperature for 30 min.", "At the end of incubation, the transfection mix was added to the cells maintained in 200 μl growth medium.", "The cells were maintained at 37° C. and 5% CO2 for four hours.", "At the end of incubation, 250 μl of growth medium containing 20% FBS and either dimethylsulfoxide (DMSO; control) or a DMSO solution of 0.2, 1, or 10 μM GS™-E [N-(2-ethyl-3-methoxybenzoyl)N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine] non-steroidal ligand was added and the cells were maintained at 37° C. and 5% CO2 for 40 hours.", "The cells were harvested and reporter activity was assayed as described above.", "Fold activities were calculated by dividing normalized relative light units (“RLU”) in ligand treated cells with normalized RLU in DMSO treated cells (untreated control).", "As discussed above, Applicants have previously shown that non-lepidopteran/non-dipteran invertebrate RXRs and RXR homologs (DSPs), referred to herein collectively as invertebrate RXRs, bind EcR and transactivate reporters at higher levels than those achieved by vertebrate RXRs in mammalian cells.", "However, vertebrate RXRs provide lower background levels in the absence of ligand.", "Applicants compared the amino acid sequences from ligand binding domains of vertebrate RXRs and invertebrate RXRs and have identified two particular amino acids that are conserved in all vertebrate RXRs but are different in invertebrate RXRs.", "Applicants determined whether replacing a vertebrate RXR amino acid with an invertebrate RXR amino acid would result in higher activity upon induction, but still provide low background in the absence of ligand.", "Applicants have now identified two amino acid residues within the EF domains of a vertebrate mouse RXR, isoform a (“MmRXRα-EF”) that, when substituted, yield a mutant RXR that exhibits increased sensitivity to a non-steroid ligand.", "The effect of these two substitution mutations: an aspartic acid substitution at amino acid residue 401 (E401D mutant), and a serine substitution at amino acid residue 429 (G429S mutant) of SEQ ID NO: 1, on the activity of the mutated MmRXRα-EF receptor is presented in FIG.", "1.In transactivation assays in NIH3T3 cells, the E401D mutant behaved more like an invertebrate RXR than a vertebrate RXR by demonstrating both higher background and higher induction levels (see FIG.", "1).", "The G429S mutant behaved more like an invertebrate RXR, demonstrating increased sensitivity to ligand but less background (see FIG.", "1).", "Thus, Applicants have demonstrated a surprising and unexpected result that a single amino acid change can drastically alter the behavior of the MmRXRα-EF in the presence and absence of ligand.", "To determine if the combination of these two mutations would provide a further improved RXR than either single point mutation alone, Applicants made a double substitution mutant MmRXRα-EF comprising a point mutation at both positions E401D and G429S.", "Three independent clones of these double mutants (DM) were analyzed in NIH3T3 cells and compared with each single point mutant alone, MmRXRα-EF, and a chimeric vertebrate RXR/invertebrate RXR (see FIG.", "2).", "Applicants' results show that these double mutants work as well as the chimeric RXR, demonstrating low background levels in the absence of ligand and increased induction levels and ligand sensitivity.", "These novel double substitution mutants provide an advantageous RXR for use in in vivo applications.", "Applicants have also determined that these MmRXRα-EF substitution mutants respond better than wild-type HsRXRβ-EF in the presence of a steroid ligand, ponasterone A (Invitrogen), similar to their response to GS™-E presented in FIGS.", "1 and 2 (data not shown).", "Example 3 As discussed above, use of an invertebrate RXR, LmUSP (also referred to herein as LmRXR) as a heterodimeric partner for CfEcR improved both the sensitivity and magnitude of induction of a CfEcR-based inducible gene expression system.", "Applicants have aligned the polypeptide sequences of the LmRXR and HsRXRβ ligand binding domains (domains EF) and identified amino acid residues that are different between these two proteins.", "This Example describes Applicants' analysis of HsRXRβ substitution mutants in which LmRXR amino acids were substituted in place of the wild-type residues in HsRXRβ.", "Applicants have now identified several substitution mutants that modify both the sensitivity and magnitude of induction of a CfEcR-based inducible gene expression system in mammalian cells.", "Briefly, Applicants constructed and analyzed substitution mutants in human RXR isoform β-EF (HsRXRβ-EF), wherein amino acids that are different in HsRXRβ-EF compared to the invertebrate RXR homolog LmUSP-EF were mutated to the LmUSP-EF amino acid residue and assayed for their effect on transactivation activity in NIH3T3 cells as described in Example 2 above in the presence of 0, 0.04, 0.2, 1, 5, or 25 μM GS™-E [N-(2-ethyl-3-methoxybenzoyl)N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine] non-steroidal ligand.", "The cells were harvested and reporter activity was assayed as described above.", "Fold activities were calculated by dividing normalized relative light units (“RLU”) in ligand treated cells with normalized RLU in DMSO treated cells (untreated control).", "As shown in FIG.", "3, HsRXRβ-EF substitution mutants T/S, DAK/EVR, SNPS/KSAQ, V/L, S/A, T/E, CKQKY/TRTTH, and EQQ/DEP improved fold induction and magnitude of induction.", "Applicants' HsRXRβ-EF substitution mutants DAK/EVR, SNPS/KSAQ, V/L, S/A, T/E, CKQKY/TRTTH, and EQQ/DEP also improved non-steroid ligand sensitivity (see FIG.", "3).", "The results presented in FIG.", "3 also show that HsRXRβ-EF substitution mutants D344N, A495S (labeled as A/S with a fold induction of 30), and A528S (labeled as A/S with a fold induction of 21) exhibit reduced fold induction, magnitude of induction, and/or ligand sensitivity compared to wild-type.", "Applicants have also determined that these HsRXRβ-EF substitution mutants respond better than wild-type HsRXRβ-EF in the presence of a steroid ligand, ponasterone A (Invitrogen), similar to their response to GS™-E presented in FIG.", "3 (data not shown).", "Example 4 As discussed above, a CfEcR-based gene regulation system is ligand dependent when RXRs are used as the heterodimeric partner and ligand independent when USPs are used as the heterodimeric partner.", "However, reporter gene expression is induced at very high levels when CfUSP is used as the heterodimeric partner.", "To improve RXR as heterodimeric partner, Applicants have aligned the polypeptide sequences of several USP and RXR ligand binding domains (domains EF) and identified amino acid residues that are different between these two nuclear receptor family members.", "In particular, Applicants identified residues that are conserved in all USPs but are different in RXRs and mutated these residues in HsRXRβ.", "These substitution mutants were analyzed in NIH3T3 cells as described in Example 2 above in the presence of 0, 0.04, 0.2, 1, 5, or 25 μM GS™-E [N-(2-ethyl-3-methoxybenzoyl)N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine] non-steroidal ligand.", "The cells were harvested and reporter activity was assayed as described above.", "Fold activities were calculated by dividing normalized relative light units (“RLU”) in ligand treated cells with normalized RLU in DMSO treated cells (untreated control).", "Briefly, this Example describes the construction and analysis of substitution mutants in human RXR isoform β-EF (HsRXRβ-EF), wherein amino acids that are different in HsRXRβ-EF compared to conserved residues of ultraspiracle proteins were mutated to the USP amino acid residue and assayed for Applicants have also determined that these HsRXRβ-EF substitution mutants respond better than wild-type HsRXRβ-EF in the presence of a steroid ligand, ponasterone A (Invitrogen), similar to their response to GS™-E presented in FIG.", "4 (data not shown)." ] ]	Patent_10468192
[ [ "Novel ecdysone receptor/invertebrate retinoid X receptor-based inducible gene expression system", "This invention relates to the field of biotechnology or genetic engineering.", "Specifically, this invention relates to the field of gene expression.", "More specifically, this invention relates to a novel ecdysone receptor/invertebrate retinoid X receptor-based inducible gene expression system and methods of modulating gene expression in a host cell for applications such as gene therapy, large-scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic organisms." ], [ "1.A gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a first hybrid polypeptide comprising: i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) an ecdysone receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide that encodes a second hybrid polypeptide comprising: i) a transactivation domain; and ii) an invertebrate retinoid X receptor ligand binding domain.", "2.The gene expression modulation system according to claim 1, further comprising a third gene expression cassette comprising: i) a response element recognized by the DNA-binding domain of the first hybrid polypeptide binds; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated.", "3.The gene expression modulation system according to claim 1, wherein the ecdysone receptor ligand binding domain (LBD) of the first hybrid polypeptide is selected from the group consisting of a spruce budworm Choristoneura fumiferana EcR (“CfEcR”) LBD, a beetle Tenebrio molitor EcR (“TmEcR”) LBD, a Manduca sexta EcR (“MsEcR”) LBD, a Heliothies virescens EcR (“HvEcR”) LBD, a midge Chironomus tentans EcR (“CtEcR”) LBD, a silk moth Bombyx mori EcR (“BmEcR”) LBD, a fruit fly Drosophila melanogaster EcR (“DmEcR”) LBD, a mosquito Aedes aegypti EcR (“AaEcR”) LBD, a blowfly Lucilia capitata (“LcEcR”) LBD, a blowfly Lucilia cuprina EcR (“LucEcR”) LBD, a Mediterranean fruit fly Ceratitis capitata EcR (“CcEcR”) LBD, a locust Locusta migratoria EcR (“LmEcR”) LBD, an aphid Myzus persicae EcR (“MpEcR”) LBD, a fiddler crab Celuca pugilator EcR (“CpEcR”) LBD, an ixodid tick Amblyomma americanum EcR (“AmaEcR”) LBD, a whitefly Bamecia argentifoli EcR (“BaEcR”) LBD, and a leafhopper Nephotetix cincticeps EcR (“NcEcR”) LBD.", "4.The gene expression modulation system according to claim 1, wherein the ecdysone receptor ligand binding domain of the first hybrid polypeptide is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 53 and SEQ ID NO: 45.5.The gene expression modulation system according to claim 1, wherein the ecdysone receptor ligand binding domain of the first hybrid polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 43, and SEQ ID NO: 59.6.The gene expression modulation system according to claim 1, wherein the invertebrate retinoid X receptor ligand binding domain of the second hybrid polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.7.The gene expression modulation system according to claim 1, wherein the invertebrate retinoid X receptor ligand binding domain of the second hybrid polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.8.The gene expression modulation system according to claim 1, wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising a DNA-binding domain selected from the group consisting of a GAL4 DNA-binding domain and a LexA DNA-binding domain, and an ecdysone receptor ligand binding domain.", "9.The gene expression modulation system according to claim 1, wherein the second gene expression cassette comprises a polynucleotide that encodes a second hybrid polypeptide comprising a transactivation domain selected from the group consisting of a VP16 transactivation domain and a B42 acidic activator transactivation domain, and an invertebrate retinoid X receptor ligand binding domain.", "10.The gene expression modulation system according to claim 1, wherein the second gene expression cassette comprises a polynucleotide that encodes a second hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a VP16 AD (SEQ ID NO: 37) and a B42 AD (SEQ ID NO: 39), and an invertebrate retinoid X receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.11.The gene expression modulation system according to claim 1, wherein the second gene expression cassette comprises a polynucleotide that encodes a second hybrid polypeptide comprising a transactivation domain comprising an amino acid sequence selected from the group consisting of a VP16 AD (SEQ ID NO: 38) and a B42 AD (SEQ ID NO: 40), and an invertebrate retinoid X receptor ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.12.A gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a first hybrid polypeptide comprising: i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) an invertebrate retinoid X receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide that encodes a second hybrid polypeptide comprising: i) a transactivation domain; and ii) an ecdysone receptor ligand binding domain.", "13.The gene expression modulation system according to claim 12, further comprising a third gene expression cassette comprising: i) a response element recognized by the DNA-binding domain of the first hybrid polypeptide binds; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated.", "14.The gene expression modulation system according to claim 12, wherein the invertebrate retinoid X receptor ligand binding domain of the first hybrid polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.15.The gene expression modulation system according to claim 12, wherein the invertebrate retinoid X receptor ligand binding domain of the first hybrid polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.16.The gene expression modulation system according to claim 12, wherein the ecdysone receptor ligand binding domain of the second hybrid polypeptide is selected from the group consisting of a spruce budworm Choristoneura fumiferana EcR (“CfEcR”) LBD, a beetle Tenebrio molitor EcR (“TmEcR”) LBD, a Manduca sexta EcR (“MsEcR”) LBD, a Heliothies virescens EcR (“HvEcR”) LBD, a midge Chironomus tentans EcR (“CtEcR”) LBD, a silk moth Bombyx mori EcR (“BmEcR”) LBD, a fruit fly Drosophila melanogaster EcR (“DmEcR”) LBD, a mosquito Aedes aegypti EcR (“AaEcR”) LBD, a blowfly Lucilia capitata (“LcEcR”) LBD, a blowfly Lucilia cuprina EcR (“LucEcR”) LBD, a Mediterranean fruit fly Ceratitis capitata EcR (“CcEcR”) LBD, a locust Locusta migratoria EcR (“LmEcR”) LBD, an aphid Myzus persicae EcR (“MpEcR”) LBD, a fiddler crab Celuca pugilator EcR (“CpEcR”) LBD, an ixodid tick Amblyomma americanum EcR (“AmaEcR”) LBD, a whitefly Bamecia argentifoli EcR (“BaEcR”) LBD, and a leafhopper Nephotetix cincticeps EcR (“NcEcR”) LBD.", "17.The gene expression modulation system according to claim 12, wherein the ecdysone receptor ligand binding domain of the second hybrid polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 53 and SEQ ID NO: 45.18.The gene expression modulation system according to claim 12, wherein the ecdysone receptor ligand binding domain of the second hybrid polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 43, and SEQ ID NO: 59.19.The gene expression modulation system according to claim 12, wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising a DNA-binding domain selected from the group consisting of a GAL4 DNA-binding domain and a LexA DNA-binding domain, and an invertebrate retinoid X receptor ligand binding domain.", "20.The gene expression modulation system according to claim 12, wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 33) or a LexA DBD (SEQ ID NO: 35) and an invertebrate retinoid X receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.21.The gene expression modulation system according to claim 12, wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising a DNA-binding domain comprising an amino acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 34) and a LexA DBD (SEQ ID NO: 36), and an invertebrate retinoid X receptor ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.22.The gene expression modulation system according to claim 12, wherein the second gene expression cassette comprises a polynucleotide that encodes a second hybrid polypeptide comprising a transactivation domain selected from the group consisting of a VP16 transactivation domain and a B42 acidic activator transactivation domain, and an ecdysone receptor ligand binding domain.", "23.A gene expression cassette comprising a polynucleotide that encodes a hybrid polypeptide comprising a DNA-binding domain and an invertebrate retinoid X receptor ligand binding domain, wherein the DNA binding domain is from a nuclear receptor other than an invertebrate retinoid X receptor.", "24.The gene expression cassette according to claim 23, wherein the DNA-binding domain is a GAL4 DNA-binding domain or a LexA DNA-binding domain.", "25.The gene expression cassette according to claim 23, wherein the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 33) and a LexA DBD (SEQ ID NO: 35), and an invertebrate retinoid X receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.26.The gene expression cassette according to claim 23, wherein the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide comprising a DNA-binding domain comprising an amino acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 34) and a LexA DBD (SEQ ID NO: 36), and an invertebrate retinoid X receptor ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.27.A gene expression cassette comprising a polynucleotide that encodes a hybrid polypeptide comprising a transactivation domain and an invertebrate retinoid X receptor ligand binding domain, wherein the transactivation domain is from a nuclear receptor other than an invertebrate retinoid X receptor.", "28.The gene expression cassette according to claim 27, wherein the transactivation domain is a VP16 transactivation domain or a B42 acidic activator transactivation domain.", "29.The gene expression cassette according to claim 27, wherein the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a VP16 AD (SEQ ID NO: 37) and a B42 AD (SEQ ID NO: 39), and an invertebrate retinoid X receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.30.The gene expression cassette according to claim 29, wherein the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide comprising a transactivation domain comprising an amino acid sequence selected from the group consisting of a VP16 AD (SEQ ID NO: 38) and a B42 AD (SEQ ID NO: 40), and an invertebrate retinoid X receptor ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.31.An isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a truncated invertebrate retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation reduces ligand binding activity of the truncated invertebrate retinoid X receptor ligand binding domain; (b) a polynucleotide encoding a truncated invertebrate retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation reduces steroid binding activity of the truncated invertebrate retinoid X receptor ligand binding domain; (c) a polynucleotide encoding a truncated invertebrate retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation reduces non-steroid binding activity of the truncated invertebrate retinoid X receptor ligand binding domain; (d) a polynucleotide encoding a truncated invertebrate retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation enhances ligand binding activity of the truncated invertebrate retinoid X receptor ligand binding domain; (e) a polynucleotide encoding a truncated invertebrate retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation enhances steroid binding activity of the truncated invertebrate retinoid X receptor ligand binding domain; (f) a polynucleotide encoding a truncated invertebrate retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation enhances non-steroid binding activity of the truncated invertebrate retinoid X receptor ligand binding domain; (g) a polynucleotide encoding a truncated invertebrate retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation increases ligand sensitivity of the truncated invertebrate retinoid X receptor ligand binding domain; and (h) a polynucleotide encoding a truncated invertebrate retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation increases ligand sensitivity of a heterodimer, wherein the heterodimer comprises said truncated invertebrate retinoid X receptor ligand binding domain and a dimerization partner.", "32-38.", "(canceled) 39.The isolated polynucleotide according to claim 31, wherein the dimerization partner is an ecdysone receptor polypeptide.", "40.An isolated polynucleotide encoding a truncated invertebrate retinoid X receptor ligand binding domain, wherein the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.41.An isolated polypeptide encoded by the isolated polynucleotide according to claim 40.42.An isolated truncated invertebrate retinoid X receptor ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.43.A method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell the gene expression modulation system according to claim 1; and b) introducing into the host cell a ligand; wherein the gene to be modulated is a component of a gene expression cassette comprising: i) a response element recognized by the DNA binding domain from the first hybrid polypeptide; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated; whereby upon introduction of the ligand into the host cell, expression of the gene of b)iii) is modulated.", "44.The method according to claim 43, wherein the ligand is a compound of the formula: wherein: E is a (C4-C6)alkyl containing a tertiary carbon or a cyano(C3-C5)alkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, CoCH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2; R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, CoCH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, NH—CN, or joined with R3 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R3 is H, Et, or joined with R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R4, R5, and R6 are independently H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, CoCH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.", "45.The method according to claim 43, further comprising introducing into the host cell a second ligand, wherein the second ligand is 9-cis-retinoic acid or a synthetic analog of a retinoic acid.", "46.A method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell the gene expression modulation system of claim 12; and b) introducing into the host cell a ligand; wherein the gene to be modulated is a component of a gene expression cassette comprising: i) a response element recognized by the DNA binding domain from the first hybrid polypeptide; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated; whereby upon introduction of the ligand into the host cell, expression of the gene of b)iii) is modulated.", "47.The method according to claim 46, wherein the ligand is a compound of the formula: wherein: E is a (C4-C6)alkyl containing a tertiary carbon or a cyano(C3-C5)alkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, CoCH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2; R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, CoCH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, NH—CN, or joined with R3 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R3 is H, Et, or joined with R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R4, R5, and R6 are independently H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, CoCH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.", "48.The method according to claim 46, further comprising introducing into the host cell a second ligand, wherein the second ligand is 9-cis-retinoic acid or a synthetic analog of a retinoic acid.", "49.An isolated host cell comprising the gene expression modulation system according to claim 1.50.The isolated host cell according to claim 49, wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell, and a mammalian cell.", "51.The isolated host cell according to claim 50, wherein the mammalian cell is a murine cell or a human cell.", "52.An isolated host cell comprising the gene expression modulation system according to claim 12.53.The isolated host cell according to claim 52, wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell, and a mammalian cell.", "54.The isolated host cell according to claim 53, wherein the mammalian cell is a murine cell or a human cell.", "55.A non-human organism comprising the host cell of claim 52.56.The non-human organism according to claim 55, wherein the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, an animal, and a mammal.", "57.The non-human organism according to claim 56, wherein the mammal is selected from the group consisting of a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep, a monkey, and a chimpanzee.", "58.A non-human organism comprising the host cell of claim 52.59.The non-human organism according to claim 58, wherein the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, an animal, and a mammal.", "60.The non-human organism according to claim 59, wherein the mammal is selected from the group consisting of a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep, a monkey, and a chimpanzee." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.", "However, the citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.", "In the field of genetic engineering, precise control of gene expression is a valuable tool for studying, manipulating, and controlling development and other physiological processes.", "Gene expression is a complex biological process involving a number of specific protein-protein interactions.", "In order for gene expression to be triggered, such that it produces the RNA necessary as the first step in protein synthesis, a transcriptional activator must be brought into proximity of a promoter that controls gene transcription.", "Typically, the transcriptional activator itself is associated with a protein that has at least one DNA binding domain that binds to DNA binding sites present in the promoter regions of genes.", "Thus, for gene expression to occur, a protein comprising a DNA binding domain and a transactivation domain located at an appropriate distance from the DNA binding domain must be brought into the correct position in the promoter region of the gene.", "The traditional transgenic approach utilizes a cell-type specific promoter to drive the expression of a designed transgene.", "A DNA construct containing the transgene is first incorporated into a host genome.", "When triggered by a transcriptional activator, expression of the transgene occurs in a given cell type.", "Another means to regulate expression of foreign genes in cells is through inducible promoters.", "Examples of the use of such inducible promoters include the PR1-a promoter, prokaryotic repressor-operator systems, immunosuppressive-immunophilin systems, and higher eukaryotic transcription activation systems such as steroid hormone receptor systems and are described below.", "The PR1-a promoter from tobacco is induced during the systemic acquired resistance response following pathogen attack.", "The use of PR1-a may be limited because it often responds to endogenous materials and external factors such as pathogens, UV-B radiation, and pollutants.", "Gene regulation systems based on promoters induced by heat shock, interferon and heavy metals have been described (Wurn et al., 1986, Proc.", "Natl.", "Acad.", "Sci.", "USA 83: 5414-5418; Arnheiter et al., 1990, Cell 62: 51-61; Filmus et al., 1992, Nucleic Acids Research 20: 27550-27560).", "However, these systems have limitations due to their effect on expression of non-target genes.", "These systems are also leaky.", "Prokaryotic repressor-operator systems utilize bacterial repressor proteins and the unique operator DNA sequences to which they bind.", "Both the tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systems from the bacterium Escherichia coli have been used in plants and animals to control gene expression.", "In the Tet system, tetracycline binds to the TetR repressor protein, resulting in a conformational change that releases the repressor protein from the operator which as a result allows transcription to occur.", "In the Lac system, a lac operon is activated in response to the presence of lactose, or synthetic analogs such as isopropyl-b-D-thiogalactoside.", "Unfortunately, the use of such systems is restricted by unstable chemistry of the ligands, i.e.", "tetracycline and lactose, their toxicity, their natural presence, or the relatively high levels required for induction or repression.", "For similar reasons, utility of such systems in animals is limited.", "Immunosuppressive molecules such as FK506, rapamycin and cyclosporine A can bind to immunophilins FKBP12, cyclophilin, etc.", "Using this information, a general strategy has been devised to bring together any two proteins simply by placing FK506 on each of the two proteins or by placing FK506 on one and cyclosporine A on another one.", "A synthetic homodimer of FK506 (FK1012) or a compound resulted from fusion of FK506-cyclosporine (FKCsA) can then be used to induce dimerization of these molecules (Spencer et al., 1993 , Science 262:1019-24; Belshaw et al., 1996 , Proc Natl Acad Sci USA 93:4604-7).", "Gal4 DNA binding domain fused to FKBP12 and VP16 activator domain fused to cyclophilin, and FKCsA compound were used to show heterodimerization and activation of a reporter gene under the control of a promoter containing Gal4 binding sites.", "Unfortunately, this system includes immunosuppressants that can have unwanted side effects and therefore, limits its use for various mammalian gene switch applications.", "Higher eukaryotic transcription activation systems such as steroid hormone receptor systems have also been employed.", "Steroid hormone receptors are members of the nuclear receptor superfamily and are found in vertebrate and invertebrate cells.", "Unfortunately, use of steroidal compounds that activate the receptors for the regulation of gene expression, particularly in plants and mammals, is limited due to their involvement in many other natural biological pathways in such organisms.", "In order to overcome such difficulties, an alternative system has been developed using insect ecdysone receptors (EcR).", "Growth, molting, and development in insects are regulated by the ecdysone steroid hormone (molting hormone) and the juvenile hormones (Dhadialla, et al., 1998, Annu.", "Rev.", "Entomol.", "43: 545-569).", "The molecular target for ecdysone in insects consists of at least ecdysone receptor (EcR) and ultraspiracle protein (USP).", "EcR is a member of the nuclear steroid receptor super family that is characterized by signature DNA and ligand binding domains, and an activation domain (Koelle et al.", "1991, Cell, 67: 59-77).", "EcR receptors are responsive to a number of steroidal compounds such as ponasterone A and muristerone A.", "Recently, non-steroidal compounds with ecdysteroid agonist activity have been described, including the commercially available insecticides tebufenozide and methoxyfenozide that are marketed world wide by Rohm and Haas Company (see International Patent Application No.", "PCT/EP96/00686 and U.S. Pat.", "No.", "5,530,028).", "Both analogs have exceptional safety profiles to other organisms.", "International Patent Applications No.", "PCT/US97/05330 (WO 97/38117) and PCT/US99/08381 (WO99/58155) disclose methods for modulating the expression of an exogenous gene in which a DNA construct comprising the exogenous gene and an ecdysone response element is activated by a second DNA construct comprising an ecdysone receptor that, in the presence of a ligand therefor, and optionally in the presence of a receptor capable of acting as a silent partner, binds to the ecdysone response element to induce gene expression.", "The ecdysone receptor of choice was isolated from Drosophila melanogaster .", "Typically, such systems require the presence of the silent partner, preferably retinoid X receptor (RXR), in order to provide optimum activation.", "In mammalian cells, insect ecdysone receptor (EcR) heterodimerizes with retinoid X receptor (RXR) and regulates expression of target genes in a ligand dependent manner.", "International Patent Application No.", "PCT/US98/14215 (WO 99/02683) discloses that the ecdysone receptor isolated from the silk moth Bombyx mori is functional in mammalian systems without the need for an exogenous dimer partner.", "U.S. Pat.", "No.", "5,880,333 discloses a Drosophila melanogaster EcR and ultraspiracle (USP) heterodimer system used in plants in which the transactivation domain and the DNA binding domain are positioned on two different hybrid proteins.", "Unfortunately, this system is not effective for inducing reporter gene expression in animal cells (for comparison, see Example 1.2, below).", "In each of these cases, the transactivation domain and the DNA binding domain (either as native EcR as in International Patent Application No.", "PCT/US98/14215 or as modified EcR as in International Patent Application No.", "PCT/US97/05330) were incorporated into a single molecule and the other heterodimeric partners, either USP or RXR, were used in their native state.", "Drawbacks of the above described EcR-based gene regulation systems include a considerable background activity in the absence of ligands and non-applicability of these systems for use in both plants and animals (see U.S. Pat.", "No.", "5,880,333).", "For most applications that rely on modulating gene expression, these EcR-based systems are undesirable.", "Therefore, a need exists in the art for improved systems to precisely modulate the expression of exogenous genes in both plants and animals.", "Such improved systems would be useful for applications such as gene therapy, large-scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic animals.", "Improved systems that are simple, compact, and dependent on ligands that are relatively inexpensive, readily available, and of low toxicity to the host would prove useful for regulating biological systems.", "Recently, Applicants have shown that an ecdysone receptor-based inducible gene expression system in which the transactivation and DNA binding domains are separated from each other by placing them on two different proteins results in greatly reduced background activity in the absence of a ligand and significantly increased activity over background in the presence of a ligand (pending application PCT/US01/09050, incorporated herein in its entirety by reference).", "This two-hybrid system is a significantly improved inducible gene expression modulation system compared to the two systems disclosed in applications PCT/US97/05330 and PCT/US98/14215.Applicants previously demonstrated that an ecdysone receptor-based gene expression system in partnership with a dipteran ( Drosophila melanogaster ) or a lepidopteran ( Choristoneura fumiferana ) ultraspiracle protein (USP) is constitutively expressed in mammalian cells, while an ecdysone receptor-based gene expression system in partnership with a vertebrate retinoid X receptor (RXR) is inducible in mammalian cells (pending application PCT/US01/09050).", "Applicants have now made the surprising discovery that a non-dipteran and non-lepidopteran invertebrate RXR homolog can function similar to vertebrate RXR in an ecdysone receptor-based inducible gene expression system.", "As described herein, Applicants' novel ecdysone receptor/invertebrate retinoid X receptor-based inducible gene expression system provides an improved inducible gene expression system in yeast and mammalian cells that is characterized by increased ligand sensitivity and magnitude of transactivation." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to a novel ecdysone receptor/invertebrate retinoid X receptor-based inducible gene expression system, novel receptor polynucleotides and polypeptides for use in the novel inducible gene expression system, and methods of modulating the expression of a gene within a host cell using this inducible gene expression system.", "In particular, Applicants' invention relates to an improved gene expression modulation system comprising a polynucleotide encoding a ligand binding domain of an invertebrate retinoid X receptor (RXR) polypeptide.", "Specifically, the present invention relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a first hybrid polypeptide comprising: i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) an ecdysone receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising: i) a transactivation domain; and an invertebrate retinoid X receptor ligand binding domain.", "The present invention also relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a first hybrid polypeptide comprising: i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) an invertebrate retinoid X receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising: i) a transactivation domain; and an ecdysone receptor ligand binding domain.", "The present invention also relates to a gene expression modulation system according to the invention further comprising c) a third gene expression cassette comprising: i) a response element to which the DNA-binding domain of the first hybrid polypeptide binds; a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated.", "The present invention also relates to a gene expression cassette that is capable of being expressed in a host cell, wherein the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide comprising either i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated, or ii) a transactivation domain; and an invertebrate retinoid X receptor ligand binding domain.", "The present invention also relates to an isolated polynucleotide that encodes a hybrid polypeptide comprising either i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated, or ii) a transactivation domain; and an invertebrate retinoid X receptor ligand binding domain.", "The present invention also relates to an isolated polynucleotide encoding a truncated invertebrate RXR polypeptide, wherein the truncation mutation affects ligand binding activity or ligand sensitivity of the invertebrate RXR polypeptide.", "The present invention also relates to an isolated polynucleotide encoding a truncated invertebrate RXR polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the truncated invertebrate RXR polypeptide and a dimerization partner.", "In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide.", "The present invention also relates to an isolated polypeptide encoded by a polynucleotide according to Applicants' invention.", "The present invention also relates to an isolated hybrid polypeptide comprising either i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated, or ii) a transactivation domain; and an invertebrate retinoid X receptor ligand binding domain.", "The present invention relates to an isolated truncated invertebrate RXR polypeptide comprising a truncation mutation, wherein the invertebrate RXR polypeptide is encoded by a polynucleotide according to the invention.", "Thus, the present invention also relates to an isolated truncated invertebrate RXR polypeptide comprising a truncation mutation that affects ligand binding activity or ligand sensitivity of said invertebrate RXR polypeptide.", "The present invention also relates to an isolated truncated invertebrate RXR polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the truncated invertebrate RXR polypeptide and a dimerization partner.", "In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide.", "Applicants' invention also relates to methods of modulating gene expression in a host cell using a gene expression modulation system according to the invention.", "Specifically, Applicants' invention provides a method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; b) introducing into the host cell a gene expression cassette comprising i) a response element comprising a domain to which the DNA binding domain from the first hybrid polypeptide of the gene expression modulation system binds; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide of the gene expression modulation system; and iii) a gene whose expression is to be modulated; and c) introducing into the host cell a ligand; whereby upon introduction of the ligand into the host cell, expression of the gene is modulated.", "Applicants' invention also provides a method of modulating the expression of a gene in a host cell comprising a gene expression cassette comprising a response element comprising a domain to which the DNA binding domain from the first hybrid polypeptide of the gene expression modulation system binds; a promoter that is activated by the transactivation domain of the second hybrid polypeptide of the gene expression modulation system; and a gene whose expression is to be modulated; wherein the method comprises the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; and b) introducing into the host cell a ligand; whereby upon introduction of the ligand into the host, expression of the gene is modulated.", "Applicants' invention also provides an isolated host cell comprising an inducible gene expression system according to the invention.", "The present invention also relates to an isolated host cell comprising a gene expression cassette, a polynucleotide, or a polypeptide according to the invention.", "Accordingly, Applicants' invention also relates to a non-human organism comprising a host cell according to the invention." ], [ "FIELD OF THE INVENTION This invention relates to the field of biotechnology or genetic engineering.", "Specifically, this invention relates to the field of gene expression.", "More specifically, this invention relates to a novel ecdysone receptor/invertebrate retinoid X receptor-based inducible gene expression system and methods of modulating the expression of a gene within a host cell using this inducible gene expression system.", "BACKGROUND OF THE INVENTION Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.", "However, the citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.", "In the field of genetic engineering, precise control of gene expression is a valuable tool for studying, manipulating, and controlling development and other physiological processes.", "Gene expression is a complex biological process involving a number of specific protein-protein interactions.", "In order for gene expression to be triggered, such that it produces the RNA necessary as the first step in protein synthesis, a transcriptional activator must be brought into proximity of a promoter that controls gene transcription.", "Typically, the transcriptional activator itself is associated with a protein that has at least one DNA binding domain that binds to DNA binding sites present in the promoter regions of genes.", "Thus, for gene expression to occur, a protein comprising a DNA binding domain and a transactivation domain located at an appropriate distance from the DNA binding domain must be brought into the correct position in the promoter region of the gene.", "The traditional transgenic approach utilizes a cell-type specific promoter to drive the expression of a designed transgene.", "A DNA construct containing the transgene is first incorporated into a host genome.", "When triggered by a transcriptional activator, expression of the transgene occurs in a given cell type.", "Another means to regulate expression of foreign genes in cells is through inducible promoters.", "Examples of the use of such inducible promoters include the PR1-a promoter, prokaryotic repressor-operator systems, immunosuppressive-immunophilin systems, and higher eukaryotic transcription activation systems such as steroid hormone receptor systems and are described below.", "The PR1-a promoter from tobacco is induced during the systemic acquired resistance response following pathogen attack.", "The use of PR1-a may be limited because it often responds to endogenous materials and external factors such as pathogens, UV-B radiation, and pollutants.", "Gene regulation systems based on promoters induced by heat shock, interferon and heavy metals have been described (Wurn et al., 1986, Proc.", "Natl.", "Acad.", "Sci.", "USA 83: 5414-5418; Arnheiter et al., 1990, Cell 62: 51-61; Filmus et al., 1992, Nucleic Acids Research 20: 27550-27560).", "However, these systems have limitations due to their effect on expression of non-target genes.", "These systems are also leaky.", "Prokaryotic repressor-operator systems utilize bacterial repressor proteins and the unique operator DNA sequences to which they bind.", "Both the tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systems from the bacterium Escherichia coli have been used in plants and animals to control gene expression.", "In the Tet system, tetracycline binds to the TetR repressor protein, resulting in a conformational change that releases the repressor protein from the operator which as a result allows transcription to occur.", "In the Lac system, a lac operon is activated in response to the presence of lactose, or synthetic analogs such as isopropyl-b-D-thiogalactoside.", "Unfortunately, the use of such systems is restricted by unstable chemistry of the ligands, i.e.", "tetracycline and lactose, their toxicity, their natural presence, or the relatively high levels required for induction or repression.", "For similar reasons, utility of such systems in animals is limited.", "Immunosuppressive molecules such as FK506, rapamycin and cyclosporine A can bind to immunophilins FKBP12, cyclophilin, etc.", "Using this information, a general strategy has been devised to bring together any two proteins simply by placing FK506 on each of the two proteins or by placing FK506 on one and cyclosporine A on another one.", "A synthetic homodimer of FK506 (FK1012) or a compound resulted from fusion of FK506-cyclosporine (FKCsA) can then be used to induce dimerization of these molecules (Spencer et al., 1993, Science 262:1019-24; Belshaw et al., 1996, Proc Natl Acad Sci USA 93:4604-7).", "Gal4 DNA binding domain fused to FKBP12 and VP16 activator domain fused to cyclophilin, and FKCsA compound were used to show heterodimerization and activation of a reporter gene under the control of a promoter containing Gal4 binding sites.", "Unfortunately, this system includes immunosuppressants that can have unwanted side effects and therefore, limits its use for various mammalian gene switch applications.", "Higher eukaryotic transcription activation systems such as steroid hormone receptor systems have also been employed.", "Steroid hormone receptors are members of the nuclear receptor superfamily and are found in vertebrate and invertebrate cells.", "Unfortunately, use of steroidal compounds that activate the receptors for the regulation of gene expression, particularly in plants and mammals, is limited due to their involvement in many other natural biological pathways in such organisms.", "In order to overcome such difficulties, an alternative system has been developed using insect ecdysone receptors (EcR).", "Growth, molting, and development in insects are regulated by the ecdysone steroid hormone (molting hormone) and the juvenile hormones (Dhadialla, et al., 1998, Annu.", "Rev.", "Entomol.", "43: 545-569).", "The molecular target for ecdysone in insects consists of at least ecdysone receptor (EcR) and ultraspiracle protein (USP).", "EcR is a member of the nuclear steroid receptor super family that is characterized by signature DNA and ligand binding domains, and an activation domain (Koelle et al.", "1991, Cell, 67: 59-77).", "EcR receptors are responsive to a number of steroidal compounds such as ponasterone A and muristerone A.", "Recently, non-steroidal compounds with ecdysteroid agonist activity have been described, including the commercially available insecticides tebufenozide and methoxyfenozide that are marketed world wide by Rohm and Haas Company (see International Patent Application No.", "PCT/EP96/00686 and U.S. Pat.", "No.", "5,530,028).", "Both analogs have exceptional safety profiles to other organisms.", "International Patent Applications No.", "PCT/US97/05330 (WO 97/38117) and PCT/US99/08381 (WO99/58155) disclose methods for modulating the expression of an exogenous gene in which a DNA construct comprising the exogenous gene and an ecdysone response element is activated by a second DNA construct comprising an ecdysone receptor that, in the presence of a ligand therefor, and optionally in the presence of a receptor capable of acting as a silent partner, binds to the ecdysone response element to induce gene expression.", "The ecdysone receptor of choice was isolated from Drosophila melanogaster.", "Typically, such systems require the presence of the silent partner, preferably retinoid X receptor (RXR), in order to provide optimum activation.", "In mammalian cells, insect ecdysone receptor (EcR) heterodimerizes with retinoid X receptor (RXR) and regulates expression of target genes in a ligand dependent manner.", "International Patent Application No.", "PCT/US98/14215 (WO 99/02683) discloses that the ecdysone receptor isolated from the silk moth Bombyx mori is functional in mammalian systems without the need for an exogenous dimer partner.", "U.S. Pat.", "No.", "5,880,333 discloses a Drosophila melanogaster EcR and ultraspiracle (USP) heterodimer system used in plants in which the transactivation domain and the DNA binding domain are positioned on two different hybrid proteins.", "Unfortunately, this system is not effective for inducing reporter gene expression in animal cells (for comparison, see Example 1.2, below).", "In each of these cases, the transactivation domain and the DNA binding domain (either as native EcR as in International Patent Application No.", "PCT/US98/14215 or as modified EcR as in International Patent Application No.", "PCT/US97/05330) were incorporated into a single molecule and the other heterodimeric partners, either USP or RXR, were used in their native state.", "Drawbacks of the above described EcR-based gene regulation systems include a considerable background activity in the absence of ligands and non-applicability of these systems for use in both plants and animals (see U.S. Pat.", "No.", "5,880,333).", "For most applications that rely on modulating gene expression, these EcR-based systems are undesirable.", "Therefore, a need exists in the art for improved systems to precisely modulate the expression of exogenous genes in both plants and animals.", "Such improved systems would be useful for applications such as gene therapy, large-scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic animals.", "Improved systems that are simple, compact, and dependent on ligands that are relatively inexpensive, readily available, and of low toxicity to the host would prove useful for regulating biological systems.", "Recently, Applicants have shown that an ecdysone receptor-based inducible gene expression system in which the transactivation and DNA binding domains are separated from each other by placing them on two different proteins results in greatly reduced background activity in the absence of a ligand and significantly increased activity over background in the presence of a ligand (pending application PCT/US01/09050, incorporated herein in its entirety by reference).", "This two-hybrid system is a significantly improved inducible gene expression modulation system compared to the two systems disclosed in applications PCT/US97/05330 and PCT/US98/14215.Applicants previously demonstrated that an ecdysone receptor-based gene expression system in partnership with a dipteran (Drosophila melanogaster) or a lepidopteran (Choristoneura fumiferana) ultraspiracle protein (USP) is constitutively expressed in mammalian cells, while an ecdysone receptor-based gene expression system in partnership with a vertebrate retinoid X receptor (RXR) is inducible in mammalian cells (pending application PCT/US01/09050).", "Applicants have now made the surprising discovery that a non-dipteran and non-lepidopteran invertebrate RXR homolog can function similar to vertebrate RXR in an ecdysone receptor-based inducible gene expression system.", "As described herein, Applicants' novel ecdysone receptor/invertebrate retinoid X receptor-based inducible gene expression system provides an improved inducible gene expression system in yeast and mammalian cells that is characterized by increased ligand sensitivity and magnitude of transactivation.", "SUMMARY OF THE INVENTION The present invention relates to a novel ecdysone receptor/invertebrate retinoid X receptor-based inducible gene expression system, novel receptor polynucleotides and polypeptides for use in the novel inducible gene expression system, and methods of modulating the expression of a gene within a host cell using this inducible gene expression system.", "In particular, Applicants' invention relates to an improved gene expression modulation system comprising a polynucleotide encoding a ligand binding domain of an invertebrate retinoid X receptor (RXR) polypeptide.", "Specifically, the present invention relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a first hybrid polypeptide comprising: i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) an ecdysone receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising: i) a transactivation domain; and an invertebrate retinoid X receptor ligand binding domain.", "The present invention also relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a first hybrid polypeptide comprising: i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) an invertebrate retinoid X receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising: i) a transactivation domain; and an ecdysone receptor ligand binding domain.", "The present invention also relates to a gene expression modulation system according to the invention further comprising c) a third gene expression cassette comprising: i) a response element to which the DNA-binding domain of the first hybrid polypeptide binds; a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated.", "The present invention also relates to a gene expression cassette that is capable of being expressed in a host cell, wherein the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide comprising either i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated, or ii) a transactivation domain; and an invertebrate retinoid X receptor ligand binding domain.", "The present invention also relates to an isolated polynucleotide that encodes a hybrid polypeptide comprising either i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated, or ii) a transactivation domain; and an invertebrate retinoid X receptor ligand binding domain.", "The present invention also relates to an isolated polynucleotide encoding a truncated invertebrate RXR polypeptide, wherein the truncation mutation affects ligand binding activity or ligand sensitivity of the invertebrate RXR polypeptide.", "The present invention also relates to an isolated polynucleotide encoding a truncated invertebrate RXR polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the truncated invertebrate RXR polypeptide and a dimerization partner.", "In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide.", "The present invention also relates to an isolated polypeptide encoded by a polynucleotide according to Applicants' invention.", "The present invention also relates to an isolated hybrid polypeptide comprising either i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated, or ii) a transactivation domain; and an invertebrate retinoid X receptor ligand binding domain.", "The present invention relates to an isolated truncated invertebrate RXR polypeptide comprising a truncation mutation, wherein the invertebrate RXR polypeptide is encoded by a polynucleotide according to the invention.", "Thus, the present invention also relates to an isolated truncated invertebrate RXR polypeptide comprising a truncation mutation that affects ligand binding activity or ligand sensitivity of said invertebrate RXR polypeptide.", "The present invention also relates to an isolated truncated invertebrate RXR polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the truncated invertebrate RXR polypeptide and a dimerization partner.", "In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide.", "Applicants' invention also relates to methods of modulating gene expression in a host cell using a gene expression modulation system according to the invention.", "Specifically, Applicants' invention provides a method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; b) introducing into the host cell a gene expression cassette comprising i) a response element comprising a domain to which the DNA binding domain from the first hybrid polypeptide of the gene expression modulation system binds; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide of the gene expression modulation system; and iii) a gene whose expression is to be modulated; and c) introducing into the host cell a ligand; whereby upon introduction of the ligand into the host cell, expression of the gene is modulated.", "Applicants' invention also provides a method of modulating the expression of a gene in a host cell comprising a gene expression cassette comprising a response element comprising a domain to which the DNA binding domain from the first hybrid polypeptide of the gene expression modulation system binds; a promoter that is activated by the transactivation domain of the second hybrid polypeptide of the gene expression modulation system; and a gene whose expression is to be modulated; wherein the method comprises the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; and b) introducing into the host cell a ligand; whereby upon introduction of the ligand into the host, expression of the gene is modulated.", "Applicants' invention also provides an isolated host cell comprising an inducible gene expression system according to the invention.", "The present invention also relates to an isolated host cell comprising a gene expression cassette, a polynucleotide, or a polypeptide according to the invention.", "Accordingly, Applicants' invention also relates to a non-human organism comprising a host cell according to the invention.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1: Transactivation of reporter genes through VP16MmRXRDEF, VP16MmRXREF, VP16LmUSP, and VP16CfUSP constructs transfected into NIH3T3 cells along with GAL4CfEcRCDEF, pFRLuc and pTKRL plasmid DNAs by a non-steroidal ligand.", "FIG.", "2: Transactivation of reporter genes through VP16MmRXRDEF, VP16MmRXREF, VP16LmUSP, and VP16CfUSP constructs transfected into NIH3T3 cells along with GAL4CfEcRDEF, pFRLuc and pTKRL plasmid DNAs by a non-steroidal ligand.", "FIG.", "3: Amino acid sequence alignments of the EF domains of six vertebrate RXRs (A) and six invertebrate RXRs (B).", "Helices 1-12 are denoted as H1-H12 and β pleated sheets are denoted as S1 and S2.F denotes the F domain junction.", "FIG.", "4: Expression data of various truncations of CfEcR, GAL4CfEcRA/BCDEF, GAL4CfEcRCDEF, GAL4CfEcR1/2CDEF, GAL4CfEcRDEF, GAL4CfEcREF, GAL4CfEcRDE transfected into NIH3T3 cells along with VP16MmRXRDEF, pFRLUc and pTKRL plasmid DNAs in the presence of non-steroidal ligand or PonA ligand.", "FIG.", "5: Expression data of various truncations of CfEcR, GAL4CfEcRA/BCDEF, GAL4CfEcRCDEF, GAL4CfEcR1/2CDEF, GAL4CfEcRDEF, GAL4CfEcREF, GAL4CfEcRDE transfected into NIH3T3 cells along with VP16MmRXREF, pFRLUc and pTKRL plasmid DNAs in the presence of non-steroidal ligand or PonA ligand.", "FIG.", "6: Expression data of various truncations of CfEcR, GAL4CfEcRA/BCDEF, GAL4CfEcRCDEF, GAL4CfEcR1/2CDEF, GAL4CfEcRDEF, GAL4CfEcREF, GAL4CfEcRDE transfected into NIH3T3 cells along with VP16LmUSPDEF, pFRLUc and pTKRL plasmid DNAs in the presence of non-steroidal ligand or PonA ligand.", "FIG.", "7: Expression data of various truncations of CfEcR, GAL4CfEcRA/BCDEF, GAL4CfEcRCDEF, GAL4CfEcR1/2CDEF, GAL4CfEcRDEF, GAL4CfEcREF, GAL4CfEcRDE transfected into NIH3T3 cells along with VP16LmUSPEF, pFRLUc and pTKRL plasmid DNAs in the presence of non-steroidal ligand or PonA ligand.", "FIG.", "8: Expression data of various truncated MmRXR/LmUSP receptor constructs transfected into NIH3T3 cells along with GAL4CfEcRDEF, pFRLUc and pTKRL plasmid DNAs in the presence of non-steroidal ligand or PonA ligand.", "FIG.", "9: Expression data of CfUSP-EF, DmUSP-EF, LmUSP-EF, MmRXRα-EF, AmaRXR1-EF and AmaRXR2-EF ligand binding domains fused to VP16 along with GAL4/CfEcR-DEF and pFRLuc in NIH3T3 cells in the presence of non-steroidal (GSE) ligand or PonA ligand.", "FIG.", "10: Expression data of GAL4:CfEcR-DEF/VP16:LmUSP-EF in stably transfected CHO cells comprising a reporter plasmid pFRLuc in the presence of non-steroidal ligand or PonA ligand.", "FIG.", "11: Expression data of a LexA:CfEcR-CDEF receptor construct transfected into NIH3T3 cells along with 8XLexAopFRLuc and VP16:CfUSP-EF, VP16:LmUSP-EF, VP16:MmRXRα-EF or VP16:DmUSP-EF in the presence of non-steroidal ligand or PonA ligand.", "FIG.", "12: NIH3T3 cells were transfected with different combinations of GAL4:CfEcR-CDEF or LexA:CfEcR-CDEF, 8XLexAopFRLuc and VP16:LmUSP-EF or B42:LmUSP-EF in the presence of non-steroidal ligand.", "FIG.", "13: Effect of 9-cis-retinoic acid on transactivation potential of the GAL4CfEcR-DEF/VP16LmUSP-EF gene switch along with pFRLuc in NIH 3T3 cells in the presence of non-steroid (GSE) and 9-cis-retinoic acid (9Cis) for 48 hours.", "DETAILED DESCRIPTION OF THE INVENTION Applicants have developed a novel ecdysone receptor-based inducible gene expression system comprising an invertebrate retinoid X receptor polypeptide.", "Applicants have also shown that truncations of an invertebrate RXR polypeptide are also functional within this gene expression system and that these mutational effects may increase or reduce ligand binding activity or ligand sensitivity and may be steroid or non-steroid specific.", "Thus, Applicants' invention provides an ecdysone receptor/invertebrate RXR-based inducible gene expression system useful for modulating expression of a gene of interest in a host cell.", "In a particularly desirable embodiment, Applicants' invention provides an inducible gene expression system that has a reduced level of background gene expression and responds to submicromolar concentrations of non-steroidal ligand.", "Thus, Applicants' novel inducible gene expression system and its use in methods of modulating gene expression in a host cell overcome the limitations of currently available inducible expression systems and provide the skilled artisan with an effective means to control gene expression.", "The present invention is useful for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics, proteomics, metabolomics, and regulation of traits in transgenic organisms, where control of gene expression levels is desirable.", "An advantage of Applicants' invention is that it provides a means to regulate gene expression and to tailor expression levels to suit the user's requirements.", "DEFINITIONS In this disclosure, a number of terms and abbreviations are used.", "The following definitions are provided and should be helpful in understanding the scope and practice of the present invention.", "In a specific embodiment, the term “about” or “approximately” means within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value or range.", "The term “substantially free” means that a composition comprising “A” (where “A” is a single protein, DNA molecule, vector, recombinant host cell, etc.)", "is substantially free of “B” (where “B” comprises one or more contaminating proteins, DNA molecules, vectors, etc.)", "when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is “A”.", "Preferably, “A” comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight.", "It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.", "The term “isolated” for the purposes of the present invention designates a biological material (nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present).", "For example, a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered “isolated”.", "The term “purified” does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds.", "It is rather a relative definition.", "A polynucleotide is in the “purified” state after purification of the starting material or of the natural material by at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.", "A “nucleic acid” is a polymeric compound comprised of covalently linked subunits called nucleotides.", "Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded.", "DNA includes but is not limited to cDNA, genomic DNA, plasmids DNA, synthetic DNA, and semi-synthetic DNA.", "DNA may be linear, circular, or supercoiled.", "A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.", "Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.", "The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.", "Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.", "In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).", "A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.", "The term “fragment” will be understood to mean a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence identical to the reference nucleic acid.", "Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.", "Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a nucleic acid according to the invention.", "As used herein, an “isolated nucleic acid fragment” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.", "An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.", "A “gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.", "“Gene” also refers to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence.", "“Native gene” refers to a gene as found in nature with its own regulatory sequences.", "“Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and/or coding sequences that are not found together in nature.", "Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.", "A chimeric gene may comprise coding sequences derived from different sources and/or regulatory sequences derived from different sources.", "“Endogenous gene” refers to a native gene in its natural location in the genome of an organism.", "A “foreign” gene or “heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer.", "Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.", "A “transgene” is a gene that has been introduced into the genome by a transformation procedure.", "“Heterologous” DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.", "Preferably, the heterologous DNA includes a gene foreign to the cell.", "The term “genome” includes chromosomal as well as mitochondrial, chloroplast and viral DNA or RNA.", "A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., 1989 infra).", "Hybridization and washing conditions are well known and exemplified in Sambrook, I., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference).", "The conditions of temperature and ionic strength determine the “stringency” of the hybridization.", "Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms.", "For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS).", "Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5× or 6×SCC.", "High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5× or 6×SCC.", "Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.", "The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another.", "For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.", "Accordingly, the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as disclosed or used herein as well as those substantially similar nucleic acid sequences.", "In a specific embodiment, the term “standard hybridization conditions” refers to a Tm of 55° C., and utilizes conditions as set forth above.", "In a preferred embodiment, the Tm is 60° C.; in a more preferred embodiment, the Tm is 65° C. Post-hybridization washes also determine stringency conditions.", "One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 minutes (min), then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 minutes, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 minutes.", "A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. Hybridization requires that the two nucleic acids comprise complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.", "The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art.", "The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences.", "The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.", "For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-0.51).", "For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8).", "In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides.", "Preferable a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least 30 nucleotides.", "Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.", "The term “probe” refers to a single-stranded nucleic acid molecule that can base pair with a complementary single stranded target nucleic acid to form a double-stranded molecule.", "As used herein, the term “oligonucleotide” refers to a nucleic acid, generally of at least 18 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule.", "Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.", "A labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid.", "Oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of a nucleic acid, or to detect the presence of a nucleic acid.", "An oligonucleotide can also be used to form a triple helix with a DNA molecule.", "Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer.", "Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.", "A “primer” is an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions.", "Such primers may be used in a polymerase chain reaction.", "“Polymerase chain reaction” is abbreviated PCR and means an in vitro method for enzymatically amplifying specific nucleic acid sequences.", "PCR involves a repetitive series of temperature cycles with each cycle comprising three stages: denaturation of the template nucleic acid to separate the strands of the target molecule, annealing a single stranded PCR oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase.", "PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.", "“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCR and means an in vitro method for enzymatically producing a target cDNA molecule or molecules from an RNA molecule or molecules, followed by enzymatic amplification of a specific nucleic acid sequence or sequences within the target cDNA molecule or molecules as described above.", "RT-PCR also provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.", "A DNA “coding sequence” is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.", "“Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence.", "Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.", "The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus.", "A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences.", "If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.", "“Open reading frame” is abbreviated ORF and means a length of nucleic acid sequence, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.", "The term “head-to-head” is used herein to describe the orientation of two polynucleotide sequences in relation to each other.", "Two polynucleotides are positioned in a head-to-head orientation when the 5′ end of the coding strand of one polynucleotide is adjacent to the 5′ end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds away from the 5′ end of the other polynucleotide.", "The term “head-to-head” may be abbreviated (5′)-to-(5′) and may also be indicated by the symbols (←→) or (3←5′5′→3′).", "The term “tail-to-tail” is used herein to describe the orientation of two polynucleotide sequences in relation to each other.", "Two polynucleotides are positioned in a tail-to-tail orientation when the 3′ end of the coding strand of one polynucleotide is adjacent to the 3′ end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds toward the other polynucleotide.", "The term “tail-to-tail” may be abbreviated (3′)-to-(3′) and may also be indicated by the symbols (→←) or (5′→3′3′←5′).", "The term “head-to-tail” is used herein to describe the orientation of two polynucleotide sequences in relation to each other.", "Two polynucleotides are positioned in a head-to-tail orientation when the 5′ end of the coding strand of one polynucleotide is adjacent to the 3′ end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds in the same direction as that of the other polynucleotide.", "The term “head-to-tail” may be abbreviated (5′)-to-(3′) and may also be indicated by the symbols (→→) or (5′→3′5′→3′).", "The term “downstream” refers to a nucleotide sequence that is located 3′ to reference nucleotide sequence.", "In particular, downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription.", "For example, the translation initiation codon of a gene is located downstream of the start site of transcription.", "The term “upstream” refers to a nucleotide sequence that is located 5′ to reference nucleotide sequence.", "In particular, upstream nucleotide sequences generally relate to sequences that are located on the 5′ side of a coding sequence or starting point of transcription.", "For example, most promoters are located upstream of the start site of transcription.", "The terms “restriction endonuclease” and “restriction enzyme” refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.", "“Homologous recombination” refers to the insertion of a foreign DNA sequence into another DNA molecule, e.g., insertion of a vector in a chromosome.", "Preferably, the vector targets a specific chromosomal site for homologous recombination.", "For specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome.", "Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.", "Several methods known in the art may be used to propagate a polynucleotide according to the invention.", "Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity.", "As described herein, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.", "A “vector” is any means for the cloning of and/or transfer of a nucleic acid into a host cell.", "A vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment.", "A “replicon” is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.", "The term “vector” includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.", "A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc.", "Possible vectors include, for example, plasmids or modified viruses including, for example bacteriophages such as lambda derivatives, or plasmids such as PBR322 or pUC plasmid derivatives, or the Bluescript vector.", "For example, the insertion of the DNA fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate DNA fragments into a chosen vector that has complementary cohesive termini.", "Alternatively, the ends of the DNA molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) into the DNA termini.", "Such vectors may be engineered to contain selectable marker genes that provide for the selection of cells that have incorporated the marker into the cellular genome.", "Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.", "Viral vectors, and particularly retroviral vectors, have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects.", "Viral vectors that can be used include but are not limited to retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors.", "Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.", "In addition to a nucleic acid, a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).", "The term “plasmid” refers to an extra chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules.", "Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.", "A “cloning vector” is a “replicon”, which is a unit length of a nucleic acid, preferably DNA, that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment.", "Cloning vectors may be capable of replication in one cell type and expression in another (“shuttle vector”).", "Vectors may be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol.", "Chem.", "267: 963-967; Wu and Wu, 1988, J. Biol.", "Chem.", "263: 14621-14624; and Hartmut et al., Canadian Patent Application No.", "2,012,311, filed Mar.", "15, 1990).", "A polynucleotide according to the invention can also be introduced in vivo by lipofection.", "For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro.", "Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et al., 1987, PNAS 84: 7413; Mackey, et al., 1988, Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 85: 8027-8031; and Ulmer et al., 1993, Science 259: 1745-1748).", "The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Felgner and Ringold, 1989, Science 337: 387-388).", "Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U.S. Pat.", "No.", "5,459,127.The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages.", "Molecular targeting of liposomes to specific cells represents one area of benefit.", "It is clear that directing transfection to particular cell types would be particularly preferred in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain.", "Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey, et al., 1988, supra).", "Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.", "Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO95/21931).", "It is also possible to introduce a vector in vivo as a naked DNA plasmid (see U.S. Pat.", "Nos.", "5,693,622, 5,589,466 and 5,580,859).", "Receptor-mediated DNA delivery approaches can also be used (Curiel et al., 1992, Hum.", "Gene Ther.", "3: 147-154; and Wu and Wu, 1987, J. Biol.", "Chem.", "262: 4429-4432).", "The term “transfection” means the uptake of exogenous or heterologous RNA or DNA by a cell.", "A cell has been “transfected” by exogenous or heterologous RNA or DNA when such RNA or DNA has been introduced inside the cell.", "A cell has been “transformed” by exogenous or heterologous RNA or DNA when the transfected RNA or DNA effects a phenotypic change.", "The transforming RNA or DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.", "“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance.", "Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.", "The term “genetic region” will refer to a region of a nucleic acid molecule or a nucleotide sequence that comprises a gene encoding a polypeptide.", "In addition, the recombinant vector comprising a polynucleotide according to the invention may include one or more origins for replication in the cellular hosts in which their amplification or their expression is sought, markers or selectable markers.", "The term “selectable marker” means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest.", "Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like.", "The term “reporter gene” means a nucleic acid encoding an identifying factor that is able to be identified based upon the reporter gene's effect, wherein the effect is used to track the inheritance of a nucleic acid of interest, to identify a cell or organism that has inherited the nucleic acid of interest, and/or to measure gene expression induction or transcription.", "Examples of reporter genes known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like.", "Selectable marker genes may also be considered reporter genes.", "“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.", "In general, a coding sequence is located 3′ to a promoter sequence.", "Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.", "It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions.", "Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.", "Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissue-specific promoters”.", "Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters”.", "Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters”.", "It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.", "A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence.", "For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.", "Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.", "A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.", "“Transcriptional and translational control sequences” are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.", "In eukaryotic cells, polyadenylation signals are control sequences.", "The term “response element” means one or more cis-acting DNA elements which confer responsiveness on a promoter mediated through interaction with the DNA-binding domains of the first chimeric gene.", "This DNA element may be either palindromic (perfect or imperfect) in its sequence or composed of sequence motifs or half sites separated by a variable number of nucleotides.", "The half sites can be similar or identical and arranged as either direct or inverted repeats or as a single half site or multimers of adjacent half sites in tandem.", "The response element may comprise a minimal promoter isolated from different organisms depending upon the nature of the cell or organism into which the response element will be incorporated.", "The DNA binding domain of the first hybrid protein binds, in the presence or absence of a ligand, to the DNA sequence of a response element to initiate or suppress transcription of downstream gene(s) under the regulation of this response element.", "Examples of DNA sequences for response elements of the natural ecdysone receptor include: RRGG/TTCANTGAC/ACYY (see Cherbas L., et.", "al., (1991), Genes Dev.", "5: 120-131); AGGTCAN(n)AGGTCA, where N(n) can be one or more spacer nucleotides (see D'Avino P P., et.", "al., (1995), Mol.", "Cell.", "Endocrinol 113: 1-9); and GGGTTGAATGAATTT (see Antoniewski C., et.", "al., (1994), Mol.", "Cell Biol.", "14: 4465-4474).", "The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.", "For example, a promoter is operably, linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).", "Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.", "The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid or polynucleotide.", "Expression may also refer to translation of mRNA into a protein or polypeptide.", "The terms “cassette”, “expression cassette” and “gene expression cassette” refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination.", "The segment of DNA comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.", "“Transformation cassette” refers to a specific vector comprising a polynucleotide that encodes a polypeptide of interest and having elements in addition to the polynucleotide that facilitate transformation of a particular host cell.", "Cassettes, expression cassettes, gene expression cassettes and transformation cassettes of the invention may also comprise elements that allow for enhanced expression of a polynucleotide encoding a polypeptide of interest in a host cell.", "These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.", "For purposes of this invention, the term “gene switch” refers to the combination of a response element associated with a promoter, and an EcR based system which, in the presence of one or more ligands, modulates the expression of a gene into which the response element and promoter are incorporated.", "The terms “modulate” and “modulates” mean to induce, reduce or inhibit nucleic acid or gene expression, resulting in the respective induction, reduction or inhibition of protein or polypeptide production.", "The plasmids or vectors according to the invention may further comprise at least one promoter suitable for driving expression of a gene in a host cell.", "The term “expression vector” means a vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence following transformation into the host.", "The cloned gene, i.e., the inserted nucleic acid sequence, is usually placed under the control of control elements such as a promoter, a minimal promoter, an enhancer, or the like.", "Initiation control regions or promoters, which are useful to drive expression of a nucleic acid in the desired host cell are numerous and familiar to those skilled in the art.", "Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to: viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue specific promoter, developmental specific promoters, inducible promoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase promoters (useful for expression in Saccharomyces); AOX1 promoter (useful for expression in Pichia); β-lactamase, lac, ara, tet, trp, lPL, lPR, T7, tac, and trc promoters (useful for expression in Escherichia coli); light regulated-promoters; animal and mammalian promoters known in the art include, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3′ long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, an elongation factor 1 alpha (EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), pathogenesis or disease related-promoters, and promoters that exhibit tissue specificity and have been utilized in transgenic animals, such as the elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region active in pancreatic beta cells, immunoglobulin gene control region active in lymphoid cells, mouse mammary tumor virus control region active in testicular, breast, lymphoid and mast cells; albumin gene, Apo AI and Apo AII control regions active in liver, alpha-fetoprotein gene control region active in liver, alpha 1-antitrypsin gene control region active in the liver, beta-globin gene control region active in myeloid cells, myelin basic protein gene control region active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region active in skeletal muscle, and gonadotropic releasing hormone gene control region active in the hypothalamus, pyruvate kinase promoter, villin promoter, promoter of the fatty acid binding intestinal protein, promoter of the smooth muscle cell α-actin, and the like.", "In addition, these expression sequences may be modified by addition of enhancer or regulatory sequences and the like.", "Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor 1 (EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.", "Termination control regions, i.e., terminator or polyadenylation sequences, may also be derived from various genes native to the preferred hosts.", "Optionally, a termination site may be unnecessary, however, it is most preferred if included.", "In a preferred embodiment of the invention, the termination control region may be comprise or be derived from a synthetic sequence, synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, or the like.", "The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)” refer to DNA sequences located downstream (3′) of a coding sequence and may comprise polyadenylation [poly(A)] recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.", "The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor.", "“Regulatory region” means a nucleic acid sequence which regulates the expression of a second nucleic acid sequence.", "A regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin that are responsible for expressing different proteins or even synthetic proteins (a heterologous region).", "In particular, the sequences can be sequences of prokaryotic, eukaryotic, or viral genes or derived sequences that stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner.", "Regulatory regions include origins of replication, RNA splice sites, promoters, enhancers, transcriptional termination sequences, and signal sequences which direct the polypeptide into the secretory pathways of the target cell.", "A regulatory region from a “heterologous source” is a regulatory region that is not naturally associated with the expressed nucleic acid.", "Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences which do not occur in nature, but which are designed by one having ordinary skill in the art.", "“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence.", "When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA.", "“Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into protein by the cell.", "“cDNA” refers to a double-stranded DNA that is complementary to and derived from mRNA.", "“Sense” RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell.", "“Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene.", "The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or the coding sequence.", "“Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet has an effect on cellular processes.", "A “polypeptide” is a polymeric compound comprised of covalently linked amino acid residues.", "Amino acids have the following general structure: Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.", "A polypeptide of the invention preferably comprises at least about 14 amino acids.", "A “protein” is a polypeptide that performs a structural or functional role in a living cell.", "An “isolated polypeptide” or “isolated protein” is a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids).", "“Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.", "“Fragment” of a polypeptide according to the invention will be understood to mean a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence.", "Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part.", "Such fragments of a polypeptide according to the invention may have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200, 240, or 300 amino acids.", "A “variant” of a polypeptide or protein is any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein.", "Different variants of the polypeptide or protein may exist in nature.", "These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification.", "The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements.", "These variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) variants in which one or more of the amino acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin.", "The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.", "), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.", "A variant polypeptide preferably comprises at least about 14 amino acids.", "A “heterologous protein” refers to a protein not naturally produced in the cell.", "A “mature protein” refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.", "“Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present.", "Pre- and propeptides may be but are not limited to intracellular localization signals.", "The term “signal peptide” refers to an amino terminal polypeptide preceding the secreted mature protein.", "The signal peptide is cleaved from and is therefore not present in the mature protein.", "Signal peptides have the function of directing and translocating secreted proteins across cell membranes.", "Signal peptide is also referred to as signal protein.", "A “signal sequence” is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell.", "This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide.", "The term “translocation signal sequence” is used herein to refer to this sort of signal sequence.", "Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.", "The term “homology” refers to the percent of identity between two polynucleotide or two polypeptide moieties.", "The correspondence between the sequence from one moiety to another can be determined by techniques known to the art.", "For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs.", "Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s) and size determination of the digested fragments.", "As used herein, the term “homologous” in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a “common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.)", "(Reeck et al., 1987, Cell 50: 667).", "Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity.", "However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and not a common evolutionary origin.", "Accordingly, the term “sequence similarity” in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., 1987, Cell 50:667).", "In a specific embodiment, two DNA sequences are “substantially homologous” or “substantially similar” when at least about 50% (preferably at least about 75%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences.", "Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.", "Defining appropriate hybridization conditions is within the skill of the art.", "See, e.g., Sambrook et al., 1989, supra.", "As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence.", "“Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology.", "“Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript.", "It is therefore understood that the invention encompasses more than the specific exemplary sequences.", "Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.", "Moreover, the skilled artisan recognizes that substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), with the sequences exemplified herein.", "Substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 70% identical to the DNA sequence of the nucleic acid fragments reported herein.", "Preferred substantially nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein.", "More preferred nucleic acid fragments are at least 90% identical to the DNA sequence of the nucleic acid fragments reported herein.", "Even more preferred are nucleic acid fragments that are at least 95% identical to the DNA sequence of the nucleic acid fragments reported herein.", "Two amino acid sequences are “substantially homologous” or “substantially similar” when greater than about 40% of the amino acids are identical, or greater than 60% are similar (functionally identical).", "Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program.", "The term “corresponding to” is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.", "A nucleic acid or amino acid sequence alignment may include spaces.", "Thus, the term “corresponding to” refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.", "A “substantial portion” of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol.", "Biol.", "215: 403-410; see also www.ncbi.nlm.nih.gov/BLAST/).", "In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.", "Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).", "In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.", "Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.", "The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.", "In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.", "“Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.)", "Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)", "Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.)", "Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.)", "Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.)", "Stockton Press, New York (1991).", "Preferred methods to determine identity are designed to give the best match between the sequences tested.", "Methods to determine identity and similarity are codified in publicly available computer programs.", "Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).", "Multiple alignment of the sequences may be performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS.", "5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).", "Default parameters for pairwise alignments using the Clustal method may be selected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences.", "“Sequence analysis software” may be commercially available or independently developed.", "Typical sequence analysis software will include but is not limited to the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol.", "Biol.", "215: 403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA).", "Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified.", "As used herein “default values” will mean any set of values or parameters which originally load with the software when first initialized.", "“Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art.", "These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene.", "“Chemically synthesized”, as related to a sequence of DNA, means that the component nucleotides were assembled in vitro.", "Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines.", "Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell.", "The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host.", "Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.", "Gene Expression Modulation System of the Invention Applicants have previously shown that separating the transactivation and DNA binding domains by placing them on two different proteins results in greatly reduced background activity in the absence of a ligand and significantly increased activity over background in the presence of a ligand (pending application PCT/US01/09050).", "This two-hybrid system is a significantly improved inducible gene expression modulation system compared to the two systems disclosed in International Patent Applications PCT/US97/05330 and PCT/US98/14215.The two-hybrid system exploits the ability of a pair of interacting proteins to bring the transcription activation domain into a more favorable position relative to the DNA binding domain such that when the DNA binding domain binds to the DNA binding site on the gene, the transactivation domain more effectively activates the promoter (see, for example, U.S. Pat.", "No.", "5,283,173).", "Briefly, the two-hybrid gene expression system comprises two gene expression cassettes; the first encoding a DNA binding domain fused to a nuclear receptor polypeptide, and the second encoding a transactivation domain fused to a different nuclear receptor polypeptide.", "In the presence of ligand, the interaction of the first polypeptide with the second polypeptide effectively tethers the DNA binding domain to the transactivation domain.", "Since the DNA binding and transactivation domains reside on two different molecules, the background activity in the absence of ligand is greatly reduced.", "The two-hybrid ecdysone receptor-based gene expression modulation system may be either heterodimeric and homodimeric.", "A functional EcR complex generally refers to a heterodimeric protein complex consisting of two members of the steroid receptor family, an ecdysone receptor protein obtained from various insects, and an ultraspiracle (USP) protein or the vertebrate homolog of USP, retinoid X receptor protein (see Yao, et al.", "(1993) Nature 366, 476-479; Yao, et al., (1992) Cell 71, 63-72).", "However, the complex may also be a homodimer as detailed below.", "The functional ecdysteroid receptor complex may also include additional protein(s) such as immunophilins.", "Additional members of the steroid receptor family of proteins, known as transcriptional factors (such as DHR38 or betaFTZ-1), may also be ligand dependent or independent partners for EcR, USP, and/or RXR.", "Additionally, other cofactors may be required such as proteins generally known as coactivators (also termed adapters or mediators).", "These proteins do not bind sequence-specifically to DNA and are not involved in basal transcription.", "They may exert their effect on transcription activation through various mechanisms, including stimulation of DNA-binding of activators, by affecting chromatin structure, or by mediating activator-initiation complex interactions.", "Examples of such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the promiscuous coactivator C response element B binding protein, CBP/p300 (for review see Glass et al., Curr.", "Opin.", "Cell Biol.", "9: 222-232, 1997).", "Also, protein cofactors generally known as corepressors (also known as repressors, silencers, or silencing mediators) may be required to effectively inhibit transcriptional activation in the absence of ligand.", "These corepressors may interact with the unliganded ecdysone receptor to silence the activity at the response element.", "Current evidence suggests that the binding of ligand changes the conformation of the receptor, which results in release of the corepressor and recruitment of the above described coactivators, thereby abolishing their silencing activity.", "Examples of corepressors include N—CoR and SMRT (for review, see Horwitz et al.", "Mol.", "Endocrinol.", "10 1167-1177, 1996).", "These cofactors may either be endogenous within the cell or organism, or may be added exogenously as transgenes to be expressed in either a regulated or unregulated fashion.", "Homodimer complexes of the ecdysone receptor protein, USP, or RXR may also be functional under some circumstances.", "The ecdysone receptor complex typically includes proteins that are members of the nuclear receptor superfamily wherein all members are generally characterized by the presence of an amino-terminal transactivation domain, a DNA binding domain (“DBD”), and a ligand binding domain (“LBD”) separated from the DBD by a hinge region.", "As used herein, the term “DNA binding domain” comprises a minimal polypeptide sequence of a DNA binding protein, up to the entire length of a DNA binding protein, so long as the DNA binding domain functions to associate with a particular response element.", "Members of the nuclear receptor superfamily are also characterized by the presence of four or five domains: A/B, C, D, E, and in some members F (see U.S. Pat.", "No.", "4,981,784 and Evans, Science 240: 889-895 (1988)).", "The “A/B” domain corresponds to the transactivation domain, “C” corresponds to the DNA binding domain, “D” corresponds to the hinge region, and “E” corresponds to the ligand binding domain.", "Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD corresponding to “F”.", "The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for ecdysone response elements.", "These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins.", "This EcR receptor, like a subset of the steroid receptor family, also possesses less well-defined regions responsible for heterodimerization properties.", "Because the domains of EcR, USP, and RXR are modular in nature, the LBD, DBD, and transactivation domains may be interchanged.", "Gene switch systems are known that incorporate components from the ecdysone receptor complex.", "However, in these known systems, whenever EcR is used it is associated with native or modified DNA binding domains and transactivation domains on the same molecule.", "USP or RXR are typically used as silent partners.", "Applicants have previously shown that when DNA binding domains and transactivation domains are on the same molecule the background activity in the absence of ligand is high and that such activity is dramatically reduced when DNA binding domains and transactivation domains are on different molecules, that is, on each of two partners of a heterodimeric or homodimeric complex (see PCT/US01/09050).", "This two-hybrid system also provides improved sensitivity to non-steroidal ligands for example, diacylhydrazines, when compared to steroidal ligands for example, ponasterone A (“PonA”) or muristerone A (“MurA”).", "That is, when compared to steroids, the non-steroidal ligands provide higher activity at a lower concentration.", "In addition, since transactivation based on EcR gene switches is often cell-line dependent, it is easier to tailor switching systems to obtain maximum transactivation capability for each application.", "Furthermore, the two-hybrid system avoids some side effects due to overexpression of RXR that often occur when unmodified RXR is used as a switching partner.", "In a specific embodiment of the two-hybrid system, native DNA binding and transactivation domains of EcR or RXR are eliminated and as a result, these hybrid molecules have less chance of interacting with other steroid hormone receptors present in the cell resulting in reduced side effects.", "Applicants have previously shown that an ecdysone receptor in partnership with a dipteran (fruit fly Drosophila melanogaster) or a lepidopteran (spruce bud worm Choristoneura fumiferana) ultraspiracle protein (USP) is constitutively expressed in mammalian cells, while an ecdysone receptor in partnership with a vertebrate retinoid X receptor (RXR) is inducible in mammalian cells (pending application PCT/US01/09050).", "Applicants have now made the surprising discovery that the ultraspiracle protein of Locusta migratoria (“LmUSP”) and the RXR homolog 1 and RXR homolog 2 of the ixodid tick Amblyomma americanum (“AmaRXR1” and “AmaRXR2”, respectively) can function similar to vertebrate retinoid X receptor (RXR) in an inducible ecdysone receptor-based inducible gene expression system.", "Thus, Applicants' findings that LmUSP, AmaRXR1, AmaRXR2, and their non-Dipteran, non-Lepidopteran homologs including, but not limited to: fiddler crab Celuca pugilator RXR homolog (“CpRXR”), beetle Tenebrio molitor RXR homolog (“TmRXR”), honeybee Apis mellifera RXR homolog (“AmRXR”), and aphid Myzus persicae RXR homolog (“MpRXR”), all of which are referred to herein collectively as invertebrate RXRs, can be substituted for vertebrate RXR in ecdysone receptor-based inducible gene expression systems can only be regarded as unexpected and surprising.", "As described herein, Applicants' novel ecdysone receptor/invertebrate RXR-based inducible gene expression system provides an improved inducible gene expression system in yeast and mammalian cells that is characterized by increased ligand sensitivity and magnitude of transactivation.", "In particular, Applicants describe herein a novel two-hybrid system that comprises an invertebrate RXR ligand binding domain.", "This novel gene expression system demonstrates for the first time that an invertebrate ultraspiracle protein/RXR homolog can function as a component of an inducible EcR-based inducible gene expression system in yeast and mammalian cells.", "As discussed herein, this finding is both unexpected and surprising.", "Specifically, Applicants' invention relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell, wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) an ecdysone receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell, wherein the second gene expression cassette comprises a polynucleotide sequence that encodes a second hybrid polypeptide comprising i) a transactivation domain; and an invertebrate retinoid X receptor ligand binding domain.", "The present invention also relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell, wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) an invertebrate retinoid X receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell, wherein the second gene expression cassette comprises a polynucleotide sequence that encodes a second hybrid polypeptide comprising i) a transactivation domain; and ii) an ecdysone receptor ligand binding domain.", "The present invention also relates to a gene expression modulation system according to the present invention further comprising c) a third gene expression cassette comprising: i) a response element to which the DNA-binding domain of the first hybrid polypeptide binds; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated.", "In a specific embodiment, the gene whose expression is to be modulated is a homologous gene with respect to the host cell.", "In another specific embodiment, the gene whose expression is to be modulated is a heterologous gene with respect to the host cell.", "The ligands for use in the present invention as described below, when combined with the ligand binding domains of an EcR and an invertebrate RXR, which in turn are bound to the response element linked to a gene, provide the means for external temporal regulation of expression of the gene.", "The binding mechanism or the order in which the various components of this invention bind to each other, that is, for example, ligand to receptor, first hybrid polypeptide to response element, second hybrid polypeptide to promoter, etc., is not critical.", "Binding of the ligand to the ligand binding domains of an EcR and invertebrate RXR enables expression or suppression of the gene.", "This mechanism does not exclude the potential for ligand binding to EcR or invertebrate RXR, and the resulting formation of active homodimer complexes (e.g.", "EcR+EcR or invertebrate RXR+invertebrate RXR).", "Preferably, one or more of the receptor domains is varied producing a chimeric or hybrid gene switch.", "Typically, one or more of the three domains, DBD, LBD, and transactivation domain, may be chosen from a source different than the source of the other domains so that the hybrid genes and the resulting hybrid proteins are optimized in the chosen host cell or organism for transactivating activity, complementary binding of the ligand, and recognition of a specific response element.", "In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein from yeast (see Sadowski, et al., (1988) Nature 335: 563-564) or LexA protein from Escherichia coli (see Brent and Ptashne, (1985), Cell 43: 729-736), or synthetic response elements specific for targeted interactions with proteins designed, modified, and selected for such specific interactions (see, for example, Kim, et al.", "(1997), Proc.", "Natl.", "Acad.", "Sci., USA 94: 3616-3620) to accommodate hybrid receptors.", "Another advantage of two-hybrid systems is that they allow choice of a promoter used to drive the gene expression according to a desired end result.", "Such double control can be particularly important in areas of gene therapy, especially when cytotoxic proteins are produced, because both the timing of expression as well as the cells wherein expression occurs can be controlled.", "When genes, operably linked to a suitable promoter, are introduced into the cells of the subject, expression of the exogenous genes is controlled by the presence of the system of this invention.", "Promoters may be constitutively or inducibly regulated or may be tissue-specific (that is, expressed only in a particular type of cells) or specific to certain developmental stages of the organism.", "Gene Expression Cassettes of the Invention The novel EcR/invertebrate RXR-based inducible gene expression system of the invention comprises gene expression cassettes that are capable of being expressed in a host cell, wherein the gene expression cassettes each comprise a polynucleotide encoding a hybrid polypeptide.", "Thus, Applicants' invention also provides novel gene expression cassettes for use in the gene expression system of the invention.", "Specifically, the present invention provides a gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide.", "In particular, the present invention provides a gene expression cassette that is capable of being expressed in a host cell, wherein the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide comprising either i) a DNA-binding domain that recognizes a response element, or a transactivation domain; and an ecdysone receptor ligand binding domain or an invertebrate retinoid X receptor ligand binding domain.", "In a specific embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain that recognizes a response element and an EcR ligand binding domain.", "In another specific embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain that recognizes a response element and an invertebrate RXR ligand binding domain.", "In another specific embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain and an EcR ligand binding domain.", "In another specific embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain and an invertebrate RXR ligand binding domain.", "In a preferred embodiment, the ligand binding domain (LBD) is an EcR LBD, an invertebrate RXR LBD, or a related steroid/thyroid hormone nuclear receptor family member LBD, or analogs, combinations, or modifications thereof.", "In a specific embodiment, the LBD is from an EcR or an invertebrate RXR.", "In another specific embodiment, the LBD is from a truncated EcR LBD or a truncated invertebrate RXR LBD.", "A truncation mutation may be made by any method used in the art, including but not limited to restriction endonuclease digestion/deletion, PCR-mediated/oligonucleotide-directed deletion, chemical mutagenesis, DNA strand breakage, and the like.", "The EcR may be an invertebrate EcR, preferably selected from the class Arthropod.", "Preferably the EcR is selected from the group consisting of a Lepidopteran EcR, a Dipteran EcR, an Orthopteran EcR, a Homopteran EcR and a Hemipteran EcR.", "More preferably, the EcR for use is a spruce budworm Choristoneura fumiferana EcR (“CfEcR”), a beetle Tenebrio molitor EcR (“TmEcR”), a Manduca sexta EcR (“MsEcR”), a Heliothies virescens EcR (“HvEcR”), a midge Chironomus tentans EcR (“CtEcR”), a silk moth Bombyx mori EcR (“BmEcR”), a fruit fly Drosophila melanogaster EcR (“DmEcR”), a mosquito Aedes aegypti EcR (“AaEcR”), a blowfly Lucilia capitata (“LcEcR”), a blowfly Lucilia cuprina EcR (“LucEcR”), a Mediterranean fruit fly Ceratitis capitata EcR (“CcEcR”), a locust Locusta migratoria EcR (“LmEcR”), an aphid Myzus persicae EcR (“MpEcR”), a fiddler crab Celuca pugilator EcR (“CpEcR”), an ixodid tick Amblyomma americanum EcR (“AmaEcR”), a whitefly Bamecia argentifoli EcR (“BaEcR”, SEQ ID NO: 57), or a leafhopper Nephotetix cincticeps EcR (“NcEcR”, SEQ ID NO: 58).", "Most preferably, the LBD is from spruce budworm (Choristoneura fumiferana) EcR (“CfEcR”), fruit fly Drosophila melanogaster EcR (“DmEcR”), whitefly Bamecia argentifoli EcR (“BaEcR”), leafhopper Nephotetix cincticeps EcR (“NcEcR”), beetle Tenebrio molitor EcR (“TmEcR”), or ixodid tick Amblyomma americanum EcR (“AmaEcR”).", "In a specific embodiment, the LBD is from a truncated EcR polypeptide.", "The EcR polypeptide truncation results in a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids.", "Preferably, the EcR polypeptide truncation results in a deletion of at least a partial polypeptide domain.", "More preferably, the EcR polypeptide truncation results in a deletion of at least an entire polypeptide domain.", "In a specific embodiment, the EcR polypeptide truncation results in a deletion of at least an A/B-domain, a C-domain, a D-domain, an F-domain, an A/B/C-domains, an A/B/1/2-C-domains, an A/B/C/D-domains, an A/B/C/D/F-domains, an A/B/F-domains, an A/B/C/F-domains, a partial E domain, or a partial F domain.", "A combination of several complete and/or partial domain deletions may also be performed.", "In one embodiment, the ecdysone receptor ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2 (DmEcR-EF), SEQ ID NO: 3 (CfEcR-DE), and SEQ ID NO: 4 (DmEcR-DE).", "In a preferred embodiment, the ecdysone receptor ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 (CfEcR-EF), SEQ ID NO: 53 (CfEcR-DEF), and SEQ ID NO: 45 (CfEcR-CDEF).", "In one embodiment, the ecdysone receptor ligand binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (DmEcR-EF), SEQ ID NO: 7 (CfEcR-DE), and SEQ ID NO: 8 (DmEcR-DE).", "In a preferred embodiment, the ecdysone receptor ligand binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 (CfEcR-EF), SEQ ID NO: 43 (CFEcR-DEF), and SEQ ID NO: 59 (CfEcR-CDEF).", "Preferably, the invertebrate RXR polypeptide is a locust Locusta migratoria ultraspiracle polypeptide (“LmUSP”), an ixodid tick Amblyomma americanum RXR homolog 1 (“AmaRXR1”), a ixodid tick Amblyomma americanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celuca pugilator RXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog (“TmRXR”), a honeybee Apis mellifera RXR homolog (“AmRXR”), an aphid Myzus persicae RXR homolog (“MpRXR”), or a non-Dipteran/non-Lepidopteran RXR homolog.", "In a specific embodiment, the LBD is from a truncated invertebrate RXR.", "The invertebrate RXR polypeptide truncation results in a deletion of at least 1, 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, or 240 amino acids.", "Preferably, the invertebrate RXR polypeptide truncation results in a deletion of at least a partial polypeptide domain.", "More preferably, the invertebrate RXR polypeptide truncation results in a deletion of at least an entire polypeptide domain.", "In a specific embodiment, the invertebrate RXR polypeptide truncation results in a deletion of at least a partial E-domain, a complete E-domain, a partial F-domain, a complete F-domain, an EF-domain helix 1, an EF-domain helix 2, an EF-domain helix 3, an EF-domain helix 4, an EF-domain helix 5, an EF-domain helix 6, an EF-domain helix 7, an EF-domain helix 8, and EF-domain helix 9, an EF-domain helix 10, an EF-domain helix 11, an EF-domain helix 12, an EF-domain β-pleated sheet, an A/B-domain, a C-domain, a D-domain, A/B/C-domains, A/B/1/2-C-domains, A/B/C/D-domains, A/B/C/D/F-domains, A/B/F-domains, or A/B/C/F-domains.", "A combination of several complete and/or partial domain deletions may also be performed.", "In a preferred embodiment, the invertebrate RXR ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9 (LmUSP-EF), SEQ ID NO: 10 (AmaRXR1-EF), SEQ ID NO: 11 (AmaRXR2-EF), SEQ ID NO: 12 (CpRXR-EF), SEQ ID NO: 13 (TmRXR-EF), SEQ ID NO: 14 (AmRXR-EF), SEQ ID NO: 15 (LmUSP-EF, BamHI-deleted), SEQ ID NO: 16 (AmaRXR1-EF, BamHI-deleted), SEQ ID NO: 17 (AmaRXR2-EF, BamHI-deleted), SEQ ID NO: 18 (CpRXR-EF, BamHI-deleted), SEQ ID NO: 19 (TmRXR-EF, BamHI-deleted), and SEQ ID NO: 20 (AmRXR-EF, BamHI-deleted).", "In another preferred embodiment, the invertebrate RXR ligand binding domain comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 21 (LmUSP-EF), SEQ ID NO: 22 (AmaRXR1-EF), SEQ ID NO: 23 (AmaRXR2-EF), SEQ ID NO: 24 (CpRXR-EF), SEQ ID NO: 25 (TmRXR-EF), SEQ ID NO: 26 (AmRXR-EF), SEQ ID NO: 27 (LmUSP-EF, BamHI-deleted), SEQ ID NO: 28 (AmaRXR1-EF, BamHI-deleted), SEQ ID NO: 29 (AmaRXR2-EF, BamHI-deleted), SEQ ID NO: 30 (CpRXR-EF, BamHI-deleted), SEQ ID NO: 31 (TmRXR-EF, BamHI-deleted), and SEQ ID NO: 32 (AmRXR-EF, BamHI-deleted).", "For purposes of this invention, EcR and invertebrate RXR also include synthetic and chimeric EcR and invertebrate RXR and their homologs.", "The DNA binding domain can be any DNA binding domain with a known response element, including synthetic and chimeric DNA binding domains, or analogs, combinations, or modifications thereof.", "Preferably, the DBD is a GAL4 DBD, a LexA DBD, a transcription factor DBD, a steroid/thyroid hormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD, or a yeast put DBD.", "More preferably, the DBD is a GAL4 DBD [SEQ ID NO: 33 (polynucleotide) or SEQ ID NO: 34 (polypeptide)] or a LexA DBD [(SEQ ID NO: 35 (polynucleotide) or SEQ ID NO: 36 (polypeptide)].", "The transactivation domain (abbreviated “AD” or “TA”) may be any steroid/thyroid hormone nuclear receptor AD, synthetic or chimeric AD, polyglutamine AD, basic or acidic amino acid AD, a VP16 AD, a GAL4 AD, an NF-κB AD, a BP64 AD, a B42 acidic activation domain (B42AD), or an analog, combination, or modification thereof.", "In a specific embodiment, the AD is a synthetic or chimeric AD, or is obtained from a VP16, GAL4, NF-kB, or B42 acidic activation domain AD.", "Preferably, the AD is a VP16 AD [SEQ ID NO: 37 (polynucleotide) or SEQ ID NO: 38 (polypeptide)] or a B42 AD [SEQ ID NO: 39 (polynucleotide) or SEQ ID NO: 40 (polypeptide)].", "In a preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GAM DBD (SEQ ID NO: 33) and a LexA DBD (SEQ ID NO: 35), and an EcR ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 53, and SEQ ID NO: 45.In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain comprising an amino acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 34) and a LexA DBD (SEQ ID NO: 36), and an EcR ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 43, and SEQ ID NO: 59.In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 33) or a LexA DBD (SEQ ID NO: 35) and an invertebrate RXR ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain comprising an amino acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 34) and a LexA DBD (SEQ ID NO: 36), and an invertebrate RXR ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 37 or SEQ ID NO: 39, and an EcR ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 53, and SEQ ID NO: 45.In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40, and an EcR ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 43, and SEQ ID NO: 59.In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 37 or SEQ ID NO: 39, and an invertebrate RXR ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40 and an invertebrate RXR ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ BD NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.The response element (“RE”) may be any response element with a known DNA binding domain, or an analog, combination, or modification thereof.", "A single RE may be employed or multiple REs, either multiple copies of the same RE or two or more different REs, may be used in the present invention.", "In a specific embodiment, the RE is an RE from GAL4 (“GAL4RE”), LexA, a steroid/thyroid hormone nuclear receptor RE, or a synthetic RE that recognizes a synthetic DNA binding domain.", "Preferably, the RE is a GALORE comprising a polynucleotide sequence of SEQ ID NO: 41 or a LexA RE (operon, “op”) comprising a polynucleotide sequence of SEQ ID NO: 42 (“2XLexAopRE”).", "Preferably, the first hybrid protein is substantially free of a transactivation domain and the second hybrid protein is substantially free of a DNA binding domain.", "For purposes of this invention, “substantially free” means that the protein in question does not contain a sufficient sequence of the domain in question to provide activation or binding activity.", "Thus, the present invention also relates to a gene expression cassette comprising: i) a response element comprising a domain to which a polypeptide comprising a DNA binding domain binds; ii) a promoter that is activated by a polypeptide comprising a transactivation domain; and iii) a gene whose expression is to be modulated.", "Genes of interest for use in Applicants' gene expression cassettes may be endogenous genes or heterologous genes.", "Nucleic acid or amino acid sequence information for a desired gene or protein can be located in one of many public access databases, for example, GENBANK, EMBL, Swiss-Prot, and PIR, or in many biology related journal publications.", "Thus, those skilled in the art have access to nucleic acid sequence information for virtually all known genes.", "Such information can then be used to construct the desired constructs for the insertion of the gene of interest within the gene expression cassettes used in Applicants' methods described herein.", "Examples of genes of interest for use in Applicants' gene expression cassettes include, but are not limited to: genes encoding therapeutically desirable polypeptides or products that may be used to treat a condition, a disease, a disorder, a dysfunction, a genetic defect, such as monoclonal antibodies, enzymes, proteases, cytokines, interferons, insulin, erthropoietin, clotting factors, other blood factors or components, viral vectors for gene therapy, virus for vaccines, targets for drug discovery, functional genomics, and proteomics analyses and applications, and the like.", "Polynucleotides of the Invention The novel ecdysone receptor/invertebrate retinoid X receptor-based inducible gene expression system of the invention comprises a gene expression cassette comprising a polynucleotide that encodes a hybrid polypeptide comprising a) a DNA binding domain or a transactivation domain, and b) an EcR ligand binding domain or an invertebrate RXR ligand binding domain.", "These gene expression cassettes, the polynucleotides they comprise, and the hybrid polypeptides they encode are useful as components of an EcR-based gene expression system to modulate the expression of a gene within a host cell.", "Thus, the present invention provides an isolated polynucleotide that encodes a hybrid polypeptide comprising a) a DNA binding domain or a transactivation domain according to the invention, and b) an EcR ligand binding domain or an invertebrate RXR ligand binding domain according to the invention.", "The present invention also relates to an isolated polynucleotide that encodes a truncated EcR or a truncated invertebrate RXR polypeptide comprising a truncation mutation according to the invention.", "Specifically, the present invention relates to an isolated polynucleotide encoding an EcR or an invertebrate RXR polypeptide comprising a truncation mutation that affects ligand binding activity or ligand sensitivity that is useful in modulating gene expression in a host cell.", "In a specific embodiment, the isolated truncated EcR polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 53 and SEQ ID NO: 45.In another specific embodiment, the isolated truncated EcR polynucleotide encodes a truncated ecdysone receptor polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 43 and SEQ ID NO: 59.In another specific embodiment, the isolated truncated invertebrate RXR polynucleotide according to the invention comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.In another specific embodiment, the isolated truncated invertebrate RXR polynucleotide according to the invention encodes a truncated invertebrate RXR polypeptide comprising an amino acid sequence consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.In particular, the present invention relates to an isolated polynucleotide encoding an invertebrate RXR polypeptide comprising a truncation mutation, wherein the mutation reduces ligand binding activity or ligand sensitivity of the invertebrate RXR polypeptide.", "In a specific embodiment, the present invention relates to an isolated polynucleotide encoding an invertebrate RXR polypeptide comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of the invertebrate RXR polypeptide.", "In another specific embodiment, the present invention relates to an isolated polynucleotide encoding an invertebrate RXR polypeptide comprising a truncation mutation that reduces non-steroid binding activity or non-steroid sensitivity of the invertebrate RXR polypeptide.", "The present invention also relates to an isolated polynucleotide encoding an invertebrate RXR polypeptide comprising a truncation mutation, wherein the mutation enhances ligand binding activity or ligand sensitivity of the invertebrate RXR polypeptide.", "In a specific embodiment, the present invention relates to an isolated polynucleotide encoding an invertebrate RXR polypeptide comprising a truncation mutation that enhances steroid binding activity or steroid sensitivity of the invertebrate RXR polypeptide.", "In another specific embodiment, the present invention relates to an isolated polynucleotide encoding an invertebrate RXR polypeptide comprising a truncation mutation that enhances non-steroid binding activity or non-steroid sensitivity of the invertebrate RXR polypeptide.", "The present invention also relates to an isolated polynucleotide encoding an invertebrate retinoid X receptor polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the mutated invertebrate retinoid X receptor polypeptide and a dimerization partner.", "Preferably, the isolated polynucleotide encoding an invertebrate retinoid X receptor polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 9 (LmUSP-EF), SEQ ID NO: 10 (AmaRXR1-EF), SEQ ID NO: 11 (AmaRXR2-EF), SEQ ID NO: 12 (CpRXR-EF), SEQ ID NO: 13 (TmRXR-EF), and SEQ ID NO: 14 (AmRXR-EF).", "In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide.", "Preferably, the dimerization partner is a truncated EcR polypeptide.", "More preferably, the dimerization partner is an EcR polypeptide in which domain A/B has been deleted.", "Even more preferably, the dimerization partner is an EcR polypeptide comprising an amino acid sequence of SEQ ID NO: 5 (CfEcR-EF), SEQ ID NO: 43 (CfEcR-DEF) or SEQ ID NO: 59 (CfEcR-CDEF).", "Polypeptides of the Invention The novel ecdysone receptor/invertebrate retinoid X receptor-based inducible gene expression system of the invention comprises a gene expression cassette comprising a polynucleotide that encodes a hybrid polypeptide comprising a) a DNA binding domain or a transactivation domain, and b) an EcR ligand binding domain or an invertebrate RXR ligand binding domain.", "These gene expression cassettes, the polynucleotides they comprise, and the hybrid polypeptides they encode are useful as components of an EcR-based gene expression system to modulate the expression of a gene within a host cell.", "Thus, the present invention also relates to a hybrid polypeptide comprising a) a DNA binding domain or a transactivation domain according to the invention, and b) an EcR ligand binding domain or an invertebrate RXR ligand binding domain according to the invention.", "The present invention also relates to an isolated truncated EcR or an isolated truncated invertebrate RXR polypeptide comprising a truncation mutation according to the invention.", "Specifically, the present invention relates to an isolated truncated EcR or an isolated truncated invertebrate RXR polypeptide comprising a truncation mutation that affects ligand binding activity or ligand sensitivity.", "In a specific embodiment, the isolated truncated EcR polypeptide is encoded by a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 53 and SEQ ID NO: 45.In another specific embodiment, the isolated truncated EcR polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 43 and SEQ ID NO: 59.In another specific embodiment, the isolated truncated invertebrate RXR polypeptide is encoded by a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.In another specific embodiment, the isolated truncated invertebrate RXR polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.The present invention relates to an isolated invertebrate RXR polypeptide comprising a truncation mutation that reduces ligand binding activity or ligand sensitivity of the invertebrate RXR polypeptide, wherein the polypeptide is encoded by a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.Thus, the present invention relates to an isolated invertebrate RXR polypeptide comprising a truncation mutation that reduces ligand binding activity or ligand sensitivity of the invertebrate RXR polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.In a specific embodiment, the present invention relates to an isolated invertebrate RXR polypeptide comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of the invertebrate RXR polypeptide.", "In another specific embodiment, the present invention relates to an isolated invertebrate RXR polypeptide comprising a truncation mutation that reduces non-steroid binding activity or non-steroid sensitivity of the invertebrate RXR polypeptide.", "In addition, the present invention relates to an isolated invertebrate RXR polypeptide comprising a truncation mutation that enhances ligand binding activity or ligand sensitivity of the invertebrate RXR polypeptide.", "The present invention relates to an isolated invertebrate RXR polypeptide comprising a truncation mutation that enhances ligand binding activity or ligand sensitivity of the invertebrate RXR polypeptide.", "In a specific embodiment, the present invention relates to an isolated invertebrate RXR polypeptide comprising a truncation mutation that enhances steroid binding activity or steroid sensitivity of the invertebrate RXR polypeptide.", "In another specific embodiment, the present invention relates to an isolated invertebrate RXR polypeptide comprising a truncation mutation that enhances non-steroid binding activity or non-steroid sensitivity of the invertebrate RXR polypeptide.", "The present invention also relates to an isolated invertebrate retinoid X receptor polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the mutated invertebrate retinoid X receptor polypeptide and a dimerization partner.", "Preferably, the isolated invertebrate retinoid X receptor polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9 (LmUSP-EF), SEQ ID NO: 10 (AmaRXR1-EF), SEQ ID NO: 11 (AmaRXR2-EF), SEQ ID NO: 12 (CpRXR-EF), SEQ ID NO: 13 (TmRXR-EF), and SEQ ID NO: 14 (AmRXR-EF).", "More preferably, the isolated polynucleotide encoding an invertebrate retinoid X receptor polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21 (LmUSP-EF), SEQ ID NO: 22 (AmaRXR1-EF), SEQ ID NO: 23 (AmaRXR2-EF), SEQ ID NO: 24 (CpRXR-EF), SEQ ID NO: 25 (TmRXR-EF), and SEQ ID NO: 26 (AmRXR-EF).", "In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide.", "Preferably, the dimerization partner is a truncated EcR polypeptide.", "More preferably, the dimerization partner is an EcR polypeptide in which domain A/B has been deleted.", "Even more preferably, the dimerization partner is an EcR polypeptide comprising an amino acid sequence of SEQ ID NO: 5 (CfEcR-EF), SEQ ID NO: 43 (CfEcR-DEF) or SEQ ID NO: 59 (CfEcR-CDEF).", "Method of Modulating Gene Expression of the Invention Applicants' invention also relates to methods of modulating gene expression in a host cell using a gene expression modulation system according to the invention.", "Specifically, Applicants' invention provides a method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; and b) introducing into the host cell a ligand; wherein the gene to be modulated is a component of a gene expression cassette comprising: i) a response element comprising a domain recognized by the DNA binding domain of the first hybrid polypeptide; a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and a gene whose expression is to be modulated, whereby upon introduction of the ligand into the host cell, expression of the gene is modulated.", "The invention also provides a method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; b) introducing into the host cell a gene expression cassette comprising i) a response element comprising a domain recognized by the DNA binding domain from the first hybrid polypeptide; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and a gene whose expression is to be modulated; and c) introducing into the host cell a ligand; whereby upon introduction of the ligand into the host cell, expression of the gene is modulated.", "Genes of interest for expression in a host cell using Applicants' methods may be endogenous genes or heterologous genes.", "Nucleic acid or amino acid sequence information for a desired gene or protein can be located in one of many public access databases, for example, GENBANK, EMBL, Swiss-Prot, and PIR, or in many biology related journal publications.", "Thus, those skilled in the art have access to nucleic acid sequence information for virtually all known genes.", "Such information can then be used to construct the desired constructs for the insertion of the gene of interest within the gene expression cassettes used in Applicants' methods described herein.", "Examples of genes of interest for expression in a host cell using Applicants' methods include, but are not limited to: genes encoding therapeutically desirable polypeptides or products that may be used to treat a condition, a disease, a disorder, a dysfunction, a genetic defect, such as monoclonal antibodies, enzymes, proteases, cytokines, interferons, insulin, erthropoietin, clotting factors, other blood factors or components, viral vectors for gene therapy, virus for vaccines, targets for drug discovery, functional genomics, and proteomics analyses and applications, and the like.", "Acceptable ligands are any that modulate expression of the gene when binding of the DNA binding domain of the two-hybrid system to the response element in the presence of the ligand results in activation or suppression of expression of the genes.", "Preferred ligands include ponasterone, muristerone A, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N′-diacylhydrazines such as those disclosed in U.S. Pat.", "Nos.", "6,013,836; 5,117,057; 5,530,028; and 5,378,726; dibenzoylalkyl cyanohydrazines such as those disclosed in European Application No.", "461,809; N-alkyl-N,N′-diaroylhydrazines such as those disclosed in U.S. Pat.", "No.", "5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those disclosed in European Application No.", "234,994; N-aroyl-N-alkyl-N′-aroylhydrazines such as those described in U.S. Pat.", "No.", "4,985,461; each of which is incorporated herein by reference and other similar materials including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, and the like.", "In a preferred embodiment, the ligand for use in Applicants' method of modulating expression of gene is a compound of the formula: wherein: E is a (C4-C6)alkyl containing a tertiary carbon or a cyano(C3-C5)alkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, CoCH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2; R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, CoCH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, NH—CN, or joined with R3 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R3 is H, Et, or joined with R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R4, R5, and R6 are independently H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, CoCH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.", "In another preferred embodiment, a second ligand may be used in addition to the first ligand discussed above in Applicants' method of modulating expression of a gene, wherein the second ligand is 9-cis-retinoic acid or a synthetic analog of retinoic acid.", "Applicants' invention provides for modulation of gene expression in prokaryotic and eukaryotic host cells.", "Thus, the present invention also relates to a method for modulating gene expression in a host cell selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell, and a mammalian cell.", "Preferably, the host cell is a yeast cell, a hamster cell, a mouse cell, a monkey cell, or a human cell.", "Expression in transgenic host cells may be useful for the expression of various polypeptides of interest including but not limited to therapeutic polypeptides, pathway intermediates; for the modulation of pathways already existing in the host for the synthesis of new products heretofore not possible using the host; cell based assays; functional genomics assays, biotherapeutic protein production, proteomics assays, and the like.", "Additionally the gene products may be useful for conferring higher growth yields of the host or for enabling an alternative growth mode to be utilized.", "Host Cells and Non-Human Organisms of the Invention As described above, the gene expression modulation system of the present invention may be used to modulate gene expression in a host cell.", "Expression in transgenic host cells may be useful for the expression of various genes of interest.", "Thus, Applicants' invention provides an isolated host cell comprising a gene expression system according to the invention.", "The present invention also provides an isolated host cell comprising a gene expression cassette according to the invention.", "Applicants' invention also provides an isolated host cell comprising a polynucleotide or a polypeptide according to the invention.", "The isolated host cell may be either a prokaryotic or a eukaryotic host cell.", "Preferably, the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell, and a mammalian cell.", "Examples of preferred host cells include, but are not limited to, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterial species such as those in the genera Synechocystis, Synechococcus, Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella, animal, and mammalian host cells.", "In a specific embodiment, the host cell is a yeast cell selected from the group consisting of a Saccharomyces, a Pichia, and a Candida host cell.", "In another specific embodiment, the host cell is a hamster cell.", "In another specific embodiment, the host cell is a murine cell.", "In another specific embodiment, the host cell is a monkey cell.", "In another specific embodiment, the host cell is a human cell.", "Host cell transformation is well known in the art and may be achieved by a variety of methods including but not limited to electroporation, viral infection, plasmid/vector transfection, non-viral vector mediated transfection, particle bombardment, and the like.", "Expression of desired gene products involves culturing the transformed host cells under suitable conditions and inducing expression of the transformed gene.", "Culture conditions and gene expression protocols in prokaryotic and eukaryotic cells are well known in the art (see General Methods section of Examples).", "Cells may be harvested and the gene products isolated according to protocols specific for the gene product.", "In addition, a host cell may be chosen which modulates the expression of the inserted polynucleotide, or modifies and processes the polypeptide product in the specific fashion desired.", "Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification [e.g., glycosylation, cleavage (e.g., of signal sequence)] of proteins.", "Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.", "For example, expression in a bacterial system can be used to produce a non-glycosylated core protein product.", "However, a polypeptide expressed in bacteria may not be properly folded.", "Expression in yeast can produce a glycosylated product.", "Expression in eukaryotic cells can increase the likelihood of “native” glycosylation and folding of a heterologous protein.", "Moreover, expression in mammalian cells can provide a tool for reconstituting, or constituting, the polypeptide's activity.", "Furthermore, different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent.", "Applicants' invention also relates to a non-human organism comprising an isolated host cell according to the invention.", "Preferably, the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, an animal, and a mammal.", "More preferably, the non-human organism is a yeast, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep, a monkey, or a chimpanzee.", "In a specific embodiment, the non-human organism is a yeast selected from the group consisting of Saccharomyces, Pichia, and Candida.", "In another specific embodiment, the non-human organism is a Mus musculus mouse.", "Measuring Gene Expression/Transcription One useful measurement of Applicants' methods of the invention is that of the transcriptional state of the cell including the identities and abundances of RNA, preferably mRNA species.", "Such measurements are conveniently conducted by measuring cDNA abundances by any of several existing gene expression technologies.", "Nucleic acid array technology is a useful technique for determining differential mRNA expression.", "Such technology includes, for example, oligonucleotide chips and DNA microarrays.", "These techniques rely on DNA fragments or oligonucleotides which correspond to different genes or cDNAs which are immobilized on a solid support and hybridized to probes prepared from total mRNA pools extracted from cells, tissues, or whole organisms and converted to cDNA.", "Oligonucleotide chips are arrays of oligonucleotides synthesized on a substrate using photolithographic techniques.", "Chips have been produced which can analyze for up to 1700 genes.", "DNA microarrays are arrays of DNA samples, typically PCR products, that are robotically printed onto a microscope slide.", "Each gene is analyzed by a full- or partial-length target DNA sequence.", "Microarrays with up to 10,000 genes are now routinely prepared commercially.", "The primary difference between these two techniques is that oligonucleotide chips typically utilize 25-mer oligonucleotides which allow fractionation of short DNA molecules whereas the larger DNA targets of microarrays, approximately 1000 base pairs, may provide more sensitivity in fractionating complex DNA mixtures.", "Another useful measurement of Applicants' methods of the invention is that of determining the translation state of the cell by measuring the abundances of the constituent protein species present in the cell using processes well known in the art.", "Where identification of genes associated with various physiological functions is desired, an assay may be employed in which changes in such functions as cell growth, apoptosis, senescence, differentiation, adhesion, binding to a specific molecules, binding to another cell, cellular organization, organogenesis, intracellular transport, transport facilitation, energy conversion, metabolism, myogenesis, neurogenesis, and/or hematopoiesis is measured.", "In addition, selectable marker or reporter gene expression may be used to measure gene expression modulation using Applicants' invention.", "Other methods to detect the products of gene expression are well known in the art and include Southern blots (DNA detection), dot or slot blots (DNA, RNA), northern blots (RNA), RT-PCR (RNA), western blots (polypeptide detection), and ELISA (polypeptide) analyses.", "Although less preferred, labeled proteins can be used to detect a particular nucleic acid sequence to which it hybridizes.", "In some cases it is necessary to amplify the amount of a nucleic acid sequence.", "This may be carried out using one or more of a number of suitable methods including, for example, polymerase chain reaction (“PCR”), ligase chain reaction (“LCR”), strand displacement amplification (“SDA”), transcription-based amplification, and the like.", "PCR is carried out in accordance with known techniques in which, for example, a nucleic acid sample is treated in the presence of a heat stable DNA polymerase, under hybridizing conditions, with one pair of oligonucleotide primers, with one primer hybridizing to one strand (template) of the specific sequence to be detected.", "The primers are sufficiently complementary to each template strand of the specific sequence to hybridize therewith.", "An extension product of each primer is synthesized and is complementary to the nucleic acid template strand to which it hybridized.", "The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers.", "Following a sufficient number of rounds of synthesis of extension products, the sample may be analyzed as described above to assess whether the sequence or sequences to be detected are present.", "The present invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention.", "EXAMPLES General Methods Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) and by T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc.", "and Wiley-Interscience (1987).", "Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art.", "Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A.", "Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass.", "(1989).", "All reagents, restriction enzymes and materials used for the growth and maintenance of host cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.", "), or Sigma Chemical Company (St. Louis, Mo.)", "unless otherwise specified.", "Manipulations of genetic sequences may be accomplished using the suite of programs available from the Genetics Computer Group Inc. (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).", "Where the GCG program “Pileup” is used the gap creation default value of 12, and the gap extension default value of 4 may be used.", "Where the CGC “Gap” or “Bestfit” program is used the default gap creation penalty of 50 and the default gap extension penalty of 3 may be used.", "In any case where GCG program parameters are not prompted for, in these or any other GCG program, default values may be used.", "The meaning of abbreviations is as follows: “h” means hour(s), “min” means minute(s), “sec” means second(s), “d” means day(s), “μl” means microliter(s), “ml” means milliliter(s), “L” means liter(s), “μM” means micromolar, “mM” means millimolar, “μg” means microgram(s), “mg” means milligram(s), “A” means adenine or adenosine, “T” means thymine or thymidine, “G” means guanine or guanosine, “C” means cytidine or cytosine, “×g” means times gravity, “nt” means nucleotide(s), “aa” means amino acid(s), “bp” means base pair(s), “kb” means kilobase(s), “k” means kilo, “μ” means micro, and “° C.” means degrees Celsius.", "Example 1 Applicants' EcR/invertebrate RXR-based inducible gene modulation system is useful in various applications including gene therapy, expression of proteins of interest in host cells, production of transgenic organisms, and cell-based assays.", "In various cellular backgrounds, including mammalian cells, invertebrate EcR heterodimerizes with vertebrate RXR and, upon binding of ligand, transactivates genes under the control of ecdysone response elements.", "Applicants have made the surprising discovery that invertebrate RXR can substitute for vertebrate RXR and provide a novel inducible gene expression system for yeast and animal cell applications.", "This Example describes the construction of several gene expression cassettes for use in the EcR-based inducible gene expression system of the invention.", "Applicants constructed several EcR-based gene expression cassettes based on the spruce budworm Choristoneura fumiferana EcR (“CfEcR”), C. fumiferana ultraspiracle (“CfUSP”), Drosophila melanogaster USP (“DmUSP”), mouse Mus musculus retinoid X receptor α (“MmRXRα”), locust Locusta migratoria USP (“LmUSP”), an invertebrate homolog of vertebrate RXR, Amblyomma americanum RXR homolog 1 (“AmaRXR1”), an invertebrate homolog of vertebrate RXR, and Amblyomma americanum RXR homolog 2 (“AmaRXR2”), an invertebrate homolog of vertebrate RXR.", "The prepared receptor constructs comprise a ligand binding domain of either an EcR, a vertebrate RXR, an invertebrate USP, or an invertebrate RXR; and a GAL4 or LexA DNA binding domain (DBD) or a VP16 or B42 acidic activator transactivation domain (AD).", "The reporter constructs include a reporter gene, luciferase or LacZ, operably linked to a synthetic promoter construct that comprises either a GAL4 response element or a LexA response element to which the Gal4 DBD or LexA DBD binds, respectively.", "Various combinations of these receptor and reporter constructs were cotransfected into mammalian cells as described in Examples 2-9 infra.", "Gene Expression Cassettes: Ecdysone receptor-based gene expression cassettes (switches) were constructed as followed, using standard cloning methods available in the art.", "The following is brief description of preparation and composition of each switch used in the Examples described herein.", "1.1—GAL4CfEcR-CDEF/VP16MmRXRα-DEF: The C, D, E, and F domains from spruce budworm Choristoneura fumiferana EcR (“CfEcR-CDEF”; SEQ ID NO: 45) were fused to a GAL4 DNA binding domain (“Gal4DNABD” or “Gal4 DBD”; SEQ ID NO: 33) and placed under the control of an SV40e promoter (SEQ ID NO: 46).", "The DEF domains from mouse (Mus musculus) RXRα (“MmRXRα-DEF”; SEQ ID NO: 47) were fused to the transactivation domain from VP16 (“VP16AD”; SEQ ID NO: 37) and placed under the control of an SV40e promoter (SEQ ID NO: 46).", "Five consensus GAL4 response element binding sites (“5XGAL4RE”; comprising 5 copies of a GAL4RE comprising SEQ ID NO: 41) were fused to a synthetic E1b minimal promoter (SEQ ID NO: 48) and placed upstream of the luciferase gene (SEQ ID NO: 49).", "1.2—GAL4CfEcR-CDEF/VP16MmRXRα-EF: This construct was prepared in the same way as in switch 1.1 above except MmRXRα-DEF was replaced with MmRXRα-EF (SEQ ID NO: 50).", "1.3—GAL4CfEcR-CDEF/VP16CfUSP-DEF: This construct was prepared in the same way as in switch 1.1 above except MmRXRα-DEF was replaced with the D, E and F domains from spruce budworm USP (“CfUSP-DEF”; SEQ ID NO: 51).", "The constructs used in this example are similar to those disclosed in U.S. Pat.", "No.", "5,880,333 except that Choristoneura fumiferana USP rather than Drosophila melanogaster USP was utilized.", "1.4—GAL4CfEcR-CDEF/VP16LmUSP-DEF: This construct was prepared in the same way as in switch 1.1 above except MmRXRα-DEF was replaced with the D, E and F domains of Locusta migratoria ultraspiracle (“LmUSP-DEF”; SEQ ID NO: 52).", "1.5—GAL4CfEcR-DEF/VP16MmRXRα-DEF: This construct was prepared in the same way as switch 1.1 except CfEcR-CDEF was replaced with CfEcR-DEF (SEQ ID NO: 53).", "1.6—GAL4CfEcR-DEF/VP16MmRXRα-EF: This construct was prepared in the same way as switch 1.5 except MmRXRα-DEF was replaced with MmRXRα-EF (SEQ ID NO: 50).", "1.7—GAL4CfEcR-DEF/VP16CfUSP-DEF: This construct was prepared in the same way as in switch 1.5 above except MmRXRα-DEF was replaced with the D, E and F domains from spruce budworm C. fumiferana USP (“CfUSP-DEF”; SEQ ID NO: 51).", "1.8—GAL4CfEcR-DEF/VP16LmUSP-DEF: This construct was prepared in the same way as in switch 1.5 above except MmRXRα-DEF was replaced with the D, E, and F domains of Locusta migratoria ultraspiracle (“LmUSP-DEF”; SEQ ID NO: 52).", "1.9—Gal4CfEcR-A/BCDEF/VP16LmUSP-DEF: The full-length spruce budworm Choristoneura fumiferana EcR (“CfEcR-A/BCDEF”; SEQ ID NO: 54) was fused to a GAL4 DNA binding domain (“Gal4DNABD” or “Gal4 DBD”; SEQ ID NO: 33) and placed under the control of an SV40e promoter (SEQ ID NO: 46).", "The DEF domains from Locusta migratoria ultraspiracle (“LmUSP-DEF”; SEQ ID NO: 52) were fused to the transactivation domain from VP16 (“VP16AD”; SEQ ID NO: 37) and placed under the control of an SV40e promoter (SEQ ID NO: 46).", "Five consensus GAL4 response element binding sites (“5XGAL4RE”; comprising 5 copies of a GAL4RE comprising SEQ ID NO: 41) were fused to a synthetic E1b minimal promoter (SEQ ID NO: 48) and placed upstream of the luciferase gene (SEQ ID NO: 49).", "1.10—Gal4CfEcR-1/2CDEF/VP16LmUSP-DEF: This construct was prepared in the same way as switch 1.9 except CfEcR-A/BCDEF was replaced with CfEcR-1/2CDEF (SEQ ID NO: 55).", "1.11—Gal4CfEcR-CDEF/VP16LmUSP-DEF: This construct was prepared in the same way as switch 1.9 except CfEcR-A/BCDEF was replaced with CfEcR-CDEF (SEQ ID NO: 45).", "1.12—Gal4CfEcR-DEF/VP16LmUSP-DEF: This construct was prepared in the same way as switch 1.9 except CfEcR-A/BCDEF was replaced with CfEcR-DEF (SEQ ID NO: 53).", "1.13—Gal4CfEcR-EF/VP16LmUSP-DEF: This construct was prepared in the same way as switch 1.9 except CfEcR-A/BCDEF was replaced with CfEcR-EF (SEQ ID NO: 1).", "1.14—Gal4CfEcR-DE/VP16LmUSP-DEF: This construct was prepared in the same way as switch 1.9 except CfEcR-CDEF was replaced with CfEcR-DE (SEQ ID NO: 3).", "1.15—Gal4CfEcR-A/BCDEF/VP16LmUSP-EF: The full-length spruce budworm Choristoneura fumiferana EcR (“CfEcR-A/BCDEF”; SEQ ID NO: 54) was fused to a GAL4 DNA binding domain (“Gal4DNABD” or “Gal4 DBD”; SEQ ID NO: 33) and placed under the control of an SV40e promoter (SEQ ID NO: 46).", "The EF domains from Locusta migratoria ultraspiracle (“LmUSP-EF”; SEQ ID NO: 9) were fused to the transactivation domain from VP16 (“VP16AD”; SEQ ID NO: 37) and placed under the control of an SV40e promoter (SEQ ID NO: 46).", "Five consensus GAL4 response element binding sites (“5XGAL4RE”; comprising 5 copies of a GAL4RE comprising SEQ ID NO: 41) were fused to a synthetic E1b minimal promoter (SEQ ID NO: 48) and placed upstream of the luciferase gene (SEQ ID NO: 49).", "1.16—Gal4CfEcR-1/2CDEF/VP16LmUSP-EF: This construct was prepared in the same way as switch 1.15 except CfEcR-A/BCDEF was replaced with CfEcR-1/2CDEF (SEQ ID NO: 55).", "1.17—Gal4CfEcR-CDEF/VP16LmUSP-EF: This construct was prepared in the same way as switch 1.15 except CfEcR-A/BCDEF was replaced with CfEcR-CDEF (SEQ ID NO: 45).", "1.18—Gal4CfEcR-DEF/VP16LmUSP-EF: This construct was prepared in the same way as switch 1.15 except CfEcR-A/BCDEF was replaced with CfEcR-DEF (SEQ ID NO: 53).", "1.19—Gal4CfEcR-EF/VP16LmUSP-EF: This construct was prepared in the same way as switch 1.15 except CfEcR-A/BCDEF was replaced with CfEcR-EF (SEQ ID NO: 1).", "1.20—Gal4CfEcR-DE/VP16LmUSP-EF: This construct was prepared in the same way as switch 1.15 except CfEcR-CDEF was replaced with CfEcR-DE (SEQ ID NO: 3).", "1.21—Gal4CfEcR-DEF/VP16AmaRXR1-EF: This construct was prepared in the same way as switch 1.18 except LmUSP-EF was replaced with the E and F domains of ixodid tick Amblyomma americanum RXR homolog 1 (“AmaRXR1-EF”; SEQ ID NO: 10).", "1.22—Gal4CfEcR-DEF/VP16AmaRXR2-EF: This construct was prepared in the same way as switch 1.21 except AmaRXR1-EF was replaced with the E and F domains of ixodid tick Amblyomma americanum RXR homolog 2 (“AmaRXR2-EF”; SEQ ID NO: 11).", "1.23—LexACfEcR-CDEF/VP16CfUSP-EF: The C, D, E, and F domains from spruce budworm Choristoneura fumiferana EcR (“CfEcR-CDEF”; SEQ ID NO: 45) were fused to a LexA DNA binding domain (“LexADNABD” or “LexADBD”; SEQ ID NO: 35) and placed under the control of an SV40e promoter (SEQ ID NO: 46).", "The E and F domains from spruce budworm C. fumiferana USP (“CfUSP-EF”; SEQ ID NO: 56) were fused to the transactivation domain from VP16 (“VP16AD”; SEQ ID NO: 37) and placed under the control of an SV40e promoter (SEQ ID NO: 46).", "Eight consensus LexA response element binding sites (“8XLexAop”; comprising 4 copies of a LexA response element binding site comprising SEQ ID NO: 42) were fused to a synthetic E1b minimal promoter (SEQ ID NO: 48) and placed upstream of the luciferase gene (SEQ ID NO: 49).", "1.24—LexACfEcR-CDEF/VP16LmUSP-EF: This construct was prepared in the same way as switch 1.23 except CfUSP-EF was replaced with LmUSP-EF (SEQ ID NO: 9).", "1.25—LexACfEcR-CDEF/VP16MmRXRα-EF: This construct was prepared in the same way as switch 1.23 except CfUSP-EF was replaced with MmRXRα-EF (SEQ ID NO: 50).", "1.26—LexACfEcR-CDEF/VP16DmUSP-EF: This construct was prepared in the same way as switch 1.23 except CfUSP-EF was replaced with the corresponding EF domains of DmUSP-EF (SEQ ID NO: 60).", "1.27—Gal4CfEcR-CDEF/B42LmUSP-EF: The C, D, E, and F domains from spruce budworm Choristoneura fumiferana EcR (“CfEcR-CDEF”; SEQ ID NO: 45) were fused to a GAL4 DNA binding domain (“GAL4DNABD” or “GAL4 DBD”; SEQ ID NO: 33) and placed under the control of an SV40e promoter (SEQ ID NO: 46).", "The E and F domains from locust Locusta migratoria USP (“LmUSP-EF”; SEQ ID NO: 9) were fused to the transactivation domain from B42 (“B42AD”; SEQ ID NO: 39) and placed under the control of an SV40e promoter (SEQ ID NO: 46).", "Five consensus GAL4 response element binding sites (“5XGAL4RE”; comprising 5 copies of a GAL4RE comprising SEQ ID NO: 41) were fused to a synthetic E1b minimal promoter (SEQ ID NO: 48) and placed upstream of the luciferase gene (SEQ ID NO: 49).", "1.28—LexACfEcR-CDEF/B42LmUSP-EF: This construct was prepared in the same way as switch 1.27 except the GAL4 DNA binding domain was replaced with a LexA DNA binding domain (SEQ ID NO: 35).", "1.29—GAL4CfEcR-DEF/VP16DmUSP-EF: This construct was prepared in the same way as switch 1.7 except CfUSP-DEF was replaced with the corresponding EF domains of DmUSP-EF (SEQ ID NO: 60).", "1.30—GAL4CfEcR-DEF/VP16CfUSP-EF: This construct was prepared in the same way as switch 1.7 except CfUSP-DEF was replaced with CfUSP-EF (SEQ ID NO: 56).", "Example 2 In a two-hybrid switch format, CfUSP and DmUSP in partnership with CfEcR are constitutively active in both yeast and mammalian cells.", "On the other hand, vertebrate RXR in partnership with CfEcR is a ligand dependent transactivator in mammalian cells.", "Applicants tested an invertebrate RXR, LmUSP in a two-hybrid format in mouse NIH3T3 cells to determine if it would function as a USP (constitutively) or as a vertebrate RXR (inducibly) in mammalian cells.", "Gal4:CfEcR-CDEF (FIG.", "1) or Gal4:CfEcR-DEF (FIG.", "2) were paired with VP16:MmRXR-DEF; VP16:MmRXR-EF; VP16:LmUSP-DEF; or VP16:CfUSP-EF and analyzed in mammalian cells.", "Briefly, gene induction potential (magnitude of induction) and ligand specificity and sensitivity were examined using two different ligands: a steroidal ligand (Ponasterone A, “PonA”) and a non-steroidal ligand [N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N-tert-butylhydrazine] in a dose-dependent induction of reporter gene expression in the transfected NIH3T3 cells.", "Reporter gene expression activities were assayed at 48 hours after ligand addition.", "Standard methods for culture and maintenance of the cells were followed.", "Transfections: DNAs corresponding to the various switch constructs outlined in Example 1, specifically switches 1.1 through 1.8, were transfected into mouse NIH3T3 cells (ATCC) as follows.", "Cells were harvested when they reached 50% confluency and plated in 6-, 12- or 24-well plates at 125,000, 50,000, or 25,000 cells, respectively, in 2.5, 1.0, or 0.5 ml of growth medium containing 10% fetal bovine serum (PBS), respectively.", "The next day, the cells were rinsed with growth medium and transfected for four hours.", "Superfect™ (Qiagen Inc.) was found to be the best transfection reagent for 3T3 cells.", "For 12-well plates, 4 μl of Superfect™ was mixed with 100 μl of growth medium.", "1.0 μg of reporter construct and 0.25 μg of each receptor construct of the receptor pair to be analyzed were added to the transfection mix.", "A second reporter construct was added [pTKRL (Promega), 0.1 μg/transfection mix] that comprises a Renilla luciferase gene operably linked and placed under the control of a thymidine kinase (TK) constitutive promoter and was used for normalization.", "The contents of the transfection mix were mixed in a vortex mixer and let stand at room temperature for 30 min.", "At the end of incubation, the transfection mix was added to the cells maintained in 400 μl growth medium.", "The cells were maintained at 37° C. and 5% CO2 for four hours.", "At the end of incubation, 500 μl of growth medium containing 20% FBS and either dimethylsulfoxide (DMSO; control) or a DMSO solution of 0.1, 1, 5, 10, and 50 μM PonA steroidal ligand or N-(2-ethyl-3-methoxybenzoyl)N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine non-steroidal ligand was added and the cells were maintained at 37° C. and 5% CO2 for 48 hours.", "The cells were harvested and reporter activity was assayed.", "The same procedure was followed for 6 and 24 well plates as well except all the reagents were doubled for 6 well plates and reduced to half for 24-well plates.", "Ligands: The steroidal ligand Ponasterone A (PonA) was purchased from Sigma Chemical Company.", "The non-steroidal ligand N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-t-butylhydrazine (GS™-E non-steroidal ligand) is a synthetic stable ecdysteroid ligand synthesized at Rohm and Haas Company.", "All ligands were dissolved in DMSO and the final concentration of DMSO was maintained at 0.1% in both controls and treatments.", "Reporter Assays: Cells were harvested 48 hours after adding ligands.", "125, 250, or 500 μl of passive lysis buffer (part of Dual-luciferase™ reporter assay system from Promega Corporation) were added to each well of 24- or 12- or 6-well plates respectively.", "The plates were placed on a rotary shaker for 15 minutes.", "Twenty μl of lysate were assayed.", "Luciferase activity was measured using Dual-luciferase™ reporter assay system from Promega Corporation following the manufacturer's instructions.", "13-Galactosidase was measured using Galacto-Star™ assay kit from TROPIX following the manufacturer's instructions.", "All luciferase and β-galactosidase activities were normalized using Renilla luciferase as a standard.", "Fold activities were calculated by dividing normalized relative light units (“RLU”) in ligand treated cells with normalized RLU in DMSO treated cells (untreated control).", "Results: As shown in FIGS.", "1 and 2, LmUSP in partnership with CfEcR functions as a ligand-inducible gene expression system in mammalian cells.", "This result is surprising since Applicants' previous experiments with CfUSP and DmUSP in partnership with CfEcR demonstrated constitutive expression activity (see PCT/US01/09050 application and FIGS.", "1 and 2 for CfUSP results; DmUSP results are not shown).", "In addition, LmUSP worked better than vertebrate RXR as a CfEcR partner.", "In particular, both the sensitivity, i.e.", "the concentration of ligand required for transactivation, and the magnitude of transactivation were increased with LmUSP compared to vertebrate RXR.", "Thus, Applicants have demonstrated for the first time that invertebrate RXRs can function effectively in partnership with an ecdysone receptor in an inducible gene expression system in mammalian cells.", "This EcR/invertebrate RXR inducible gene expression system is an improvement over the EcR/vertebrate RXR gene expression system since less ligand is required for transactivation and increased levels of transactivation can be achieved.", "Based upon Applicant's discovery described herein, one of ordinary skill in the art is able to predict that other invertebrate RXRs and their homologs, with the exception of Dipteran RXR homologs (example DmUSP) and Lepidopteran RXR homologs (example CfUSP), will also function in Applicants' EcR/invertebrate RXR-based inducible gene expression system.", "In addition, one of ordinary skill in the art is also able to predict that Applicants' novel inducible gene expression system will also work to modulate gene expression in yeast cells.", "Since the Dipteran RXR homolog/and Lepidopteran RXR homolog/EcR gene expression systems function constitutively in yeast cells (data not shown), similar to how they function in mammalian cells, and Applicants have shown herein that non-Dipteran and non-Lepidopteran invertebrate RXRs function inducibly in partnership with an EcR in mammalian cells, the EcR/invertebrate RXR-based gene expression system is also predicted to function inducibly in yeast cells.", "Thus, the EcR/invertebrate RXR inducible gene expression system of the present invention is useful in applications where modulation of gene expression levels is desired in both yeast and mammalian cells.", "Further, there is no reason not to expect that the present invention would also work in other cells.", "Example 3 This Example describes the comparison of vertebrate RXR and invertebrate RXR-based two-hybrid gene expression systems comprising full length or truncated EcR, vertebrate RXR, and invertebrate RXR polypeptides.", "An amino acid sequence alignment, comparing the EF domains of twelve different vertebrate and invertebrate RXRs is shown in FIGS.", "3A and B.", "As described below, Applicants compared different GAL4/CfEcR-based switches comprising MmRXRα-EF (a vertebrate RXR), LmUSP-EF (an invertebrate RXR), AmaRXR1-EF (an invertebrate RXR), and AmaRXR2-EF (an invertebrate RXR) fused to a VP16 activation domain to identify the receptors that give a switch with a) maximum induction in the presence of ligand; b) minimum background in the absence of ligand; c) highly sensitive to ligand concentration; and/or d) minimum cross-talk among ligands and receptors in mammalian cells.", "Briefly, full-length EcR and truncated EcRs, created by a truncation mutation at the junctions of A/B, C, D, E and F domains and fused to a GAL4 DNA binding domain encoding polynucleotide (SEQ ID NO: 33) as described in Example 1 above.", "A VP16 activation domain encoding polynucleotide (SEQ ID NO: 37) was fused to the E and F domains of MmRXRα, LmUSP, AmaRXR1, and AmaRXR2 as described in Example 1.The resulting hybrid EcR/vertebrate or invertebrate RXR-encoding gene expression cassettes were assayed in NIH3T3 cells in pairwise comparisons.", "Plasmid pFRLUC (Stratagene) encoding a luciferase polypeptide was used as a reporter gene construct and pTKRL (Promega) encoding a Renilla luciferase polypeptide under the control of the constitutive TK promoter was used to normalize the transfections as described above.", "The transfected cells were grown in the presence 0, 1, 5 or 25 μM of the non-steroid N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine or the steroid PonA for 48 hours.", "The cells were harvested, lysed and luciferase reporter activity was measured in the cell lysates.", "Total fly luciferase relative light units are presented.", "The number on the top of each bar is the maximum fold induction for that treatment.", "The analysis was performed in triplicate and mean luciferase counts [total relative light units (RLU)] were determined as described above.", "As shown in the FIGS.", "4-7, CfEcR-CDEF performs better than any other CfEcR truncation.", "In particular, Gal4CfEcR-CDEF showed better induction than Gal4CfEcR-DEF using VP16LmUSP-EF.", "The EF domain of CfEcR in combination with LmUSP-DEF showed fairly good induced levels with very low uninduced levels.", "Most of EcR-EF domains described in patents and publications include D, E, and F domains (about 300 amino acids).", "This particular truncation includes only 230 amino acids and may rely on the D domain of LmUSP for heterodimerization.", "Of all the truncations of LmUSP tested, Applicants' results show that the VP16LmUSP-EF hybrid receptor polypeptide was the best partner for Gal4CfEcR-based hybrid polypeptides, with GAL4CfEcRCDEF/VP16LmUSP-EF (switch 1.17) performing better than any other receptor combination and more sensitive to non-steroids than steroids (FIGS.", "6 and 7).", "In general, the CfEcR/LmUSP-based switch was more sensitive to the non-steroid N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine than to the steroid PonA.", "Thus, the EF domain of LmUSP is sufficient and performs better than DEF domains of this receptor in partnership with CfEcR constructs.", "Applicants' results show that the magnitude and fold induction of MmRXRα and LmUSP are similar but LmUSP improves sensitivity to ligand by at least 10 fold.", "Thus, the EcR/invertebrate system is an improvement over the EcR/vertebrate system.", "Example 4 This Example describes Applicants' further analysis of gene expression cassettes encoding truncated EcR or RXR receptor polypeptides that affect either ligand binding activity or ligand sensitivity, or both.", "Briefly, eleven different combinations of two-hybrid receptor pairs, constructed as described in Example 1, were further analyzed in a single experiment in NIH3T3 cells.", "These eleven receptor pair combinations and their corresponding sample numbers are depicted in Table 1.TABLE 1 CfEcR + MmRXRα/LmUSP Truncation Receptor Combinations in NIH3T3 Cells FIG.", "8 CfEcR Polypeptide MmRXRα or LmUSP X-Axis Sample No.", "Construct Polypeptide Construct Samples 1 and 2 GAL4CfEcR-CDEF VP16MmRXRα-A/BCDEF Samples 3 and 4 GAL4CfEcR-CDEF VP16MmRXRα-DEF Samples 5 and 6 GAL4CfEcR-CDEF VP16MmRXRα-EF Samples 7 and 8 GAL4CfEcR-DEF VP16MmRXRα-A/BCDEF Samples 9 and 10 GAL4CfEcR-DEF VP16MmRXRα-DEF Samples 11 and 12 GAL4CfEcR-DEF VP16MmRXRα-EF Samples 13 and 14 GAL4:CfEcR-CDEF VP16:LmUSP-DEF Samples 15 and 16 GAL4:CfEcR-CDEF VP16:LmUSP-EF Samples 17 and 18 GAL4:CfEcR-DEF VP16:LmUSP-DEF Samples 19 and 20 GAL4:CfEcR-DEF VP16:LmUSP-EF Samples 21 and 22 GAL4:CfEcR-EF VP16:LmUSP-DEF The above receptor construct pairs, along with the reporter plasmid pFRLuc were constructed as described above and transfected into NIH3T3 cells as described above.", "The eleven CfEcR truncation receptor combinations were duplicated into two groups and treated with either steroid (odd numbers on x-axis of FIG.", "8) or non-steroid (even numbers on x-axis of FIG.", "8).", "In particular, the cells were cultured in media containing 0, 1, 5 or 25 uM PonA (steroid) or N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine (non-steroid) ligand.", "The reporter gene activity was measured and total RLU are shown.", "The number on top of each bar is the maximum fold induction for that treatment and is the mean of three replicates.", "As shown in FIG.", "8, the CfEcR-CDEF/LmUSP-EF receptor combination (columns 15 and 16) was the best format both in terms of total RLU and fold induction.", "This result is consistent with Applicants' results presented above in Example 3.These eleven receptor pair combinations were also assayed in a human lung carcinoma cell line A549 (ATCC) and similar results were observed (data not shown).", "Example 5 This Example describes Applicants' analysis of additional invertebrate retinoid X receptor homologs for use within the EcR/invertebrate RXR-based inducible gene expression system of the present invention.", "Briefly, two-hybrid receptor gene switches were constructed as described in Example 1 comprising a GAL4/CfEcR-DEF gene expression cassette and VP16AmaRXR1-EF or a VP16AmaRXR2-EF.", "These AmaRXR1- and AmaRXR2-based gene switches (switches 1.21 and 1.22 of Example 1) were compared to GAL4/CfEcR-DEF gene switches comprising VP16MmRXRα-EF (switch 1.6), VP16LmUSP-EF (switch 1.18), VP16DmUSP-EF (switch 1.29), and VP16CfUSP-EF (switch 1.30) along with pFRLuc in NIH3T3 cells.", "The above receptor construct pairs, along with the reporter plasmid pFRLuc were constructed as described above and transfected into NIH3T3′ cells as described above.", "The transactivation potential of these six CfEcR-DEF receptor-based gene switches were determined in the transfected cells in the presence of 0, 0.2, 1, or 10 μM PonA (steroid) or 0, 0.4, 0.2, 1, or 10 μM non-steroid ligand N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine.", "The reporter gene activity was measured and total RLU are shown.", "The number on top of each bar is the maximum fold induction for that treatment and is the mean of three replicates.", "As shown in FIG.", "9, both AmaRXR1-EF and AmaRXR2-EF based switches performed better than the vertebrate MmRXRα-EF based switch, demonstrating that these non-dipteran, non-lepidopteran invertebrate RXR homologs can also function in the EcR/invertebrate RXR-based inducible gene expression system of the present invention.", "Thus, based upon Applicants' surprising discovery that an invertebrate RXR (LmUSP) can substitute for a vertebrate RXR and the findings borne out in this Example regarding additional invertebrate species RXRs, one of ordinary skill in the art is able to predict that other invertebrate species, non-dipteran and non-lepidopteran RXR homologs will work in Applicant's gene expression system.", "Example 6 This Example describes the construction of host cells comprising the EcR/invertebrate RXR-based gene expression modulation system according to the invention.", "To make stable cells expressing GAL4:CfEcR-DEF/VP16:LmUSP-EF (switch 1.18, prepared as described in Example 1), Applicants transfected the gene expression cassettes encoding the hybrid GAL4:CfEcR-DEF and VP16:LmUSP-EF polypeptides into Chinese hamster ovary CHO cells comprising a stably transfected reporter plasmid pFRLuc.", "Briefly, CHO cells were harvested when they reach 60-80% confluency and plated in 6- or 12- or 24-well plates at 250,000, 100,000, or 50,000 cells in 2.5, 1.0, or 0.5 ml of growth medium containing 10% Fetal bovine serum respectively.", "The next day, the cells were rinsed with growth medium and transfected for four hours.", "LipofectAMINE™ 2000 (Life Technologies Inc,) was found to be the best transfection reagent for these cells.", "For 12-well plates, 4 μl of LipofectAMINE™ 2000 was mixed with 100 μl of growth medium.", "1.0 μg of reporter construct and 0.25 μg of each receptor construct GAL4:CfEcR-DEF and VP16:LmUSP-EF were added to the transfection mix.", "A second reporter construct was added (0.1 μg/transfection mix) and comprised a Renilla luciferase gene operably linked and placed under the control of a thymidine kinase (TK) constitutive promoter and was used for normalization.", "The contents of the transfection mix were mixed in a vortex mixer and let stand at room temperature for 30 min.", "At the end of incubation, the transfection mix was added to the cells maintained in 400 μl growth medium.", "The cells were maintained at 37° C. and 5% CO2 for four hours.", "At the end of incubation, 500 μl of growth medium containing 20% FBS and either DMSO (control) or a DMSO solution of appropriate ligands were added and the cells were maintained at 37° C. and 5% CO2 for 24-48 hr.", "The cells were harvested and reporter activity was assayed.", "The same procedure was followed for 6 and 24 well plates as well except all the reagents were doubled for 6 well plates and reduced to half for 24-well plates.", "The transfected CHO cells were grown in the presence of 0, 1, 5, or 25 μM PonA steroid ligand or GS™-E non-steroid ligand for 48 hours.", "The cells were harvested, lysed and the reporter activity was measured.", "Total fly luciferase relative light units (RLU) are presented.", "The numbers on the top of the bars correspond to the maximum fold induction for each treatment.", "Bulk populations of cells were selected for resistance to the antibiotic neomycin (VP16:LmUSP/VP16:RXR constructs have the neomycin resistance gene incorporated).", "Several clones from each population were isolated by end point dilution.", "Three clones of stably transfected GAL4:CfEcR-DEF/VP16:LmUSP-EF cells were analyzed (see FIG.", "10, clone 1A2; the data related to the two other clones are not shown).", "Of the three clones analyzed, the GAL4:CfEcR-DEF/VP16:LmUSP-EF stable clone 1A2 exhibited the highest fold induction, 162 fold, in the presence of non-steroidal ligand and 42 fold induction in the presence of steroid PonA (see FIG.", "10).", "Example 7 This Example describes the development of another embodiment of the EcR/invertebrate RXR gene expression modulation system of the invention.", "Specifically, Applicants have constructed LexA DNA binding domain (DBD) based-EcR/invertebrate RXR gene switches for use in the gene expression modulation system of the invention.", "This embodiment can be useful as an alternate switch for a GAL4 DBD-based switch and can also be used in multiple switch formats.", "While the LexA DBD has been used in yeast and plant expression systems, Applicants are not aware of its use in mammalian applications.", "Briefly, a gene expression cassette comprising the LexA DNA binding domain (SEQ ID NO: 35) fused to CfEcR-CDEF domains (SEQ ID NO: 45) was prepared as described in Example 1.The LexA:CfEcR-CDEF gene expression cassette, along with a VP16:MmRXRα-EF, a VP16:CfUSP-EF, a VP16:DmUSP-EF, or a VP16:LmUSP-EF gene expression cassette, and a reporter construct (8opFRLuc) comprising an 8XLexA operator (4 copies of LexA response element; SEQ ID NO: 42), a minimal promoter (synthetic E1b minimal promoter SEQ ID NO: 48), and a luciferase gene (SEQ ID NO: 49) were transfected into mouse NIH3T3 cells.", "The transfected cells were cultured in the presence of 0, 0.1, 1, 5, 10, and 50 μM GS™-E non-steroidal ligand or PonA steroid ligand for 48 hours as described above.", "The cells were harvested, lysed and reporter activity was measured and total relative light units (RLU) are presented in FIG.", "11.The number on the top of each bar corresponds to the maximum fold induction of each treatment.", "The LexA:CfEcR-DEF construct functioned well in these mammalian cells with all partners examined (see FIG.", "11).", "The fold induction is comparable to what was observed with Applicants' GAL4 system (see FIG.", "4).", "The 8opFRLuc reporter (control) showed very little activity in these cells.", "The results presented in FIG.", "11 show that the LexA DNA binding domain functions well in Applicants' two-hybrid system, demonstrating that the DNA binding domain is portable in these gene expression cassettes.", "Example 8 This Example describes the development of another embodiment of the EcR/invertebrate RXR gene expression modulation system of the invention.", "Specifically, Applicants have constructed gene expression cassettes for use in the gene modulation system of the invention comprising a B42 acidic activator domain as a transactivation domain.", "The B42 acidic activator domain (“B42AD”; see Gyuris et al., (1993) Cell 75: 791-803) works well as a transactivator in yeast and as Applicants have now shown, works well in mammalian cells.", "The B42 acidic activator domain may be used in the gene expression cassettes of the present invention as an alternative to VP16 transactivation domain.", "Briefly, Applicants have constructed a gene expression cassette comprising a polynucleotide encoding a B42AD (SEQ ID NO: 39) fused to a polynucleotide encoding LmUSP-EF domains (SEQ ID NO: 9) as described in Example 1.This B42AD:LmUSP-EF gene expression cassette was evaluated in mouse NIH3T3 cells in partnership with either a GAL4:CfEcR-CDEF or a LexA:CfEcR-CDEF gene expression cassette and compared to a VP16:LmUSP-EF-based switch.", "All gene expression cassettes were prepared as described in Example 1.The appropriate reporter constructs were transfected into NIH3T3 cells.", "The transfected cells were cultured in the presence of 0, 0.1, 1, 5, 10, and 50 μM GS™-E non-steroidal ligand for 48 hours as described above.", "Reporter activity is plotted as total RLU (see FIG.", "12).", "The numbers on the top of the bars correspond to the maximum fold induction observed for that combination.", "The results show that the B42 acidic activation domain works as well as the VP16 transactivation domain in Applicants' two-hybrid system, demonstrating that the transactivation domain is also portable in these gene expression cassettes.", "Example 9 This Example demonstrates the effect of introduction of a second ligand into the host cell comprising an EcR/invertebrate RXR-based inducible gene expression modulation system of the invention.", "In particular, Applicants have determined the effect of 9-cis-retinoic acid on the transactivation potential of the GAL4CfEcR-DEF/VP16LmUSP-EF (switch 1.18) gene switch along with pFRLuc in NIH3T3 cells in the presence of non-steroid (GSE) for 48 hours.", "Briefly, GAL4CfEcR-DEF, pFRLuc and VP16LmUSP-EF were transfected into NIH3T3 cells and the transfected cells were treated with 0, 0.04, 0.2, 1, 5 and 25 μM non-steroidal ligand (GSE) and 0, 1, 5 and 25 μM 9-Cis-retinoic acid (Sigma Chemical Company).", "The reporter activity was measured at 48 hours after adding ligands.", "As shown in FIG.", "13, the presence of retinoic acid increased the sensitivity of CfEcR-DEF to non-steroidal ligand.", "At a non-steroid ligand concentration of 1 μM or less, there is very little induction in the absence of 9-Cis-retinoic acid, but when 1 μM 9-Cis-retinoic acid is added in addition to non-steroid, induction is greatly increased." ] ]	Patent_10468200
[ [ "Method and apparatus to stress test medicament inhalation aerosol device by inductive heating", "The invention relates to a method and apparatus for heat stress testing medicament aerosol inhalation devices, such as metered dose inhaler.", "An electrical induction work coil (24, 26, 28) is used to heat the inhalers to 55° C.±5 C.°.", "Heated inhalers are subsequently weighed to detect and reject nonconforming inhalers where a nonconforming amount of propellant has leaked." ], [ "1.An apparatus for heating medicinal inhalation devices comprising: an electrical power supply, and one or more induction coils.", "2.The apparatus of claim 1, further including a microprocessor for controlling the power supply in the range of 100-130 amps to heat the inhalation devices to a temperature in the range of 50-60° C. 3.The apparatus of claim 2, further including a cooling system comprising a condenser and a pump, wherein the apparatus includes a single induction coil, and wherein a liquid coolant is pumped through the induction coil, and wherein the electrical power supply has a maximum power output of 20 kilowatts and operates in the range of 80-90% efficiency.", "4.The apparatus of claim 2, wherein the power supply provides up to 20 kva and 50-450 kHz, and wherein 140 volts is applied to the one or more induction coils.", "5.The apparatus of claim 2, wherein the induction coil is constructed from copper tubing and silver soldered joints.", "6.The apparatus of claim 5, wherein the induction coil is a single, continuous coil in the shape of a loop having first and second bridges on first and second ends of the loop, respectively, and wherein the first and second bridges are suitably sized and configured to directly heat each entire inhalation device.", "7.The apparatus of claim 6 further including: a conveyor, and a computer-controlled gating assembly for indexing a predetermined number of medicinal inhalation devices along the conveyor between the first and second bridges of the induction coil, wherein the medicinal inhalation devices are metered dose inhalers.", "8.The apparatus of claim 7, wherein the gating assembly further includes first and second heat exchangers suitably adapted and positioned to heat a first and last metered dose inhaler indexed within the one or more induction coils.", "9.The apparatus of claim 8, wherein the first and second heat exchanger each comprise an aluminum canister adapted to be cooled.", "10.The apparatus of claim 7, wherein the gating assembly is adapted to index 32 metered dose inhalers per heating cycle, wherein 4 slugs of 8 metered dose inhalers per slug are indexed each cycle, and wherein the cycle time is in the range of 30-40 seconds.", "11.The apparatus of claim 7, wherein the conveyor has a line speed in the range of 100-140 metered dose inhalers per minute.", "12.The apparatus of claim 7 further including an infrared thermometer to measure the temperature of the metered dose inhalers.", "13.The apparatus of claim 12, wherein the measured temperatures are fed to the microprocessor, and wherein the microprocessor adjusts the power supply to heat the metered dose inhalers to a temperature of about 55° C. 14.The apparatus of claim 7 further including a weighing device to check the weight of the heated metered dose inhalers.", "15.An apparatus for heating medicinal inhalation devices comprising: an electrical power supply means, and one or more induction coil means.", "16.A process of heat stress testing medicinal inhalation devices to detect and reject nonconforming or leaking devices comprising the acts of: providing one or more inhalation devices, and induction heating the one or more inhalation devices.", "17.The process of claim 16, wherein the inhalation devices are metered dose inhalers, and wherein the metered dose inhalers are provided continuously at a line speed in the range of 100-140 inhalers per minute.", "18.The process of claim 17 further including the act of: indexing the continuously provided metered dose inhalers.", "19.The process of claim 18, wherein the metered dose inhalers are indexed in slugs of 8 providing 32 metered dose inhalers per induction heating cycle.", "20.The process of claim 17, wherein the metered dose inhalers are heated to a temperature in the range of 50-60° C. 21.The process of claim 20, wherein the metered dose inhalers are heated to a temperature of about 55° C. 22.The process of claim 21, wherein the temperature of the metered dose inhalers, the indexing and the induction heating is computer process controlled.", "23.The process of claim 18 further including the acts of: check weighing the heated metered dose inhalers, and rejecting any nonconforming or leaking metered dose inhalers.", "24.A process of heat stress testing medicinal inhalation devices to detect and reject nonconforming or leaking devices comprising the steps of: providing one or more inhalation devices, and induction heating the one or more inhalation devices." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>In the art of manufacturing medicament aerosol inhalation devices, recent attention has been aimed at detecting and rejecting potential and actual nonconforming devices.", "For example, a small percentage of aerosol inhalation devices leak or will leak due to manufacturing defects, such as broken or tom gaskets, loss of proper sealing, defective valve, swollen gasket(s), etc.", "Such defects cause a loss of aerosol propellant, which adversely affects or otherwise alters the performance of the inhalation device.", "Recently, the Food and Drug Administration (“FDA”) has become very concerned with leaking (or otherwise nonconforming or defective) aerosol inhalation devices, particularly MDI's.", "The performance of an MDI can be significantly altered when the propellant leaks, particularly where the propellant leaks in significantly amounts or at a significant rate.", "For example, a “gross leaker” may not deliver the medicament at all to the patient.", "MDI's that leak at more than an insignificant rate may under deliver the medicament.", "In other words, the medicament delivery from a leaking MDI does not conform to the dosing regimen set forth and approved by the FDA.", "In that case, the patient may not even realize that the insufficient medicament is being delivered to the lungs.", "As a result, the FDA has begun requiring manufacturers to stress test aerosol inhalation devices and subsequently weigh the stressed devices to detect actual and potential nonconforming devices.", "Leaking MDI's will weigh less than what they are supposed to weigh.", "Stressed MDI's that lose a predetermined amount of propellant are rejected as nonconforming.", "The FDA has set conforming standards.", "The FDA has also set a temperature standard of 55° C.±5 C.° for heat stress testing the MDI's.", "Many methods and apparatus are available for heating MDI's.", "The present invention involves using electromagnetic induction heating to heat the electro-conductive materials present in the MDI, such as primarily the aluminum canister.", "Electromagnetic induction is a method of henerating heat within a metal part.", "Any electrical conductor can be heated by electromagnetic induction.", "As alternating current from the generator flows through the inductor, or work coil, a highly concentrated, rapidly alternating magnetic field is established within the coil.", "The magnetic field thus induces an electric potential in the part to be heated.", "The part represents a closed circuit.", "The induced voltage causes current to flow within the part.", "Eddy currents are typically established.", "The resistance of the part to the flow of the induced current causes heating.", "The pattern of heating obtained by induction is determined by a number of factors: the shape of the induction coil producing the magnetic field, the number of turns in the coil, the operating frequency, the alternating current power input, and the nature of the work pieces.", "The rate of heating obtained by the coils depends on the strength of the magnetic field to which the part is exposed.", "In the work piece, this becomes a function of the induced currents and the resistance to electrical flow.", "The depth of current penetration depends upon work piece permeability, resistivity, and the alternating current frequency.", "Since the first two factors vary comparatively little, the greatest variable is frequency.", "Depth of current penetration decreases as frequency increases.", "High frequency current is generally used when shallow heating is desired.", "Intermediate and low frequencies are used in applications requiring deeper heating.", "The induction coil and associated components and the processes thereof of the present invention are advantageous.", "The present invention advantageously heats and stress tests MDI's in a relatively short period of time (i.e., dwell time), which permits in-line processing at high throughput.", "The present invention also advantageously employs a few relatively simple processing and material handling equipment resulting in low investment, reduced maintenance, high efficiency, and reliability.", "The present invention is further advantageous in that only the electroconductive portions of the MDI are heated significantly reducing the heating of plastic components (e.g., gaskets and valve components) that are susceptible to being unnecessarily damaged by heat.", "Still further, the present invention advantageously heat stress tests the MDI's without exposing the MDI's to steam or moisture (except any negligible moisture associated with the sealed heat exchangers) which can ingress into the MDI reducing product performance.", "Further benefits and advantages of the present invention are set forth herein." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>One aspect of the invention is an apparatus for heating medicinal inhalation devices.", "The apparatus includes an electrical power supply and one or more induction coils.", "Preferably, the apparatus further includes a microprocessor for controlling the power supply in the range of 100-130 amps, preferably 110-115 amps, and more preferably about 113 amps, to heat the inhalation devices to a temperature in the range of 50-60° C., preferably around 55° C. By “about” or the like language as used herein, it is meant to include those values surrounding the recited value or range of values that achieve substantially the same desired ultimate result.", "The apparatus also preferably further includes a cooling system including a condenser and a pump, wherein a liquid coolant is pumped through the induction coil.", "Preferably, the power supply provides up to 20 kilowatts (kW) at a frequency in the range of 240-440 kHz.", "A voltage of 140 volts may be applied to the induction coils.", "The electrical power supply may be operated in the range of 80-90% efficiency, preferably about 85%.", "The induction coil may be constructed from copper tubing and silver soldered joints.", "Preferably, the induction coil is a single, continuous coil in the shape of a loop having two bridges, one at each end of the loop, respectively.", "The bridges are suitably sized and configured to directly heat each entire inhalation device.", "In other words, the bridges of the induction coil may be 4 inches in height for heating metered dose inhalers (“MDI's”) that are 2-3 inches in height.", "Examples of MDI's that may be stress tested by this invention include those disclosed in U.S. Pat.", "Nos.", "6,170,717; 6,131,566; 6,143,277; and 6,149,892, which are incorporated herein by reference.", "Another aspect of the invention is an apparatus including the electrical powers supply and induction coil(s) described herein above, a conveyor, and a gating assembly for indexing a predetermined number of medicinal inhalation devices along the conveyor between the bridges of the induction coil.", "Preferably, the gating assembly further includes two heat exchangers suitably adapted and positioned to heat the first and last metered dose inhaler cycled and indexed within the induction coil.", "The heat exchanger may include an aluminum canister (similar to the canister used in the MDI's) adapted to be cooled by circulating cooling water.", "Preferably, the gating assembly is adapted and controlled to index 32 metered dose inhalers per heating cycle.", "The MDI's are indexed one slug at a time where each slug may include 8 MDI's.", "Each heating cycle may include 4 slugs of 8 metered dose inhalers per slug.", "The heating cycle time may be in the range of 30-40 seconds.", "As such, the conveyor may have a line speed in the range of 100-140 metered dose inhalers per minute.", "An infrared thermometer may also be employed to measure the temperature of the metered dose inhalers.", "Preferably, the measured temperatures are fed to the microprocessor, whereby the microprocessor adjusts the power supply to heat the metered dose inhalers to the desired temperature, for example 55° C.±5 C.°.", "A check weighing device is also preferably employed to check the weight of the heated metered dose inhalers.", "Any nonconforming and/or leaking (e.g., gross leakers) MDI's are detected and summarily rejected/discarded.", "Another aspect of the invention is a process or method of heat stress testing medicinal inhalation devices to detect and reject nonconforming or leaking devices.", "In general, the process includes providing one or more inhalation devices, and induction heating the one or more inhalation devices.", "Preferably, the inhalation devices are metered dose inhalers, whereby the metered dose inhalers are provided continuously (e.g., continuous runs) at a line speed in the range of 100-140 inhalers per minute.", "Preferably, the process also includes indexing the continuously provided metered dose inhalers.", "The metered dose inhalers may be indexed in slugs of 8 providing 32 metered dose inhalers per induction heating cycle.", "During the heating cycle, the metered dose inhalers are preferably heated to a temperature in the range of about 50-60° C., preferably about 55° C. The temperature of the metered dose inhalers, the indexing, the induction heating, and other steps in the process may be computer process controlled by a microprocessor and other suitable electronic (e.g., sensors) and electromechanical equipment and instruments (e.g., pneumatic actuators).", "The process preferably also includes the steps of check weighing the heated metered dose inhalers, and rejecting any nonconforming or leaking metered dose inhalers." ], [ "FIELD OF THE INVENTION The present invention generally relates to the field of medicament inhalation devices, specifically metered dose inhalers, and to heat stress testing of such inhalation devices to detect and reject nonconforming inhalers.", "BACKGROUND OF THE INVENTION In the art of manufacturing medicament aerosol inhalation devices, recent attention has been aimed at detecting and rejecting potential and actual nonconforming devices.", "For example, a small percentage of aerosol inhalation devices leak or will leak due to manufacturing defects, such as broken or tom gaskets, loss of proper sealing, defective valve, swollen gasket(s), etc.", "Such defects cause a loss of aerosol propellant, which adversely affects or otherwise alters the performance of the inhalation device.", "Recently, the Food and Drug Administration (“FDA”) has become very concerned with leaking (or otherwise nonconforming or defective) aerosol inhalation devices, particularly MDI's.", "The performance of an MDI can be significantly altered when the propellant leaks, particularly where the propellant leaks in significantly amounts or at a significant rate.", "For example, a “gross leaker” may not deliver the medicament at all to the patient.", "MDI's that leak at more than an insignificant rate may under deliver the medicament.", "In other words, the medicament delivery from a leaking MDI does not conform to the dosing regimen set forth and approved by the FDA.", "In that case, the patient may not even realize that the insufficient medicament is being delivered to the lungs.", "As a result, the FDA has begun requiring manufacturers to stress test aerosol inhalation devices and subsequently weigh the stressed devices to detect actual and potential nonconforming devices.", "Leaking MDI's will weigh less than what they are supposed to weigh.", "Stressed MDI's that lose a predetermined amount of propellant are rejected as nonconforming.", "The FDA has set conforming standards.", "The FDA has also set a temperature standard of 55° C.±5 C.° for heat stress testing the MDI's.", "Many methods and apparatus are available for heating MDI's.", "The present invention involves using electromagnetic induction heating to heat the electro-conductive materials present in the MDI, such as primarily the aluminum canister.", "Electromagnetic induction is a method of henerating heat within a metal part.", "Any electrical conductor can be heated by electromagnetic induction.", "As alternating current from the generator flows through the inductor, or work coil, a highly concentrated, rapidly alternating magnetic field is established within the coil.", "The magnetic field thus induces an electric potential in the part to be heated.", "The part represents a closed circuit.", "The induced voltage causes current to flow within the part.", "Eddy currents are typically established.", "The resistance of the part to the flow of the induced current causes heating.", "The pattern of heating obtained by induction is determined by a number of factors: the shape of the induction coil producing the magnetic field, the number of turns in the coil, the operating frequency, the alternating current power input, and the nature of the work pieces.", "The rate of heating obtained by the coils depends on the strength of the magnetic field to which the part is exposed.", "In the work piece, this becomes a function of the induced currents and the resistance to electrical flow.", "The depth of current penetration depends upon work piece permeability, resistivity, and the alternating current frequency.", "Since the first two factors vary comparatively little, the greatest variable is frequency.", "Depth of current penetration decreases as frequency increases.", "High frequency current is generally used when shallow heating is desired.", "Intermediate and low frequencies are used in applications requiring deeper heating.", "The induction coil and associated components and the processes thereof of the present invention are advantageous.", "The present invention advantageously heats and stress tests MDI's in a relatively short period of time (i.e., dwell time), which permits in-line processing at high throughput.", "The present invention also advantageously employs a few relatively simple processing and material handling equipment resulting in low investment, reduced maintenance, high efficiency, and reliability.", "The present invention is further advantageous in that only the electroconductive portions of the MDI are heated significantly reducing the heating of plastic components (e.g., gaskets and valve components) that are susceptible to being unnecessarily damaged by heat.", "Still further, the present invention advantageously heat stress tests the MDI's without exposing the MDI's to steam or moisture (except any negligible moisture associated with the sealed heat exchangers) which can ingress into the MDI reducing product performance.", "Further benefits and advantages of the present invention are set forth herein.", "SUMMARY OF THE INVENTION One aspect of the invention is an apparatus for heating medicinal inhalation devices.", "The apparatus includes an electrical power supply and one or more induction coils.", "Preferably, the apparatus further includes a microprocessor for controlling the power supply in the range of 100-130 amps, preferably 110-115 amps, and more preferably about 113 amps, to heat the inhalation devices to a temperature in the range of 50-60° C., preferably around 55° C. By “about” or the like language as used herein, it is meant to include those values surrounding the recited value or range of values that achieve substantially the same desired ultimate result.", "The apparatus also preferably further includes a cooling system including a condenser and a pump, wherein a liquid coolant is pumped through the induction coil.", "Preferably, the power supply provides up to 20 kilowatts (kW) at a frequency in the range of 240-440 kHz.", "A voltage of 140 volts may be applied to the induction coils.", "The electrical power supply may be operated in the range of 80-90% efficiency, preferably about 85%.", "The induction coil may be constructed from copper tubing and silver soldered joints.", "Preferably, the induction coil is a single, continuous coil in the shape of a loop having two bridges, one at each end of the loop, respectively.", "The bridges are suitably sized and configured to directly heat each entire inhalation device.", "In other words, the bridges of the induction coil may be 4 inches in height for heating metered dose inhalers (“MDI's”) that are 2-3 inches in height.", "Examples of MDI's that may be stress tested by this invention include those disclosed in U.S. Pat.", "Nos.", "6,170,717; 6,131,566; 6,143,277; and 6,149,892, which are incorporated herein by reference.", "Another aspect of the invention is an apparatus including the electrical powers supply and induction coil(s) described herein above, a conveyor, and a gating assembly for indexing a predetermined number of medicinal inhalation devices along the conveyor between the bridges of the induction coil.", "Preferably, the gating assembly further includes two heat exchangers suitably adapted and positioned to heat the first and last metered dose inhaler cycled and indexed within the induction coil.", "The heat exchanger may include an aluminum canister (similar to the canister used in the MDI's) adapted to be cooled by circulating cooling water.", "Preferably, the gating assembly is adapted and controlled to index 32 metered dose inhalers per heating cycle.", "The MDI's are indexed one slug at a time where each slug may include 8 MDI's.", "Each heating cycle may include 4 slugs of 8 metered dose inhalers per slug.", "The heating cycle time may be in the range of 30-40 seconds.", "As such, the conveyor may have a line speed in the range of 100-140 metered dose inhalers per minute.", "An infrared thermometer may also be employed to measure the temperature of the metered dose inhalers.", "Preferably, the measured temperatures are fed to the microprocessor, whereby the microprocessor adjusts the power supply to heat the metered dose inhalers to the desired temperature, for example 55° C.±5 C.°.", "A check weighing device is also preferably employed to check the weight of the heated metered dose inhalers.", "Any nonconforming and/or leaking (e.g., gross leakers) MDI's are detected and summarily rejected/discarded.", "Another aspect of the invention is a process or method of heat stress testing medicinal inhalation devices to detect and reject nonconforming or leaking devices.", "In general, the process includes providing one or more inhalation devices, and induction heating the one or more inhalation devices.", "Preferably, the inhalation devices are metered dose inhalers, whereby the metered dose inhalers are provided continuously (e.g., continuous runs) at a line speed in the range of 100-140 inhalers per minute.", "Preferably, the process also includes indexing the continuously provided metered dose inhalers.", "The metered dose inhalers may be indexed in slugs of 8 providing 32 metered dose inhalers per induction heating cycle.", "During the heating cycle, the metered dose inhalers are preferably heated to a temperature in the range of about 50-60° C., preferably about 55° C. The temperature of the metered dose inhalers, the indexing, the induction heating, and other steps in the process may be computer process controlled by a microprocessor and other suitable electronic (e.g., sensors) and electromechanical equipment and instruments (e.g., pneumatic actuators).", "The process preferably also includes the steps of check weighing the heated metered dose inhalers, and rejecting any nonconforming or leaking metered dose inhalers.", "DESCRIPTION OF THE DRAWINGS FIG.", "1 is a top view of one aspect of the present invention.", "FIG.", "2 is a side view of a portion of the gating assembly and heat exchanger of the present invention.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Shown in FIG.", "1 is a top view of various aspects of the invention.", "A plurality of medicament inhalation aerosol devices 10, such as MDI's, are conveyed along a conveyor 12.The MDI's 10 are loaded onto the conveyor 12 from a tray unloading station 14.From there, the MDI's 10 pass along the conveyor 12 past a backup cue sensor 16 to between rails 18.A can count sensor 20 keeps track of the number of MDI's 10 passing that point on the conveyor 12.A computer-controlled gating assembly 22a,22b,22c indexes a predetermined number of MDI's 10 (called a slug, e.g., 8 MDI's per slug) into a zone above a channel portion 24 of the induction coil and between the end bridge portions 26,28 of the induction coil.", "Preferably, the induction coil is a continuous loop, single tube, single turn, channel/radiused type induction coil fabricated from copper tubing and silver soldered joints, such as those available from Pillar Industries of Brookfield, Wis.", "The coil 24,26,28 is water cooled using a chiller (not shown), and is pressure rated for 100 psi.", "Preferably, the channel 24 is about 4 feet long and the coil has a 0.75 inch face width to accommodate various MDI 10 sizes and configurations.", "One of gating assembly levers 22c accumulates the MDI's 10 prior to entering the induction zone, and the other gating assembly levers 22a,22b are preferably pneumatically actuated during the induction heating cycle.", "The MDI's 10 can be alternatively indexed using a feedscrew or starwheel assembly.", "Preferably, 4 slugs of 8 MDI's 10 are indexed into the induction heating zone per heating cycle, thus having 32 MDI's heated per cycle.", "Preferably, the dwell time of the MDI's 10 in the heating induction cycle is around 9 seconds for 32 MDI's.", "A single slug of 8 MDI's may be indexed in 5 seconds.", "The heated MDI's 10 may be removed from the induction zone in around 3 seconds.", "Thus, line speeds of around 120 or greater MDI's per minute are preferably achieved.", "The gating assembly desirably allows the conveyor to run at a constant line speed.", "The number of MDI's in each heat induction cycle affects the heating efficiency.", "In general, coil efficiency is improved by increasing the number of MDI's.", "In general, MDI's heat faster in a static mode, so the indexing method is more effective than where the MDI's are moving.", "A safety enclosure 30 constructed from LEXAN® is also preferably provided.", "A controlled electrical power supply 32 having a control panel 34 supplies electrical power to the bus bar 36 which, in turn, supplies power to the induction coil 24,26,28.Preferably, the power supply 32 is a 20 kW, 400 kHz solid state RF generator, such as those available from Pillar Industries, MK-20, Model 7500.After completion of the heating cycle, the gating assembly 22a,22b releases the MDI's 10 conveying the MDI's 10 along the conveyor 12 to the accumulation table 38.A check weighing device (not shown) may be employed prior to or after the accumulation table 38 to detect and reject nonconforming (e.g., marginal sealing, defective valve, poor crimp, cut gasket, faulty component, etc.)", "or otherwise leaking (e.g., gross leaker) MDI's 10.Preferably, the power supply 32 has a built-in timer that can be set according to the time necessary to heat a predetermined number of MDI's to a desired temperature or temperature range.", "For example, it may take 4 second to induction heat 32 MDI's 10 to 54-60° C. The weight may be checked in-line or offline.", "A suitable check-weighing device is available from Anritsu.", "An infrared thermometer 38 may be used to measure the temperature of the MDI's 10 to ensure proper heating.", "Such measurements may be fed to the computer controlled power supply 32 to control heating.", "An irreversible temperature indicator may also be used by putting the test strip inside the MDI 10 and removing it after the heating cycle to determine the maximum temperature reached in the MDI.", "The infrared thermometer (Model OS91) and temperature test strips (Model U-08068-22) are available from Cole-Parmer Shown in FIG.", "2 is a preferred embodiment of the outer canister heat exchangers 40,42 of the present invention.", "The heat exchangers 40,42 are fixedly or removably attached to the non-conductive, placement arm of the gating assembly 22a,22b.", "The heat exchangers 40,42 include a sealed, aluminum canister 44 similar or identical in construction to the aluminum canister employed in the MDI's 10.The internal geometry of the heat exchangers is preferably designed for turbulent flow therein.", "A liquid, preferably water, is supplied to the heat exchangers 40,42 from a supply feed line 46 (from a chiller which is not shown) to a non-conductive, flexible line 48 to an internal supply tube 50 fixedly or removably attached to the removable canister lid 52.The lid 52 is removable to inspect,.clean, or repair the interior of the heat exchanger 40,42.An internal return tube 54 is similarly fixedly or removably attached to the lid 52.Cooling water is returned to the chiller (not shown) through a cooling return line 56 that also includes a nonconductive, flexible line 48′.", "In a linear configuration of objects, it is known in the art that induction heating efficiency is improved where each conductive object is proximate (preferably touching) to other conductive objects.", "This phenomenon is known as an electromagnetic effect inherent in induction heating.", "Thus, in this case, the first and last MDI's 10′,10″ are proximate to only one other MDI 10 whereas the other MDI's 10 are proximate to two other MDI's.", "It was determined that this electromagnetic effect reduced the induction heating efficiency of the first and last MDI's 10′,10″ such that they were 5 C.° cooler.", "This would be problematic since the FDA has required stress testing of all MDI's in the range of 55° C.±5 C.°.", "The present invention overcomes this potential problem.", "The heat exchangers 40,42 are preferably employed to straddle the first and last MDI's 10′,10″ (preferably 32 total MDI's) so that they are positioned proximate (touching or close) to the first and last MDI's during the heating cycle.", "During the induction heating cycle, the first and last MDI's 10′,10″ are heated sufficiently the same as the other MDI's because each MDI 10′,10″ is proximate to another conductive MDI 10 and a respective canister 44 of each respective heat exchanger 40,42.The heat exchangers 40,42 are continuously or intermittently cooled with cooling water to control their temperature.", "Cooling is needed so that a more than insignificant amount of heat is not transferred to the first and last MDI 10′,10″ by conductive and/or convection.", "Such non-induction forms of heat transfer may occur without cooling because the heat exchangers 40,42 are repetitively heated whereas the MDI's 10, 10′,10″ should be heated by induction only once." ] ]	Patent_10468219
[ [ "Detachable substrate or detachable structure and method for the production thereof", "The invention relates to the preparation of a thin layer comprising a step in which an interface is created between a layer used to create said thin layer and a substrate, characterized in that said interface is made in such way that it is provided with at least one first zone (Z1) which has a first level of mechanical strength, and a second zone (Z2) which has a level of mechanical strength which is substantially lower than that of the first zone.", "Said interface can be created by glueing surfaces which are prepared in a differentiated manner, by a layer which is buried and embrittled in a differentiated manner in said zones, or by an intermediate porous layer." ], [ "1.A method of preparing a thin layer comprising a step of producing an interface between a layer intended to form part of said thin layer and a substrate, wherein said interface is produced so as to have at least a first region having a first level of mechanical strength and a second region having a second level of mechanical strength significantly greater than the first level of mechanical strength, and wherein the first region is included within the second.", "2.A method according to claim 1, wherein the second region constitutes a periphery of a wafer of which the first region constitutes a core of the wafer.", "3.A method according to claim 1, wherein the first region is divided into fragments, each fragment being surrounded by the second region.", "4.A method according to claim 1 wherein the interface is produced between a surface of the substrate and a surface of the layer and wherein the step of producing the interface includes a step of preparing at least one of the surfaces, and a bonding step during which the surface is of the substrate and the surface of the layer are bonded by molecular adhesion bonding.", "5.A method according to claim 4, wherein the step of producing the interface includes a step of preparing the surface of the substrate and the surface of the layer.", "6.A method according to claim 4, wherein the step of preparing at least one of the surface includes a treatment step of locally increasing the roughness of the surface in the first region.", "7.A method according to claim 6, wherein the treatment step comprises localized acid etching of the surface in the first region.", "8.A method according to claim 7, wherein the localized acid etching comprises etching with hydrofluoric acid, the surface in said second region being protected from said etching by a nitride layer, that is eliminated after etching.", "9.A method according to claim 4, wherein the step of preparing at least one of the surfaces comprises a step in which the surface is roughened entirely and a step in which the roughness of some portions is enhanced to obtain greater bonding forces.", "10.A method according to claim 9, wherein the roughness of some portions is reduced by one or more of chemical polishing, mechanical treatment, chemical-mechanical treatment, or dry etching.", "11.A method according to claim 1 wherein the step of producing the interface includes a step of weakening a buried layer in a starting substrate, whereby at least a first region is made weaker than a second region, said buried layer being disposed between the layer and the substrate.", "12.A method according to claim 11, wherein the weakening step includes a step of implanting at least one gaseous element, said implantation step being carried out differently for the first and second regions.", "13.A method according to claim 1, wherein the step of producing the interface comprises a treatment step adapted to render a surface layer of the substrate porous, said treatment step being carried out in such a way as to render the first region more porous than the second region, and being followed by a covering step during which the layer is produced on top of said porous layer.", "14.A method according to claim 13, wherein the substrate comprises silicon and the treatment step comprises electrolysis in an hydrofluoric acid medium.", "15.A method according to claim 1, wherein the step of producing the interface is followed by a step of separating the layer from the substrate.", "16.A method according to claim 15, wherein the method further comprises, after the step of producing the interface, a step of cutting at least one fragment of the layer containing the first region and the second region so that the second region extends along a periphery of the at least one fragment and a separation step during which the substrate and the thin layer are lifted off.", "17.A method according to claim 15, wherein the method further comprises, between the step of producing the interface and the step of separating the layer from the substrate, a step of cutting the second region relative to the first region.", "18.A method according to claim 15, wherein the method further comprises, between the step of producing the interface and the lift-off step, a step of producing in the layer all or part of microelectronic, optical or mechanical components.", "19.A method according to claim 18, wherein each component is produced facing the first region of low mechanical strength surrounded by the second region of higher mechanical strength.", "20.A method according to claim 15, wherein the method further comprises between the step of producing the interface and the separation step, there is a bonding step during which the layer is bonded to a second substrate.", "21.A method according to claim 15, wherein said bonding step includes molecular adhesion bonding.", "22.A method according to claim 15, wherein said bonding step comprises adhesive bonding.", "23.A method according to claim 22, wherein said adhesive bonding comprises an adhesive that is hardened by UV radiation.", "24.A method according to claim 15, wherein the lift-off step is carried out by comprises acid etching and application of mechanical stresses.", "25.A method according to claim 1, wherein the layer comprises silicon.", "26.An assembly comprising a layer on a substrate, said layer being connected to said substrate at an interface of which at least a selected first region has a first level of mechanical strength and a selected second region has a second level of mechanical strength significantly greater than the first level, and wherein the first region is included in the selected second region.", "27.An assembly according to claim 26, wherein the selected second region constitutes a periphery of a wafer of which the selected first region constitutes a core of the wafer.", "28.An assembly according to claim 26, wherein the selected first region is divided into fragments, each fragment being surrounded by a selected second region.", "29.An assembly according to claim 26, wherein a fragment cut out in the layer contains the selected first region and the second region so that the selected second region extends along a periphery of said fragment.", "30.An assembly according to any one of claims 26 to 29, wherein the layer includes all or part of a micro-electronic, optical or mechanical component.", "31.An assembly according to claim 30, wherein said component faces the selected first region of low mechanical strength surrounded by the selected second region of higher mechanical strength.", "32.An assembly according to claim 26, wherein the interface is produced between a surface of the substrate and a surface of the layer that and wherein the surfaces are molecular adhesion bonded.", "33.An assembly according to claim 26, wherein at least one surface of the interface has greater roughness in said selected first region than in the selected second region.", "34.An assembly according to claim 26, wherein the interface is formed by a buried layer in a starting substrate, the selected first region being weakened more than the selected second region.", "35.An assembly according to claim 26, wherein the interface is formed by a porous layer between the layer and said substrate, the layer having different porosities in the selected first and selected second regions.", "36.An assembly according to claim 26, wherein the layer is additionally bonded to a second substrate.", "37.An assembly according to claim 36, wherein the second substrate is bonded by molecular adhesion.", "38.An assembly according to claim 36, wherein the second substrate is adhesive bonded.", "39.An assembly according to claim 38, wherein adhesive bonding comprises an adhesive that is hardened by UV radiation.", "40.An assembly according to claim 26, wherein the layer comprises silicon." ], [ "<SOH> FIELD OF THE INVENTION <EOH>The invention relates to the production of components from a thin layer on a substrate and the production of this thin layer/substrate assembly.", "The substrate can be an initial or intermediate substrate and can be detachable, i.e.", "adapted to be separated from the thin layer." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>Objects, features and advantages of the invention will emerge from the following description, which is given by way of non-limiting illustrative example and with reference to the accompanying drawings, in which: FIG.", "1 is a diagrammatic partial plan view of a surface on which two regions are prepared so that they have different molecular bonding characteristics, FIG.", "2 is a view in section taken along the line II-II in FIG.", "1 , FIG.", "3 is a view of a combination comprising a thin layer on a substrate with an interface of the FIG.", "1 type, FIG.", "4 is a diagrammatic view in section of the whole of a thin layer on a substrate, FIG.", "5 is a view in section of a substrate wafer provided with a protective layer, FIG.", "6 is a view of the FIG.", "5 wafer after recessing it, FIG.", "7 is a view of the same wafer after filling the recess with a deposit of oxide, FIG.", "8 is a view of said wafer covered with the oxide deposit after polishing, FIG.", "9 is a view of the wafer after elimination of surplus oxide coating, FIG.", "10 is a view of the wafer after fixing a thin layer or after fixing a thick layer and then thinning it, FIG.", "11 is a variant of FIG.", "10 , in which the oxide coating penetrates into the substrate and into the thin layer, FIG.", "12 is a variant of FIG.", "10 , in which the oxide coating is on the thin layer, FIG.", "13 is a variant of FIG.", "11 , in which the oxide occupies different regions in the substrate and in the thin layer, FIG.", "14 shows a variant in which the interface is formed of a layer formed by regions of different materials (in this example SiO 2 and Si 3 N 4 ), FIG.", "15 is a variant of FIG.", "10 , showing a plurality of regions formed by the oxide, FIG.", "16 is a variant of FIG.", "14 , showing an intermediate layer formed of a plurality of regions of different materials, FIG.", "17 shows concentric and alternating rings Z 1 and Z 2 , FIG.", "18 shows an array of regions Z 1 in an overall region Z 2 , FIG.", "19 is a diagrammatic view of the assembly from FIG.", "4 after depositing a surface layer, FIG.", "20 is another view after molecular bonding of a final substrate, FIG.", "21 is another view after application of a lift-off operation, FIG.", "22 is a diagrammatic view of a wafer obtained after lifting off and polishing, FIG.", "23 is a view of a separable assembly of the FIG.", "4 type, FIG.", "24 is a view thereof after production of some or all of the components, for example a first transistor gate, FIG.", "25 is a view thereof after deposition of oxide, FIG.", "26 is a view thereof after planarization by CMP, FIG.", "27 is a view thereof after molecular adhesion bonding (including heat treatment), FIG.", "28 is a view thereof after separation and deoxidization, FIG.", "29 is a view of a separable assembly of the FIG.", "4 type, FIG.", "30 is a view thereof after production of components, FIG.", "31 is a view thereof after separation with no transfer to a target substrate, by hydrofluoric etching and/or application of mechanical forces, FIG.", "32 is a view thereof after separation into a final substrate and a substrate that can be recycled, FIG.", "33 is a variant of FIG.", "30 , after cutting of trenches or notches between components, FIG.", "34 is a view thereof showing a component in the process of being lifted off, for example after hydrofluoric etching, FIG.", "35 is a view analogous to FIG.", "4 , FIG.", "36 is a diagrammatic view in section of the FIG.", "35 assembly after adhesive bonding of a transparent substrate, FIG.", "37 is a view of the upper portion of this assembly after lifting off and polishing, FIG.", "38 is a view of the upper portion of this assembly after lifting off and polishing, FIG.", "39 is a view of an assembly analogous to that of FIG.", "4 showing regions eliminated by chemical and mechanical cutting, FIG.", "40 is a view thereof after bonding an upper substrate, FIG.", "41 is a view to a larger scale of an assembly including interleaved regions Z 1 and Z 2 , FIG.", "42 is a view of an assembly analogous to that of FIG.", "4 , FIG.", "43 is a view thereof after deposition of an epitaxial stack based on GaN, FIG.", "44 is a view thereof after bonding a substrate, FIG.", "45 is a view thereof at the time of lifting off, FIG.", "46 is a view of the upper portion thereof after polishing, FIG.", "47 is a view thereof after removal of the layer under the stack, FIG.", "48 is a view of a substrate including a buried weak layer, and FIG.", "49 is a view of a substrate including a buried weak layer portion.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The invention relates to the production of components from a thin layer on a substrate and the production of this thin layer/substrate assembly.", "The substrate can be an initial or intermediate substrate and can be detachable, i.e.", "adapted to be separated from the thin layer.", "DESCRIPTION OF THE PRIOR ART Increasingly, components must be integrated onto supports different from those used to produce them.", "Components on plastics material substrates or on flexible substrates may be cited, for example.", "By component is meant any microelectronic, opto-electronic or sensor (for example chemical, mechanical, thermal, biological or biochemical sensor) device that is completely or partly “processed”, i.e.", "completely or partly produced.", "A layer transfer method can be used to integrate the components onto flexible supports.", "There are many other examples of applications in which layer transfer techniques can provide a suitable solution for integrating components or layers onto a support that is a priori unsuited to their production.", "In the same line of thinking, layer transfer techniques are also very useful when it is required to isolate a thin layer, with or without components, from its original substrate, for example by separating or eliminating the latter.", "Still in the same line of thinking, turning over a thin layer and transferring it onto another support provides engineers with valuable freedom to design structures that would otherwise be impossible.", "Sampling and turning over thin films can be used to produce buried structures, for example, such as buried capacitors for dynamic random access memory (DRAM) where, in contradistinction to the usual situation, the capacitors are formed first and then transferred onto another silicon substrate before fabricating the remainder of the circuits on the new substrate.", "Another example relates to the production of transistor structures referred to as double gate structures.", "The first gate of the CMOS transistor is produced on a substrate using a conventional technology and then turned over and transferred to a second substrate to produce the second gate and finish the transistor, thereby leaving the first gate buried in the structure (see, for example, K. Suzuki, T. Tanaka, Y. Tosaka, H. Horie and T. Sugii, “High-Speed and Low-Power n+-p+Double-Gate SOI CMOS”, IEICE Trans.", "Electron., vol.", "E78-C, 1995, pp.", "360-367).", "The requirement to isolate a thin layer from its original substrate is encountered in the field of light-emitting diodes (LED), for example, for instance as reported in the documents W. S Wong et al., Journal of Electronic MATERIALS, page 1409, Vol.", "28, No.", "12, 1999 and I. Pollentier et al., page 1056, SPIE Vol.", "1361 Physical Concepts of Materials for Novel Opto-electronic Device Applications I (1990).", "One of the objects here is to improve the control of extraction of the emitted light.", "Another object relates to the fact that in this particular example the sapphire substrate used to produce the epitaxial stack is a posteriori bulky, in particular because it is electrically insulative, which prevents making electrical contacts to its rear face.", "To be able to remove afterwards the sapphire substrate, which was advantageous for the phase of growing the material, would thus appear to be desirable.", "An identical situation is encountered in the field of applications to telecommunications and microwaves, for example.", "In this situation, it is preferable for the components to be finally integrated onto a support having a high resistivity, typically at least several kohms.cm.", "However, a highly resistive substrate is not necessarily available at the same cost and with the same quality as the standard substrates usually employed.", "In the case of silicon, 200 and 300 mm silicon wafers of standard resistivity are available, whereas for resistivities greater than 1 kohm.cm, there is very little on offer in the 200 mm size and nothing at all in the 300 mm size.", "One solution consists in producing the components on standard substrates and then, during the final process steps, transferring a thin layer containing the components onto a glass, quartz, sapphire, etc.", "insulative substrate.", "From a technical point of view, the major benefit of these transfer operations is to decorrelate the properties of the layer in which the components are formed and those of the final support layer, and they are consequently beneficial in many other situations.", "There may also be cited situations in which the substrate that is beneficial for the production of the components is excessively costly.", "In this case, for example that of silicon carbide, offering improved performance (higher temperatures of use, significantly improved maximum powers and frequencies of use, etc.", "), but whose cost compared to silicon is very high, it would be beneficial to transfer a thin layer of the costly substrate (in this instance silicon carbide) onto the inexpensive substrate (here silicon), and to recover the remainder of the costly substrate for re-use, possibly after a recycling operation.", "The transfer operation can be carried out before, during or after the production of the components.", "The above techniques can also prove beneficial in all fields in which obtaining a thin substrate is important for the final application.", "Power applications in particular may be cited, whether for reasons associated with the evacuation of heat, which is improved if the substrate is thin, or because in some cases the current must flow through the thickness of the substrates with losses that are to a first approximation proportional to the thickness through which the current passes.", "Smart card applications for which a thin substrate is required for reasons of flexibility may also be cited.", "For these applications, the circuits are produced on thick or standard thickness substrates, which has the advantage, firstly, of good mechanical resistance to the various process steps, and, secondly, of conforming to standards with regard to their use on certain production equipment.", "The final thinning is achieved by separation.", "This separation can be accompanied by transfer to another support.", "In some cases the transfer to another support is not indispensable, especially if the final thickness aimed at during thinning is sufficient to produce self-supporting structures.", "Various techniques can be used to transfer layers from one support to another.", "The techniques disclosed in 1985 by T. Hamaguchi et al., Proc.", "IEDM, 1985, p. 688, may be cited, for example.", "These techniques are of great benefit since they enable a layer to be transferred from one substrate to another, but they necessarily consume the basic substrate (which is destroyed during the process) and cannot achieve homogeneous transfer of a thin film unless a stop layer is present (i.e.", "a non-homogeneous layer in the substrate material).", "Of the transfer methods known to the person skilled in the art, it is also possible to use one of transferring thin layers of materials that may contain all or part of a microelectronic component.", "Some of these methods are based on creating a buried weak layer within a material, by introducing one or more gaseous substances.", "On this subject see the documents U.S. Pat.", "No.", "5,374,564 (or EP-A-533551), US-A-6020252 (or EP-A-807970), FR-A-2767416 (or EP-A-1010198), FR-A-2748850 (or EP-A-902843), and FR-A-2773261 (or EP-A-963598), which disclose such methods.", "These methods are generally used with the objective of detaching the whole of a film from an original substrate to transfer it onto a support.", "The thin film obtained may then contain a portion of the original substrate.", "These films can serve as active layers for the production of electronic or optical components.", "They may contain some or all of a component.", "These methods in particular enable reuse of the substrate after separation, very little of the substrate being consumed on each cycle.", "This is because the thickness removed is frequently no more than a few micrometers, whereas substrate thicknesses are typically several hundred micrometers.", "It is therefore possible to obtain substrates that are similar to substrates that are “demountable” (that is to say detachable) by mechanical stress, in particular in the case of the method disclosed in the document US-A-6020252 (or EP-A-807970).", "This particular process is based on implantation to form a fragile buried region which is cut at the time of final transfer.", "Other methods, based on the “lift-off” principle, also separate a thin layer from the remainder of its original support, again without the latter necessarily being consumed.", "These methods generally use selective chemical etching of a buried intermediate layer, possibly associated with mechanical forces.", "This type of method is very widely used to transfer III-V elements to different types of support (see C. Camperi et al., IEEE Transactions on photonics technology, vol.", "3, 12 (1991), 1123).", "As explained in the paper by P. Demeester et al., Semicond.", "Sci.", "Technol.", "8 (1993), 1124-1135, the transfer, which generally takes place after an epitaxial growth step, can be carried out before or after the production of the components (by “post-processing” or preprocessing, respectively).", "Of the methods using a (pre-existing) buried layer of lower mechanical strength than the remainder of the substrate to obtain localized separation, the ELTRAN® method may be cited (Japanese Patent Publication Number 07302889).", "In this case, a stack based on monocrystalline silicon is locally weakened by the formation of a porous region.", "Another similar situation consists in exploiting the presence of a buried oxide in the case of a silicon on insulator (SOI) structure, however standard the latter may otherwise be (i.e.", "produced without seeking any particular detachability effect).", "If the structure is bonded sufficiently strongly to another substrate and a high stress is applied to the structure, localized fracture, preferentially achieved in the oxide, can lead to a cutting effect on the scale of the entire substrate.", "The document “PHILIPS Journal of Research”, vol.", "49, N° 1/2, 1995, shows an example of this on pages 53 to 55.Unfortunately, the fracture is difficult to control and necessitates high mechanical stresses to bring it about, which is not free of the risk of breaking of the substrate or damaging the components.", "The advantage of such buried fragile layer methods is that they can be used to produce layers based on crystalline silicon (or SiC, InP, AsGa, LiNbO3, LiTaO3, etc.)", "in a range of thickness from a few tens of angstrom units (Å) to a few micrometers (μm), with very good homogeneity.", "Greater thicknesses can also be achieved.", "To fabricate detachable structures for possible subsequent transfer of a layer onto another support or substrate, it is known to the person skilled in the art to control the energies of the bonds between the layer and the substrate, as indicated in the document EP 0702609 A1.The inventors of this patent are also aware that, to produce a detachable substrate, it is also possible to use methods involving the control of bonding forces existing at the “detachment” surface to assemble together temporarily the thin layer and the substrate from which it is subsequently to be detached.", "The situation in which the bonding is obtained by molecular adhesion is of particular benefit.", "Of the categories of assemblies obtained by molecular bonding, silicon on insulator (SOI) substrates produced by these bonding techniques constitute a particularly beneficial category.", "The category encompasses a number of variants, the principles of which are described in the book “Semiconductor Wafer bonding Science and Technology”, Q.-Y.", "Tong and U. Gösele, a Wiley Interscience publication, John Wiley & Sons, Inc.", "Some variants are known as bonded SOI (BSOI) or bond and etch back SOI (BESOI).", "Apart from bonding involving molecular adhesion, these variants rely on physical removal of the original substrate, by polishing techniques and/or chemical etching techniques.", "Other variants, described in part heretofore as layer transfer techniques, are additionally based on bonding by molecular adhesion and separation by “cutting” along a region that has been weakened, for example as in the methods described in the documents U.S. Pat.", "No.", "5,374,564 (or EP-A-533551) and U.S. Pat.", "No.", "6,020,252 (or EP-A-807970) (separation along an implanted region) or in the document EP 0925888 (separation by fracture along a buried layer that has been rendered porous).", "Whatever the exact technique used, the common feature of these variants is their use of molecular bonding, in most of the cases encountered in the literature between two substrates having silicon (Si) or silicon oxide (SiO2) at the surfaces to be brought into contact.", "Other materials are sometimes encountered (nitrides, silicides, etc.).", "If non-detachable SOI structures are to be obtained, the surface preparation operations are intended finally to provide, and often with the aid of annealing carried out after bonding, high bonding energies typically from 1 to 2 J/m2.Conventionally, with standard preparation operations, the bonding energy of the structure is of the order of 100 mJ/m2 at room temperature and 500 mJ/m2 after annealing at 400° C. for 30 minutes, in the case of SiO2/SiO2 bonding (bonding energy determined by the blade method developed by Maszara (see: Maszara et al., J. Appl.", "Phys., 64 (10), p. 4943, 1988)).", "When the structure is annealed at a high temperature (1 100° C.), the bonding energy can be as high as 2 J/m2 (C. Maleville et al., Semiconductor wafer bonding, Science Technology and Application IV, PV 97-36, 46, The Electrochemical Society Proceedings Series, Pennington, N.J. (1998)).", "Other forms of preparation prior to bonding exist, for example exposure of the surfaces to be bonded to a plasma (for example an oxygen plasma), and can yield equivalent bonding energies without always necessitating such annealing (YA, Li and R. W. Bower, Jpn.", "J: Appl.", "Phys., vol.", "37, p. 737, 1998).", "In contradistinction to the above, the inventors are interested in detachable SOI structures.", "It has been shown that different mechanical strengths can be obtained by modifying the hydrophilic properties and the roughness of the surfaces.", "For example, as indicated in a paper by O. Rayssac et al.", "(Proceedings of the 2nd International Conference on Materials for Microelectronics, IOM Communications, p. 183, 1998), hydrofluoric etching increases the roughness of a layer of silicon oxide.", "The paper describes how an 8 000 Å etch increases the RMS roughness from 0.1 nm to 0.625 nm.", "It has been verified that SiO2/SiO2 bonding with RMS roughnesses of 0.625 nm and 0.6.25 nm for the facing surfaces yields a bonding energy value of the order of 500 mJ/m2 after annealing at 1 100° C., i.e.", "much lower than in the standard situation previously cited.", "The inventors have shown that in this case the roughening can be exploited to provide detachable bonding interfaces, even after annealing at high temperatures, as high as 1 100° C. By astutely combining roughness preparation before bonding with suitable thermal annealing treatment, it has been demonstrated that detachable SOI substrates can withstand, without untimely separation at the assembly interface, most steps of a process for producing CMOS transistors (including in particular heat treatment steps at high temperatures, typically 1 100° C., as well as steps of depositing constrained layers, for example layers of nitride), and could a posteriori be detached at the bonding interface by the intentional application of controlled mechanical stress.", "Technical Problem and Solution in Accordance with the Invention Delamination is a well-known problem in the context of multilayer structures, in particular in the field of fabrication techniques for microelectronic components, sensors, etc.", "This is because heat treatment, chemical treatment (hydrofluoric etching, etc.", "), mechanical and/or physical operations of removing materials (polishing, etc.)", "necessary for producing components, deposition and/or epitaxial growth steps, and the mechanical stresses generated during the production of non-homogeneous stacks, often generate cleavages at the layer edge or start lift-off at the edges of the structures.", "In the case of SOI, for example, the many hydrofluoric treatments used to deoxidize the Si surface can in some cases lead to important overetching of the buried oxide and thereby weaken the surface film at the edge of the wafer.", "Layer transfer techniques (with or without components) based on the production of detachable substrates by forming an intermediate layer or fragile interface (whether by weakening by implantation of substances, by formation of a porous region, by control of the bonding energy, etc.)", "come up against certain problems associated with untimely delamination if the processing prior to intentional separation is too aggressive.", "Cracking can start unintentionally at the edges, during the process of fabrication of the whole or part of the component, and can reduce the yield.", "Apart from the reduction of the surface area of the active film caused by peeling of the film at the edge, these problems can lead to a high increase in particulate contamination of the wafers, and therefore a high reduction of yield in the fabrication of components and contamination of the plant used (in particular furnaces).", "An object of the invention is to alleviate the drawbacks previously cited thanks to a layer/substrate interface that reliably combines the imperative of easy separation at the right time and the imperative of, where necessary, withstanding the application of heat or mechanical treatment necessary for the production of all or some of the microelectronic, optical, or acoustic components or sensors, or epitaxial growth steps, without causing premature separation or delamination.", "More generally, the invention consists in a combination comprising a thin layer on a substrate, said layer being connected to said substrate by an interface or an intermediate layer having a controlled level of mechanical strength.", "To this end it proposes, firstly, a method of preparing a thin layer including a step of producing an interface or an intermediate layer between said thin layer and a substrate, characterized in that said interface is produced so as to have a first region having a first level of mechanical strength and at least one second region having a second level of mechanical strength significantly greater than the first level.", "In other words, the invention proposes a method of obtaining a structure having a buried structure (interface or layer) whose mechanical strength is higher in one region than in another.", "Thus the interface (or the intermediate layer) can be optimized as a function of requirements, allowing for processing that it is intended to apply to it.", "Throughout this document, the expression “mechanical strength” means the mechanical strength in the “strength of materials” sense, but can also refer more generally to the susceptibility to rupture or dissociation of a continuous or discontinuous medium (such as an interface or a stack, which could suffer delamination, for example, etc.", "), whether this is in response to pure mechanical stress (traction, bending, shear, compression, torsion, etc.", "), during heat treatment, or during chemical attack, as well as all feasible combinations.", "In the case of the particular problem discussed hereinabove, if it is required to detach the substrate from the layer at the correct time, and not before, the first region having a lower mechanical strength is a region included within the second region.", "Especially when the whole of the thin layer is to be transferred, at the wafer scale, the region with the highest mechanical strength is preferably a ring (an annular ring in the case of round substrates), whose width can vary from a few hundred micrometers to a few millimeters, or even be of the order of one centimeter.", "Thus the ring can constitute the periphery of a round, square, polygonal or other shape plate whose core is of lower mechanical strength.", "In a situation corresponding instead to separation at the level of portions of the thin layer (by die, by component, set or subset of dies, etc.", "), the preparation of said layer preferably includes a step wherein at least one fragment of said layer is isolated on the substrate and said second region extends along the contour of said fragment.", "Thus the first region can be fragmented, each fragment being surrounded by a region of greater mechanical strength.", "Accordingly, the interface or intermediate layer produced between the thin layer and the substrate is mechanically weaker in its central portion that at its periphery.", "This greatly reduces the risk of untimely delamination.", "Said interface or buried layer can take various forms.", "It can in particular be defined as: a bonding interface (with or without adhesive, for example bonding by molecular adhesion), and with or without an intermediate layer (oxide, nitride), a layer of microcavities (and/or gas microbubbles and/or platelets), and more generally a layer of defects, an intermediate layer having characteristics different from those of the substrate and the layer, for example a porous silicon layer, which can be differentiated in terms of its mechanical strength or its susceptibility to chemical etching (chemical and mechanical), etc., an intermediate layer of different chemical composition intended to be selectively chemically etched.", "The differentiation between connecting regions of higher or lower mechanical strength or other different kinds of connections can be effected: in the case of a bonded interface, by connecting energies obtained, for example, by different preparation before bonding (roughness, different hydrophilic properties, chemical surface connecting states, etc.)", "and/or by heat treatment differences, in particular after bringing into contact for bonding, in the case of a layer of microcavities, by reduced implantation dosing in the second region or by preferential growth of microcracks in the first region, in the case of a porous layer, by modifying the percentage porosity so that it is higher in the first region, in the case of an intermediate layer of different chemical composition, intended to be selectively chemically etched, by varying the chemical composition, which can be a doping difference or a composition percentage difference for a semiconductor substance, for example, which would have a direct impact on any variation in sensitivity to chemical etching.", "To be more precise, in the situation where there are only two regions, in accordance with preferred features of the invention, possibly in combination (in which case the second region surrounds the first region): After the step of producing the interface there are effected a step of isolating a fragment of the layer containing said first region and said second region so that said second region runs along the periphery of said fragment, followed by a separation operation in which the substrate and the thin layer are lifted off; in this case, the lift-off step is sometimes advantageously preceded by a step of physical delimitation of the second region vis àvis the first region, for example after partial or total cutting, by total or partial chemical etching, by total or partial mechanical fracture, etc.", "The interface is produced between a surface of the substrate and a surface of the layer, and the step of producing the interface includes a step of preparing at least one of these surfaces and a bonding step during which that surface is bonded to the other surface by molecular adhesion bonding; the step of producing the interface preferably and advantageously includes a preparation step for each of the substrate and layer surfaces.", "The surface preparation step preferably includes a treatment step during which said surface in said first region is made less hydrophilic or its roughness is locally increased, for example; this refers, for example, to localized acid etching of the surface in this first region; to be more precise, it is even more preferable if at least one surface includes an oxide layer and the acid etching is effected with hydrofluoric acid, the surface in this second region being protected from said etching by a protection (for example nitride) layer which is eliminated afterwards.", "In another variant, the surface of at least one of the two wafers is entirely roughened and in some portions the roughness is then modified, essentially improved, to obtain greater bonding forces, for example with the aid of chemical polishing treatment, mechanical or chemical-mechanical or ionic treatment, or by dry etching.", "To prepare the surface and control the roughness, especially if one of the surfaces is an oxide, partial acid etching can be effected using hydrofluoric acid.", "The step of producing the interface includes a step of weakening a buried layer in a starting substrate, whereby at least the first region is made weaker than the second region, said buried layer being disposed between a portion forming the layer and a portion forming the substrate.", "The weakening step preferably includes a step of implanting at least one element, preferably a gas, this implantation step being conducted in a different way for the first and second regions.", "The step of producing the interface or intermediate layer includes a treatment step adapted to render a surface layer of the substrate porous, this treatment step being conducted in a different manner for the first and second regions, followed by a coverage step in which the layer is produced on top of said porous layer.", "In the particular case where the substrate is of silicon, this treatment step advantageously includes electrolysis in a hydrofluoric acid medium.", "The mechanical and/or chemical strength of the interface or connecting layer is modified by non-uniform heat treatment to selectively strengthen or selectively weaken some regions compared to others, according to whether it is a matter of bonding roughened surfaces, porous materials, buried defects, gaseous or other microcavities.", "The step of producing the interface is followed by a step of separating the layer from the substrate.", "Between the step of producing the interface and the lifting off step, there is advantageously a bonding step during which the layer is bonded to a second substrate.", "Said bonding step advantageously consists of molecular adhesion bonding or adhesive bonding, in the latter case using an adhesive hardened by UV, a resin, or a polymer adhesive, for example.", "In these cases, the separation step is advantageously effected by acid etching and/or application of mechanical stress.", "The layer is of semiconductor materials (Si, Ge, SiGe, SiC, GaN and other equivalent nitrides, AsGa, InP, Ge, etc.)", "or ferro-electric materials or piezo-electric materials (LiNbO3, LiTaO3), or processed or unprocessed magnetic materials.", "The thin layer on the separable substrate was obtained by thinning an original semiconductor material substrate.", "The thinning is obtained by machining and/or chemical-mechanical or other polishing and/or chemical etching.", "The thin layer on the separable substrate was obtained by cutting into the original semiconductor material substrate.", "The cutting is achieved by cutting at the level of a buried weak layer.", "The buried weak layer is obtained by implantation and separation is obtained by heat and/or mechanical and/or chemical treatment.", "The substance implanted is a gas (hydrogen, helium, etc.).", "In terms of product, the invention proposes an assembly comprising a layer on a substrate, said layer being connected to said substrate by an interface of which at least a first chosen region has a first level of mechanical strength and a second chosen region has a level of mechanical strength significantly higher than the first level, and the second region surrounding the first region.", "According to preferred features, which may be combined: A fragment is totally or partially delimited (by cutting, etching, etc.)", "in said layer containing said first region and said second region so that said second region runs along the periphery of said fragment.", "The interface is produced between a surface of the substrate and a surface of the layer that are bonded by molecular adhesion.", "At least one surface of the interface has a lower roughness in said second region than in the first region.", "The interface is formed by a buried layer in an original substrate, the first region being weakened more than the second region.", "The interface is formed by a porous layer between said layer and said substrate, said layer having differences of porosity between said first and second regions.", "Said layer is further bonded to a second substrate, advantageously by molecular adhesion bonding or adhesive bonding, for example by an adhesive hardened by UV radiation.", "The mechanical and/or chemical strength of the interface or connecting layer is selectively modified with the aid of treatments (heat treatments, UV exposure treatments, laser irradiation treatments, etc.)", "that are localized or non-uniform to selectively strengthen or selectively weaken some regions more than others, according to whether it is a question of bonding roughened surfaces, porous materials, buried defects, or gaseous or non-gaseous microcavities.", "The layer is of semiconductor materials (Si, Ge, SiGe, SiC, GaN and other equivalent nitrides, AsGa, InP, etc.)", "or ferro-electric or piezo-electric materials (LiNbO3, LiTaO3), or processed or unprocessed magnetic materials.", "BRIEF DESCRIPTION OF THE DRAWINGS Objects, features and advantages of the invention will emerge from the following description, which is given by way of non-limiting illustrative example and with reference to the accompanying drawings, in which: FIG.", "1 is a diagrammatic partial plan view of a surface on which two regions are prepared so that they have different molecular bonding characteristics, FIG.", "2 is a view in section taken along the line II-II in FIG.", "1, FIG.", "3 is a view of a combination comprising a thin layer on a substrate with an interface of the FIG.", "1 type, FIG.", "4 is a diagrammatic view in section of the whole of a thin layer on a substrate, FIG.", "5 is a view in section of a substrate wafer provided with a protective layer, FIG.", "6 is a view of the FIG.", "5 wafer after recessing it, FIG.", "7 is a view of the same wafer after filling the recess with a deposit of oxide, FIG.", "8 is a view of said wafer covered with the oxide deposit after polishing, FIG.", "9 is a view of the wafer after elimination of surplus oxide coating, FIG.", "10 is a view of the wafer after fixing a thin layer or after fixing a thick layer and then thinning it, FIG.", "11 is a variant of FIG.", "10, in which the oxide coating penetrates into the substrate and into the thin layer, FIG.", "12 is a variant of FIG.", "10, in which the oxide coating is on the thin layer, FIG.", "13 is a variant of FIG.", "11, in which the oxide occupies different regions in the substrate and in the thin layer, FIG.", "14 shows a variant in which the interface is formed of a layer formed by regions of different materials (in this example SiO2 and Si3N4), FIG.", "15 is a variant of FIG.", "10, showing a plurality of regions formed by the oxide, FIG.", "16 is a variant of FIG.", "14, showing an intermediate layer formed of a plurality of regions of different materials, FIG.", "17 shows concentric and alternating rings Z1 and Z2, FIG.", "18 shows an array of regions Z1 in an overall region Z2, FIG.", "19 is a diagrammatic view of the assembly from FIG.", "4 after depositing a surface layer, FIG.", "20 is another view after molecular bonding of a final substrate, FIG.", "21 is another view after application of a lift-off operation, FIG.", "22 is a diagrammatic view of a wafer obtained after lifting off and polishing, FIG.", "23 is a view of a separable assembly of the FIG.", "4 type, FIG.", "24 is a view thereof after production of some or all of the components, for example a first transistor gate, FIG.", "25 is a view thereof after deposition of oxide, FIG.", "26 is a view thereof after planarization by CMP, FIG.", "27 is a view thereof after molecular adhesion bonding (including heat treatment), FIG.", "28 is a view thereof after separation and deoxidization, FIG.", "29 is a view of a separable assembly of the FIG.", "4 type, FIG.", "30 is a view thereof after production of components, FIG.", "31 is a view thereof after separation with no transfer to a target substrate, by hydrofluoric etching and/or application of mechanical forces, FIG.", "32 is a view thereof after separation into a final substrate and a substrate that can be recycled, FIG.", "33 is a variant of FIG.", "30, after cutting of trenches or notches between components, FIG.", "34 is a view thereof showing a component in the process of being lifted off, for example after hydrofluoric etching, FIG.", "35 is a view analogous to FIG.", "4, FIG.", "36 is a diagrammatic view in section of the FIG.", "35 assembly after adhesive bonding of a transparent substrate, FIG.", "37 is a view of the upper portion of this assembly after lifting off and polishing, FIG.", "38 is a view of the upper portion of this assembly after lifting off and polishing, FIG.", "39 is a view of an assembly analogous to that of FIG.", "4 showing regions eliminated by chemical and mechanical cutting, FIG.", "40 is a view thereof after bonding an upper substrate, FIG.", "41 is a view to a larger scale of an assembly including interleaved regions Z1 and Z2, FIG.", "42 is a view of an assembly analogous to that of FIG.", "4, FIG.", "43 is a view thereof after deposition of an epitaxial stack based on GaN, FIG.", "44 is a view thereof after bonding a substrate, FIG.", "45 is a view thereof at the time of lifting off, FIG.", "46 is a view of the upper portion thereof after polishing, FIG.", "47 is a view thereof after removal of the layer under the stack, FIG.", "48 is a view of a substrate including a buried weak layer, and FIG.", "49 is a view of a substrate including a buried weak layer portion.", "DESCRIPTION OF THE INVENTION 1—Molecular Adhesion Bonding Interface The preferred examples selected for the detailed description primarily relate to silicon, which is generally available in the form of round substrates, for example of 200 mm diameter.", "These methods transfer readily to other systems characterized in particular by materials other than silicon, in a nonlimiting manner and without departing from the scope of the invention.", "Some embodiments of the method according to the invention tend to encourage lifting of the layer off its substrate at the overall level, i.e.", "on the scale of the whole of the substrate, while others tend to delimit fragments.", "An assembly to be produced in the former case is shown diagrammatically in FIGS.", "1 and 2, the interface or intermediate layer shown diagrammatically in FIG.", "2 representing the region in which local bonding differences are to be created.", "In addition to these figures, FIG.", "3 in particular shows one example of the preparation of a surface intended to participate in an interface having, in accordance with the invention, two regions having different mechanical strengths.", "To be more precise, in the example shown, the object is to obtain a central region Z1 whose mechanical strength Ec1 is less than that Ec2 of a peripheral region Z2 around the central region.", "Different methods can be used to obtain a higher bonding energy in the peripheral region Z2 than in the central region Z1.Examples of SiO2/SiO2 and Si/SiO2 bonding are considered.", "In the case of layers of different kinds (Si3N4 is another conventional example, but there are also silicides), and by analogy with what is described hereinafter, it suffices to use appropriate chemical treatment (for example NH4OH/H2O2/H2O (also known as SC1) for Si and H3PO4 or HF for Si3N4).", "FIG.", "4 shows the option in which the substrate 11 and the thin layer 14 are of monocrystalline silicon and two intermediate layers 12 and 13 are formed prior to bonding on the substrate 11 and the thin layer 14, respectively.", "Of course, only one of the two intermediate layers 12 or 13 may suffice, and the situation in which neither of them exists (i.e.", "that of Si/Si bonding) must be considered (i.e.", "there are two special cases).", "If the intermediate layers 12 and 13 exist and are both of SiO2, the system is referred to as SiO2/SiO2 bonding.", "If only one of the two exists, and consists of SiO2, then the system is referred to as Si/SiO2 bonding.", "A number of techniques can be employed to produce a structure like that shown in FIG.", "4, in addition to technical aspects related to molecular adhesion bonding, including those previously cited, for the production of non-separable SOI substrates (see Semiconductor Wafer Bonding, Science and Technology, Q. Y. Tong and U. Gösele, Wiley Interscience Publications).", "Hereinafter the layer 14 is referred to as the active layer, being the layer comprising the components, except for certain special cases in which an additional, for example epitaxial, layer is deposited onto the layer 14.In some variants of the invention, the thin layer is obtained by thinning the structure chemically and/or mechanically.", "These variants are known as bonded SOI (BSOI) and bond and etch back SOI (BESOI).", "In addition to molecular adhesion bonding, these variants are based on physical removal of the original substrate by polishing techniques and/or chemical etching techniques.", "Other variants, partially described hereinbefore as layer transfer techniques, are based, in addition to molecular adhesion bonding, on separation by “cutting” or cleaving along a weakened region, such as the methods described in the documents U.S. Pat.", "No.", "5,374,564 (or EP-A-533551) and U.S. Pat.", "No.", "6,020,252 (or EP-A-807970): separation along an implanted region, or in the document EP 0925888: separation by fracture along a buried layer that has been rendered porous.", "With reference to specific aspects related to bonding for the production of separable substrates, in the case of SiO2/SiO2 (or even Si/SiO2) bonding, a temporary mask can first be used to deposit a protection layer, for example a layer of silicon nitride Si3N4, onto only the ring of the silicon oxide SiO2 layer(s) 12 and/or 13.The oxide layer can be prepared in several ways (deposition, thermal oxidation of the silicon) and can have a thickness that varies according to the application.", "For this example, a 1 μm thick thermal oxide is chosen.", "The following structure is therefore obtained: the surface of the central disc is formed of oxide only and the surface of the external ring (which is typically a few mm wide) is formed of the oxide covered with an additional protection layer (for example of nitride).", "This is followed by hydrofluoric acid etching to roughen the surface of the oxide, the roughening required increasing with the thickness of oxide removed.", "For each application, the roughness can be optimized, in particular as a function of the method of producing the components (or of epitaxial growth), which must be formed without delamination, and the method adopted for the final separation.", "Typically, hydrofluoric acid etching to remove a thickness of oxide of the order of a few hundred to a few thousand Å is a good starting compromise.", "A nitride thickness of the order of 1 000 Å protects the underlying oxide from hydrofluoric acid etching, which increases the roughness of the central region of the oxide layer(s) 12 and/or 13.The nitride is subsequently removed by etching with hot (>110° C.) orthophosphoric acid (H3PO4), for example.", "This can be combined with selective wet or dry cleaning to obtain different hydrophilic properties between the regions Z1 and Z2.The resulting effect is one of weaker bonding than the standard bonding at the center and bonding identical to the standard bonding at the location of the ring.", "If the oxide removed is thick, in particular because the application requires this (especially in the range of a few thousand A, knowing that 5 000 Å is a good starting compromise in many cases), it must be noted that in addition to the required effect of locally increased roughness, a difference in level is created between the central region (which has been hydrofluoric acid etched) and the ring (which has been protected from this etching).", "To obtain bonding of very high quality in the ring, in combination with bonding of satisfactory quality in the central region, it may be necessary to eliminate or reduce the difference in level in some cases.", "It can be appropriate to use polishing for this (for example chemical/mechanical polishing).", "Because of the difference in level, polishing that is a priori uniform results, through a planarizing effect that is well known to the person skilled in the art of microelectronics, in preferential polishing of the emergent region, i.e.", "the region of the ring in the present instance.", "However, the polishing can also be intentionally localized to the ring.", "If the substrate is circular, the ring can be preferentially polished by using a polishing cloth with a central opening, for example, thereby reducing the level of the ring to that of the central region that has been hydrofluoric acid etched.", "Moreover, it is known that polishing leads to higher bonding energies than are obtained by conventional molecular adhesion bonding (with no polishing).", "This therefore combines two effects tending toward weaker bonding than the standard bonding at the center and stronger bonding at the location of the ring.", "This combination is another special case of what is shown in FIG.", "3.Another way to reduce the level of the ring to that of the center is localized wet or dry etching.", "An alternative to this is to roughen all of the intermediate layer(s) 12 and/or 13, i.e.", "without protecting the ring, but to add treatment localized to the ring that significantly increases the bonding strength.", "These treatments include, for example, the use of an oxygen plasma or localized annealing, one aim of which would be, through a flow effect, to reconstruct and reduce the roughness of the surface of the oxide, or any other treatment known to the person skilled in the art to improve the cohesion of the assembly.", "These treatments have the advantage that they do not create any relief.", "Another alternative relates to the use of localized thermal annealing (laser beam, non-uniform furnaces, heating by lamps, etc.)", "after the bonding operation.", "As reported in C. Malevill et al., Semiconductor wafer bonding, Science Technology and Applications IV, PV 97-36, 46, The Electrochemical Society Proceedings Series, Pennington, N.J. (1998), a 100° C. annealing temperature difference after bonding can significantly vary the bonding energy, especially in the range of temperatures beyond 800° C. This alternative can be used in combination with roughening at least one intermediate layer 12 or 13, or alone (i.e.", "with no step of roughening everything).", "One very specific example, that must not in any way be regarded as limiting on the invention, applied to SiO2/SiO2 bonding is to anneal the whole of the structure at 1 000° C. and to heat the ring selectively to 1 200° C. Another is not to anneal the whole of the ring at a temperature of 1 000° C. For this selective annealing alternative, it must be understood that, because of thermal conduction phenomena and the difficulty of producing equipment for localized heating with perfect selectivity, localized heating can result in a heat input gradient.", "In this case, the annealing temperature can be considered to be at a maximum at the edges of the substrate and to fall on approaching the center of the substrate.", "Another variant of the method of producing a separable substrate can be based on a difference in chemical nature between the region Z1 and the region Z2.The following pairs may be cited by way of non-exhaustive example: Z1=SiO2 and Z2=Si; Z1=Si3N4 and Z2=SiO2; Z1=Si3N4 and Z2=Si, etc.", "Only the pair Z1=SiO2 and Z2=Si is described here.", "As shown in FIG.", "5, a protective ring (of resin, deposited PECVD, etc.)", "a few mm wide is deposited onto a silicon substrate to define the dimensions of the region Z2.The structure is then etched (wet or dry, by any of the techniques conventionally employed to etch silicon) so that the etching attacks only the unprotected region.", "Mechanical attack (milling, polishing, etc.)", "can also be envisaged so that only the center is machined, by virtue of the configuration and/or size of the tool employed.", "In the above cases, the operation of depositing a protection layer is no longer indispensable.", "Regardless of the method employed (possibly involving a step of removing the protective ring), a silicon wafer is obtained with a recess at the center, as shown in FIG.", "6.This defines the location of the region Z1 (disc with recess) and the region Z2 (outer ring).", "An oxide is deposited by the CVD process on the resulting substrate with a recess.", "The thickness of the layer of oxide deposited is much greater than the depth of the recess, producing the structure shown in FIG.", "7.Planarizing by polishing eliminates the difference in level between the ring and the center of the structure as well as the high inherent roughness of this type of deposit (see FIG.", "8).", "Hydrofluoric acid etching then produces the structure shown in FIG.", "9.In this case, hydrofluoric acid etching is stopped when the silicon is flush with the ring.", "This produces a “silicon ring and roughened oxide at the center” configuration, which defines an energy difference between the regions Z1 and Z2, firstly because these two regions have different roughnesses and secondly because the nature of the material is different (different molecular adhesion bonding properties).", "Depending on the energy difference required between the regions Z1 and Z2, the above sequence can be modified to yield a “silicon ring and non-roughened oxide at the center” configuration.", "To this end, the hydrofluoric acid etching step can be eliminated, for example, and the previous polishing step extended so that polishing the silicon ring results in a flush surface.", "In this case, the energy difference between the regions Z1 and Z2 is essentially caused by the difference in the nature of the materials.", "In this case, the difference is smaller.", "A recess and an oxide deposit have been cited in the situation where they are produced on the substrate 11 (with the result that the FIG.", "10 configuration is obtained after bonding).", "A variant consists in carrying out these operations on the side of the thin layer 14 (see FIG.", "12), or even on both sides (see FIG.", "11).", "Methods for other pairs of materials that can exhibit a difference in mechanical strength can include a small number of additional steps.", "For example, if the aim is to obtain the pair Z1=Si3N4 and Z2=SiO2, Si3N4 must be deposited in addition to the oxide, taking care to define the required ring for the whole of the structure using conventional masking techniques (photolithography, mechanical methods, etc.).", "This structure has the advantage of excellent chemical etching (for example hydrofluoric acid etching) selectivity between the two materials concerned, which could facilitate separation through easy and selective removal of the ring by chemical etching.", "Other configurations are possible, for example: FIG.", "13 is a variant of FIG.", "11 in which the central layer does not have the same dimensions in the substrate and in the thin layer, FIG.", "14 shows an assembly in which the interface layer between the substrate and the thin layer has a central portion Z1 of a material (Si3N4) different from that (SiO2) of the peripheral portion Z2, these two portions being of different materials (here Si) than the substrate and the thin layer, FIG.", "15 is a variant of FIG.", "10 in which there is a plurality of regions Z1 of a material different from that of the support, FIG.", "16 is a variant of FIG.", "14 in which there is a plurality of regions Z1 and a plurality of regions Z2 of materials (here Si3N4 and SiO2) different from those (here Si) of the substrate and the thin layer.", "FIGS.", "15 and 16 can correspond to a plurality of geometries of which FIGS.", "17 and 18 are examples (at the die level or otherwise): FIG.", "17 shows a configuration with a plurality of concentric bands Z1 or Z2, and FIG.", "18 shows a configuration having an array (made up of rows and columns) of regions of one type (here Z1) in an overall region of the other type (Z2).", "Apart from the production of the separable substrate itself, using techniques based on molecular bonding, there are several means as to its use and to the means to implement it.", "The benefit of the separable substrate, depending on the thickness of the unprocessed active layer or of the processed active layer (i.e.", "its thickness when it has been processed to produce all or part of a component), is that it allows separation of the active layer to obtain either a self-supporting layer (a relatively thick layer, whether the thickness is already present in the separable substrate as manufactured or obtained during deposition steps subsequent to its fabrication, as is the case in an epitaxial growth step) or a surface layer, which is generally thinner, transferred onto a target support, whether the latter is the final support or merely a temporary support which is itself intended to be detached.", "There are various ways to transfer the surface layer to the target substrate.", "First of all, transfer can be effected by molecular adhesion bonding of what is to become the thin layer to be transferred to another substrate.", "By way of illustration, a separation method is described next in the context of producing a new SOI structure that is referred to herein as the second SOI structure.", "This kind of method, a priori less direct than the techniques previously mentioned, nevertheless has a number of benefits.", "The example chosen here relates to the production of a second SOI substrate with a buried oxide layer 500 Å thick, which thickness is difficult to obtain using this kind of method directly.", "The first structure is obtained by one of the methods previously described, yielding a separable substrate corresponding to FIG.", "4.In this example the monocrystalline silicon layer 14 is to become the active layer.", "Onto this separable substrate, in which the central region of the connecting layer has been roughened before bonding and has not undergone any strengthening heat treatment at very high temperatures (preferably less than 1 100° C. and even better less than 1 000° C. or even 900° C.), a 500 Å oxide layer is formed by thermal oxidation to yield the structure shown in FIG.", "20.This oxide is to become the buried oxide of the second SOI structure.", "In the present example, the separable substrate (11+12+13+14+15) is molecular adhesion bonded to a silicon substrate 16 that is to become the final support of the active layer (see FIG.", "2).", "The stack obtained is preferably stabilized at a high temperature (1 100° C.) to consolidate strongly the second bond at the interface of the layers 15 and 16.This second bond is conventional in the sense that there is no differentiation between regions of different mechanical strength.", "The first bond, if subjected to the same treatment, nevertheless has, at least in its central portion, corresponding to the region Z1, a mechanical strength less than that of the second bond.", "A chemical and/or mechanical separation method can be used.", "For example, the stack obtained as above is first immersed in a hydrofluoric acid bath, one object of which is to overetch the oxide layers 12 and 13, starting from the edges of the assembly, to eliminate the ring corresponding to the region Z2 and reach the region Z1.The two interfaces 12/13 and 15/16 are preferentially etched.", "Moreover, the interface 12/13 of the separable substrate is advantageously an oxide/oxide interface.", "It therefore yields more readily than the interface 15/16 between the oxide and the silicon.", "Accordingly, during this step of lifting off the ring of higher mechanical strength, there is less surface etching at the interface of the second bond than at the interface of the separable substrate.", "When the acid reaches the low energy region (the central region, see FIG.", "21), mechanical separation (by pressurized water jet, as in the document EP 0925888, by compressed air jet, as in the document FR 2796491, by traction, as in the document WO 00/26000, by insertion of a blade, etc.)", "completely frees the final structure 13+14+15+16 (see FIG.", "22).", "Following removal of the oxide 13, for example by hydrofluoric etching, the final SOI structure is obtained.", "The Si wafer 11 that served as a substrate within the separable substrate can be recycled and reused, for example to produce another separable substrate (preferably after eliminating the layer 12).", "Other means of eliminating a ring to obtain access to the region Z1 can at least partially eliminate the ring.", "Wet or dry chemical etching techniques or other mechanical polishing, laser cutting, etc.", "techniques can be employed for this, localized to the ring (see FIGS.", "39 and 40, which correspond to the production of a mechanical or chemical cut (cross-hatched area), before bonding the second substrate).", "Note that the 500 Å thick buried oxide layer 15 formed previously on the layer 14 could have been formed on the substrate 16 before bonding rather than on the layer 14.Another variant divides the 500 Å thickness into two portions, with one portion on the substrate 16, for example 250 Å thick, and the other portion on the layer 14, also 250 Å thick in this example.", "Note that if the two molecular adhesion bonded interfaces are both of the oxide/oxide type, stabilization of the second bond at high temperature can be carried out in such a way as to guarantee that preferential hydrofluoric acid etching occurs at the first interface.", "In this method, the creation of a mechanically weak region Z1 achieves preferential separation of the complete stack at the first bond interface, and the region Z2 firstly yields an active layer 14 of good quality and secondly prevents the onset of cracks that can cause losses of yield and a reduction in the surface of the active film caused by peeling of the film at the edge, and thus a strong increase in particulate contamination of the wafers.", "Another example of the use of the method according to the invention relates to the production of double-gate transistor structures.", "The first operations relating to the fabrication of the transistors essentially consist in producing the first CMOS transistor gate (see FIG.", "24) on a separable substrate using conventional technology (see FIG.", "23), as shown in FIG.", "4, for example, in exactly the same way as previously described for the production of SOI structures with a thin buried oxide layer.", "The bonding stabilization temperature can be reduced in a temperature range of the order of 900/1 000° C. An oxide layer with a thickness of the order of 1 μm is then deposited onto this substrate (see FIG.", "25) using a conventional deposition technique (for example CVD).", "The oxide is planarized using a conventional chemical/mechanical polishing technique (see FIG.", "26).", "This is followed by molecular adhesion bonding to another silicon substrate 16 (see FIG.", "27).", "The bonding is preferentially stabilized at a temperature from 1 000 to 1 100° C. if the structures formed for the first gate can withstand this high a temperature, or at temperatures of the order of 900/1 000° C. otherwise.", "Finally (see FIG.", "28), separation is carried out in exactly the same way as previously (insertion of a blade, pressurized water jet, compressed air jet, etc.).", "Before resuming the transistor fabrication process, in particular to produce the second gate (on the new “substrate” shown in FIG.", "28), the remainder of the oxide layer 13 is removed by chemical etching.", "Because the oxide is etched with a hydrofluoric acid solution of known etching selectivity with respect to silicon, etching stops naturally once the oxide has been entirely etched, revealing a silicon surface.", "The major advantage of this technique over other fracture techniques, for example obtained by way of implantation, is that it does not necessitate excessively complex finishing sequences that are problematic in terms of their potential to create defects, as for a final polishing operation, for example.", "The remainder of the double-gate transistor fabrication process will be obvious to the person skilled in the art.", "The same process can be used in many other applications.", "If the first SOI structure (see FIG.", "4) is used to produce transistors, circuits, components, etc., these can be finally transferred onto many types of dedicated support.", "For example, the substrate 16 can be chosen for its electrical insulation properties (high-resistivity silicon, quartz, sapphire, etc.)", "to provide an ideal support for microwave and telecommunication circuits, thereby limiting losses in the substrate.", "For applications relating to flat screens, a transparent substrate is chosen for the final support.", "Another example of separation is briefly described here (see FIGS.", "29 to 32) in the case of producing circuits on thin substrates.", "The final thicknesses of interest are typically less than a few hundred μm, even of the order of a few tens of μm.", "They relate, for example, to power applications or applications to smart cards and other circuits for which some flexibility is required (plastics material supports, curved supports, etc.).", "One variant relates to a type of separation necessitating no transfer to a target substrate.", "Here the object is to achieve separation without transferring the layer 14 after producing the circuits or components C, when the layer 14 is sufficiently thick to be self-supporting but too thin to withstand without damage a circuit production process (typically less than a few hundred μm thick, and even of the order of a few tens of μm).", "The method of producing the separable substrate is identical in every respect to any of those described previously to produce the FIG.", "4 structure, for example.", "In the case of 200 mm diameter silicon wafers, the standard substrate thickness is 725 μm.", "If the application requires a final substrate thickness of 80 μm, for example, a silicon substrate 725−80=645 μm is chosen for the support substrate 11.This 645 μm thick substrate is then molecular adhesion bonded, for example, to a 0.725 μm wafer, forming areas of lower mechanical strength.", "The 725 μm wafer is then thinned to the required thickness of 80 μm, for example by planing and chemical/mechanical polishing.", "The resulting assembly therefore corresponds to the standards and offers sufficient resistance to the processes for fabricating some or all of the components.", "After production of the latter, one of the separation techniques previously cited may be used (see FIG.", "31, hydrofluoric acid etching and mechanical stresses), except that the substrate 16 can be omitted.", "Its presence can be beneficial in some cases, however.", "After separation, the self-supporting layer 14 alone represents the final substrate of interest, characterized by a substrate thickness of 80 μm, including the components.", "The remainder of the substrate can be recycled.", "If required, the dies can be cut before separation, as is clear from FIGS.", "33 and 34: FIG.", "33 shows cuts between the components, extending to just below the future separation region, and FIG.", "34 shows a pull-off support SA bonded to one of the fragments and adapted to allow pulling off after hydrofluoric acid etching, if any.", "The weakening parameters have to be adapted to the technological operations that have to be carried out on the separable substrate prior to separation, in particular thermal and chemical treatments, and according to the nature of the mechanical stresses.", "For example, if the separable substrate consists of a surface layer of germanium having an SiO2—SiO2 bonding interface that has to withstand an epitaxial growth temperature of 550° C. (which is typical in the case of growing GaInAs to constitute a solar cell for use in space), then the rms roughness must advantageously be 0.4 nm for the substrate to be separable.", "Another example of use concerns the production of circuits for smart cards, where the flexibility of the support becomes critical, firstly because of the increase in the size of the circuits, and secondly because the trend is to require cards with ever higher resistance to deformation.", "A monocrystalline silicon support whose thickness is greater than around 50 μm is too fragile in this context, because its thickness is too high if it is subjected to a bending force, as can happen regularly with a smart card.", "FIG.", "35 shows a starting assembly similar to that shown in FIG.", "4, with a starting substrate 11′ covered with a layer 12′ of silicon oxide that is molecular adhesion bonded to a second layer 13′ of silicon oxide, in turn covered with a silicon layer 14′.", "The circuits are produced within the silicon layer 14′.", "Then, for assembly to the second support 16′ (see FIG.", "36), an adhesive is preferably chosen that produces a very thin layer 15, whilst having the highest possible mechanical strength at low temperature, for example <400° C., so as not to risk damaging the components of the active layer produced after this bonding step.", "These can advantageously be adhesives hardened by heat or by exposure to UV radiation (in this latter case, it is sufficient to choose a final substrate 16′ that is transparent to UV).", "When it comes to cutting along the weakened area of the separable substrate (here the bonding interface 12′/13′), it may appear difficult to produce purely chemical lift-off because adhesives and substrates transparent to UV (in practice made of quartz and glass) are not totally inert to the chemical products (hydrofluoric acid, solvents, etc.).", "On the other hand, a purely mechanical action may be sufficient to lift off the structure at the weak interface 12′/13′ if the bonding energy of the ring is lower than the strength of the adhesive and the various layers constituting the integrated circuits (this can be achieved relatively easily).", "It is then possible to use the substrate of the separable substrate several times.", "Apart from limited chemical etching of the ring, it is further possible to eliminate the ring by making a circular cut in the structure after adhesive bonding.", "The cutting can advantageously be effected by a laser at the boundary between the high bonding energy region and the low bonding energy region.", "If the ring is only a few mm wide, or even narrower, it is then possible to reuse the substrate.", "As a general rule, the residue of the first substrate is obtained, the support of the substrate referred to until now as “separable” (see FIG.", "37) and reusable, preferably after polishing the layer 12′ and the active layer 14′ (FIG.", "38) that is transferred onto the other support 16′ or alternatively is free and self-supporting if the thickness of the layer 14′ is appropriate.", "Unlike the preceding examples, the second substrate 16′ can instead be merely an intermediate substrate in a much longer process that will continue either with the pure and simple elimination of the intermediate substrate 16′ or another layer transfer operation onto a yet further support, generally involving elimination of the substrate 16′.", "The separable substrate obtained with the aid of the technique previously described is bonded to the intermediate substrate after it has been “processed”.", "The intermediate substrate can be rigid or flexible (see the above examples).", "If it is rigid, it can even be a silicon wafer.", "The use of adhesive films known to the person skilled in the art can be envisaged for the adhesive bonding, in particular for the operations of cutting silicon wafers and encapsulating integrated circuits or packaging or back-ending (“Blu Tak”, Teflon® adhesive film, etc.).", "If the adhesive film is double-sided, it may be as well to stick an intermediate substrate to the rear face of the film, to act as a substrate or support for stiffening the assembly at cutting time.", "The lift-off techniques that can be envisaged include the application of traction and/or shear and/or bending forces.", "It may be as well to combine the application of force with chemical etching of the interface, or other means, such as ultrasound.", "If the interface to be lifted off is of the oxide type, etching the low energy interface makes it easier for the bonding interface to yield and therefore facilitates the transfer of the processed layer to the intermediate substrate.", "Under these conditions, it is advantageous for the processed layers to be protected (for example by an additional deposit of nitride in the case of hydrofluoric acid etching).", "Stresses can be applied directly to the thin film or to the intermediate substrate (known as a handle).", "They can be mechanical stresses (in particular applied by inserting a blade at the bonding interface), and/or use a lift-off tool (see WO 00/26000), and/or a jet, or involve inserting a gas flow, as described in the document FR 2796491, and/or a liquid (see EP 0925888, EP 0989593).", "In the case of a gas flow (or even a liquid flow, for example a flow of hydrofluoric acid if the interface is of oxide), the separable substrate can advantageously be prepared beforehand (for example by chemical etching) to feed the fluid locally to the bonding interface.", "This facilitates preferential lifting off at the multilayer structure bonding interface, where the lifting off must take place, by protecting the various layers of the structure incorporating the components.", "Thus it is possible to lift off the bonding interface even when the adhesion between the internal component layers is weak.", "In this regard, see FIGS.", "39 and 40.The intermediate substrate, which is sometimes referred to as a “handle”, can then be cut, totally or in part (to form notches or cutting precursors), into elements corresponding to the electronic components, which can be transferred to different supports.", "The transfer can be collective, in which case all of the components, even if they are interconnected only by a support, are transferred at the same time in the same technological operation, or component by component (or die by die), if the latter are transferred one after the other.", "The supports can be of plastics material, as in the case of a smart card, and in this case an adhesive is advantageously used for the transfer.", "The elements can also be transferred onto a wafer incorporating other electronic or opto-electronic devices, in which case the transfer can again use a molecular adhesion technique (see FIGS.", "4 and 19 to 22, imagining the additional presence of components formed in the layer 14).", "The elements can be transferred by conventional means such as pick and place means.", "The elements can also be transferred onto another support to improve the thermal properties, for example.", "Then, by applying stress or localized heating (for example using a laser), the thin layer, previously bonded to its final support, can be separated (element by element, or globally) from its handle by means of mechanical forces.", "As indicated with reference to FIGS.", "17 and 18, the regions Z1 and Z2 may not define a system comprising a central circular region Z1 surrounded by an outer ring Z2.A multitude of other structures can be envisaged.", "The array of squares in FIG.", "18 can be replaced by arrays of other shapes (rows, columns, concentric circles, etc.)", "whose pitch and other geometrical dimensions can vary as a function of the application and the separation technique employed.", "The benefit of adopting an array may prove to be preferable if the structure to be produced imposes high mechanical stresses that are transmitted to the connecting interface (in this case short-range repetition of areas of good mechanical strength is preferable), or if the layer 14 may feature holes, whether intentional or otherwise, locally uncovering the connecting region, which may in turn be subject to untimely delamination whose epicenter is the hole, or to simplify the method (for example for cutting into individual dies) in the case of producing dies.", "In principle, dimensions of the order of 1 μm are preferred for structures having to withstand high stresses (for example hetero-epitaxy or any other deposition or technological step applying significant tension or compression to the structure), or in the case of small circuits, or of circuits produced with submicron photolithographic resolution.", "In the contrary situation, scales of the order of 1 mm or even 1 cm are preferable.", "Many combinations of more than two regions can of course be envisaged (Z1; Z2, Z3, etc.).", "Rather than a discrete number of types of regions of particular mechanical strength, a continuum of regions characterized by a continuous variation of the mechanical strength can be envisaged.", "For example, the mechanical strength decreasing continuously from a maximum value in the vicinity of the edge to a minimum value at its center can be envisaged.", "The variation can optionally be circularly symmetrical about an axis perpendicular to the plane of the substrate.", "Production processes yielding this type of substrate and their use in separation are in all respects similar to those described with reference to FIGS.", "1 to 8, provided that the geometrical shapes employed are suitable (mask shapes for formation of protection layers for selective roughness, mask shapes for selective deposition, shape of polishing tissues for preferentially polishing eccentric rings, etc.).", "The above geometrical shapes are compatible with the transfer of a layer 14 as a whole from the separable original substrate 11+12+13+14 to the target substrate 16.As opposed to embodiments of the method according to the invention that tend to encourage separation of the layer from its substrate at the global level, i.e.", "on the scale of the whole of the substrate, others tend to delimit fragments, the shape of which is clearly related to the dies or components to be produced from the active layer.", "FIG.", "41 shows one example of this, with enlargement of the region that subsequently accommodates the die (or any other component).", "This structure can be repeated as many times as there are components to be separated on the original substrate.", "Ideally, each region Z2 surrounds or simply extends along the contour of each fragment of the region Z1.The surface area of the component (or die, etc.)", "can correspond exactly to the region Z1, or be greater than or less than the latter, in configurations where one of the surfaces of the component and of the region Z1 encompasses the other.", "The configuration adopted depends on the separation technique to be used, which can be the same as those previously described for separating a substrate having only a ring Z2 around a central disc Z1.One interesting variant consists in using conventional component cutting techniques (sawing, laser cutting, etc.)", "to cut or delimit trenches, at least partially around dies, fragments, etc.", "Another beneficial variant is based on the use of chemical etching associated with a photolithography operation to produce identical trenches and/or to remove the connecting region corresponding to the region Z2.As FIG.", "41 shows in the case of one particular example, the vertical dashed lines indicate the contour of the required fragments.", "For example, after preparing molecular adhesion bonding over a large area, only layers 3 and 4 are cut to the contour shown, after which each fragment is lifted off the substrate, which amounts to considering that the substrate is lifted off each fragment (as an alternative, all the layers or all of the fragments can be cut at one and the same time).", "To the extent that, after cutting, the high mechanical strength region is at the periphery, the risk of delamination during treatment steps, for example to produce electronic, optical or other components on the layers 3 or 4, is reduced, whereas, when lifting off is required, it propagates without difficulty, in a controlled manner, in the central region (it may have been initiated at the periphery).", "Combining the distribution of the regions Z1 and Z2 with the aim of forming an outer ring at the level of the substrate, as shown in FIG.", "4, with a distribution intended to protect each of the dies, as shown in FIG.", "13, is one beneficial combination.", "Of course, the embodiments previously described are not limited to the single case of monocrystalline silicon, and can be extended to many materials, such as other semiconductor materials (Ge, SiGe, SiC, GaN and other equivalent nitrides, AsGaInP, etc.", "), ferro-electric and piezo-electric materials (LiNbO3, LiTaO3), and “treated” magnetic materials, whether components are produced before separation or not.", "As already mentioned, for the situation of a separable substrate consisting of a surface layer of germanium having an SiO2—SiO2 bonding interface that has to be subjected to an epitaxial growth temperature of 550° C. (as is typical in the case of growing GaInAs to constitute a solar cell for use in space), then the rms roughness should advantageously be 0.4 nm for the substrate to be separable.", "Another example is that of epitaxially growing an epitaxial stack on a separable substrate.", "This applies in particular to the production of blue and white LEDs and thin layer laser diodes (for example, for improved extraction of the light emitted or improved evacuation of heat, thanks to transfer onto a substrate that is a good conductor of heat, such as copper or diamond).", "In this case, the epitaxial stack concerned is based on composite semiconductors derived from GaN (AIN, GaAIN, GaAIInN, etc.).", "One method consists of using one of the processes previously described to form a separable structure equivalent to that of FIG.", "4 (or FIG.", "26 or FIG.", "35) in which the layer 14 is of SiC6H (the transferred Si face is at the top in the figures), the layers 12 and 13 are of silicon oxide, as in the FIG.", "4 example, and the substrate 11 is of polycrystalline SiC (or sapphire).", "The stack 15″ based on nitrides (FIG.", "43) is grown epitaxially on this structure (FIG.", "42).", "The epitaxial techniques used can be those well known to the person skilled in the art such as molecular beam epitaxy (MBE) for one category, and metallo-organic chemical vapor deposition (MOCVD) for another category.", "In the former case, the epitaxial growth temperatures rarely exceed 600° C., whereas the typical temperatures for the second category are of the order of 1 050 to 1 100° C. For each of the above two techniques, the choice of the pairs or sets of regions Z1, Z2, etc.", "must be optimized.", "In the second case, one of the processes previously described based on roughening of the two oxide layers 12 and 13 by hydrofluoric acid etching is chosen, for example, and another variant consists in forming a 5 mm wide ring from the edge of the substrate.", "Subsequent MOCVD epitaxial growth at 1 100° C. produces a stack whose thickness is of the order of 1 μm.", "The structure is optionally annealed before the epitaxial growth stage, typically in the temperature range from 900 to 1 200° C., in order to consolidate strongly the mechanical strength of the ring.", "After the growth step, the assembly is subjected to deposition of oxide, planarization by CMP, molecular adhesion bonding (for example onto a silicon substrate), and annealing at 1 100° C. to strength this latter bonding.", "The layer 16 is produced (FIG.", "44).", "Finally, there is separation at the bonding interface (FIG.", "45).", "A few hours in a 50% hydrofluoric acid bath are sufficient to etch the oxide layer laterally to a depth of a few mm where this interface corresponds to the region Z2, thereby uncovering the region Z1.This is followed by separation by means of mechanical forces, for example by inserting a blade, applying a pressurized water jet, or applying a compressed air jet, using the techniques previously described, for example.", "A final deoxidation step removes the residue of the oxide layer 13 (FIG.", "46).", "At least the SiC layer 14 that served as a nucleation layer for the epitaxial stack can optionally be eliminated by etching (FIG.", "41).", "Diodes can be produced before or after the final transfer.", "2—Weak Buried Layer Incorporating Microcavities, Microbubbles or Platelets The interface between the substrate and what is to form the “active” layer can instead take the form of a buried weak layer, formed of microcavities, microbubbles, or platelets, for example.", "This process can be used with many semiconductor and other materials.", "The difference between regions with different mechanical strengths is obtained by different levels of weakening; in the case where, as in FIGS.", "1 to 3, a central region is required to be surrounded by a peripheral region of greater mechanical strength, it is sufficient to weaken the structure less at the periphery than at the center, for example, by introducing at the periphery smaller amounts of the substances for weakening the region (for example by hydrogen implantation); this can be obtained by masking a portion of the layer during a portion of the implantation operation, for example, or by modifying the sweep during implantation; it can also be achieved by carrying out successive implantations, under different conditions, in the various regions whose properties are to be different.", "Regions can also be weakened differently by applying different heat treatments to them to obtain different levels of weakening.", "With regard to the distribution of the regions Z1 and Z2 (a single ring, fragments, array of regions, multitude of regions, etc.", "), the techniques for separating these structures (by means of mechanical stresses, chemical treatment, heat treatment, selective removal of rings and regions Z2, etc.)", "and how they are reduced to practice, the elements and examples given in section 1 relating to an interface or intermediate layer produced by molecular adhesion remain valid.", "3—Interface Formed of a Porous Layer The person skilled in the art knows, in particular from the document EP 0843346A2, that a porous layer can be obtained from the following materials in particular: Si, GaAs, InP, GaAsP, GaAIAs, InAs, AIGaSb, ZnS, CdTe and SiGe, in monocrystalline, polycrystalline or amorphous forms.", "Thus it is only by way of example that the remainder of this description refers to silicon.", "It is possible to obtain porous silicon by electrolysis in hydrofluoric acid.", "The person skilled in the art knows how to vary the porosity of the Si by modifying the concentration of the hydrofluoric acid or by varying the current.", "For example, reducing the hydrofluoric acid concentration from 50% to 20% changes the porosity from 2.1 g/cm3 to 0.6 g/cm3.Thus by protecting the outer ring of the Si wafer with a thick deposit (of nitride, oxide or polycrystalline silicon, for example) it is possible to make a central region of the silicon layer porous whilst preserving the periphery intact.", "It then suffices to eliminate the layer deposited at the periphery and to electrolyze the entire wafer.", "The consequence of these two electrolysis operations is to produce a higher porosity at the center of the wafer than at the periphery.", "A monocrystalline layer of Si is then produced on this porous layer by epitaxial growth (see FIG.", "48, which represents a structure comprising a substrate 21, the porous layer 22, having controlled differences in porosity between the regions Z1′ and Z2′, and a thin layer 23).", "Smoothing annealing of the hydrogen annealing type is advantageously employed before the epitaxial growth step to stop up the surface of the pores at least in part.", "There can be crystal lattice continuity between the various layers if they have the same constituent.", "The thickness of this monocrystalline layer, which thus becomes the active layer, depends on the intended applications.", "Depending on the thickness of the active layer, after separation it can become a self-supporting layer (if it is relatively thick) or it can be transferred to a target substrate (for example by molecular adhesion), especially if the layer is thin.", "The active layer can be lifted off its substrate by chemical means or by introducing a fluid locally into the porous layer.", "An alternative is to render the periphery porous only to a distance of a few mm (as in FIG.", "49, with a substrate 21′, a layer 22′, some regions of which are porous, and a monocrystalline layer 23′).", "It then becomes appropriate to eliminate this ring by cutting (by mechanical or laser cutting or by chemical processing using HF/HNO3 or TMAH).", "With regard to the distribution of the regions Z1 and Z2 (a single ring, fragments, array of regions, multitude of regions, etc.", "), the techniques for separating these structures (by mechanical stresses, chemical treatment, heat treatment, selective removal of rings and regions Z2, etc.)", "and how they are reduced to practice, the elements and examples given in section 1 relating to an interface or intermediate layer produced by molecular adhesion remain valid.", "Note that: the interface can be a simple contact surface or a connecting layer, the fragments can be distributed in a network of squares, lines, concentric circles, etc., the geometrical definition of the fragments is preferably related to the location and the size of the dies (to the nearest region Z2 or the nearest sawing line), the surface preparation step before bonding can include roughening of the oxide layer, the preparation before bonding can affect the roughness but also the hydrophilic properties, to obtain a greater roughness of the first region relative to that of the second region, both regions can be roughened and the roughness of only the second region reduced, the difference between Z1, Z2 can result from heat treatment after localized molecular bonding; alternatively, localized thermal annealing can be used after the bonding operation (laser beam, non-uniform furnace, heating by lamps, etc.", "), when the interface is a buried layer weakened by implanting a gaseous element, the latter preferably remains gaseous, to obtain lift-off, portions of material corresponding to the region Z2 can advantageously be eliminated by chemical etching and/or by mechanical removal of material (machining), the lift-off step can be effected by etching in a vacuum and/or application of stresses; a jet of water, air or pressurized fluid can be used, the preceding description has emphasized a silicon layer, but SiC, GaN, GaAs, InP, SiGe and semiconductors derived therefrom are also relevant, there can be a step of epitaxial growth on the layer before separation; likewise components can be produced completely or in part before separation, there can be regions Z3, and even Z4, having levels of mechanical strength different from those of the regions Z1 and Z2, there can be a continuous evolution of the mechanical strength between extreme values, rather than in plateaux linked by discrete steps, the difference in mechanical strength between the regions can result from a difference of composition (interface formed of a layer with one region of one material and the other region of another material, or an interface formed in part by the material of the substrate or the layer and in part by a transferred layer)." ] ]	Patent_10468223
[ [ "Elastic meth(acrylic) adhesive compositions", "Described is an adhesive composition with a high elasticity, measured in terms of elongation at break, and an elastic behavior approaching the ideal Hook law in the stress-strain diagram.", "Said adhesive composition comprises (a) at least one mono-functional meth(acrylic) monomer A whose homo-polymer or co-polymer exhibit a glass transition temperature (Tg) between 40° C. and 140° C., (b) at least one monofunctional meth(acrylic) monomer B of the following structure (B): wherein R is H or CH3, R′ is H or (CH2)nCH3 with n=0 to 2, in particular CH2CH3 or H, and R″ is C3-C20-alkyl or phenoxy or O—(CH2)n—H3 or O—[(CH2)2—O]s; 0 to 2, in particular (CH2)nCH3 wherein n is 3 or 10 to 13, and c) at least one liquid elastomer C in the molecular weight range of 1000 to 9000 with (meth)acrylic derivative groups, preferably (meth)acrylic ester groups, whereby the components A, B, and C are present in the following amounts: B based on the total weight of A+B+C=5 to 20% by weight, C based on the total weight of A+C=30 to 70% by weight." ], [ "1.An adhesive composition comprising (a) at least one monofunctional meth(acrylic) monomer A whose homopolymer or co-polymer exhibit a glass transition temperature (Tg) between 40° C. and 140° C., (b) at least one monofunctional meth(acrylic) monomer B of the following structure (B): wherein R is H or CH3, R′ is H or (CH2)nCH3, with n=0 to 2, in particular CH2CH3 or H, and R″ is C3-C20-alkyl or phenoxy or O—(CH2)n—CH3, or O-[(CH2)2—O]n—CH2—CH3 with n=0 to 2, in particular (CH2)nCH3 with n=3 or 10 to 13, and c) at least one liquid elastomer C in the molecular weight range of 1000 to 9000 with (meth)acrylic derivative groups, whereby the components A, B, and C are present in the following amounts: B based on the total weight of A+B+C=5 to 20% by weight, C based on the total weight of A+C=30 to 70% by weight.", "2.The adhesive composition of claim 1, wherein the at least one monomer A is selected from (meth)acrylic esters, in particular linear or branched or cyclic C1-C6-alkyl esters, or heterocyclic or aromatic esters, much preferred the group consisting of methyl methacrylate, tetrahydrofurfuryl methacrylate, cyclohexylmethacrylate, cyclic trimethylolpropane formal acrylate, isobornylmethacrylate, benzylmethacrylate, dicyclopentadienyloxyethylmethacrylate, t-butylmethacrylate, isobornylacrylate, dihydrodicyclopentadienylacrylate (DHDCPA), and mixtures thereof, in particular methyl methacrylate, tetrahydrofurfuryl methacrylate, and mixtures thereof.", "3.The adhesive composition of claim 1, wherein the at least one monomer B is selected from linear or branched C6-C15-alkyl esters, in particular from the esters wherein R′ is CH2CH3 and R″ is (CH2)3CH3, or R′ is H and R″ is (CH2)nCH3 with n=10-13, much preferred n=10, or mixtures thereof.", "4.The adhesive composition of claim 3 wherein the one or more (meth)acrylate ester(s) are one or more acrylate ester(s), preferably lauryl acrylate, 2-ethyl hexyl acrylate, or mixtures thereof.", "5.The adhesive composition of anyone claim 1, wherein the ethylenically unsaturated (meth)acrylic groups of the liquid elastomer C are chosen from the group consisting of (meth)acrylic functionalized butadiene, isoprene based polymer or block-copolymer, PU-(meth)acrylate obtainable through the syntheses of a polyethylene polyol or polypropylene polyol, a diisocyanate and a hydroxy functionalyzed ethylenically unsaturated monomer.", "6.The adhesive composition of claim 5, wherein the PU-(meth)acrylate is obtainable through a synthesis using polyols with low unsaturation and narrow molecular weight distribution as obtainable through double metal cyanide complex catalysis.", "7.The adhesive composition of claim 1, wherein the amount of B based on the total weight of A+B+C=5 to 12% by weight.", "8.The adhesive composition of claim 1, wherein the amount of C based on the total weight of A+C=40 to 60% by weight.", "9.The adhesive composition of claim 1 that furthermore contains at least one initiator, preferably an organic peroxide and/or at least one catalyst, preferably selected from the group consisting of tertiary amines and salts of transition metals and complexes of transition metals.", "10.The adhesive composition of claim 9 wherein the initiator is benzoylperoxide, and/or the tertiary amine is selected from the group consisting of N,N-dimethylaniline, N,N-dimethyl-p-toluidine, N,N-diethylaniline, N,N-diethyltoluidine, N,N-bis(2-hydroxyethyl)-p-toluidine, N-ethoxylated p-toluidine, N-alkylmorpholine or mixtures thereof, or the salts and complexes of transition metals are selected from the group consisting of salts and complexes of cobalt, nickel, copper and mixtures thereof.", "11.The adhesive composition of claim 1 that contains at least one further compound, in particular a filler or thixotropic agent.", "12.Use of an adhesive composition of anyone claim 1 for bonding applications with materials having different thermal expansion coefficients, in particular for bonding applications in motor vehicles including trucks, and in rail cars, e.g.", "for bonding of side panels of trailers or direct glazing.", "13.A method for bonding materials having different thermal expansion coefficients, wherein said bonding is performed by application of an adhesive composition of claim 1 between said materials." ], [ "<SOH> BACKGROUND ART <EOH>The long term performance of bonding is dependent on the elastic behavior of the adhesive.", "The first-generation meth(acrylic) adhesives are brittle, with very low elongation at break.", "Many approaches are known to increase the flexibility of methacrylic adhesives.", "One of said efforts to improve flexibility has led to the addition of solid non-reactive elastomers that are dissolved in the meth(acrylic) monomers.", "Such compositions are called rubber-toughened adhesive compositions.", "The elastomers are solids at room temperature, and commercially available as large particles or granulates.", "This approach is described in several patent documents, such as: U.S. Pat.", "No.", "3,890,407, U.S. Pat.", "No.", "4,106,971, U.S. Pat.", "No.", "4,263,419, U.S. Pat.", "No.", "3,725,504, U.S. Pat.", "No.", "4,200,480, U.S. Pat.", "No.", "3,994,764, EP 0 641 846.The disadvantage of such formulations is that they can only be mixed homogeneously with special equipment like extruders or kneaders due to the high viscosity of the polymer that is formed in the monomer solution.", "Moreover, this type of compositions suffers from the limitation that the monomer, or the monomer mixture, must be chosen such that the non-reactive thermoplastic polymer is soluble therein.", "Practically, methyl methacrylate is the only monomer with high dissolving properties, and thus the only monomer that allows a polymer-in-monomer composition with up to 30% polymer content.", "Such low molar mass monomers, like methyl methacrylate, have a strong odor and are highly flammable.", "Polymer-in-monomer compositions resulting from this approach have a rubbery, stringy consistency and a high viscosity that makes their handling difficult.", "Due to the high viscosity of the mixture, only a limited amount of fillers can be used making the formulation expensive.", "A high viscosity is also limiting the adhesion of the formulation, as it limits the wetting of the substrate.", "A further approach is the addition of liquid, low-molar mass elastomers that dissolve in the monomers.", "A number of patent documents describe such approach to increase the flexibility of the systems by adding liquid reactive elastomers to the reactive monomer (mixtures) to increase the flexility of the adhesives, namely U.S. Pat.", "No.", "4,769,419; U.S. Pat.", "No.", "4,331;765 and EP 0,561,352 disclosing mixtures of monomers and liquid rubber.", "DE 2,610,423; U.S. Pat.", "No.", "4,439,600; EP 0,640,672; DE 2,319,637 and U.S. Pat.", "No.", "4,223,115 disclose mixtures of monomers and acrylic functionalised polyurethanes.", "In U.S. Pat.", "No.", "4,223,115 the composition optionally contains a dissolved elastomer, e.g.", "NBR (nitrile-butadiene-rubber), polychloroprene.", "Flexibility is measured in terms of elongation at break.", "During deformation, however, all the above mentioned prior-art adhesives show a visco-elastic behavior with a significant viscous component.", "This means that during the deformation the binder matrix of the adhesive shows plastic flow and is being damaged.", "The visco-elastic behavior of the material is reflected in the shape of the stress-strain curve, measured at an elongation speed relevant to the practical application.", "The plot of the prior-art adhesives is not linear, and this even for the compositions of U.S. Pat.", "No.", "4,439,600 (see above) although elastic behavior is claimed.", "The plastic flow component during deformation reduces the number of deformation cycles an adhesive can perform before failing.", "This significantly reduces the life span of an adhesive bond under dynamic load.", "It was therefore an object of the present invention to provide meth(acrylic) adhesive compositions with a high elasticity, measured in terms of elongation at break, and an elastic behavior approaching the ideal Hook law in the stress-strain diagram." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following description thereof and the annexed Figures, wherein: FIG.", "1 shows a stress-strain diagram for the inventive composition of Example 1.FIG.", "2 shows a stress-strain diagram for the inventive composition of Example 2.FIG.", "3 shows a stress-strain diagram for the inventive composition of Example 3.FIG.", "4 shows a stress-strain diagram for the comparative composition of Example 4.FIG.", "5 shows a stress-strain diagram for the state of the art composition of Example 5.detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "CROSS REFERENCES TO RELATED APPLICATIONS This application claims the priority of European patent application No.", "01 105 351.9, filed Mar.", "8, 2001, the disclosure of which is incorporated herein by reference in its entirety.", "TECHNICAL FIELD The present invention relates to elastic adhesive compositions.", "BACKGROUND ART The long term performance of bonding is dependent on the elastic behavior of the adhesive.", "The first-generation meth(acrylic) adhesives are brittle, with very low elongation at break.", "Many approaches are known to increase the flexibility of methacrylic adhesives.", "One of said efforts to improve flexibility has led to the addition of solid non-reactive elastomers that are dissolved in the meth(acrylic) monomers.", "Such compositions are called rubber-toughened adhesive compositions.", "The elastomers are solids at room temperature, and commercially available as large particles or granulates.", "This approach is described in several patent documents, such as: U.S. Pat.", "No.", "3,890,407, U.S. Pat.", "No.", "4,106,971, U.S. Pat.", "No.", "4,263,419, U.S. Pat.", "No.", "3,725,504, U.S. Pat.", "No.", "4,200,480, U.S. Pat.", "No.", "3,994,764, EP 0 641 846.The disadvantage of such formulations is that they can only be mixed homogeneously with special equipment like extruders or kneaders due to the high viscosity of the polymer that is formed in the monomer solution.", "Moreover, this type of compositions suffers from the limitation that the monomer, or the monomer mixture, must be chosen such that the non-reactive thermoplastic polymer is soluble therein.", "Practically, methyl methacrylate is the only monomer with high dissolving properties, and thus the only monomer that allows a polymer-in-monomer composition with up to 30% polymer content.", "Such low molar mass monomers, like methyl methacrylate, have a strong odor and are highly flammable.", "Polymer-in-monomer compositions resulting from this approach have a rubbery, stringy consistency and a high viscosity that makes their handling difficult.", "Due to the high viscosity of the mixture, only a limited amount of fillers can be used making the formulation expensive.", "A high viscosity is also limiting the adhesion of the formulation, as it limits the wetting of the substrate.", "A further approach is the addition of liquid, low-molar mass elastomers that dissolve in the monomers.", "A number of patent documents describe such approach to increase the flexibility of the systems by adding liquid reactive elastomers to the reactive monomer (mixtures) to increase the flexility of the adhesives, namely U.S. Pat.", "No.", "4,769,419; U.S. Pat.", "No.", "4,331;765 and EP 0,561,352 disclosing mixtures of monomers and liquid rubber.", "DE 2,610,423; U.S. Pat.", "No.", "4,439,600; EP 0,640,672; DE 2,319,637 and U.S. Pat.", "No.", "4,223,115 disclose mixtures of monomers and acrylic functionalised polyurethanes.", "In U.S. Pat.", "No.", "4,223,115 the composition optionally contains a dissolved elastomer, e.g.", "NBR (nitrile-butadiene-rubber), polychloroprene.", "Flexibility is measured in terms of elongation at break.", "During deformation, however, all the above mentioned prior-art adhesives show a visco-elastic behavior with a significant viscous component.", "This means that during the deformation the binder matrix of the adhesive shows plastic flow and is being damaged.", "The visco-elastic behavior of the material is reflected in the shape of the stress-strain curve, measured at an elongation speed relevant to the practical application.", "The plot of the prior-art adhesives is not linear, and this even for the compositions of U.S. Pat.", "No.", "4,439,600 (see above) although elastic behavior is claimed.", "The plastic flow component during deformation reduces the number of deformation cycles an adhesive can perform before failing.", "This significantly reduces the life span of an adhesive bond under dynamic load.", "It was therefore an object of the present invention to provide meth(acrylic) adhesive compositions with a high elasticity, measured in terms of elongation at break, and an elastic behavior approaching the ideal Hook law in the stress-strain diagram.", "DISCLOSURE OF THE INVENTION Thus one object of the present invention is an adhesive composition comprising (a) at least one monofunctional meth(acrylic) monomer A whose homo-polymer or co-polymer exhibit a glass transition temperature (Tg) between 40° C. and 140° C., (b) at least one monofunctional meth(acrylic) monomer B of the following structure (B): wherein R is H or CH3, R′ is H or (CH2)nCH3, with n=0 to 2, in particular CH2CH3 or H, and R″ is C3-C20-alkyl or phenoxy or O—(CH2)n CH3, or O—[(CH2)2—O]n—CH2—CH3 with n=0 to 2, in particular (CH2)nCH3, wherein n is 3 or 10-13, and c) at least one liquid elastomer C in the molecular weight range of 1000 to 9000 with (meth)acrylic derivative groups, preferably (meth)acrylic ester groups, whereby the components A, B, and C are present in the following amounts: B based on the total weight of A+B+C=5 to 20% by weight, preferably 5 to 12% by weight, C based on the total weight of A+C=30 to 70% by weight, preferably 40 to 60% by weight.", "Much preferred components of structure (B) are those wherein R′ is CH2CH3 and R′ is (CH2)3CH3 or R′ is H and R″ is (CH2)nCH3 with n=10-13.BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following description thereof and the annexed Figures, wherein: FIG.", "1 shows a stress-strain diagram for the inventive composition of Example 1.FIG.", "2 shows a stress-strain diagram for the inventive composition of Example 2.FIG.", "3 shows a stress-strain diagram for the inventive composition of Example 3.FIG.", "4 shows a stress-strain diagram for the comparative composition of Example 4.FIG.", "5 shows a stress-strain diagram for the state of the art composition of Example 5.MODES FOR CARRYING OUT THE INVENTION In a preferred embodiments of the inventive adhesive composition, the monofunctional meth(acrylic) monomer A or mixture of meth(acrylic) monomers A whose homo-polymer or co-polymer exhibit a glass transition temperature (Tg) between 40° C. and 140° C. is a methacrylate ester, preferably an alkyl ester with a linear or branched or cyclic C1-C6 alkyl, or a heterocyclic or aromatic ester, much preferred a methacrylate ester selected from the group consisting of methyl methacrylate (MMA), tetrahydrofurfuryl methacrylate (THFMXA), cyclohexylmethacrylate (CHMA), cyclic trimethylolpropane formal acrylate (CTFA), isobornylmethacrylate (IBMA), benzyl-methacrylate (BMA), dicyclopentadienyloxyethylmethacrylate (DCPOEMA), t-butylmethacrylate (tBMA), isobornylacrylate (IBA), dihydrodicyclopentadienylacrylate (DHDCPA), and mixtures thereof.", "In another preferred embodiment of the inventive composition, the monofunctional (meth)acrylic monomer B of formula (B) is a (meth)acrylic ester, such as an ester selected from the group consisting of linear and branched C6-C15-alkyl esters, in particular an acrylic ester such as lauryl acrylate, 2-ethyl hexyl acrylate, and mixtures thereof.", "Preferred (meth)acrylates lead to homopolymers and copolymers with a preferred Tg of <40° C. In yet another preferred embodiment, the inventive adhesive composition contains a liquid elastomer C with ethylenically unsaturated groups which is chosen from the group consisting of (meth)acrylic functionalized butadiene, isoprene based polymer or block-copolymer, PU-(meth)acrylate obtainable through the syntheses of a polyethylene polyol or polypropylene polyol, a diisocyanate and a hydroxy functionalyzed ethylenically unsaturated monomer, whereby said PU (meth)acrylate is preferably obtainable through a synthesis using polyols with low unsaturation and narrow molecular weight distribution as obtainable through double metal cyanide complex catalysis (such polyols are e.g.", "known as Acclaim polyols).", "The compositions of the present invention usually also comprise at least one initiator and/or catalyst, as well as preferably also at least one organic or inorganic filler or thixotropic agent.", "The compositions can also comprise further substances, such as stabilizers, additives, toughening agents, adhesion promoters, impact modifiers, core-shell polymers, defoaming agents, thickeners, plasticizers, wetting agents, wax compounds, cross-linking agents, inhibitors etc.", "Such additional substances are known to the skilled person.", "Examples for free radical initiators are organic peroxides, in particular benzoylperoxide, and examples for catalysts are tertiary amines and/or salts and/or complexes of transition metals.", "Examples for tertiary amines are e.g N,N-dimethylaniline, N,N-dimethyl-p-toluidine, N,N-diethylaniline, N,N-diethyltoluidine, N,N-bis(2-hydroxyethyl)-p-toluidine, N-ethoxylated p-toluidine, N-alkylmorpholine or mixtures thereof, and examples for the salts and complexes of transition metals are salts and complexes of cobalt, nickel and/or copper.", "Examples for inhibitors are hydrochinone, methylhydrochinone, t-butyl-p-cresol and for thixotropic agents e.g.", "Aerosil.", "A further advantage of the compositions of the present invention is that they can be easily manufactured, namely by simply mixing monomer(s) A, monomer(s) B and liquid elastomer and an initiator and/or catalyst.", "Said mixture is preferably obtained at ambient temperature for a time sufficient to get a homogeneous liquid (usually about 10 to 20 minutes).", "Obtaining a homogeneous liquid, e.g.", "at 40° C., usually takes about 15 minutes.", "Then, preferably also present filler(s) are added and the composition is mixed at high revolutions per minute until the material shows thixotropic behavior.", "The compositions of the present invention are especially suitable for binding applications, in particular bonding applications with materials having different thermal expansion coefficients as they are e.g.", "found in motor vehicles, e.g.", "trucks, and rail cars.", "Examples of such applications are bonding of side panels of trailers or direct glazing.", "The ideal elastic behavior over a wide range of elongations obtained with the compositions of the present invention increases significantly the performance of the bond under dynamic load.", "Furthermore, the compositions show an excellent adhesion on many substrates without pre-treatment, and thus are easy to manufacture due to their low viscosity.", "The exceptionally high degree of elasticity imparts them excellent impact resistance at low temperatures, even without impact modifiers.", "Also their close to ideal elastic behavior imparts them an excellent recovery behavior.", "Their very high elasticity can be maintained even when the formulations are filled with commonly known fillers.", "EXAMPLES In the compositions used in the examples 1 to 5, the abbreviations have the following meanings: MMA=methyl methacrylate THFMA=tetrahydrofuryl methacrylate LA=lauryl acrylate PUE=prepolymer PUA p-Tol=N,N-bis(2-dihydroxyethyl)-p-toluidine BPO paste=a past of 40% benzoyl peroxide in phthalate plasticizer The compounds used to formulate the adhesive compositions of examples 1 to 5 and their amounts are listed in Table 1.Preparation of Prepolymer PUA To 800 g of a diol with mw 4000 (e.g.", "Acclaim 4200 from Bayer), 88.6 g of IPDI (e.g.", "Vestanat IPDI from Bayer) together with 0.05% of dibutyltin dilaurate (DBTL) are added.", "The reaction mixture is stirred at 80° C. for about 4 hours under nitrogen.", "Then 64 g of 2-hydroxy-ethyl methacrylate (HEMA) are added at once, and the mixture is stirred again at 80° C. for another 45 minutes.", "After cooling the NCO value of the reaction product is measured as 0.01.Preparation of the Adhesive Compositions of Examples 1 to 5 Methyl methacrylate, prepolymer PUA, lauryl methacrylate and N,N-bis-(2-hydroxyethyl)-p-toluidine were mixed in a dissolver at 40° C. for 15 minutes, until a homogeneous liquid was obtained.", "Then Aerosil was added and mixed at high revolutions per minute until the material showed thixotropic behavior.", "The adhesives were cured by adding 4% of the BPO paste.", "The test results obtained for the cured adhesives are also listed in Table 1.The adhesives were tested as follows: Tensile Strength and Elongation at Break (DIN 53504) The adhesive was cured to form a sheet of approximately 2.5 mm thickness from which tensile test dumbbells were cut.", "The stress-strain tests were performed using a rate of 200 mm per minute for measurements at room temperature.", "Tensile Shear Strength (TSS) (DIN EN 1465) Substrates with a dimension of 100 mm×25 mm and 2 mm thick in case of aluminium and 4 mm thick in case of ABS were cleaned with isopropanol.", "The bond thickness is 1.5 mm and the overlap 11.5 mm.", "The tensile shear strength was measured at 10 mm/min.", "TABLE 1 Designa- Compo- Example 1 Example 2 Example 3 Example 4 Example 5 tion nents [w/w %] [w/w %] [w/w %] [w/w %] [w/w %] A MMA 40 38 20 A THFMA 40 70 B LA 8 10 8 10 C PUA 40 42 41 60 18 p-Tol 2 2 2 2 2 Aerosil 10 9 8 10 200 Tensile 9.5 7 7.8 13 9.8 Strength [MPa] Elongation 243 224 293 224 140 at Break [%] TSS 6.4 n.d. n.d. n.d. n.d. ALMg3 [MPa] TSS ABS 4.5 n.d. n.d. n.d. n.d. [MPa] n.d. = not determined Examples 1 to 4 all have a high tensile strength and a high elongation at break.", "Only examples 1 to 3 exhibit an elastic behaviour which approaches the ideal Hook law.", "The stress strain curves shown in FIGS.", "1 to 3 are linear.", "Example 4 contains a larger amount of liquid elastomer C than is described in the present invention.", "The composition of example 4 has a stress strain curve which is only linear up to about 5%.", "Although this composition has an elongation at break of up to 200%, it does not exhibits an elastic behaviour.", "Example 5 represents a state of the art composition.", "The stress strain plot of Example 5 is not linear, and therefore, this adhesive is not elastic.", "While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims." ] ]	Patent_10468299
[ [ "Fluorescent life hammer", "The invention relates to safety device (1) for breaking glass, provided with a housing (2) and a head (4), wherein the head is provided with a relatively hard end (14), wherein at least a part of the housing is provided with a fluorescent outer surface (30).", "The invention also relates to a method for manufacturing a safety device, wherein at least two housing parts (2) are injection-molded, provided at least a part of the outer surface with an in-mold label (20) which is of fluorescent design, which housing parts are assembled for forming the housing of the safety device, wherein between at least a part of the parts of the housing a head is included extending at least partly outside the housing and being provided with at least one relatively hard end." ], [ "1.A safety device for breaking glass, provided with a housing (2) built up from at least two housing parts and a head (4), the head (4) being provided with a relatively hard end (14), characterized in that at least a part of the housing (2) is provided with a fluorescent outer surface.", "2.A safety device according to claim 1, wherein the housing (2) is manufactured from plastic which is of fluorescent design.", "3.A safety device according to claim 2, wherein the plastic is fluorescent.", "4.A safety device according to claim 1, wherein at least a part of the housing (2) is provided with a fluorescent print (30).", "5.A safety device according to claim 4, wherein at least a part of the fluorescent print (30) has been provided with the aid of in-mold-labeling technique.", "6.A safety device according to claim 1, wherein the entire housing (2) on the outside is of fluorescent design.", "7.A safety device according to claim 1, wherein the housing comprises two parts (2), wherein each of the parts (2) is provided at the outside with a fluorescent surface.", "8.A safety device according to claim 1, wherein at least a part of the fluorescent surface has been obtained by injection or printing with the aid of paint or ink after formation of the respective parts (2) of the housing.", "9.A method for manufacturing a safety device (1), wherein at least two housing parts (2) are injection-molded, provided with at least a part of an outside surface with an in-mold label (20) which is of fluorescent design, which housing parts (2) are assembled for forming the housing of the safety device (1), wherein between at least a part of the parts of the housing (2) a head (4) is included extending at least partly outside the housing and being provided with at least one relatively hard end (14).", "10.A safety device according to claim 7, wherein the parts are mirror symmetrical." ], [ "The invention relates to a safety device for the breaking of glass.", "Such a safety device is known as a safety hammer and is marketed under the name life hammer.", "U.S. Pat.", "No.", "5,952,916 (Yamabe) discloses a safety device, provided with a hammer head and a handle.", "The handle houses a light bulb, several LEDs and a battery.", "A fluorescent material may be applied to an inner surface of the handle.", "As long as the device is mounted on a holder the light and LEDs are switched off.", "At removal of the device from said holder, the light and LEDs are automatically activated, thus enabling the device to serve as an emergency signal instrument.", "In emergencies, it has been found that escaping from, for instance, vehicles or buildings can be made impossible or considerably more difficult by closed windows or windows which cannot or insufficiently easily be opened.", "To prevent this problem, the known safety device has been put on the market.", "Herewith, in a simple manner, the obstructing glass can be broken, whereupon escape is still possible.", "Such a safety device seems very effective in itself.", "However, it has been found that the position of this safety device is not always sufficiently clear.", "In particular in dark surroundings, the use of such safety devices is rendered difficult in that its position is not always clear.", "The invention contemplates a safety device of the type described in the preamble, wherein tho drawbacks mentioned of the known safety device have been obviated, while maintaining its advantages.", "To that end, a safety device according to the invention is characterized by the features of claim 1.With a safety device according to the present invention, it has been found that, precisely by using a fluorescent surface or at least a part thereof, location of these safety devices is considerably simplified, without, to that end, for instance, electric provisions being required.", "The fact is that during at least a part of the time, in the normal waiting position, there where it has been suspended clearly visible in case of emergencies, this safety device is exposed to (day) light.", "Usually, this time will be sufficient to obtain the desired fluorescent action, in particular when a relatively bright color is used, such as orange, red or yellow which are generally experienced as being alarming.", "Furthermore, also in normal (day) light circumstances such coloration offers an enhanced visibility.", "Through the use of a fluorescent surface, no external light source is required for considerably increasing the visibility, which would be necessary, for instance, when using a reflecting surface.", "In particular in situations when lighting has completely ceased, for instance in case of fire or in a vehicle fallen into the water, with failing electric provisions or the like, the safety of persons present is thereby enhanced such that lives will thus be saved.", "With a safety device according to the present invention, it is preferred to provide the or each fluorescent surface by the use of in-mold labeling techniques, whereby a label with a fluorescent print is placed in a mold cavity of, for instance, an injection mold, whereupon at least a part of the safety device, for instance a housing part, is injection-molded against it, so that an integral connection is obtained.", "Thus, the fluorescent action ceasing to exist is prevented in a simple manner.", "The fact is that the fluorescent surfaces can no longer be separated from the safety device.", "It is noted, for that matter, that a comparable effect can be achieved by providing a suspension device for such a safety device with an at least partly fluorescent outer surface, in addition to or instead of the safety device.", "The invention further relates to a method for manufacturing a safety device for breaking glass, characterized by the features of claim 9.Such a method offers the advantage that in a particularly simple and economical manner, a safety device according to the invention can be formed which maintains its functionality for a longer period of time.", "In the further subclaims, further advantageous embodiments of a safety device according to the invention are given.", "In elucidation of the invention, exemplary embodiments of a safety device and a method according to the invention will be explained in more detail with reference to the drawing.", "In the drawing: FIG.", "1 shows, in disassembled condition, a safety device according to the invention, in the shape of a hammer; FIG.", "2 shows a safety device according to FIG.", "1, in assembled condition; and FIG.", "3 shows, in cross-sectional side view, a mold part with mold cavity, an in-mold label and a product part formed against it.", "In this description, identical or corresponding parts have identical or corresponding reference numerals.", "FIG.", "1 shows, in disassembled condition, a safety device 1, according to the invention, comprising two housing parts 2 and a head 4.In this embodiment, the housing parts 2 are identical to each other and have a front view which is a substantially somewhat T-shaped.", "The housing parts 2 are dish parts, injection-molded and thin-walled.", "The horizontal beam 6 of each T-shaped housing part 2 is largely semi-cylinder-shaped with open ends 8, which open ends are bounded by an edge 10 reaching inwards.", "When the two T-shaped housing parts 2 are brought against each other as shown in FIG.", "2, where they can, for instance, be glued, sealed or welded together or be connected to each other in a different manner, the two beams 6 together form a cylinder-shape with open ends 8.The head 4 is formed by a solid metal cylinder 12 having conical ends 14.Preferably, the head 4 is hardened.", "Adjacent each conical end 14, a circular groove 16 has been provided, in which the edge 10 can engage when the head 4 is received between the beams 6.Thus, the head 4 is locked in between the housing parts 2.In the assembled condition shown in FIG.", "2, the upright legs 17 of the housing parts 2 form a handle 18.In the exemplary embodiment shown, each housing part 2 is provided at the outside with a fluorescent print 30, for instance in fluorescent orange, yellow or red.", "Here, fluorescent orange or yellow appears to lead to the best results, but any other desired fluorescent color is possible.", "This fluorescent outer surface can be provided by, for instance, printing the outer surface of each of the housing parts 2 after formation with a fluorescent ink, by spraying with fluorescent paint or by providing it with fluorescent printed labels such as stickers or the like.", "The latter technique is particularly suitable for substantially single-curved or flat parts, for instance rings on the handle 18 or around the beam 6.In a preferred embodiment, a safety device 1 according to the invention is formed with a method wherein a device is used such as, for instance, shown partly in cross section in FIG.", "3.In this method, a label 20 with fluorescent properties is placed in a mold cavity 22, whereupon the mold 24 is closed and, in a manner known per se, plastic is injected in the mold cavity 22 against the label 20.Then, partial fusion occurs between the label 20 and the plastic 26, at least such that a fixed connection therebetween is obtained.", "Upon ejection of the thus formed housing part 2, the label 20, and, hence a print 30 optionally applied thereon, forms an integral part of the respective housing part 2.The fluorescent properties of the respective label 20 can be obtained by printing it with fluorescent ink or paint, prior to placement in the mold cavity 22, but can, for instance, also be obtained by specific selection of fluorescent plastic for forming the respective label.", "If such a label is manufactured as a laminate, one of the layers can be of fluorescent design, optionally covered by a permanent transparent cover layer.", "It is preferred that, prior to placement in the mold cavity 22, the label 20 is already partly brought into the desired shape the label should eventually have on the housing part 2, for instance with the aid of vacuum forming techniques, by a deep or thin drawing technique or such deforming techniques known per se.", "Thus, relatively thin foil can be used while damages occurring to this foil during injection of the plastic in the mold can be prevented.", "Naturally, the desirability of such predeformation also depends on the degree of deformation of the label from a flat position to the final shape.", "For instance with single curved surfaces or relatively flat surfaces, such predeformation is less necessary.", "Also, the housing parts can be completely or partly manufactured from fluorescent plastic.", "The invention is not limited in any way to the exemplary embodiments given in the description and in the drawings.", "Many variations thereon are possible within the framework of the invention as outlined by the claims.", "For instance, a safety device according to the invention can be manufactured in a different manner, for instance by blow molding, rotational molding, assembly from several parts and the like.", "Also, the head can be designed differently and be secured in the housing in a different manner.", "Also, a housing of a safety device according to the invention could be designed to be wholly or partly transparent, with fluorescent means disposed therein.", "Also, such a safety device can be combined with other signaling means, for instance a breaking light included therein, which breaks at undesirably large accelerations or decelerations, so that the visibility of the safety device is thereby increased, also in case no or insufficient fluorescent action is present.", "Such a break light can also be sufficient in itself.", "The invention is not limited in any way to the exemplary embodiments presented in the description.", "Many variations thereon are possible within the framework of the invention as outlined by the claims, while all aspects mentioned can be used both separately and in combination with each other." ] ]	Patent_10468311
[ [ "Agent learning apparatus, method and program", "An agent learning apparatus comprises a sensor (301) for acquiring a sense input, an action controller (307) for creating an action output in response to the sense input and giving the action output to a controlled object, an action state evaluator (303) for evaluating the behavior of the controlled object, a selective attention mechanism (304) for storing the action output and the sense input corresponding to the action output in one of the columns according to the evaluation, calculating a probability model from the action outputs stored in the columns, and outputting, as a learning result, the action output related to a newly given sense input in the column where the highest confidence obtained by applying the newly given sense input to the probability model is stored.", "By thus learning, the selective attention mechanism (304) obtains a probability relationship between the sense input and the column.", "An action output is calculated on the basis of the column evaluated as a stable column.", "As a result, the dispersion of the action output is quickly minimized, and thereby the controlled object can be stabilized." ], [ "1.An agent learning apparatus (100) for performing optimal control for a controlled object, comprising: a sensor (301) for capturing external environmental information for conversion to sensory inputs; a behavior controller (302,307) for supplying behavior outputs to said controlled object based on results of learning performed on said sensory inputs; a behavior status evaluator (303) for evaluating behavior of the controlled object caused by said behavior outputs; and a selective attention mechanism (304) for storing said behavior outputs in one of a plurality of columns in association with corresponding sensory inputs based on the evaluation, computing probabilistic models based on the behavior outputs stored in said columns, calculating confidence for each column by applying newly given sensory inputs to said probabilistic models and outputting, as said results of learning, behavior outputs in association with newly given sensory inputs in the column having largest confidence; wherein said probabilistic model is probabilistic relationship that a sensory input belongs to each column.", "2.An agent learning apparatus (100) for performing optimal control for a controlled object, comprising: a sensor (301) for capturing external environmental information for conversion to sensory inputs; a behavior controller (302,307) for supplying behavior outputs to said controlled object based on results of learning performed on said sensory inputs; a behavior status evaluator (303) for evaluating behavior of the controlled object caused by said behavior outputs; and a selective attention mechanism (304) for storing said behavior outputs in one of a plurality of columns in association with corresponding sensory inputs based on the evaluation, computing probabilistic models based on the behavior outputs stored in said columns, and outputting, as said results of learning, behavior outputs in association with newly given sensory inputs in the column, said column containing the behavior outputs having largest evaluation; wherein said probabilistic model is probabilistic relationship that a sensory input belongs to each column.", "3.The agent learning apparatus (100) of claim 1 or 2, said computing probabilistic model comprising: representing behavior outputs stored in columns as normal distribution by using Expectation Maximization algorithm; using said normal distribution to compute a priori probability that a behavior output is contained in each column; and using said a priori probability to compute said probabilistic model by supervised learning with neural network, said probabilistic model being probabilistic relationship between any sensory input and each column.", "4.The agent learning apparatus (100) of claim 3, said confidence being calculated by applying said a priori probability and said probabilistic model to Bayes' rule.", "5.The agent learning apparatus (100) of claim 4, wherein said probabilistic model is computed in advance using data sets of relationship between sensory inputs and behavior outputs, wherein after computing said probabilistic model, said confidence is calculated using the probabilistic model for newly given sensory inputs.", "6.An agent learning method for performing optimal control for a controlled object, comprising: capturing external environmental information for conversion to sensory inputs; supplying behavior outputs to said controlled object based on results of learning performed on said sensory inputs; evaluating behavior of the controlled object caused by said behavior outputs; storing said behavior outputs in one of a plurality of columns in association with corresponding sensory inputs based on the evaluation; computing probabilistic models based on the behavior outputs stored in said columns, wherein said probabilistic model is probabilistic relationship that a sensory input belongs to each column; calculating confidence for each column by applying newly given sensory inputs to said probabilistic models, and outputting, as said results of learning, behavior outputs in association with newly given sensory inputs in the column having largest confidence.", "7.An agent learning method for performing optimal control for a controlled object, comprising: capturing external environmental information for conversion to sensory inputs; supplying behavior outputs to said controlled object based on results of learning performed on said sensory inputs; evaluating behavior of the controlled object caused by said behavior outputs; storing said behavior outputs in one of a plurality of columns in association with corresponding sensory inputs based on the evaluation; computing probabilistic models based on the behavior outputs stored in said columns, wherein said probabilistic model is probabilistic relationship that a sensory input belongs to each column; and outputting, as said results of learning, behavior outputs in association with newly given sensory inputs in the column, said column containing the behavior outputs having largest evaluation.", "8.The agent learning method of claim 6 or 7, said computing probabilistic model comprising: representing behavior outputs stored in columns as normal distribution by using Expectation Maximization algorithm; using said normal distribution to compute a priori probability that a behavior output is contained in each column; and using said a priori probability to compute said probabilistic model by supervised learning with neural network, said probabilistic model being probabilistic relationship between any sensory input and each column.", "9.The agent learning method of claim 8, said confidence being calculated by applying said a priori probability and said probabilistic model to Bayes' rule.", "10.The agent learning method of claim 9, wherein said probabilistic model is computed in advance using data sets of relationship between sensory inputs and behavior outputs, wherein after computing said probabilistic model, said confidence is calculated using the probabilistic model for newly given sensory inputs.", "11.An agent learning program for performing optimal control for a controlled object, comprising: capturing external environmental information for conversion to sensory inputs; supplying behavior outputs to said controlled object based on results of learning performed on said sensory inputs; evaluating behavior of the controlled object caused by said behavior outputs; storing said behavior outputs in one of a plurality of columns in association with corresponding sensory inputs based on the evaluation; computing probabilistic models based on the behavior outputs stored in said columns, wherein said probabilistic model is probabilistic relationship that a sensory input belongs to each column; calculating confidence for each column by applying newly given sensory inputs to said probabilistic models; and outputting, as said results of learning, behavior outputs in association with newly given sensory inputs in the column, having largest confidence.", "12.An agent learning program when executing on a computer to realize optimal control for a controlled object, comprising: capturing external environmental information for conversion to sensory inputs; supplying behavior outputs to said controlled object based on results of learning performed on said sensory inputs; evaluating behavior of the controlled object caused by said behavior outputs; storing said behavior outputs in one of a plurality of columns in association with corresponding sensory inputs based on the evaluation; computing probabilistic models based on the behavior outputs stored in said columns, wherein said probabilistic model is probabilistic relationship that a sensory input belongs to each column; and outputting, as said results of learning, behavior outputs in association with newly given sensory inputs in the column, said column containing the behavior outputs having largest evaluation.", "13.The agent learning program of claim 11 or 12, said computing probabilistic model comprising: representing behavior outputs stored in columns as normal distribution by using Expectation Maximization algorithm; using said normal distribution to compute a priori probability that a behavior output is contained in each column; and using said a priori probability to compute said probabilistic model by supervised learning with neural network, said probabilistic model being probabilistic relationship between any sensory input and each column.", "14.The agent learning program of claim 13, said confidence being calculated by applying said a priori probability and said probabilistic model to Bayes' rule.", "15.The agent learning program of claim 14, wherein said probabilistic model is computed in advance using data sets of relationship between sensory inputs and behavior outputs, wherein after computing said probabilistic model, said confidence is calculated using the probabilistic model for newly given sensory inputs." ], [ "<SOH> BACKGROUND ART <EOH>Examples of the conventional learning scheme include a supervised learning scheme for minimizing an error between model control path by the time-series representation given by an operator and predicted path (Gomi.", "H. and Kawato.", "M., Neural Network Control for a Closed-Loop System Using Feedback-Error-Learning, Neural Networks, Vol.", "6, pp.", "933-946, 1933).", "Another example is a reinforcement learning scheme, in which optimal path is acquired by iterating try and error process in given environment for control system without model control path (Doya.", "K., Reinforcement Learning In Continuous Time and Space, Neural Computation, 2000).", "However, since the environment surrounding the control system is changing constantly in real world, it is difficult for the operator to keep giving model control path to the control system and therefore such a supervised learning scheme cannot be applied.", "In the latter learning scheme, there is a problem that it takes much time for the control system to acquire the optimal path by iterating try and error process.", "Thus, it is difficult to employ the aforementioned learning schemes for controlling an object (e.g.", "a helicopter) which requires to be controlled rapidly and precisely responsive to the environment.", "On the other hand, recent research on human control mechanism proves that the human control mechanism focuses on time-series “smoothness” of behavior outputs determined by non-linear approximations of control system based on sensory inputs and symmetric nature of behavior outputs in statistical normal distribution, and acquires control path statistically and very rapidly for minimizing the variance of the behavior outputs by selecting the sensory inputs to be paid attention (Harris.", "M. C., Signal-dependent noise determines motor planning, Nature, Vol.", "394, 20 August, 1998).", "In the cognitive science field, it is considered that human being has a mechanism to realize rapid and efficient control by consciously selecting necessary information out of massive sensory information.", "It has been suggested that this mechanism should be applied to engineering, but no concrete model has been proposed to apply this mechanism to engineering.", "Therefore, it is an objective of the invention to provide an agent learning apparatus, method and program for acquiring optimal control path rapidly." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.", "1 shows a graph of an exemplary time-series data of behavior outputs; FIG.", "2 shows a histogram of the time-series data in FIG.", "1 ; FIG.", "3 shows a functional block diagram of an agent learning apparatus in advance learning stage according to the invention; FIG.", "4 shows a functional block diagram of an agent learning apparatus in behavior control stage according to the invention; FIG.", "5 is a flowchart illustrating operation of the agent learning apparatus in advance learning stage; FIG.", "6 is an example of a normal distribution curved surface showing relationship between sensory inputs and behavior outputs, which are stored in the column corresponding to “stable” reward; FIG.", "7 is an example of a normal distribution curved surface showing relationship between sensory inputs and behavior outputs, which are stored in the column corresponding to “unstable” reward; FIG.", "8 shows an example of hierarchical neural network for learning the relationship between sensory inputs and attention class; FIG.", "9 is a flowchart illustrating operation of the agent learning apparatus in behavior control stage; FIG.", "10 shows configuration of helicopter control system according to the invention; FIG.", "11 shows learning result of relationship between visual sensory inputs and attention class in the system shown in FIG.", "10 ; and FIG.", "12 shows a graph of the variance of the behavior outputs for the controlled helicopter over time when the control is executed in FIG.", "6 .", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The invention relates to an agent learning apparatus, method and program.", "More specifically, the invention relates to an agent learning apparatus, method and program for implementing the rapid and highly adaptive control for non-linear or non-stationary targets or physical system control such as industrial robots, automobiles, and airplanes with high-order cognitive control mechanism.", "BACKGROUND ART Examples of the conventional learning scheme include a supervised learning scheme for minimizing an error between model control path by the time-series representation given by an operator and predicted path (Gomi.", "H. and Kawato.", "M., Neural Network Control for a Closed-Loop System Using Feedback-Error-Learning, Neural Networks, Vol.", "6, pp.", "933-946, 1933).", "Another example is a reinforcement learning scheme, in which optimal path is acquired by iterating try and error process in given environment for control system without model control path (Doya.", "K., Reinforcement Learning In Continuous Time and Space, Neural Computation, 2000).", "However, since the environment surrounding the control system is changing constantly in real world, it is difficult for the operator to keep giving model control path to the control system and therefore such a supervised learning scheme cannot be applied.", "In the latter learning scheme, there is a problem that it takes much time for the control system to acquire the optimal path by iterating try and error process.", "Thus, it is difficult to employ the aforementioned learning schemes for controlling an object (e.g.", "a helicopter) which requires to be controlled rapidly and precisely responsive to the environment.", "On the other hand, recent research on human control mechanism proves that the human control mechanism focuses on time-series “smoothness” of behavior outputs determined by non-linear approximations of control system based on sensory inputs and symmetric nature of behavior outputs in statistical normal distribution, and acquires control path statistically and very rapidly for minimizing the variance of the behavior outputs by selecting the sensory inputs to be paid attention (Harris.", "M. C., Signal-dependent noise determines motor planning, Nature, Vol.", "394, 20 August, 1998).", "In the cognitive science field, it is considered that human being has a mechanism to realize rapid and efficient control by consciously selecting necessary information out of massive sensory information.", "It has been suggested that this mechanism should be applied to engineering, but no concrete model has been proposed to apply this mechanism to engineering.", "Therefore, it is an objective of the invention to provide an agent learning apparatus, method and program for acquiring optimal control path rapidly.", "DISCLOSURE OF INVENTION According to the invention, a selective attention mechanism is devised for creating non-observable information (attention classes) by learning and for associating sensory inputs with the attention classes.", "With this mechanism, optimal control path for minimizing the variance of the behavior outputs may be acquired rapidly.", "An agent learning apparatus according to the invention comprises a sensor for capturing external environmental information for conversion to sensory inputs, and a behavior controller for supplying behavior outputs to a controlled object based on results of learning performed on said sensory inputs.", "The apparatus further comprises a behavior status evaluator for evaluating behavior of the controlled object caused by said behavior outputs.", "The apparatus further comprises a selective attention mechanism for storing said behavior outputs in one of a plurality of columns in association with corresponding sensory inputs based on the evaluation, computing probabilistic models based on the behavior outputs stored in said columns, calculating confidence for each column by applying newly given sensory inputs to said probabilistic models, and outputting, as said results of learning, behavior outputs in association with said newly given sensory inputs in the column having largest confidence.", "The probabilistic model is probabilistic relationship that a sensory input belongs to each column.", "By such configuration, the agent learning apparatus may be applied to initiate controlling an object without advance learning.", "In this case, the instability of controlled object is large before computing the probabilistic models and the object may be damaged or so by unexpected motion of the object.", "Therefore, range of behavior outputs given to the object by the behavior controller is preferably limited forcefully for a predetermined period.", "Instead of selecting a column having largest confidence for a given sensory input, a column containing the behavior outputs having largest evaluation by the behavior status evaluator may be always selected and a behavior output in association with newly given sensory inputs in that column may be outputted.", "The computing probabilistic model comprises representing behavior outputs stored in columns as normal distribution by using Expectation Maximization algorithm, using said normal distribution to compute a priori probability that a behavior output is contained in each column, and using said a priori probability to compute said probabilistic model by supervised learning with neural network.", "The probabilistic model is probabilistic relationship between any sensory input and each column.", "Specifically, the probabilistic model may be conditional probabilistic density function p(Ii(t)\|Q1).", "The confidence may be calculated by applying the a priori probability and the probabilistic model to Bayes' rule.", "The confidence is the probability that a sensory input belongs to each attention class (column).", "As described above, controlling the object may be initiated without advance learning.", "However, it is preferable that data sets of relationship between sensory inputs and behavior outputs are prepared and probabilistic models are computed in advance by performing advance learning with the data sets.", "After computing the probabilistic models, confidence is calculated using the probabilistic model for newly given sensory inputs.", "In this case, probabilistic models same with those computed in advance learning stage are continued to be used.", "Therefore, the object may be stabilized more rapidly.", "When performing advance learning, sensory inputs are converted into behavior outputs by a behavior output generator based on the data sets and supplied to the object.", "BRIEF DESCRIPTION OF DRAWINGS FIG.", "1 shows a graph of an exemplary time-series data of behavior outputs; FIG.", "2 shows a histogram of the time-series data in FIG.", "1; FIG.", "3 shows a functional block diagram of an agent learning apparatus in advance learning stage according to the invention; FIG.", "4 shows a functional block diagram of an agent learning apparatus in behavior control stage according to the invention; FIG.", "5 is a flowchart illustrating operation of the agent learning apparatus in advance learning stage; FIG.", "6 is an example of a normal distribution curved surface showing relationship between sensory inputs and behavior outputs, which are stored in the column corresponding to “stable” reward; FIG.", "7 is an example of a normal distribution curved surface showing relationship between sensory inputs and behavior outputs, which are stored in the column corresponding to “unstable” reward; FIG.", "8 shows an example of hierarchical neural network for learning the relationship between sensory inputs and attention class; FIG.", "9 is a flowchart illustrating operation of the agent learning apparatus in behavior control stage; FIG.", "10 shows configuration of helicopter control system according to the invention; FIG.", "11 shows learning result of relationship between visual sensory inputs and attention class in the system shown in FIG.", "10; and FIG.", "12 shows a graph of the variance of the behavior outputs for the controlled helicopter over time when the control is executed in FIG.", "6.BEST MODE FOR CARRYING OUT THE INVENTION First, preliminary experiment is described using a radio-controlled helicopter (hereinafter simply referred to as a “helicopter”) shown in FIG.", "10, which will be described later.", "FIG.", "1 is a graph of the time-series data on outputs of control motor for the helicopter acquired every 30 milliseconds when the helicopter was operated to maintain stability.", "FIG.", "2 is a histogram of that data.", "As shown in FIG.", "2, control outputs for stabilizing the helicopter (hereinafter referred to as “behavior output”) may be represented in a normal distribution curve.", "To realize a stable control for various controlled objects, attention should be paid on symmetric nature of such normal distribution of the behavior outputs of the controlled objects.", "This is because most frequent behavior outputs on the normal distribution may be expected to be heavily used for realizing stability of the controlled object.", "Therefore, through the use of the symmetric nature of the normal distribution, behavior outputs to be supplied to the controlled object under ever-changing environment may be statistically predicted.", "When selecting behavior outputs supplied to the controlled object based on sensory inputs captured by a sensor or the like, the number of selectable behavior outputs may be unlimited.", "However, if learning is performed such that the variance of behavior result of the controlled object caused by supplied behavior output (hereinafter simply referred to as “behavior result”) is decreased over time, the range of selectable behavior outputs based on the captured sensory inputs come to be limited, and resultantly the controlled object will be stabilized.", "In other words, by minimizing the variance for a normal distribution of behavior outputs, stable control with lowest width or rate of fluctuation is realized.", "The agent learning apparatus according to the invention is characterized in that both statistical learning schemes based on such preliminary experiment and conventional supervised learning scheme are employed synthetically.", "Now, preferable embodiments of the invention will be described with reference to FIGS.", "1 to 12.The agent learning apparatus 100 according to the invention performs learning with prepared data sets, for example.", "Such process is referred to as “advance learning stage” herein.", "FIG.", "3 shows a functional block diagram of an agent learning apparatus 100 according to one embodiment of the invention in advance learning stage.", "The agent learning apparatus 100 is illustrated in the area enclosed with dotted lines in FIG.", "3, including one or more sensors 301, a behavior output generator 302, a behavior status evaluator 303 and a selective attention mechanism 304.The selective attention mechanism 304 includes a plurality of columns 1, 2, 3, .", ".", ".", ", m created according to rewards produced by the behavior status evaluator 303.The selective attention mechanism 304 also includes an attention class selector 306.To the sensory inputs captured by the sensor 301, the behavior output generator 302 generates behavior outputs based on the data sets and supplies them to a controlled object 308.The behavior status evaluator 303 evaluates the behavior result of the controlled object 308 to generate rewards for behavior outputs one by one.", "The selective attention mechanism 304 distributes the behavior outputs to one of columns according to each reward to create probabilistic models described later.", "Creating probabilistic models in advance enables high-accurate control.", "After the advance learning stage is completed, the agent learning apparatus 100 performs a process which is referred to as “behavior control stage” herein.", "FIG.", "4 shows a functional block diagram of an agent learning apparatus 100 according to one embodiment of the invention in behavior control stage.", "In behavior control stage, new sensory inputs captured by sensor 301 are provided directly to the attention class selector 306.The attention class selector 306 performs some process to the sensory inputs using the probabilistic model computed in advance.", "A behavior controller 307 determines behavior outputs for stabilizing the controlled object 308 and supplies them to the controlled object 308.An example of the controlled object 308 is a helicopter as noted above.", "It should be noted that advance learning stage is not necessary.", "Operation of the agent learning apparatus in such case without advance learning will be described later.", "All or part of the behavior output generator 302, the behavior status evaluator 303, the selective attention mechanism 304 and the behavior controller 307 may be implemented by, for example, executing on a general purpose computer a program configured to realize functionality of them.", "Features of each functional block and operation of the agent learning apparatus 100 in advance learning stage is described referring to flowcharts in FIGS.", "3 and 5.External environment information is captured by a sensor 301 at given interval and converted into signals as sensory inputs Ii(t) (i=1, 2, .", ".", ".", ", m), which are supplied to a behavior output generator 302.The behavior output generator 302 generates behavior outputs Qi(t) corresponding to the supplied sensory inputs Ii(t) and supplies them to a behavior status evaluator 303 and a controlled object 308.The transformation between the sensory inputs Ii(t) and the behavior outputs Qi(t) is represented by the following mapping f. f: Ii(t)Qi(t) (1) The mapping f is, for example, a non-linear approximation transformation using well-known Fourier series or the like.", "In advance learning according the embodiment, the mapping f corresponds to preparing random data sets which includes the mapping between sensory inputs Ii(t) and behavior outputs Qi(t).", "In other words, the behavior output generator 302 generates behavior outputs Qi(t) one by one corresponding to each sensory input Ii(t) based on these data sets (step S401 in FIG.", "5).", "Generated behavior outputs Qi(t) are supplied to the behavior status evaluator 303 and the controlled object 308.The controlled object 308 will work in response to the supplied behavior outputs Qi(t).", "The result of this work is supplied to the behavior status evaluator 303 (step S402 in FIG.", "5).", "The behavior status evaluator 303 then evaluates the result of this work (for example, behavior of the controlled object gets stable or not) with predetermined evaluation function and generates reward for every behavior outputs Qi(t) (step S403 in FIG.", "5).", "Such process in the behavior status evaluator 303 is considered as reinforcement learning.", "The evaluation function herein is a function that yields reward “1” if the controlled object gets stable by the supplied behavior output Qi(t) or yields reward “2” otherwise.", "Type of rewards may be selected in consideration of behavior characteristics of the controlled object 308 or the required control accuracy.", "When using the helicopter noted above, reward “1” or reward “2” is yielded according to whether the helicopter is stable or not, which may be judged on, for example, its pitching angle detected with gyro-sensor on the helicopter.", "Evaluation function is used to minimize the variance σ of the behavior outputs Qi(t).", "In other words, by using the evaluation function, sensory inputs Ii(t) unnecessary for stable control may be removed and necessary sensory inputs Ii(t) are reinforced.", "Finally, reinforcement learning satisfying σ(Q1)<σ(Q2) is attained.", "Q1 is a group of the behavior outputs Qi(t) which are given reward “1” and Q2 is a group of the behavior outputs Qi(t) which are given reward On receiving rewards from the behavior status evaluator 303, the selective attention mechanism 304 creates a plurality of columns 1, 2, 3, .", ".", ".", ", m in response to the type of the rewards.", "Then the selective attention mechanism 304 distributes the behavior outputs Qi(t) to each column (step S404 in FIG.", "5).", "In each column, behavior outputs Qi(t) are stored by rewards, being associated with the sensory inputs Ii(t) which cause that behavior outputs.", "More specifically, when the behavior status evaluator 303 generates either reward “1” or reward “2” for example, the selective attention mechanism 304 creates column 1 and column 2.Then the behavior outputs Qi(t) which are given reward “1” are stored in the column 1 (stable) and the behavior outputs Qi(t) which are given reward “2” are stored in the column 2 (unstable).", "Thus the columns 1, 2, 3, .", ".", ".", ", m correspond to cluster models of behavior outputs Qi(t) distributed by the rewards.", "The selective attention mechanism 304 performs expectation maximization (EM) algorithm and supervised learning with neural network, both will be described later, to calculate conditional probabilistic distribution (that is, probabilistic model) p(Ii(t)\|Ωl) (steps S405-S408 in FIG.", "4) for sensory inputs Ii(t).", "Ωl (l=1, 2, 3, .", ".", ".", ", n) is a parameter called “attention class” and corresponds to a column one by one.", "It should be noted that such attention class Ωl is created under the assumption that true probabilistic distribution p(Ii(t)\|Ωl) will exist.", "The attention class Ωl is used to select noticeable sensory inputs Ii(t) from among massive sensory inputs Ii(t).", "More specifically, attention class Ωl is a parameter used for modeling the behavior outputs Ωi(t) stored in each column using the probabilistic density function of the normal distribution of behavior outputs Qi(t).", "Attention classes Ωl are created as many as the number of the columns storing these behavior outputs Qi(t).", "Calculating the attention class Ωl corresponding to the behavior outputs Qi(t) stored in each column is represented by the following mapping h. h: Qi(t)Ωl(t) (2) Processes in steps S405-S408 in FIG.", "5 will be next described in detail.", "It should be noted that each process in steps S405-S408 is performed on every column.", "First, Expectation Maximization algorithm (EM algorithm) in step S405 is described.", "The EM algorithm is an iterative algorithm for estimating parameter θ which takes maximum likelihood when observed data is viewed as incomplete data.", "As noted above, since it is considered that the behavior outputs Qi(t) stored in each column appear to be the normal distribution, the parameter θ may be represented as θ(μl,Σl) with mean μl and covariance Σl.", "EM algorithm is initiated with appropriate initial values of θ(μl, Σl).", "Then the parameter θ(μl, Σl) is updated one after another by iterating Expectation (E) step and Maximization (M) step alternately.", "On the E step, conditional expected value φ(θ\|θ(k)) is calculated by following equation.", "φ ⁡ ( θ ❘ θ ( k ) ) = ∑ i ⁢ ∑ l ⁢ p ⁡ ( Q i l ⁡ ( t ) ❘ Ω l ; θ ( k ) ) ⁢ log ⁡ ( p ⁡ ( Q i l ⁡ ( t ) , Ω l ; θ ( k ) ) ) ( 3 ) Then on the M step, parameters μl and Σl for maximizing φ(θ\|θ(k)) is calculated by following equation and are set to a new estimated value θ(k+1).", "θ(k+1)=arg maxθφ(θ,θ(k)) (4) By partial differentiating the calculated φ(θ\|ƒ(k)) on θ(k) and letting a result equal to zero, parameters μl and Σl may be finally calculated.", "More detailed explanation will be omitted because this EM algorithm is well known in the art.", "Thus, behavior outputs Qi(t) stored in each column may be represented by normal distribution (step S405 in FIG.", "5).", "Calculating μl and Σl for the behavior outputs Qi(t) corresponds to calculating a posteriori probability for attention class Ωl.", "FIGS.", "6 and 7 show examples of normal distribution of behavior outputs Qi(t) included in column 1 (stable) or column 2 (unstable) respectively.", "As is apparent from the figures, the normal distribution of column 1 has shaper end of graph than that of column 2 and thus the variance of behavior outputs Qi(t) is smaller (σ(Q1)<σ(Q2)).", "A priori probability {overscore (p)}(Qil(t)\|Ωl(t)) of attention class Ωl is calculated by following equation with the calculated parameters μl and Σl (step S406 in FIG.", "5).", "p _ ⁡ ( Q i l ⁡ ( t ) ❘ Ω l ⁡ ( t ) ) = ∑ j = 1 J ⁢ α jQ i l ⁡ ( t ) ( 2 ⁢ π ) N / 2 ⁢  ∑ jQ i l ⁡ ( t ) ⁢  ⁢ exp ⁡ ( - 1 2 ⁢ ( Q i l ⁡ ( t ) - μ ) T ⁢ ∑ jQ i l ⁡ ( t ) - 1 ⁢ ( Q i l ⁡ ( t ) - μ ) ) ( 5 ) where N is dimension of the behavior outputs Qi(t).", "Supervised learning with neural network is described below.", "In this learning, conditional probabilistic density function p(Ii(t)\|Ωl) is calculated with the attention class Ωl, which has been calculated as a posteriori probability, as supervising signal (step S407 in FIG.", "5).", "FIG.", "8 shows the exemplary structure of hierarchical neural network used for the supervised learning with neural network.", "This hierarchical neural network is composed of three layers of nodes.", "The input layer 501 corresponds to sensory inputs Ii(t), middle layer 502 to behavior outputs Qi(t), and output layer 503 to attention classes Ωl, respectively.", "Although only three nodes are illustrated at the input layer 501 for simple illustration, there are actually as many nodes as the number of sensory inputs Ii(t) in the data sets.", "Likewise, in the middle layer 502, behavior output nodes Qi(t) exist as many as nodes in the input layer 501.Nodes in the middle layer 502 correspond to nodes in the input layer one by one.", "Nodes in the output layer 503 are created as many as the number of the attention classes Ωl.", "λ shown in FIG.", "8 denotes synaptic weight matrices of the hierarchical neural network.", "Since a probability that a behavior outputs Qi(t) belong to its each attention classes Ωl is computed by EM algorithm, and behavior outputs Qi(t) are stored in the column in a one-to-one correspondence with sensory inputs Ii(t) probabilistic relationship (that is, λ in FIG.", "8) between sensory inputs Ii(t) and attention classes Ωl are determined by repeating the supervised learning with attention class Ωl as a teacher signal.", "This probabilistic relationship is conditional probabilistic density function p(Ii(t)\|Ωl).", "It should be noted that the attention class Ωl may be calculated from sensory inputs Ii(t) through synthetic mapping h·f.", "More detailed explanation will be omitted because such hierarchical neural network is well known in the art.", "With such supervised learning using neural network, conditional probabilistic density function p(Ii(t)\|Ωl), which is probabilistic relationship between sensory inputs Ii(t) and attention class Ωl, may be computed.", "As noted above, learning within the selective attention mechanism 304 on advance learning stage proceed in feed back fashion.", "After the conditional probabilistic density function p(Ii(t)\|Ωl) is calculated, the probability may be determined of which attention class Ωl a new sensory input Ii(t) belongs to without calculating the mapping h·f at each time for that sensory input.", "Processes in steps S401 to S407 are performed on every pair of sensory input Ii(t) and behavior output Qi(t) in given data sets (step S408 in FIG.", "5).", "During the advance learning stage, conditional probabilistic distribution p(Ii(t)\|Ωl) continues to be updated in response to given behavior outputs Qi(t).", "An explanation of how the agent learning apparatus 100 operates in advance learning stage is finished.", "After the advance learning stage using the data sets is completed, the agent learning apparatus 100 starts to control the controlled object 308 based on the established learning result.", "Now it will be described below how the agent learning apparatus 100 operates on behavior control stage referring to FIGS.", "4 and 9.In behavior control stage, a priori probability {overscore (p)}(Qil(t)\|Ωl(t)) for each column and conditional probabilistic distribution p(Ii(t)\|Ωl), both has been calculated in advance learning stage, are used.", "New sensory inputs Ii(t) captured at sensor 301 are provided to the attention class selector 306 in the selective attention mechanism 304 (step S410 in FIG.", "9).", "Then using a priori probability {overscore (p)}(Qil(t)\|Ωl(t)) and conditional probabilistic distribution p(Ii(t)\|Ωl), confidence p(Ωl(t)) for each attention class Ωl is calculated by following Bayes' rule (step S411 in FIG.", "9).", "p ⁡ ( Ω l ⁡ ( t ) ) = p _ ⁡ ( Q i l ⁡ ( t ) ❘ Ω l ⁡ ( t ) ) ⁢ p ⁡ ( I i ⁡ ( t ) ❘ Ω l ⁡ ( t ) ) ∑ k ⁢ p _ ⁡ ( Q i l ⁡ ( t ) ❘ Ω k ⁡ ( t ) ) ⁢ p ⁡ ( I i ⁡ ( t ) ❘ Ω k ⁡ ( t ) ) ( 6 ) The confidence p(Ωl(t)) is the probability that a sensory input Ii(t) belongs to each attention class Ωl(t).", "Calculating the probability that a sensory input Ii(t) belongs to each attention class Ωl(t) with Bayes' rule means that one attention class may be identified selectively by increasing the confidence p(Ωl(t)) with learning of Bayes' rule (weight).", "In other words, with selective attention mechanism 304, the attention class Ωl, or hidden control parameter, may be directly identified based on observable sensory input Ii(t).", "The attention class selector 306 determines that the attention class Ωl with highest confidence p(Ωl(t)) is an attention class corresponding to the new sensory input Ii(t).", "The determined attention class Ωl is informed to the behavior controller 307 (step S412 in FIG.", "9).", "When informed attention class Ωl is Ωl corresponding to “stable” column, the behavior controller 307 calculates a behavior output Qi(t) corresponding to a captured sensory input Ii(t) based on the behavior outputs Qi(t) stored in the column 1 (step S413 in FIG.", "9), then provides it to the controlled object 308 (step S414).", "It should be noted that these behavior outputs Qi(t) may be calculated on the probabilistic distribution calculated by EM algorithm and not behavior outputs Qi(t) itself which was given as data sets in advance learning stage.", "When informed attention class Ωl is Ω2 corresponding to “unstable” column, the behavior controller 307 selects not column 2 but column 1 having smaller variance, and calculates a behavior output Qi(t) corresponding to a captured sensory input Ii(t) based on the behavior outputs Qi(t) stored in column 1, then provides it to the controlled object 308 (S414).", "If no corresponding behavior output is stored, last behavior output is supplied.", "If no behavior output Qi(t) associated with the sensory input Ii(t) is stored in the column, previous behavior output Qi(t) is selected and provided to the controlled object 308.By repeating such process, relation between variance of columns σ(Q1)<σ(Q2) is accomplished (that is, variance of behavior outputs in column 1 gets smaller rapidly and the stability of controlled object 308 is attained).", "Alternatively, when supplied attention class Ω1 is Ω2 which corresponds to “unstable”, the behavior controller 307 may select column 2, calculate behavior outputs Qi(t) corresponding to captured sensory inputs Ii(t) from among behavior outputs Qi(t) stored in column 2 having smaller variance, and supply them to the controlled object 308.The controlled object 308 exhibits behavior according to the provided behavior output Qi(t).", "The result of this behavior is provided to the behavior status evaluator 303 again.", "Then, when new sensory input Ii(t) is captured by sensor 301, attention class is selected using conditional probability density function p(Ii(t)\|Ωl) based on learning by Bayes' rule.", "After that, processes described above are repeated (S415).", "An explanation of how the agent learning apparatus 100 operates in behavior control stage is finished.", "In this embodiment, since conditional probabilistic density function p(Ii(t)\|Ωl) is calculated in the advance learning stage, the attention class Ωl may be directly selected corresponding to new sensory input Ii(t) using statistical learning in behavior control stage without computing mapping f and h. Generally, the information amount of sensory inputs Ii(t) from the sensor 301 is enormous.", "Therefore, if mapping f and h are calculated for all sensory inputs Ii(t), its computing amount far exceeds the processing capacity of the typical computer.", "Thus, according to the invention, appropriate filtering for sensory inputs Ii(t) with the attention classes Ωl may improve the efficiency of the learning.", "In addition, selecting attention class Ωl with highest confidence p(Ωl(t)) corresponds to selecting column which includes behavior output Qi(t) with highest reward for a sensory input Ii(t).", "Learning process is performed three times in this embodiment.", "That is, 1) reinforcement learning in the behavior status evaluator 303 (in other words, generation of cluster models by rewards), 2) learning of the relationship between attention classes Ωl and sensory inputs Ii(t) using the hierarchical neural network, and 3) selection of attention class corresponding to new sensory input Ii(t) with Bayes' rule.", "Thus, the agent learning apparatus 100 according to the invention is characterized in that supervised learning scheme and statistical learning scheme are synthetically applied.", "In conventional supervised learning scheme, optimal control given by an operator is learned by a control system, but this is not practical as noted above.", "In conventional reinforcement learning scheme, optimal control is acquired through try and error process of a control system, but this takes much processing time.", "In contrast, the agent learning apparatus 100 according to the invention can select the attention class with the selective attention mechanism 304 and learn the important sensory inputs Ii(t) selectively, resulting to reduce the processing time and to eliminate the need of the supervising information given by an operator.", "Furthermore, if the motion of the controlled object 308 has non-linear characteristics, it takes much time for learning with only the reinforcement learning scheme because complicated non-linear function approximation is required.", "On the other hand, since the agent learning apparatus 100 of the invention learns the sensory inputs according to their importance with the selective attention mechanism 304, the processing speed is improved.", "The agent learning apparatus 100 is also characterized in that feed back control is performed in advance learning stage and feed forward control is performed in behavior control stage.", "Now referring to FIG.", "10, one example of the invention is described in detail.", "FIG.", "10 shows a schematic view of a control system for a radio-controlled helicopter 601 applying an agent learning apparatus 100 of the invention.", "Mounted on a helicopter 601 is a vision sensor 602, which captures visual information every 30-90 milliseconds and sends them to a computer 603 as sensory inputs Ii(t).", "The computer 603 is programmed to implement the agent learning apparatus 100 shown in FIG.", "3 or FIG.", "4 and generates behavior outputs Qi(t) for the sensory inputs Ii(t) according to the invention.", "The behavior outputs Qi(t) are sent to a motor control unit 605 mounted on the helicopter 601 via a radio transmitter 604, rotating a rotor on the helicopter.", "In this example, the number of the attention classes Ωl is set at two.", "Total 360 data sets for advance learning were used for processing in selective attention mechanism 304.After the advance learning was over, it was confirmed whether the system could select correct attention class Ωl when another 150 test data (new sensory inputs Ii(t)) were provided to the system.", "In advance learning stage, two types of rewards (positive rewards and negative rewards) are assigned to behavior outputs Qi(t).", "The selective attention mechanism 304 distributes the behavior outputs Qi(t) to column 1 or column 2 according to their rewards.", "This process is represented by the following evaluation function.", "If \|{tilde over (Q)}i−Qi\|≦δ then Qi1Qi(Positive) else Qi2=Qi(Negative) where Q1 or Q2 representes a group of behavior outputs Qi(t) distributed to column 1 or column 2 respectively.", "In this case, the positive reward corresponds to column 1 and the negative reward correspond to column 2.", "{tilde over (Q)}i denotes the behavior output Qi(t) to keep the helicopter stable.", "{tilde over (Q)}i is a mean value of a probabilistic distribution p(Qi) and set to “82” in this example.", "δ is a threshold that represents a tolerance of stability and set to “1.0” in this example.", "The evaluation function shown above acts as reinforcement learning satisfying the relation between the variances of column σ(Q1)<σ(Q2).", "FIGS.", "11A to 11C show the relationship between the sensory inputs Ii(t) and the attention class Ωl, which was acquired from the experiment using the system in FIG.", "10.It should be noted that the actual attention class could be calculated by the data sets.", "FIG.", "11A is a diagram of the correct attention class Ωl corresponding to sensory inputs Ii(t).", "FIG.", "11B is a diagram of the experiment result when the iteration number is small (early stage) in the EM algorithm.", "FIG.", "11C is a diagram of the experiment result when the iteration number is sufficient (late stage).", "Solid lines in each figure represent that the attention class Ωl selected at its time point is shifted from one to another.", "In other words, during time steps where no solid line is shown in each figure, same attention class Ωl keeps on being selected corresponding to either column 1 or column 2.It is apparent that the diagram in late stage (FIG.", "11C) is similar to the diagram of actual attention class (FIG.", "11A) than that in early stage (FIG.", "11B).", "These results prove that the agent learning apparatus 100 according to the invention can learn the predictive relationship between the sensory inputs Ii(t) and the two attention classes Ωl.", "In other words, the result suggests that the discriminative power of the prediction between sensory inputs Ii(t) and two attention classes is “weak” when the probabilistic distribution for statistical column is in early stage of EM algorithm.", "As the iteration number in the EM algorithm is increased, the accuracy of the prediction is improved.", "The discriminative power of the prediction may be affected by the number of the normal distribution (Gaussian function) used in the EM algorithm.", "Although single Gaussian function is used in the aforementioned embodiments, mixture of Gaussian functions may be used in the EM algorithm to improve the discriminative power of the prediction.", "FIG.", "12 shows a graph of the minimum variance value of the behavior outputs Qi(t) over time when the helicopter 601 in the example is controlled.", "A dashed line represents the result when the helicopter 601 is controlled by the conventional feedback control, while a solid line represents the result when the helicopter 601 is controlled by the agent learning apparatus 100 according to the invention.", "The conventional methods have no learning process by the selective attention mechanism 304.Therefore, since it is learned through try and error process which visual information (that is, sensory inputs Ii(t)) are needed for stabilizing the helicopter 601 among sensory inputs Ii(t) captured by the helicopter 601, it takes much time until the variance becomes small, in other words, the helicopter 601 gets stabilized.", "In contrast, the agent learning apparatus 100 according to the invention acquires the sensory inputs Ii(t) needed for stabilizing the helicopter 601 not through try and error process but by the learning according to the importance of the sensory inputs Ii(t) using the selective attention mechanism 304.Therefore, minimization of the variance of the behavior outputs Qi(t) may be attained very rapidly.", "Although the vision sensor 602 is used in this example, sensory inputs Ii(t) is not limited to vision information but other input such as auditory information or tactile information may be also used.", "In addition, the example has been described for two columns and two rewards, but three or more columns and rewards may be used.", "Only one column will not accelerate the convergence of the learning because, if there is only one column, it will take much time until the normal distribution curve of behavior outputs Qi(t) contained in the column is sharpened and the variance gets small.", "One feature of the invention is that the normal distribution curve of behavior outputs Qi(t) is sharpened rapidly by generating a plurality of columns.", "The more columns are used, the more complicated and various behavior outputs may be obtained.", "In embodiments described above, a priori learning using data sets is performed.", "Such a priori learning is for stabilizing the controlled object 308 more rapidly.", "However, control for controlled object 308 (for example, a helicopter 601) may be started by applying the agent learning apparatus 100 without a priori learning.", "In this case, the behavior controller 307 supplies behavior outputs Qi(t) to the controlled object 308 in a random fashion irrespective of sensory inputs Ii(t) captured by the sensor because no probabilistic model described above is created during short period after control is started.", "The behavior status evaluator 303 supplies rewards to behavior results of the controlled object 308.The selective attention mechanism distributes behavior outputs Qi(t) to columns in association with sensory inputs Ii(t) according to the reward.", "Then, relationship between sensory inputs Ii(t) and behavior outputs Qi(t) is stored in columns according to rewards and normal distributions for behavior outputs stored in columns may be computed with EM algorithm, a priori probability {overscore (p)}(Qil(t)\|Ωl(t)) and conditional probability density function p(Ii(t)\|Ωl) may be computed according to processes described above.", "These values are applied to Bayes' rule to calculate confidence p(Ωl(t)) for each attention class.", "The behavior controller 307 computes behavior output Qi(t) corresponding to newly-captured sensory input Ii(t) based on column where confidence p(Ωl(t)) is maximum or where behavior outputs having best reward are stored and supply them to controlled object.", "Again, the behavior status evaluator supplies rewards to behavior results of the controlled object 308 and the computed behavior outputs Qi(t) are stored in one of columns.", "Based on this, a priori probability {overscore (p)}(Qil(t)\|Ωl(t)) and conditional probability density function p(Ii(t)\|Ωl) may be updated.", "Then, these updated probabilities are applied to Bayes' rule and new behavior output Qi(t) is output.", "In this way, without advance learning, a priori probability {overscore (p)}(Qil(t)\|Ωl(t)) and conditional probability density function p(Ii(t)\|Ωl) are updated one after another.", "In this case, the instability of controlled object is large when at the beginning of starting the control and the controlled object may be damaged or so by unexpected motion of the object.", "Therefore, range of behavior output Ql(t) given to the controlled object 308 by the behavior controller 307 is preferably limited forcefully until the predetermined number of the relationship between sensory inputs and behavior outputs are gained (alternatively, until predetermined period is lapsed).", "Furthermore, well-known competitive learning or learning with self-organized network may be employed instead of EM algorithm in step S405.Well-known belief network or graphical model may be employed instead of Bayes' rule in step S411.INDUSTRIAL APPLICABILITY As described above, according to the invention, variance of behavior outputs Qi(t) may be minimized rapidly to stabilize a controlled object by computing the behavior output based on a column which is estimated as stable." ] ]	Patent_10468316
[ [ "Chemical compounds", "This invention relates to polymorphisms in the human cMOAT gene and corresponding novel allelic polypeptides encoded thereby.", "The invention also relates to methods and materials for analysing allelic variation in the cMOAT gene, and to the use of cMOAT polymorphism in treatment of diseases with cMOAT transportable drugs." ], [ "1.A method for the diagnosis of a polymorphism in cMOAT in a human, which method comprises determining the sequence of the human at one or more of the following positions: positions 78, 1350, 1584, 1686, 2647, 3208, 3664 and 4391 in the coding region of the cMOAT gene as defined by the positions in SEQ ID NO: 1; positions 1349, 1875 and 1879 in the 5′UTR region of the cMOAT gene as defined by the positions in SEQ ID NO:2; positions 12704 and 29446 in the intron region of the cMOAT gene as defined by the positions in SEQ ID NO:3; positions 292 in the intron region of the cMOAT gene as defined by the position in SEQ ID NO:4; positions 232 and 457 in the intron region of the cMOAT gene as defined by the positions in SEQ ID NO:5; positions 50 and 68 in the intron region of the cMOAT gene as defined by the positions in SEQ ID NO:6; and positions 417, 495, 529, 849, 1036 and 1188 in the cMOAT polypeptide as defined by the positions in SEQ ID NO:7, and determining the status of the human by reference to the polymorphism in cMOAT.", "2.Use of a diagnostic method as defined in claim 1 to assess the pharmacogenetics of a drug transportable by cMOAT.", "3.A polynucleotide comprising at least 20 bases of the human cMOAT gene and comprising an allelic variant selected from any one of the following: Variant Region SEQ ID NO: 1 Coding 78 T 1350 A 1584 G 1686 T 2647 G 3208 C 3664 A 4391 G Region Variant SEQ ID NO: 2 5′UTR or promoter 1349 A 1875 A 1879 G Region Variant SEQ ID NO: 3 12704 T SEQ ID NO: 3 29446 T SEQ ID NO: 4 292 C SEQ ID NO: 5 232 G SEQ ID NO: 5 457 G SEQ ID NO: 6 50 A SEQ ID NO: 6 68 A 4.A nucleotide primer which can detect a polymorphism as defined in claim 1.5.An allele specific primer capable of detecting a cMOAT gene polymorphism as defined in claim 1.6.An allele-specific oligonucleotide probe capable of detecting a cMOAT gene polymorphism as defined in claim 1.7.Use of a cMOAT gene polymorphism as defined in claim 1 as a genetic marker in a linkage study.", "8.A method of treating a human in need of treatment with a drug transportable by cMOAT in which the method comprises: i) diagnosis of a polymorphism in cMOAT in the human, which diagnosis preferably comprises determining the sequence at one or more of the following positions: positions 78, 1350, 1584, 1686, 2647, 3208, 3664 and 4391 in the coding region of the cMOAT gene as defined by the positions in SEQ ID NO: 1; positions 1349, 1875 and 1879 in the 5′UTR region of the cMOAT gene as defined by the positions in SEQ ID NO:2; positions 12704 and 29446 in the intron region of the cMOAT gene as defined by the positions in SEQ ID NO:3; position 292 in the intron region of the cMOAT gene as defined by the position in SEQ ID NO:4; positions 232 and 457 in the intron region of the cMOAT gene as defined by the positions in SEQ ID NO:5; positions 50 and 68 in the intron region of the cMOAT gene as defined by the positions in SEQ ID NO:6; and positions 417, 495, 529, 849, 1036 and 1188 in the cMOAT polypeptide as defined by the positions in SEQ ID NO:7, and determining the status of the human by reference to the polymorphism in cMOAT; and ii) administering an effective amount of the drug.", "9.An allelic variant of human cMOAT polypeptide comprising at least one of the following: an isoleucine at position 417 at SEQ ID NO:7; a glutamic acid at position 495 of SEQ ID NO:7; a tryptophan at position 529 of SEQ ID NO:7; an arginine at position 849 of SEQ ID NO:7; a threonine at position 1036 of SEQ ID NO:7; a valine at position 1188 of SEQ ID NO:7; or a fragment thereof comprising at least 10 amino acids provided that the fragment comprises at least one allelic variant.", "10.An antibody specific for an allelic variant of human cMOAT polypeptide as defined in claim 9." ], [ "This invention relates to polymorphisms in the human cMOAT gene and corresponding novel allelic polypeptides encoded thereby.", "The invention also relates to methods and materials for analysing allelic variation in the cMOAT gene, and to the use of cMOAT polymorphism in treatment of diseases with cMOAT transportable drugs.", "The liver converts endogenous and xenobiotic lipophilic compounds into anionic conjugates with glutathione, glucuronate, or sulphate.", "These conjugates are transported across the canalicular (apical) membrane into bile by a 190 kDa membrane glycoprotein.", "This apical conjugate-transporting ATPase has been termed canalicular multidrug resistance protein (cMRP) because of the similarity in substrate specificity and sequence with the multidrug resistance protein (MRP1); other names include canalicular multispecific organic anion transporter (cMOAT) and multidrug resistance protein 2 (MRP2).", "The term “cMOAT” will generally be used herein.", "The amino acid sequence identity of human cMOAT and MRP1 is 49%.", "cMOAT is predominantly expressed in hepatocytes and localised to apical membrane domains.", "cMOAT is not expressed in the human Dubin-Johnson syndrome, which is therefore associated with an inherited deficiency in the secretion of amphiphilic anionic conjugates into the bile.", "The rat homolog CMOAT is absent in two mutant strains of rats with different point mutations in the corresponding gene.", "These mutant rats are hyperbilirubinemic and deficient in the ATP-dependent transport of conjugates from hepatocytes into bile.", "Impairment of bile flow (cholestasis) can be associated with a down-regulation of the expression of the conjugate export pump, and cMOAT contributes to bile flow as an important driving force (for reviews see Keppler D. and Konig J. FASEB J, 1997.11, 509-516 and Ishikawa et al.", "Biosci Rep 1997; 17:189-207).", "cMOAT has also been shown to be involved in the transport of xenobiotics, and drugs involved in lipid lowering e.g.", "statins.", "Statins have been refered to as a first-line therapy for patients with atherosclerotic vascular diseases.", "The cMOAT gene and its product is also thought to be of importance in other diseases.", "All positions herein of polymorphisms in the exon regions of the cMOAT polynucleotide relate to the position in SEQ ID NO 1 unless stated otherwise or apparent from the context.", "At the time of writing, EMBL U49248 is equivalent.", "All positions herein of polymorphisms in the 5′ UTR or promoter region of the cMOAT polynucleotide relate to the position in SEQ ID NO 2 unless stated otherwise or apparent from the context.", "At the time of writing, EMBL AF1446309 is equivalent.", "All positions herein of polymorphisms in the intron regions of the cMOAT polynucleotide relate to the position in SEQ ID NO 3, 4, 5 or 6 depending on the context used.", "At the time of writing, SEQ ID NO 3, is part of a bacterial artificial chromosome RP11-483F11 (Sanger Centre, Hinxton, Cambridgeshire, CB10 1SA, UK.", "); EMBL AL133353 wherein SEQ ID NO 3 position 1=position 129361 in published BAC sequence acc no AL133353.All positions herein of polymorphisms in the cMOAT polypeptide relate to the position in SEQ ID NO 7 unless stated otherwise or apparent from the context.", "One approach is to use knowledge of polymorphisms to help identify patients most suited to therapy with particular pharmaceutical agents (this is often termed “pharmacogenetics”).", "Pharmacogenetics can also be used in pharmaceutical research to assist the drug selection process.", "Polymorphisms are used in mapping the human genome and to elucidate the genetic component of diseases.", "The reader is directed to the following references for background details on pharmacogenetics and other uses of polymorphism detection: Linder et al.", "(1997), Clinical Chemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15, 1249; International Patent Application WO 97/40462, Spectra Biomedical; and Schafer et al.", "(1998), Nature Biotechnology, 16, 33.Clinical trials have shown that patient response to treatment with pharmaceuticals is often heterogeneous.", "Thus there is a need for improved approaches to pharmaceutical agent design and therapy.", "Point mutations in polypeptides will be referred to as follows: natural amino acid (using 1 or 3 letter nomenclature), position, new amino acid.", "For (a hypothetical) example “D25K” or “Asp25Lys” means that at position 25 an aspartic acid (D) has been changed to lysine (K).", "Multiple mutations in one polypeptide will be shown between square brackets with individual mutations separated by commas.", "The present invention is based on the discovery of polymorphisms in cMOAT.", "In particular, we have found eight polymorphisms in the coding sequence of the cMOAT gene, 6 of which lead to changes in the sequence of expressed protein; three polymorphisms in the 5′UTR; and seven intronic polymorphisms.", "According to one aspect of the present invention there is provided a method for the diagnosis of a polymorphism in cMOAT in a human, which method comprises determining the sequence of the human at at least one polymorphic position and determining the status of the human by reference to polymorphism in cMOAT.", "Preferred polymorphic positions are one or more of the following positions: positions 78, 1350, 1584, 1686, 2647, 3208, 3664 and 4391 in the coding region of the cMOAT gene as defined by the position in SEQ ID NO: 1; positions 1349, 1875 and 1879 in the 5′UTR region of the cMOAT gene as defined by the position in SEQ ID NO: 2; positions 12704 and 29446 in the intron region of the cMOAT gene as defined by the position in SEQ ID NO: 3; position 292 in the intron region of the cMOAT gene as defined by the position in SEQ ID NO: 4; positions 232 and 457 in the intron region of the cMOAT gene as defined by the position in SEQ ID NO: 5; positions 50 and 68 in the intron region of the cMOAT gene as defined by the position in SEQ ID NO: 6; and positions 417, 495, 529, 849, 1036 and 1188 in the cMOAT polypeptide as defined by the position in SEQ ID NO: 7.The term human includes both a human having or suspected of having a cMOAT mediated disease and an asymptomatic human who may be tested for predisposition or susceptibility to such disease.", "At each position the human may be homozygous for an allele or the human may be a heterozygote.", "The term polymorphism includes single nucleotide substitution, nucleotide insertion and nucleotide deletion which in the case of insertion and deletion includes insertion or deletion of one or more nucleotides at a position of a gene and corresponding alterations in expressed protein.", "In one embodiment of the invention preferably the method for diagnosis described herein is one in which the polymorphism in the coding region of the cMOAT gene as defined by the position in SEQ ID NO: 1 is any one of the following: Position DNA SEQ ID NO 1 Variation 78 C/T 1350 G/A 1584 A/G 1686 C/T 2647 T/G 3208 T/C 3664 A/T 4391 T/G In one embodiment of the invention preferably the method for diagnosis described herein is one in which the polymorphism in the in the 5′UTR region of the cMOAT gene as defined by the position in SEQ ID NO: 2 is any one of the following: Position DNA SEQ ID NO 2 polymorphism 1349 G/A 1875 G/A 1879 A/G In one embodiment of the invention preferably the method for diagnosis described herein is one in which the polymorphism in the intronic regions of the cMOAT gene is any following: Position DNA polymorphism 12704 SEQ ID NO 3 C/T 29446 SEQ ID NO 3 C/T 292 SEQ ID NO 4 T/C 232 SEQ ID NO 5 C/G 457 SEQ ID NO 5 A/G 50 SEQ ID NO 6 G/A 68 SEQ ID NO 6 G/A In one embodiment of the invention preferably the method for diagnosis described herein is one in which the polymorphism in the cMOAT protein is any one of the following in SEQ ID NO 7: Val417Ile, Lys495Glu, Arg529Trp, Leu849Arg, Ile1036Thr and Glu1188Val.", "The method for diagnosis is preferably one in which the sequence is determined by a method selected from amplification refractory mutation system, restriction fragment length polymorphism and primer extension.", "The status of the individual may be determined by reference to allelic variation at any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more positions.", "The test sample of nucleic acid is conveniently a sample of blood, bronchoalveolar lavage fluid, sputum, or other body fluid or tissue obtained from an individual.", "It will be appreciated that the test sample may equally be a nucleic acid sequence corresponding to the sequence in the test sample, that is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g.", "PCR, before analysis of allelic variation.", "It will be apparent to the person skilled in the art that there are a large number of analytical procedures which may be used to detect the presence or absence of variant nucleotides at one or more polymorphic positions of the invention.", "In general, the detection of allelic variation requires a mutation discrimination technique, optionally an amplification reaction and optionally a signal generation system.", "Table 1 lists a number of mutation detection techniques, some based on the PCR.", "These may be used in combination with a number of signal generation systems, a selection of which is listed in Table 2.Further amplification techniques are listed in Table 3.Many current methods for the detection of allelic variation are reviewed by Nollau et al., Clin.", "Chem.", "43, 1114-1120, 1997; and in standard textbooks, for example “Laboratory Protocols for Mutation Detection”, Ed.", "by U. Landegren, Oxford University Press, 1996 and “PCR”, 2nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.Abbreviations: ALEX ™ Amplification refractory mutation system linear extension APEX Arrayed primer extension ARMS ™ Amplification refractory mutation system b-DNA Branched DNA bp base pair CMC Chemical mismatch cleavage COPS Competitive oligonucleotide priming system DGGE Denaturing gradient gel electrophoresis ELISA Enzyme Linked ImmunoSorbent Assay FRET Fluorescence resonance energy transfer LCR Ligase chain reaction MASDA Multiple allele specific diagnostic assay NASBA Nucleic acid sequence based amplification OLA Oligonucleotide ligation assay PCR Polymerase chain reaction PTT Protein truncation test RFLP Restriction fragment length polymorphism SDA Strand displacement amplification SNP Single nucleotide polymorphism SSCP Single-strand conformation polymorphism analysis SSR Self sustained replication TGGE Temperature gradient gel electrophoresis Table 1—Mutation Detection Techniques General: DNA sequencing, Sequencing by hybridisation Scanning: PTT, SSCP, DGGE, TGGE, Cleavase, Heteroduplex analysis, CMC, Enzymatic mismatch cleavage Note: not useful for detection of promoter polymorphisms.", "Hybridisation Based: Solid phase hybridisation: Dot blots, MASDA, Reverse dot blots, Oligonucleotide arrays (DNA Chips).", "Solution phase hybridisation: Taqman™—U.S.", "Pat.", "No.", "5,210,015 & U.S. Pat.", "No.", "5,487,972 (Hoffmann-La Roche), Molecular Beacons—Tyagi et al (1996), Nature Biotechnology, 14, 303; WO 95/13399 (Public Health Inst., New York) Extension Based: ARMS™, ALEX™—European Patent No.", "EP 332435 B1 (Zeneca Limited), COPS—Gibbs et al (1989), Nucleic Acids Research, 17, 2347.Incorporation Based: Mini-sequencing, APEX Restriction Enzyme Based: RFLP, Restriction site generating PCR Ligation Based: OLA Other: Invader assay Table 2—Signal Generation or Detection Systems Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation—United Kingdom Patent No.", "2228998 (Zeneca Limited) Other: Chemiluminescence, Electrochemiluminescence, Raman, Radioactivity, Colorimetric, Hybridisation protection assay, Mass spectrometry Table 3—Further Amplification Methods SSR, NASBA, LCR, SDA, b-DNA Table 4—Protein Variation Detection Methods Immunoassay Immunohistology Peptide sequencing Preferred mutation detection techniques include ARMS™, ALEX™, COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques.", "Immunoassay techniques are known in the art e.g.", "A Practical Guide to ELISA by D M Kemeny, Pergamon Press 1991; Principles and Practice of Immunoassay, 2nd edition, C P Price & D J Newman, 1997, published by Stockton Press in USA & Canada and by Macmillan Reference in the United Kingdom.", "Particularly preferred methods include ARMS™ and RFLP based methods.", "ARMS™ is an especially preferred method.", "In a further aspect, the diagnostic methods of the invention are used to assess the pharmacogenetics of a drug transported by cMOAT.", "Assays, for example reporter-based assays, may be devised to detect whether one or more of the above polymorphisms affect transcription levels and/or message stability.", "Individuals who carry particular allelic variants of the cMOAT gene may therefore exhibit differences in their ability to regulate protein biosynthesis under different physiological conditions and will display altered abilities to react to different diseases.", "In addition, differences arising as a result of allelic variation may have a direct effect on the response of an individual to drug therapy.", "The diagnostic methods of the invention may be useful both to predict the clinical response to such agents and to determine therapeutic dose.", "In a further aspect, the diagnostic methods of the invention, are used to assess the predisposition and/or susceptibility of an individual to diseases mediated by cMOAT.", "This may be particularly relevant in the development of hyperlipoproteinemia and cardiovascular disease and the present invention may be used to recognise individuals who are particularly at risk from developing these conditions.", "In a further aspect, the diagnostic methods of the invention are used in the development of new drug therapies which selectively target one or more allelic variants of the cMOAT gene.", "Identification of a link between a particular allelic variant and predisposition to disease development or response to drug therapy may have a significant impact on the design of new drugs.", "Drugs may be designed to regulate the biological activity of variants implicated in the disease process whilst minimising effects on other variants.", "In a further diagnostic aspect of the invention the presence or absence of variant nucleotides is detected by reference to the loss or gain of, optionally engineered, sites recognised by restriction enzymes.", "According to another aspect of the present invention there is provided a human cMOAT gene or its complementary strand comprising a variant allelic polymorphism at one or more of positions defined herein or a fragment thereof of at least 20 bases comprising at least one novel polymorphism.", "Fragments are at least 17 bases, more preferably at least 20 bases, more preferably at least 30 bases.", "According to another aspect of the present invention there is provided a polynucleotide comprising at least 20 bases of the human cMOAT gene and comprising an allelic variant selected from any one of the following: Variant Region SEQ ID NO: 1 coding 78 T 1350 A 1584 G 1686 T 2647 G 3208 C 3664 A 4391 G Variant Region SEQ ID NO: 2 5′UTR or 1349 A promoter 1875 A 1879 G Region Variant SEQ ID NO 3 12704 T SEQ ID NO 3 29446 T SEQ ID NO 4 292 C SEQ ID NO 5 232 G SEQ ID NO 5 457 G SEQ ID NO 6 50 A SEQ ID NO 6 68 A According to another aspect of the present invention there is provided a human cMOAT gene or its complementary strand comprising a polymorphism, preferably corresponding with one or more the positions defined herein or a fragment thereof of at least 20 bases comprising at least one polymorphism.", "Fragments are at least 17 bases, more preferably at least 20 bases, more preferably at least 30 bases.", "The invention further provides a nucleotide primer which can detect a polymorphism of the invention.", "According to another aspect of the present invention there is provided an allele specific primer capable of detecting a cMOAT gene polymorphism, preferably at one or more of the positions as defined herein.", "An allele specific primer is used, generally together with a constant primer, in an amplification reaction such as a PCR reaction, which provides the discrimination between alleles through selective amplification of one allele at a particular sequence position e.g.", "as used for ARMS™ assays.", "The allele specific primer is preferably 17-50 nucleotides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides.", "An allele specific primer preferably corresponds exactly with the allele to be detected but derivatives thereof are also contemplated wherein about 6-8 of the nucleotides at the 3′ terminus correspond with the allele to be detected and wherein up to 10, such as up to 8, 6, 4, 2, or 1 of the remaining nucleotides may be varied without significantly affecting the properties of the primer.", "Primers may be manufactured using any convenient method of synthesis.", "Examples of such methods may be found in standard textbooks, for example “Protocols for Oligonucleotides and Analogues; Synthesis and Properties,” Methods in Molecular Biology Series; Volume 20; Ed.", "Sudhir Agrawal, Humana ISBN: 0-89603-247-7; 1993; 1st Edition.", "If required the primer(s) may be labelled to facilitate detection.", "According to another aspect of the present invention there is provided an allele-specific oligonucleotide probe capable of detecting a cMOAT gene polymorphism, preferably at one or more of the positions defined herein.", "The allele-specific oligonucleotide probe is preferably 17-50 nucleotides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides.", "The design of such probes will be apparent to the molecular biologist of ordinary skill.", "Such probes are of any convenient length such as up to 50 bases, up to 40 bases, more conveniently up to 30 bases in length, such as for example 8-25 or 8-15 bases in length.", "In general such probes will comprise base sequences entirely complementary to the corresponding wild type or variant locus in the gene.", "However, if required one or more mismatches may be introduced, provided that the discriminatory power of the oligonucleotide probe is not unduly affected.", "The probes of the invention may carry one or more labels to facilitate detection.", "According to another aspect of the present invention there is provided an allele specific primer or an allele specific oligonucleotide probe capable of detecting a cMOAT gene polymorphism at one of the positions defined herein.", "According to another aspect of the present invention there is provided a diagnostic kit comprising an allele specific oligonucleotide probe of the invention and/or an allele-specific primer of the invention.", "The diagnostic kits may comprise appropriate packaging and instructions for use in the methods of the invention.", "Such kits may further comprise appropriate buffer(s) and polymerase(s) such as thermostable polymerases, for example taq polymerase.", "In another aspect of the invention, the single nucleotide polymorphisms of this invention may be used as genetic markers in linkage studies.", "This particularly applies to the polymorphisms at 78 and 1350 in SEQ ID NO 1 because of their relatively high frequencies (see below).", "The cMOAT gene has been mapped to chromosome 10q24 (Tanaguchi et al (1996).", "Cancer Res.", "56, 4124-4129.).", "Low frequency polymorphisms may be particularly useful for haplotyping as described below.", "A haplotype is a set of alleles found at linked polymorphic sites (such as within a gene) on a single (paternal or maternal) chromosome.", "If recombination within the gene is random, there may be as many as 2n haplotypes, where 2 is the number of alleles at each SNP and n is the number of SNPs.", "One approach to identifying mutations or polymorphisms which are correlated with clinical response is to carry out an association study using all the haplotypes that can be identified in the population of interest.", "The frequency of each haplotype is limited by the frequency of its rarest allele, so that SNPs with low frequency alleles are particularly useful as markers of low frequency haplotypes.", "As particular mutations or polymorphisms associated with certain clinical features, such as adverse or abnormal events, are likely to be of low frequency within the population, low frequency SNPs may be particularly useful in identifying these mutations (for examples see: Linkage disequilibrium at the cystathionine beta synthase (CBS) locus and the association between genetic variation at the CBS locus and plasma levels of homocysteine.", "Ann Hum Genet (1998) 62:481-90, De Stefano V, Dekou V, Nicaud V, Chasse J F, London J, Stansbie D, Humphries S E, and Gudnason V; and Variation at the von willebrand factor (vWF) gene locus is associated with plasma vWF:Ag levels: identification of three novel single nucleotide polymorphisms in the vWF gene promoter.", "Blood (1999) 93:4277-83, Keightley A M, Lam Y M, Brady J N, Cameron C L, Lillicrap D).", "According to another aspect of the present invention there is provided a computer readable medium comprising at least one novel sequence of the invention stored on the medium.", "The computer readable medium may be used, for example, in homology searching, mapping, haplotyping, genotyping or pharmacogenetic analysis.", "According to another aspect of the present invention there is provided a method of treating a human in need of treatment with a drug transportable by cMOAT in which the method comprises: i) diagnosis of a polymorphism in cMOAT in the human, which diagnosis preferably comprises determining the sequence at one or more of the following positions: positions 78, 1350, 1584, 1686, 2647, 3208, 3664 and 4391 in the coding region of the cMOAT gene as defined by the position in SEQ ID NO: 1; positions 1349, 1875 and 1879 in the 5′UTR region of the cMOAT gene as defined by the position in SEQ ID NO: 2; positions 12704 and 29446 in the intron region of the cMOAT gene as defined by the position in SEQ ID NO: 3; position 292 in the intron region of the cMOAT gene as defined by the position in SEQ ID NO: 4; positions 232 and 457 in the intron region of the cMOAT gene as defined by the position in SEQ ID NO: 5; positions 50 and 68 in the intron region of the cMOAT gene as defined by the position in SEQ ID NO: 6; and positions 417, 495, 529, 849, 1036 and 1188 in the cMOAT polypeptide as defined by the position in SEQ ID NO: 7.and determining the status of the human by reference to polymorphism in the cMOAT gene; and ii) administering an effective amount of the drug.", "Preferably determination of the status of the human is clinically useful.", "Examples of clinical usefulness include deciding which statin drug or drugs to administer and/or in deciding on the effective amount of the statin drug or drugs.", "Statins already approved for use in humans include atorvastatin, cerivastatin, fluvastatin, pravastatin and simvastatin.", "The reader is referred to the following references for further information: Drugs and Therapy Perspectives (12th May 1997), 9: 1-6; Chong (1997) Pharmacotherapy 17: 1157-1177; Kellick (1997) Formulary 32: 352; Kathawala (1991) Medicinal Research Reviews, 11: 121-146; Jahng (1995) Drugs of the Future 20: 387-404, and Current Opinion in Lipidology, (1997), 8, 362-368.Another statin drug of note is compound 3a (S4522) in Watanabe (1997) Bioorganic and Medicinal Chemistry 5: 437-444.The term “drug transportable by cMOAT” means that transport by cMOAT in humans is an aspect of a drug exerting its pharmaceutical effect in man.", "For example, some statins may be transported by cMOAT as conjugates in the human body.", "According to another aspect of the present invention there is provided an allelic variant of human cMOAT polypeptide comprising at least one of the following: a isoleucine at position 417 of SEQ ID NO 7; a glutamic acid at position 495 of SEQ ID NO 7; a tryptophan at position 529 of SEQ ID NO 7; a arginine at position 849 of SEQ ID NO 7; a threonine at position 1036 of SEQ ID NO 7; and a valine at position 1188 of SEQ ID NO 7; or a fragment thereof comprising at least 10 amino acids provided that the fragment comprises at least one allelic variant.", "Fragments of polypeptide are at least 10 amino acids, more preferably at least 15 amino acids, more preferably at least 20 amino acids.", "According to another aspect of the present invention there is provided an antibody specific for an allelic variant of human cMOAT polypeptide as described herein.", "Antibodies can be prepared using any suitable method.", "For example, purified polypeptide may be utilized to prepare specific antibodies.", "The term “antibodies” is meant to include polyclonal antibodies, monoclonal antibodies, and the various types of antibody constructs such as for example F(ab′)2, Fab and single chain Fv.", "Antibodies are defined to be specifically binding if they bind the allelic variant of cMOAT with a Ka of greater than or equal to about 107 M−1.Affinity of binding can be determined using conventional techniques, for example those described by Scatchard et al., Ann.", "N.Y. Acad.", "Sci., 51:660 (1949).", "Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats, using procedures that are well-known in the art.", "In general, antigen is administered to the host animal typically through parenteral injection.", "The immunogenicity of antigen may be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant.", "Following booster immunizations, small samples of serum are collected and tested for reactivity to antigen.", "Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.", "), Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S. Pat.", "Nos.", "4,376,110 and 4,486,530.Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the procedures described in U.S. Pat.", "Nos.", "RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.", "), (1980).", "The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., “Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas”, Strategies in Molecular Biology 3: 1-9 (1990) which is incorporated herein by reference.", "Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody.", "Such a technique is described in Larrick et al., Biotechnology, 7: 394 (1989).", "Once isolated and purified, the antibodies may be used to detect the presence of antigen in a sample using established assay protocols, see for example “A Practical Guide to ELISA” by D. M. Kemeny, Pergamon Press, Oxford, England.", "According to another aspect of the invention there is provided a diagnostic kit comprising an antibody of the invention.", "The invention will now be illustrated but not limited by reference to the following Examples.", "All temperatures are in degrees Celsius.", "In the Examples below, unless otherwise stated, the following methodology and materials have been applied.", "AMPLITAQ™ available from Perkin-Elmer Cetus, is used as the source of thermostable DNA polymerase.", "General molecular biology procedures can be followed from any of the methods described in “Molecular Cloning—A Laboratory Manual” Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989) or in “Current Protocols in Molecular Biology Volumes 1-3, edited by F M Asubel, R Brent and R E Kingston; published by John Wiley, 1998.Electropherograms were obtained in a standard manner: data was collected by ABI377 data collection software and the wave form generated by ABI Prism sequencing analysis (2.1.2).", "EXAMPLE 1 Identification of Polymorphisms 1.Methods DNA Preparation DNA was prepared from frozen blood samples collected in EDTA following protocol I (Molecular Cloning: A Laboratory Manual, p392, Sambrook, Fritsch and Maniatis, 2nd Edition, Cold Spring Harbor Press, 1989) with the following modifications.", "The thawed blood was diluted in an equal volume of standard saline citrate instead of phosphate buffered saline to remove lysed red blood cells.", "Samples were extracted with phenol, then phenol/chloroform and then chloroform rather than with three phenol extractions.", "The DNA was dissolved in deionised water.", "Template Preparation Templates were prepared by PCR using the oligonucleotide set out below.", "The annealing temperature for all sets of primers was 58° C. followed by an extension temperature was 72° C. and a denaturation temperature of 94° C. Generally 50 ng of genomic DNA was used in each reaction and subjected to 35 cycles of PCR.", "All primers used for amplification of the exons are as published by Toh et al.", "(Am.", "J. Hum.", "Genet., 1999, 64:739-746).", "Primers for the amplification of the promoter and 5′UTR are listed below: Primer sequences are based on SEQ ID NO 2.Fragment Forward Oligo Reverse Oligo Annealing Temp Time 1164-1729 1164-1183 1710-1729 58° C. 60 s 1622-2164 1622-1641 2145-2164 58° C. 60 s 2061-2677 2061-2080 2658-2677 58° C. 60 s Primers for the amplification of the 3′UTR are listed below and are based on SEQ ID NO 1.Fragment Forward Oligo Reverse Oligo Annealing Temp Time 4707-5207 4707-4726 5188-5207 58° C. 60 s For dye-primer sequencing these primers were modified to include M13 forward and reverse primer sequences (ABI protocol P/N 402114, Applied Biosystems) at the 5′ end of the forward and reverse oligonucleotides respectively.", "Dye Primer Sequencing Dye primer sequencing using M13 forward and reverse primers was as described in the ABI protocol P/N 402114 for the ABI Prism™ dye primer cycle sequencing core kit with “AmpliTaq FS” DNA polymerase, modified in that the annealing temperature was 45° C. and DMSO was added to the cycle sequencing mix to a final concentration of 5%.", "The extension reactions for each base were pooled, ethanol/sodium acetate precipitated, washed and resuspended in formamide loading buffer.", "4.25% Acrylamide gels were run on an automated sequencer (ABI 377, Applied Biosystems).", "2.Results Polymorphisms Protein Position DNA polymorphism Allele Allele SEQ ID 1 polymorphism SEQ ID NO 7 frequency frequency 78 C/T none C 72.7% T 27.3% 1350 G/A Val417Ile GTT 77.5% ATT 22.5% 1584 A/G Lys495Glu AAA 97.8% GAA 2.2% 1686 C/T Arg529Trp CGG 97.8% TGG 2.2% 2647 T/G Leu849Arg CTG 94.4% CGG 5.6% 3208 T/C Ile1036Thr ATA 93.5% ACA 6.5% 3664 T/A Val1188Glu GTG 95.5% GAG 4.5% 4391 T/G none T 4.3% G 95.7% Position DNA Allele Allele SEQ ID NO 2 polymorphism frequency frequency 1349 G/A G 57.5% A 42.5% 1875 G/A G 80% A 20% 1879 A/G A 52.6% G 47.4% Position DNA SEQ ID NO 3 polymorphism Allele frequency Allele frequency 12704 C/T C 58.7% T 41.3% 29446 T/C T 95.6% C 4.4% Position DNA Allele Allele SEQ ID NO 4 polymorphism frequency frequency 292 T/C T 58.9% C 41.1% SEQ ID NO 4 is exon 15 and intronic sequence flanking both ends thereof.", "Position DNA Allele Allele SEQ ID NO 5 polymorphism frequency frequency 232 C/G C 60.9% G 39.1% 457 A/G A 93.8% G 6.2% SEQ ID NO 5 is exon 27/intron 27/exon 28 and intronic sequence flanking both ends thereof.", "Position DNA Allele Allele SEQ ID NO 6 polymorphism frequency frequency 50 G/A G 97.8% A 2.2% 68 G/A G 89.3% A 10.7% SEQ ID NO 6 is exon 30 and intronic sequence flanking both ends thereof.", "Allele frequencies were based on analysis of 20 to 23 individuals.", "EXAMPLE 2 Diagnostic Assays for Polymorphisms within the 5′End of the cMOAT Gene.", "The cMOAT 5′end is set out in SEQ ID NO 2 and all positions in this Example 2 relate to the position therein.", "DNA and template preparation as before; see Example 1.Position 2874 C/T Diagnostic Primer 2875-2903 The diagnostic primer contains a 2 mismatches from the wild type sequence at positions 25 (G→T) and position 27 (A→T).", "Constant Primer 2624-2643 PCR amplification using these primers will generate a product of 280 bp.", "The use of the diagnostic primer on a template creates a Bsr GI recognition sequence (TGTACA) at the site of the polymorphism.", "Bsr GI (New England Biolabs) can therefore distinguish the two polymorphic variants.", "Digestion of PCR products was performed according to the manufacturers instructions (New England Biolabs).", "...CTT CGT ACA... wild type uncut ...CTT TGT ACA... variant cut Position 1875 G/A Diagnostic Primer 1849-1874 The diagnostic primer contains a 2 mismatches from the wild type sequence at positions 22 (C→T) and position 25 (A→T).", "Constant Primer 2145-2164 PCR amplification using these primers will generate a product of 315 bp.", "The use of the diagnostic primer on a template creates a Bsp H1 recognition sequence (TCATGA) at the site of the polymorphism.", "Bsp H1 (New England Biolabs) can therefore distinguish the two polymorphic variants.", "Digestion of PCR products was performed according to the manufacturers instructions (New England Biolabs).", "...AGG TCA TGG... wild type uncut ...AGG TCA TGA... variant cut Position 1879 A/G Diagnostic Primer 1880-1903 The diagnostic primer contains a 2 mismatches from the wild type sequence at positions 22 (C→G) and position 24 (T→A).", "Constant Primer 1622-1641 PCR amplification using these primers will generate a product of 315 bp.", "The use of the diagnostic primer on a template creates a NsiI recognition sequence (ATGCAT) at the site of the polymorphism.", "NsiI (New England Biolabs) can therefore distinguish the two polymorphic variants.", "Digestion of PCR products was performed according to the manufacturers instructions (New England Biolabs).", "...ACA ATC CTT... wild type uncut ...ACA ATG CAT... variant cut EXAMPLE 3 Diagnostic Assays for Polymorphisms within the Coding Sequence of the cMOAT Gene.", "The cMOAT gene sequence is set out in SEQ ID NO 1 and all positions in this Example 3 relate to the position therein unless stated otherwise.", "Position 1350 G/A Diagnostic Primer 1352-1379 (SEQ ID NO: 1) The diagnostic primer contains 1 mismatch from the wild type sequence at position 28 (T→C) Constant Primer 23107-23128 (SEQ ID NO: 3) PCR amplification using these primers will generate a product of 201 bp.", "The use of the diagnostic primer on a template creates a Nco I recognition sequence (CCATGG) at the site of the polymorphism.", "Nco I (New England Biolabs) can therefore distinguish the two polymorphic variants.", "Digestion of PCR products was performed according to the manufacturers instructions (New England Biolabs).", "...ACA CCG TTG... wild type uncut ...ACA CCA TTG... variant cut Position 1686 C/T Amplification is carried out using primers described below for the amplification of exon 12.PCR amplification with these primers will generate a product of approximately 500 bp.", "The polymorphism destroys a natural AccIII recognition sequence (TCCGGA) at the site of the polymorphism.", "AccIII (New England Biolabs) can therefore distinguish the two polymorphic variants.", "Digestion of PCR products was performed according to the manufacturers instructions (New England Biolabs).", "...ACC TCC GGA... wild type cut ...ACC TCT GGA... variant uncut Position 2647 T/G Amplification is carried out using primers described below for the amplification of exons 18 and 19.PCR amplification with these primers will generate a product of approximately 780 bp.", "The polymorphism destroys a natural EcoRII recognition sequence (CCWGG where W represents A or T) at the site of the polymorphism.", "EcoRII (New England Biolabs) can therefore distinguish the two polymorphic variants.", "Digestion of PCR products was performed according to the manufacturers instructions (New England Biolabs).", "...CTC TCC TGG... wild type cut ...CTC TCC GGG... variant uncut Position 3208 T/C Amplification is carried out using primers described below for the amplification of exons 22 and 23.PCR amplification with these primers will generate a product of approximately 750 bp.", "The polymorphism creates a natural RsaI recognition sequence (GTAC) at the site of the polymorphism.", "RsaI (New England Biolabs) can therefore distinguish the two polymorphic variants.", "Digestion of PCR products was performed according to the manufacturers instructions (New England Biolabs).", "...AAT GTA TCT... wild type uncut ...AAT GTA CCT... variant cut Nucleotide 3664 T/A Diagnostic Primer 3665-3691 The diagnostic primer contains 1 mismatch from the wild type sequence at position 25 (C→G) Constant Primer within intron 24 was as published in Toh et al.", "Am.", "J. Hum.", "Genet., 1999, 64:739-746.PCR amplification using these primers will generate a product of 241 bp.", "The use of the diagnostic primer on a template destroys a Tsp 45I recognition sequence (GTSAC where S is either G or C) at the site of the polymorphism.", "Tsp 45I (New England Biolabs) can according to the manufacturers instructions (New England Biolabs).", "...GAG GTG ACG... wild type cut ...GAG GAG ACG... variant uncut Position 4391 T/G Diagnostic Primer 4392-4112 The diagnostic primer contains 2 mismatches from the wild type sequence at positions 18 (C→A) and 19 (G→C).", "Constant Primer 4252-4272 PCR amplification using these primers will generate a product of 163 bp.", "The use of the diagnostic primer on a template creates a Sna BI recognition sequence (TACGTA) at the site of the polymorphism.", "Sna BI (New England Biolabs) can therefore distinguish the two polymorphic variants.", "Digestion of PCR products was performed according to the manufacturers instructions (New England Biolabs).", "...AGT TAC TTA... wild type uncut ...AGT TAC GTA... variant cut cMOAT Exon-specific oligos were from Toh et al.", "Am.", "J. Hum.", "Genet., 1999, 64:739-746 Promoter & 5′UTR Oligonucleotides (SEQ ID NO 2; F=Forward, R=Reverse) Product 1F (1164-1183) Product 1R (1710-1729) Promoter 2F (1622-1641) Product 2R (2145-2164) Product 3F (2061-2080) Product 3R (2658-2677) 3′UTR Oligonucleotides (SEQ ID NO: 1; F=Forward, R=Reverse) 3′UTR F (4707-4726) 3′UTR R (5188-5207)" ] ]	Patent_10468326
[ [ "Methods", "The molecules of formula (I) are useful in treating diabetes, obesity, hypercholesterolaemia, hyperlipidaemia, cancer, inflammation or other conditions in which modulation of lipid or eicosanoid status or functions may be desirable.", "Formula (I): Z1-X-Z2 wherein a) Z1 represents CO2H or a derivative thereof; b) Z2 represents F, H, —CO2H or a derivative thereof; and c) X represents fluorinated alkylene; or a solvate thereof, for example a perfluorinated fatty acid or derivative thereof." ], [ "1.A method of treatment of a patient in need of modulation of body mass or modulation of increase in body mass, and/or in need of modulation of plasma insulin, plasma glucose, plasma triglycerides, plasma cholesterol and/or leptin, comprising administering to the patient an effective amount of a compound of formula I, Z1-X-Z2 I wherein Z1 represents —CO2H or a derivative thereof; Z2 represents F, H, —CO2H or a derivative thereof; and X represents fluorinated alkylene; or a solvate thereof.", "2.A method of treatment of a patient in need of an antitumour agent or an antiinflammatory agent, or in need of modulation in lipid or eicosanoid status, comprising administering to the patient an effective amount of a compound of formula I as defined in claim 1.3.A method of treatment of a patient who is overweight or obese and/or has diabetes, hyperlipidaemia, atherosclerosis, coronary heart-disease, stroke, obstructive sleep apnoea, arthritis and/or reduced fertility, or is at risk of developing such a condition, comprising administering to the patient an effective amount of a compound of formula I as defined in claim 1.4.A method of treatment of a patient in need of modulation of PPAR (for example PPARα) activity, comprising administering to the patient an effective amount of a compound of formula I as defined in claim 1.5.A method of treatment of a patient in need of modulation of lipid or eicosanoid status or function, comprising administering to the patient an effective amount of a compound of formula I as defined in claim 1.6.Use of a compound of formula I as defined in claim 1 in the manufacture of a medicament for treating a patient in need of modulation of PPAR (for example PPARα) activity.", "7.The method of claim 4 wherein the patient is in need of an increase in PPAR activity and the compound is a PPAR agonist.", "8.The method of claim 7 wherein the PPAR is PPARα or PPARγ.", "9.The use of a compound of formula I as defined in claim 1 in the manufacture of a medicament for the treatment of a patient in need of modulation of body mass or modulation of increase in body mass, and/or in need of modulation of plasma insulin, plasma glucose, plasma triglycerides, plasma cholesterol and/or leptin.", "10.The method of claim 1 wherein the patient is in need of reduction of body mass or prevention of increase in body mass, and/or in need of reduction of plasma insulin, plasma glucose, plasma triglycerides, plasma cholesterol and/or leptin.", "11.The use of a compound of formula I as defined in claim 1 in the manufacture of a medicament for the treatment of a patient who is overweight or obese and/or has diabetes, hyperlipidaemia, atherosclerosis, coronary heart disease, stroke, obstructive sleep apnoea, arthritis and/or reduced fertility, or is at risk of developing such a condition.", "12.The use of a compound of formula I in the manufacture of a medicament for the treatment of a patient in need an antitumour agent or an antiinflammatory agent or of modulation of lipid or eicosanoid status or function, or of modulation of a lipid metabolising or binding entity activity.", "13.The method of treatment of claim 1 wherein the compound is or comprises a perfluorinated fatty acid or derivative thereof.", "14.The method of treatment of claim 1 wherein the compound is or comprises a fluorinated carboxylic acid or perfluorinated carboxylic acid or pharmaceutically acceptable salt, ester or halide thereof.", "15.The method of claim 13 wherein the compound is perfluorooctanoic acid (PFOA) or a pharmaceutically acceptable salt, ester or halide thereof, for example ammonium perfluorooctanoate (APFO).", "16.The method of claim 1 wherein the patient is human.", "17.A screening method for identifying a drug-like compound or lead compound for the development of a drug-like compound in which (1) a mammal is exposed to a compound of formula I as defined in claim 1 or derivative thereof (2) the plasma insulin, glucose, cholesterol, triglyceride and/or leptin level of the mammal is measured, and/or bodyweight of the mammal is measured, and/or lipid or eicosanoid status or function of the mammal is measured.", "18.The method of claim 17 comprising the step of selecting a compound on exposure to which the plasma insulin, glucose, cholesterol, triglyceride and/or leptin level of the mammal is changed or reduced, and/or bodyweight or bodyweight increase of the mammal is changed or reduced.", "19.A screening method for identifying a drug-like compound or lead compound for the development of a drug-like compound in which (1) a compound of formula I as defined in claim 1 or derivative thereof is exposed to a PPAR polypeptide (2) the binding of the compound to the PPAR polypeptide is measured or the change in the activity of the PPAR polypeptide is measured.", "20.A screening method for identifyg a drug-like compound or lead compound for the development of a drug-like compound in which (1) a compound of formula I as defined in claim 1 or derivative thereof is exposed to a lipid metabolising or binding entity, for example cycloxygenase (for example cyclooxygenase I or cyclooxygenase II) or phospholipase A (for example phospholipase A2) (2) the binding of the compound to the lipid metabolising or binding entity is measured or the change in the activity of the lipid metabolising or binding entity is measured.", "21.A screening method for identifying a drug-like compound or lead compound for the development of a drug-like compound in which (1) a cell is exposed to a compound of formula I as defined herein (for example a perfluorinated fatty acid) or derivative thereof (2) the phenotype (for example differentiation) and/or eicosanoid biosynthesis of the cell is measured.", "22.The method of claim 21 further comprising the step of selecting a compound on exposure to which the phenotype, for example differentiation, of the cell is changed, and/or eicosanoid biosynthesis of the cell is changed, preferably reduced.", "23.A compound identifiable or identified by a screening method according to claim 17.24.A compound identified or identifiable by a screening method of according to claim 17 for use in medicine.", "25.The use of a compound identified or identifiable by a screening method according to claim 17 in the manufacture of a medicament for the treatment of a patient in need of modulation of body mass or modulation of increase in body mass, and/or in need of modulation of plasma insulin, plasma glucose, plasma triglycerides, plasma cholesterol and/or leptin.", "26.Use of a compound identified or identifiable by a screening method according to claim 17 in the manufacture of a medicament for the treatment of a patient who is overweight or obese and/or has diabetes, hyperlipidaemia, atherosclerosis, coronary heart disease, stroke, obstructive sleep apnoea, arthritis and/or reduced fertility, or is at risk of developing such a condition.", "27.The use of a compound identified or identifiable by a screening method according to claim 17 in the manufacture of a medicament for treating a patient in need of modulation of PPAR (for example PPARα) activity, or in need of an antitumour agent or an anninflammatory agent, or in need of modulation of lipid or eicosanoid status or function, or in need of modulation of lipid metabolising or binding entity activity.", "28.The use of claim 27 wherein the patient is in need of an increase in PPAR activity and the compound is a PPAR (for example a PPARα) agonist.", "29.A method of treatment of a patient in need of modulation of body mass or modulation of increase in body mass, and/or in need of modulation of plasma insulin, plasma glucose, plasma triglycerides, plasma cholesterol and/or leptin, comprising administering to the patient an effective amount of a compound identified or identifiable by the screening method of claim 17.30.A method of treatment of a patient who is overweight or obese and/or has diabetes, hyperlipidaemia, atherosclerosis, coronary heart disease, stroke, obstructive sleep apnoea, arthritis and/or reduced fertility, or is at risk of developing such a condition, comprising administering to the patient an effective amount of a compound identified or identifiable by the screening method of claim 17.31.A method of treatment of a patient in need of modulation of PPAR (for example PPARα) activity, or in need of an antitumour agent or an antiinflammatory agent, or in need of modulation of lipid or eicosanoid status or function, or of a lipid metabolising or binding entity activity, comprising administering to the patient an effective amount of a compound identified or identifiable by the screening method of claim 17.32.A compound identified or identifiable by the screening method of claim 17 for use in the manufacture of a composition for use as a food supplement or a food additive.", "33.A food product comprising a foodstuff and a compound of formula I as defined in claim 1 or a compound identified or identifiable by the screening method of claim 17, wherein the food is not laboratory rodent feed.", "34.A kit of parts of screening system comprising (1) a library of compounds each of formula I as herein defined or a derivative thereof, and (2) a PPAR polypeptide or polynucleotide encoding a PPAR polypeptide, and/or a test mammal.", "35.A kit of parts of screening system comprising (1) a library of compounds each of formula I as herein defined or a derivative thereof and (2) a lipid metabolising or binding entity (for example COXI or COXII or phospholipase A2 or lipoxygenase) or polynucleotide encoding a lipid metabolising or binding entity.", "36.", "(canceled)" ], [ "This invention relates to the medical use of compounds, and to methods of identifying further useful compounds, particularly in the treatment of diabetes, obesity, hyperlipidaemia, hypercholesterolaemia, atherosclerosis, cancer and inflammation, or other conditions where alterations in lipid or eicosanoid status may be desirable.", "Perfluorooctanoic acid (PFOA) and other perfluorinated fatty acids or fluoroalkyl molecules are synthetic molecules used in industrial applications, principally as surfactants.", "The effects of these compounds on laboratory animals and cells has been studied, as have the effects of occupational exposure in humans (see, for example, Gilliland & Mandel (1993) J Occup Med 35(9), 950-954; Kees et al (1992) J Med Chem 35, 944-953).", "U.S. Pat.", "No.", "4,624,851 suggests treatment of symptoms of cancer using fluorine containing acids; no experimental data is presented.", "We have surprisingly found that such compounds may have beneficial effects.", "We have found that such compounds may be useful in treatment of diabetes, obesity, hyperlipidaemia, hypercholesterolaemia, atherosclerosis, cancer and inflammation, or other conditions where alterations in lipid or eicosanoid status may be desirable.", "A first aspect of the invention provides a method of treatment of a patient in need of modulation (preferably reduction) of body mass or modulation (preferably prevention or reduction) of increase in body mass, and/or in need of modulation (preferably reduction) of plasma insulin, plasma glucose, plasma triglycerides and/or plasma cholesterol, comprising administering to the patient an effecfive amount of a compound of formula I as defined herein.", "The compound of formula I is Z1-X-Z2 I wherein Z1 represents —CO2H or a derivative thereof; Z2 represents F, H, —CO2H or a derivative thereof; and X represents fluorinated alkylene; or a solvate thereof; which compounds are referred to hereinafter as “the compounds of the invention”.", "A further aspect of the invention provides a method of treatment of a patient in need of an antitumour agent or an antiinflammatory agent, or in need of modulation in lipid or eicosanoid status, comprising administering to the patient an effective amount of a compound of formula I as defined herein.", "A compound of formula I is considered to be effective as an antitumour agent or an anfiinflammatory agent or in modulating lipid or eicosanoid status (ie type and concentration of lipid or eicosanoid, either systemically or in a particular locus or tissue).", "The patient may be a patient with or at risk of excessive inflammation, for example with or at risk of arthritis, or a patient with or at risk of developing a tumour.", "The compound may reduce the development, growth or metastasis of a tumour.", "The compound may be useful in treating any condition or disorder in which the patient has or is at risk of excessive inflammation.", "The patient may have an allergic or autoimmune disease.", "The patient may have, for example, psoriasis, inflammatory bowel disease, asthma or rheumatism.", "A further aspect of the invention provides a method of treatment of a patient who is overweight or obese and/or has diabetes, hyperlipidaemia, atherosclerosis, coronary heart disease, stroke, obstructive sleep apnoea, arthritis (for example osteoarthritis) and/or reduced fertility, or is at risk of developing such a condition, comprising administering to the patient an effective amount of a compound of formula I as defined herein.", "A further aspect of the invention provides a method of treatment of a patient in need of modulation of PPAR (for example PPARα, δ or γ) activity, comprising administering to the patient an effective amount of a compound of formnula I as defined herein.", "The compound may be a PPAR agonist or a PPAR antagonist; it may be an agonist for one PPAR and an antagonist for a different PPAR.", "Preferably the patient is in need of an increase in PPARα or PPARγ activity and the compound is a PPARα or PPARγ agonist.", "Alternatively, the patient may be in need of a decrease in PPARα or PPARγ activity and the compound may be a PPARα or PPARγ antagonist.", "In a further alternative, the patient may be in need of an increase in PPARδ activity and the compound is a PPARδ agonist.", "In a still further alternative, the patient may be in need of a decrease in PPARδ activity and the compound may be a PPARδ antagonist.", "PPARδ may have opposing effects to PPARα or PPARγ (see, for example, WO 01/07066).", "A further aspect of the invention provides a method of treatment of a patient in need of modulation of lipid or eicosanoid status or function, for example in need of modulation of the activity of a lipid metabolising or binding entity (including a lipid metabolising enzyme and a lipid binding polypeptide, for example a lipid transporting polypeptide), for example cycloxygenase (for example cyclooxygenase I or cyclooxygenase II) activity or phospholipase A (for example phospholipase A2) or lipoxygenase, comprising administering to the patient an effective amount of a compound of formula I as defined herein.", "Preferably the patient is in need of a decrease in a lipid metabolising or binding activity, for example cycloxygenase (for example cyclooxygenase I or cyclooxygenase II) activity or phospholipase A or lipoxygenase and the compound is an inhibitor of such activity.", "For example, inappropriate lipoxygenase activity may be involved in inflammation, hypersensitivity, asthma and some vascular diseases; thus a decrease in a lipoxygenase activity may be useful in such a condition.", "Alternatively, the patient may be in need of an increase in such activity and the compound may be an activator of such activity.", "Further preferences in relation to the patient and compound are indicated below.", "Compounds of formula I may exhibit tautomerism.", "All tautomeric forms and mixtures thereof are included within the scope of the invention.", "Compounds of formula I may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism.", "Diastereoisomers may be separated using conventional techniques, e.g.", "chromatography or fractional crystallisation.", "The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g.", "fractional crystallisation or HPLC, techniques.", "Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric esters by conventional means (e.g.", "HPLC, chromatography over silica).", "All stereoisomers are included within the scope of the invention.", "When referred to herein, derivatives of —CO2H groups include groups which are commonly derived from a carboxylic acid and/or groups that contain a central carbon atom (i.e.", "the carbon atom that is attached to X) that is at the same oxidation state as —C(O)OH.", "Derivatives of —CO2H groups therefore includes groups such as: (i) esters, e.g.", "those formed with an alcohol of formula R1OH, wherein R1 represents aryl or alkyl; (ii) thioesters, e.g.", "those formed with a thiol of formula R1SH, wherein R1 is as hereinbefore defined; and (iii) salts, e.g.", "those formed with a nitrogen-containing base such as ammonia, an alkylamine, a dialkylamine, a trialkylamine and pyridine or alkali or alkaline earth metal salts (e.g.", "Na, K, Cs, Mg or Ca salts).", "Preferred derivatives of —CO2H groups include those that are pharmaceutically acceptable.", "Where the term fluorine is used herein, it is intended (where appropriate) that reference to other halogens, for example chlorine or bromine or more than one halogen, is included.", "However, it is strongly preferred that the halogen is fluorine.", "It is preferred that the compound of Formula I comprises at least two fluorine atoms, preferably at least three, four, five, six, seven or eight fluorine atoms.", "The term “aryl”, when used herein, includes C6-10 aryl groups such as phenyl, naphthyl and the like.", "Aryl groups may be substituted by one or more substituents including —OH, cyano, halo, nitro, amino, alkyl and alkoxy.", "When substituted, aryl groups are preferably substituted by between one and three substituents.", "The term alkyl, when used herein, refers to alkyl groups of 1 to 16, preferably 1 to 10 (e.g.", "1 to 6) carbon atoms.", "The term alkoxy, when used herein, refers to alkoxy groups of 1 to 16, preferably 1 to 10 (e.g.", "1 to 6) carbon atoms.", "Alkyl and alkoxy groups as defined herein may be straight-chain or, when there is a sufficient number (i.e.", "a minimum of three) of carbon atoms, be branched-chain and/or cyclic and/or heterocyclic.", "Further, when there is a sufficient number (i.e.", "a minimum of four) of carbon atoms, such alkyl and alkoxy groups may also be part cyclic/acyclic.", "Such alkyl and alkoxy groups may also be saturated or, when there is a sufficient number (i.e.", "a minimum of two) of carbon atoms, be unsaturated and/or interrupted by one or more oxygen and/or sulfur atoms.", "Alkyl and alkoxy groups may also be substituted by one or more halo, and especially fluoro, atoms.", "The term “halo”, when used herein, includes fluoro, chloro, bromo and iodo.", "The terms alkylamine, dialkylamine and trialkylamine, when used herein, refer to amines bearing one, two or three alkyl groups as defined herein, respectively.", "The term alkylene, when used herein, refers to alkylene groups of 1 to 20, preferably 2 to 17 (e.g.", "6 to 12) carbon atoms.", "Alkylene groups may be straight-chain or, when there is a sufficient number (i.e.", "a minimum of two or three, as appropriate) of carbon atoms, be branched-chain and/or cyclic and/or heterocyclic.", "Further, when there is a sufficient number (i.e.", "a minimum of four) of carbon atoms, such alkylene groups may also be part cyclic/acyclic.", "Such alkylene chains may also be saturated or, when there is a sufficient number (i.e.", "a minimum of two) of carbon atoms, be unsaturated and/or interrupted by one or more oxygen and/or sulfur atoms.", "Preferred compounds of formula I include those in which: alkylene group X is at least 50% fluorinated; alkylene group X contains between 2 and 17 carbon atoms; Z1 represents —CO2H, an ammonium, (C1-10 alkyl)ammonium, di-(C1-10 alkyl)ammonium or tri-(C1-10 alkyl)ammonium salt of —CO2H, or —CO2R1; Z2 represents F, H, —CO2H or —CO2R1; R1 represents C1-6 alkyl.", "More preferred compounds of formula I include those in which: alkylene group X is at least 75% fluorinated; alkylene group X is straight-chain, saturated and contains between 4 and 14 carbon atoms; Z1 represents —CO2H, an ammonium, (C1-6 alkyl)ammonium, di-(C1-6 alkyl)ammonium or tri-(C1-6 alkyl)ammonium salt of —CO2H, or —CO2R1; Z2 represents F, —CO2H or —CO2R1; R1 represents straight-chain, unsubstituted, saturated C1-4 alkyl.", "Even more preferred compounds of formula I include those in which: alkylene group X is at least 90% fluorinated; alkylene group X is straight-chain, saturated and contains between 6 and 12 carbon atoms; Z1 represents —CO2H, an ammonium salt of —CO2H, or —CO2R1; R1 represents straight-chain, unsubstituted, saturated C1-2 alkyl.", "Particularly preferred compounds of formula I may be or comprise a member of the following group: Perfluoroheptanoic acid; perfluorooctanoic acid; perfluorononanoic acid; perfluorodecanoic acid; perfluoroundecanoic acid; perfluorododecanoic acid; perfluorotetradecanoic acid; perfluorohexadecanoic acid; perfluorooctadecanoic acid; perfluorosuccinic acid; perfluoroglutaric acid; perfluoroadipic acid; perfluorosuberic acid; perfluoroazelaic acid; perfluorosebacic acid; perfluoro-1,10-decanedicarboxylic acid; methyl perfluoroheptanoate; methyl perfluorooctanoate; methyl perfluorononanoate; methyl perfluorodecanoate; methyl perfluoroundecanoate; methyl perfluorododecanoate; methyl perfluorotridecanoate; methyl perfluorotetradecanoate; methyl perfluoropentadecanoate; methyl perfluorohexadecanoate; methyl perfluorooctadecanoate; dimethyl perfluorosuccinate; dimethyl perfluoroglutarate; dimethyl perfluoroadipate; dimethyl perfluorosuberate; dimethyl perfluoroazelate; dimethyl perfluorosebacate; perfluoro-1,10-decanedicarboxylic acid, dimethyl ester; and dimethyl perfluorododecanedioate.", "These compounds may be obtained from any suitable supplier, for example 3M, DuPont, Miteni or Dyneon.", "Examples of fluoroalkyl carbonyl compounds that may be useful include alpha-branched fluoroalkylcarbonyl fluorides and derivatives thereof, as described in U.S. Pat.", "No.", "6,013,795 or U.S. Pat.", "No.", "6,015,838 (both incorporated herein by reference) and references given therein, for example U.S. Pat.", "No.", "2,567,011 (incorporated herein by reference).", "Methods of preparing same are also described.", "These compounds may also be useful in the synthesis of further compounds of the invention.", "It is preferred that the compound is not a compound as discussed in U.S. Pat.", "No.", "6,028,109, which are indicated to be PPAR agonists.", "Thus, it is preferred that the compound is not a compound represented by formula (I) of U.S. Pat.", "No.", "6,028,109 (shown in FIG.", "8).", "Particularly preferred compounds of formula I include fluorinated fatty acids, such as perfluorinated fatty acids, for example perfluorooctanoic acid (PFOA) or a derivative or pharmaceutically acceptable salt or ester thereof (e.g.", "ammonium perfluorooctanoate (APFO)).", "The chemical formula for APFO is CF3(CF2)6COO− NH4+ (octanoic acid, pentadecafluoro-, ammonium salt; C-8, FC-143; CAS Registry No 3825-26-1).", "It may be obtained from DuPont (DuPont Chemical Solutions Enterprise, DuPont-Strassel, D-61343 Bad Homburg, Germany).", "Common contaminants of APFO include ammonium perfluoroheptanoate (CAS 6130-43-4), ammonium perfluorohexanoate (CAS 68259-11-0), ammonium perfluoropentanoate (CAS 21615-47-4), and branched chain homologs that are generically known as ammonium perfluoroisooctanoate, ammonium perfluoroisoheptanoate, ammonium perfluoroisohexanoate and ammonium perfluoroisopentanoate.", "Whilst it is considered that the effects observed in Example 1 using an APFO preparation arise from the administration of APFO itself, it will be appreciated that one or more contaminants, for example one or more of the possible contaminants listed above, may contribute to the effects observed.", "It is preferred that the compound is not PFOS (perfluorooctylsulphonate) or perfluorodecanoic acid or a derivative or salt or ester thereof; these compounds may have toxic or environmentally undesirable effects.", "It is preferred that the compound of formula I (or identified or identifiable by a screening method of the invention, as discussed below) is metabolically stable; for example it is preferred that the compound has a similar rate of metabolism to perfluorooctanoic acid.", "The compound may be considered to be a lipid mimetic which may be metabolically stable.", "A further aspect of the invention provides the use of a compound of formula I as defined herein in the manufacture of a medicament for the treatment of a patient in need of modulation (preferably reduction) of body mass or modulation (preferably prevention or reduction) of increase in body mass, and/or in need of modulation (preferably reduction) of plasma insulin, plasma glucose, plasma triglycerides, leptin and/or plasma cholesterol.", "The patient may (for example in relation to a decrease in the above-listed parameters) be overweight or obese and/or have diabetes, hyperlipidaemia and/or atherosclerosis, or be at risk of developing such a condition.", "The risk may arise from genetic factors, age, or environmental factors, such as diet.", "The patient may have other condition(s) associated with obesity, for example coronary heart disease, stroke, obstructive sleep apnoea, arthritis (for example osteoarthritis) or reduced fertility.", "Accordingly, a further aspect of the invention provides the use of a compound of formula I as defined herein in the manufacture of a medicament for the treatment of a patient who is overweight or obese and/or has diabetes, hyperlipidaemia, atherosclerosis, coronary heart disease, stroke, obstructive sleep apnoea, arthritis (for example osteoarthritis) and/or reduced fertility, or is at risk of developing such a condition.", "A further aspect of the invention provides the use of a compound of formula I in the manufacture of a medicament for the treatment of a patient in need an antitumour agent or an antiinflammatory agent or of modulation of lipid or eicosanoid status.", "Preferences in relation to such a patient are noted above.", "As is well known to those skilled in the art, obesity may be described as a state of excessive accumulation of body fat.", "Obesity may be determined by determining the body mass index (BMI) for a patient, and/or by measuring subcutaneous fat deposits in the arm using a “pinch test”.", "The BMI is defined as weight (in kilograms) divided by the square of the height in metres.", "A BMI of 25-30 is considered as overweight and more than 30 as obese.", "Preferably, treatment leads to lowering of the BMI to less than about 29 to 31, or to a point at which health risks from being overweight are no longer significant.", "It will be appreciated that the treatment of the invention may be used in combination with other treatments for the relevant condition.", "For example in relation to obesity, the patient may follow a calorie-restricted diet and/or follow a program of physical exercise.", "The medicament may comprise more than one (e.g.", "two) compounds of formula I (such as perfluorinated fatty acids or their derivatives).", "The medicament may comprise a prodrug, for example a molecule which is converted to a molecule with the required biological activity following administration of the medicament to the patient.", "Compounds of formula I may be particularly usefiul in the treatment of patients with diabetes.", "For example, the compound may be useful in treating type II diabetes.", "In type I diabetes, the compounds may be useful as an insulin sensitiser and may therefore allow the dose of insulin administered to be reduced, thereby lowering costs and potentially reducing side effects of insulin administration.", "Existing anti-diabetic agents, for example the thiazolidinedione class of agents, may have the undesirable effect of stimulating weight gain.", "Compounds of formula I, such as perfluoroalkyl carboxylic acid compounds and their derivatives (e.g.", "PFOA or APFO or derivatives thereof), are considered to have the desirable effect of preventing weight gain as well as being useful as anti-diabetic agents.", "Compounds of the invention may be useful in the concomitant treatment of a number of abnormalities, for example diabetes, obesity and hyperlipidaemia.", "Thus, it may be possible to treat a patient with these conditions (which may often occur together) with a single compound or preparation.", "This may have benefits, for example in relation to patient compliance, the avoidance of drug interactions, ease of formulation and marketing.", "It is preferred that the patient is mammalian, most preferably human or, less preferably a domesticated animal, for example an animal kept as a pet or in agriculture, for example horse, cow, cat or dog.", "It is preferred that the compound is a compound wherein the plasma insulin levels are modulated (preferably reduced) in a mammal following administration of the compound to the mammal, relative to either preadministration levels or a control mammal which has not been administered the compound.", "It is particularly preferred that plasma insulin levels are modulated (for example reduced) (for example relative to a control animal) in a male Fischer 344 rat following administration of the compound to the rat, as described in Example 1.It is sufficient for a reduction to be found at any time following first administration of the compound to the mammal (preferably rat), but it is preferred that such reduction is found (ie appears or is still present) at least seven days after first administration of the compound.", "It is preferred that a reduction of insulin levels of at least 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80% is achieved.", "It will be appreciated that the comparative measurements are made on animals at substantially the same stage of feeding, ie at substantially the same time of day or at substantially the same time following ingestion of food.", "When the mammal is a rat, it is preferred that plasma insulin levels are modulated (preferably reduced) following administration of the compound at a level of between 30 and 5000 or 3000 ppm of the diet, preferably between about 50 and 500 ppm, still more preferably about 300 ppm.", "It is preferred that the test is conducted using the methods and conditions described in Example 1.It is preferred that the change in insulin level is not accompanied by any adverse clinical symptoms or change in behaviour/activity of the mammal.", "Thus, the animals may be observed in relation to standard clinical chemistry analyses, blood pressure and/or dizziness.", "Thus, it is preferred that insulin levels are modulated (preferably reduced) following administration of an amount of the compound that does not produce a significant adverse effect on the animal.", "Tests may be performed on more than one animal for a compound or given dose of a compound, as known to those skilled in the art.", "Alternatively or in addition, it is preferred that plasma cholesterol, glucose and/or trigylceride levels are modulated (preferably reduced), and/or leptin levels modulated, in a mammal following administration of the compound to the mammal, relative to either preadministration levels or a control mammal which has not been administered the compound.", "Preferences indicated above in relation to modulation (for example reduction) of insulin levels apply similarly in relation to modulation (for example reduction) of plasma cholesterol, glucose and trigylceride levels, and to modulation of leptin levels.", "Alternatively or in addition, eicosanoid status (ie type or concentration) may be modulated (preferably reduced) in a mammal (or in a particular locus or tissue) following administration of the compound to the mammal, relative to either preadministration levels or a control mammal which has not been administered the compound.", "Alternatively or in addition, it is preferred that bodyweight or bodyweight gain is modulated (preferably reduced) in a mammal following administration of the compound to the mammal, relative to either preadministration levels (for bodyweight) or a control mammal (for bodyweight or bodyweight gain) which has not been administered the compound.", "Preferences indicated above in relation to modulation (for example reduction) of insulin levels apply similarly in relation to modulation (for example reductions) in bodyweight or bodyweight gain.", "Food consumption (expressed as weight of food consumed per unit bodyweight) may be increased following administration of the compound to the mammal, relative to either preadministration levels or a control mammal which has not been administered the compound.", "The increase may not be seen immediately after commencing administration of the compound; an initial decrease may be seen, which may be followed by an increase.", "Whilst not wishing to be bound by theory, it is considered that a compound of the invention, for example a perfluorinated fatty acid, for example APFO or PFOA, may bind to a peroxisome proliferator activated receptor (PPAR), for example PPARα, PPARδ or PPARγ (Kliewer et al (1994) PNAS, 91, 7355-7359; reviewed in Gelman et al (1999) Cell Mol Life Sci 55, 932-943; Kersten et al (2000) Nature 405, 421-424 and Issemann & Green (1990) Nature 347, 645-650) and may be a PPAR agonist or antagonist.", "It is preferred that the compound binds to a peroxisome proliferator activated receptor (PPAR), for example PPARα, PPARδ or PPARγ.", "It is further preferred that the compound is a PPARα, PPARδ or PPARγ modulator, for example a PPARα, PPARδ or PPARγ agonist or antagonist.", "It is particularly preferred that the compound is a PPARα or PPARγ agonist or a PPARδ antagonist.", "Any suitable method may be used for determining whether a compound binds to and/or is a modulator, for example an agonist or antagonist of a PPAR.", "Also whilst not wishing to be bound by theory, a compound of the invention, for example a perfluorinated fatty acid, for example APFO or PFOA, may bind to a lipid metabolising or binding entity, for example a cycloxygenase, for example COXI or COXII, or phospholipase A, for example Phospholipase A2, or lipoxygenase and may be a modulator, for example an activator or inhibitor, of such an entity's activity (including degree of activation).", "It is preferred that the compound binds to a lipid metabolising enzyme, for example a cycloxygenase, for example COXI or COXII.", "It is further preferred that the compound is a modulator of the activity of a lipid metabolising enzyme, for example a cycloxygenase, for example COXI or COXII.", "Any suitable method may be used for determining whether a compound binds to and/or is a modulator, for example an activator or inhibitor of a lipid binding or metabolising entity.", "A further aspect of the invention provides the use of a compound of formula I as defined herein in the manufacture of a medicament for treating a patient in need of modulation of PPAR (for example PPARα, PPARδ (also known as β) or PPARγ) activity.", "Preferably the patient is in need of an increase in PPAR (preferably PPARα and/or PPARγ) activity and the compound of formula I as defined herein is a PPAR (for example a PPARα or γ) agonist.", "Alternatively, the patient is in need of a decrease in PPAR (preferably PPARδ) activity and the compound of formula I as defined herein is a PPAR (for example a PPARδ) antagonist.", "A further aspect of the invention provides the use of a compound of formula I as defined herein in the manufacture of a medicament for treating a patient in need of modulation of a lipid metabolising entity activity, for example cycloxygenase (for example cyclooxygenase I or cyclooxygenase II) activity or phospholipase A (for example phospholipase A2) activity or lipoxygenase activity.", "A further aspect of the invention provides a screening method for identifying a drug-like compound or lead compound for the development of a drug-like compound in which (1) a mammal is exposed to a compound of formula I as defined herein (for example a perfluorinated fatty acid) or derivative thereof (2) the plasma insulin, glucose, cholesterol, triglyceride and/or leptin level of the mammal is measured, and/or bodyweight of the mammal is measured, and/or lipid or eicosanoid status (ie type and level of at least one lipid or eicosanoid) or function (for example assessed by degree of responsiveness to the mammal to a lipid or eicosanoid) of the mammal is measured.", "The method preferably comprises the step of selecting a compound on exposure to which the plasma insulin, glucose, cholesterol, and/or triglyceride level of the mammal is changed, preferably reduced, and/or leptin level of the mammal is modulated, and/or bodyweight or bodyweight increase is changed, preferably reduced.", "Preferences for this aspect of the invention include those indicated above in relation to investigating effects on insulin, cholesterol, glucose, triglyceride or leptin levels, or on bodyweight.", "For example, it is preferred that the mammal is a rodent, for example a rat or a mouse, or other laboratory animal such as a dog.", "A further aspect of the invention provides a screening method for identifying a drug-like compound or lead compound for the development of a drug-like compound in which (1) a mammal is exposed to a compound of formula I as defined herein (for example a perfluorinated fatty acid) or derivative thereof (2) the plasma insulin, glucose, cholesterol, triglyceride and/or leptin level of the mammal is measured, and/or bodyweight of the mammal is measured, and/or lipid or eicosanoid status (ie type and level of at least one lipid or eicosanoid) or function (for example assessed by degree of responsiveness to the mammal to a lipid or eicosanoid) of the mammal is measured.", "A further aspect of the invention provides a screening method for identifying a drug-like compound or lead compound for the development of a drug-like compound in which (1) a compound of formula I as defined herein or related compound is exposed to a PPAR polypeptide (2) the binding of the compound to the PPAR polypeptide is measured or the change in the activity of the PPAR polypeptide is measured.", "Suitable methods by which binding of the compound to the PPAR polypeptide or effect on activity of the PPAR polypeptide may be measured are described, for example, in U.S. Pat.", "No.", "6,028,109.The method may comprise the step of selecting a compound that binds to the PPAR polypeptide and/or changes its activity, for example nucleic acid binding activity and/or transcription factor activity.", "It is preferred that the selected compound increases PPARα or PPARγ activity ie acts as a PPARα or PPARγ agonist, or decreases PPARδ activity, ie acts as a PPARk antagonist.", "A further aspect of the invention provides a screening method for identifying a drug-like compound or lead compound for the development of a drug-like compound in which (1) a compound of formula I as defined herein or related compound is exposed to a lipid metabolising or binding entity, for example cycloxygenase (for example cyclooxygenase I or cyclooxygenase II) or phospholipase A (for example phospholipase A2) (2) the binding of the compound to the lipid metabolising or binding entity is measured or the change in the activity of the lipid metabolising or binding entity is measured.", "Suitable methods by which binding of the compound to the lipid metabolising or binding entity or effect on activity of the lipid metabolising or binding entity may be measured will be well known to those skilled in the art.", "Methods similar to those described in, for example, U.S. Pat.", "No.", "6,028,109, may be suitable, as noted above.", "The method may comprise the step of selecting a compound that binds to the lipid metabolising or binding entity and/or changes its activity, for example production of arachidonic acid from appropriate phospholipid (phospholipase A) or production of prostaglandin from arachidonic acid (cyclooxygenase).", "It is preferred that the selected compound decreases the enzymic or binding activity ie acts as an inhibitor of the enzyme or binding entity.", "A screening method of the invention may involve comparing the effect achieved using the test compound with that achieved using APFO or PFOA or other compound with desirable properties, as indicated above.", "A screening method of the invention may involve determining whether the test compound is able to compete with APFO or PFOA or other compound with desirable properties, as indicated above, for example whether it competes with APFO or PFOA for binding to a PPAR polypeptide, for example PPARα, or other lipid metabolising or binding entity, for example COXI, COXII or phospholipase A2.Useful screening methods (for example in which the effect of the test compound is compared with that of APFO or PFOA or other compound with desirable properties, as indicated above) also include lipid displacement assays, cell (for example adipocyte) differentiation assays, or other phenotypic assays, insulin sensitisation assays, antiniflammatory screen, or investigation of effects on eicosanoid biosynthesis.", "The compound may be tested in animal models useful in investigating conditions of interest as noted above, such as obesity, diabetes, hyperlipidaemia or carcinogenesis.", "Such models include obese (ob/ob) or diabetic (db/db) mice, APC/min mice, BB rat or human tumour xenograft models, as known to those skilled in the art.", "A further aspect of the invention provides a screening method for identifying a drug-like compound or lead compound for the development of a drug-like compound in which (1) a cell is exposed to a compound of formula I as defined herein (for example a perfluorinated fatty acid) or derivative thereof (2) the phenotype (for example differentiation) and/or eicosanoid biosynthesis of the cell is measured.", "The method preferably comprises the step of selecting a compound on exposure to which the phenotype, for example differentiation, of the cell is changed, and/or eicosanoid biosynthesis of the cell is changed, preferably reduced.", "The screening methods may be useful in identifing a drug-like compound or lead compound for the development of a drug-like compound for treating diabetes, obesity, hypercholesterolaemia and/or hyperlipidaemia.", "The methods may further comprise the step of determining whether the compound is toxic or carcinogenic, for example at a concentration sufficient to elicit a change in bodyweight or bodyweight gain, plasma insulin, glucose, cholesterol, triglyceride and/or leptin levels.", "Such methods will be well known to those skilled in the art.", "It will be appreciated that the compound may preferably be tested in more than of the screening methods of the invention.", "For example, a compound may be tested for its effect on a PPAR polypeptide, and for its effect on a mammal to which it is administered.", "The toxicity or carcinogenicity of the compound may also be determined.", "The term “drug-like compound” is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament.", "Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons molecular weight.", "A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate cellular membranes, but it will be appreciated that these features are not essential.", "The term “lead compound” is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.", "These “lead” compounds may then be developed further, for example by molecular modelling/and or experiments to determine a structure activity relationship, in order to develop more efficacious compounds, for example by improving potency, selectivity/specificity and pharmacokinetic properties.", "The methods may be performed ini vitro, either in intact cells or tissues (for example liver cells or adipocytes), with broken cell or tissue preparations or at least partially purified components.", "Alternatively, they may be performed in vivo.", "The cells tissues or organisms in/on which the use or methods are performed may be transgenic.", "In particular they may be transgenic for a PPAR polypeptide or lipid metabolising or binding entity.", "It will be appreciated that the polynucleotide encoding the PPAR (for example PPARα, β or γ) or lipid metabolising or binding entity may be mutated in order to encode a variant of the PPAR, for example by insertion, deletion, substitution, truncation or fusion, as known to those skilled in the art.", "It is preferred that the PPAR or lipid metabolising or binding entity is not mutated in a way that may materially affect its biological behaviour, for example its nucleic acid binding or transcription factor activity or lipid metabolising or binding activity, as appropriate.", "The following references relate to the sequences and tissue distribution of PPARs: Auboeuf et al (1997) Diabetes 46(8), 1319-1327; Braissant et al (1996) Endocrinol 137(1), 354-366; Mukherjee et al (1994) J Steroid Biochem Mol Biol 51, 157-166; Mukerjee et al (1997) J Biol Chem 272, 8071-8076.The following references and GenBank Accession numbers relate to the sequences and/or tissue distribution of the indicated polypeptides.", "U63846 Human COX-1 cDNA (PTSG1); Hla (1996) Prostaglandins 51, 81-85.NM000963 Human COX2 cDNA (PTSG2); Hla & Neilson (1992) PNAS 89(16), 7384-7388; Jones et al (1993) J Biol Chem 268(12), 9049-9054; Appleby et al (1994) Biochem J 302, 723-727; Kosaka et al (1994) Eur J Biochem 221(3), 889-897.AF306566 Human phospholipase A2 (secreted form); Valentin et al (2000) Biochem Biophys Res Commun 279(1), 223-228.NM021628 Human lipogenase ALOXE3.XM005818 Human lipoxygenase ALOXE5.XM008328 Human lipoxegenase ALOX12.NM001141 Human lipoxegenase ALOX15; Brash et al (1997) PNAS 94(12), 6148-6152.NM005090 and NM 003706 Human phospholipase A2 (cPLA2-gamma) Underwood et al (1998) J Biol Chem 273(34), 21926-21932 and Pickard et al (1999) J Biol Chem 274(13), 8823-8831 M68874 Human phospholipase A2 (cPLA2) Sharp et al (1991) J Biol Chem 266(23), 14850-14853.It will be appreciated that such a compound may be an agonist or antagonist of the PPAR polypeptide used in the screen and that the intention of the screen is to identify compounds that act as agonists or antagonists of the PPAR, even if the screen makes use of a binding assay rather than an activity assay, for example transcription factor activity or nucleic acid (for example DNA) binding activity.", "It will be appreciated that the action of a compound found to bind the PPAR polypeptide may be confirmed by performing an assay of transcription factor activity or nucleic acid binding activity in the presence of the compound.", "Likewise, such a compound may be an inhibitor or activator of the lipid metabolising or binding entity used in the screen and that the intention of the screen is to identify compounds that act as inhibitors or activators of the lipid metabolising or binding entity, even if the screen makes use of a binding assay rather than an activity assay, for example lipid metabolising activity, for example prostaglandin production from arachidonic acid for COXI or COXII.", "It will be appreciated that the action of a compound found to bind the lipid metabolising or binding entity may be confirmed by performing an assay of the appropriate enzyme or binding activity in the presence of the compound.", "It is preferred that the assay is capable of being performed in a “high throughput” format.", "This may require substantial automation of the assay and minimisation of the quantity of a particular reagent or reagents required for each individual assay.", "A scintillation proximity assay (SPA) based system, as known to those skilled in the art, may be beneficial.", "Combinatorial chemistry techniques may be used in generating compounds to be tested.", "A further aspect of the invention provides a kit of parts of screening system comprising (1) a library of compounds each of formula I as herein defined or a derivative thereof, and (2) a PPAR polypeptide or polynucleotide encoding a PPAR polypeptide, and/or a test mammal.", "The kit may optionally comprise reagents useful in measuring plasma insulin, glucose, triglyceride and/or cholesterol levels, or in measuring PPAR activity, for example nucleic acid binding.", "Such reagents will be apparent to those skilled in the art, and may include reagents useful in performing transactivation assays or DNA binding assays.", "A further aspect of the invention provides a kit of parts of screening system comprising (1) a library of compounds each of formula I as herein defined or a derivative thereof, and (2) a lipid metabolising or binding entity (for example COXI or COXII or phospholipase A2 or lipoxygenase) or polynucleotide encoding a lipid metabolising or binding entity.", "The kit may optionally comprise reagents useful in measuring plasma insulin, glucose, triglyceride, cholesterol and/or leptin levels, or in measuring the activity of the lipid metabolising or binding entity, for example a substrate of the lipid metabolising or binding entity (for example arachidonic acid in the case of COXII or lipoxygenase) or reagent useful in measuring a product of a lipid metabolising enzyme, for example in assessing eicosanoid biosynthesis.", "As well known to those skilled in the art, reagents may include labelled ligand, for example radiolabelled or fluorescently labelled.", "Direct binding or displacement of ligand may be measured.", "Binding may be measured using fluorescence resonance energy transfer (FRET) techniques.", "The kit may optionally include reagents useful in cell differentiation assays, for example adipocyte differentiation assays, as will be known to those skilled in the art.", "A further aspect of the invention provides a compound identifiable or identified by a screening method of the invention.", "A further aspect of the invention provides a compound identified or identifiable by a screening method of the invention for use in medicine.", "A further aspect of the invention provides a pharmaceutical composition comprising a compound identified or identifiable by a screening method of the invention and a pharmaceutically acceptable excipient.", "Preferences in relation to properties of such compounds are as indicated above and in relation to the first aspect of the invention.", "A compound identified or identifiable by a screening method of the invention is also provided for use in the manufacture of a composition for use as a food supplement or a food additive.", "The invention also relates to a food product comprising a foodstuff and a compound of formula I as defined herein or a compound identified or identifiable by a screening method of the invention, wherein the food is not laboratory rodent, for example rat or mouse, feed.", "It is preferred that the food is not laboratory animal feed.", "Preferably, the food (the term including food product and foodstuff) is suitable for administration to an animal (for example a domesticated animal as discussed above but not a laboratory rodent) or human, for example an adult human, baby or infant.", "A further aspect of the invention provides the use of a compound identified or identifiable by a screening method of the invention in the manufacture of a medicament for the treatment of a patient in need of modulation (for example reduction) of body mass or modulation (preferably reduction or prevention) of increase in body mass, and/or in need of modulation (preferably reduction) of plasma insulin, plasma glucose, plasma triglycerides and/or plasma cholesterol, and/or in need of modulation of plasma leptin.", "The patient may be obese and/or have diabetes, hyperlipidaemia and/or atherosclerosis, or be at risk of developing such a condition.", "A further aspect of the invention provides the use of a compound identified or identifiable by a screening method of the invention in the manufacture of a medicament for the treatment of a patient who is overweight or obese and/or has diabetes, hyperlipidaemia, atherosclerosis, coronary heart disease, stroke, obstructive sleep apnoea, arthritis (for example osteoarthritis) and/or reduced fertility, or is at risk of developing such a condition.", "A further aspect of the invention provides the use of compound identified or identifiable by a screening method of the invention in the manufacture of a medicament for treating a patient in need of modulation of PPAR (for example PPARα) or modulation of lipid or eicosanoid status or function, or of lipid metabolising or binding entity (for example COXI, COXII, phospholipase A or lipoxygenase) activity.", "Preferably the patient is in need of an increase in PPAR (preferably PPARα or PPARγ) activity and the compound is a PPAR (for example a PPARα or PPARγ) agonist.", "Alternatively, the patient may be in need of a decrease in activity of a lipid metabolising or binding entity and the compound is an inhibitor of that lipid metabolising or binding entity (or entities).", "The compounds may be administered in any suitable way, usually parenterally, for example intravenously, intraperitoneally or intravesically, in standard sterile, non-pyrogenic formulations of diluents and carriers.", "The compounds may also be administered topically.", "The compounds of the invention may also be administered in a localised manner, for example by injection.", "Preferably, the compounds are administered orally.", "The compounds may be administered as a tablet or capsule or as a supplement added to food or drink.", "A slow-release formulation may be used.", "A further aspect of the invention provides a method of treatment of a patient in need of modulation (preferably reduction) of body mass or modulation (for example reduction or prevention) of increase in body mass, and/or in need of modulation (for example reduction) of plasma insulin, plasma glucose, plasma triglycerides, plasma cholesterol and/or leptin, comprising administering to the patient an effective amount of a compound identified or identifiable by the screening method of the invention.", "A further aspect of the invention provides a method of treatment of a patient who is overweight or obese and/or has diabetes, hyperlipidaemia, atherosclerosis, coronary heart disease, stroke, obstructive sleep apnoea, arthritis (for example osteoarthritis) and/or reduced fertility, or is at risk of developing such a condition, comprising administering to the patient an effective amount of a compound identified or identifiable by the screening method of the invention.", "Preferences in relation to the patient and compound are as indicated above.", "A further aspect of the invention provides a method of treatment of a patient in need of modulation of PPAR (for example PPARα, δ or γ) activity, or of lipid or eicosanoid status or function, or of a lipid metabolising or binding entity activity, comprising administering to the patient an effective amount of a compound identified or identifiable by the screening method of the invention.", "Preferably the patient is in need of an increase in PPAR (preferably PPARα or γ) activity and the compound is a PPAR (for example a PPARα or γ) agonist.", "Further preferences in relation to the patient and compound are as indicated above.", "Alternatively, the patient may be in need of a decrease in activity of a lipid metabolising or binding entity and the compound is an inhibitor of that lipid metabolising or binding entity or entities.", "The invention is now described in more detail by reference to the following, non-limiting, Figures and Examples: FIG.", "1: Growth curves for male Fischer 344 rats administered APFO in diet.", "FIG.", "2: Food consumption by Fischer 344 rats administered APFO in diet.", "FIG.", "3: Food utilisation in Fischer 344 rats administered APFO in diet.", "FIG.", "4: Effect of APFO treatment on plasma insulin concentration.", "FIG.", "5: Effect of APFO on plasma cholesterol concentration.", "FIG.", "6: Effect of APFO on plasma glucose concentration.", "FIG.", "7: Effect of APFO on plasma triglyceride concentration.", "FIG.", "8: compounds indicated to be PPAR agonists in U.S. Pat.", "No.", "6,028,109 FIG.", "9: Effect of APFO treatment on mouse body weights FIG.", "10: Effect of APFO treatment on mouse food consumption FIG.", "11: Effect of APFO treatment on mouse food consumption (expressed as grams food consumed per unit body weight).", "FIG.", "12: Effect of APFO treatment on mouse plasma insulin concentration.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "13: Effect of APFO treatment on mouse plasma triglyceride concentration.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "14: Effect of APFO on mouse plasma glucose concentration.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "15: Effect of APFO treatment on mouse plasma cholesterol.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "16: Effect of APFO treatment on mouse plasma leptin.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "17: Effect of APFO treatment of mouse epididimal fat pad weight.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "18: Effect of APFO treatment on rat body weight.", "FIG.", "19: Effect of APFO treatment on rat food consumption.", "FIG.", "20: Effect of APFO on rat food consumption (expressed as grams food consumed per unit body weight).", "FIG.", "21: Effect of APFO treatment on plasma insulin concentration.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "22: Effect of APFO treatment on rat plasma glucose concentration.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "23: Effect of APFO treatment on rat plasma triglyceride concentration.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "24: Effect of APFO treatment on rat plasma cholesterol.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "25: Effect of APFO treatment on rat plasma leptin concentration.", "FIG.", "26: Cytotoxic effect of APFO on HepG2 cells (A), HT-29 cells (B) and MCF7 cells (C).", "FIG.", "27: Effect of prophylactic APFO treatment on tumour volume in an HT-29 xenograft model.", "=APFO administration.", "Arrow indicates the point of tumour cell implantation.", "Tumour measurement began on day 1.FIG.", "28: Effect of prophylactic APFO administration on tumour volume between day 1 and day 15 of treatment.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); p<0.05; p<0.01; * p<0.001.FIG.", "29: Effect of prophylactic APFO administration on tumour weight.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "30: Effect of therapeutic APFO treatment on tumour volume in an HT-29 xenograft model.", "=APFO administration.", "Arrow indicates the point of tumour cell implantation.", "Tumour measurement and APFO administration began on day 1.FIG.", "31: Effect of therapeutic APPO administration on tumour volume between day 1 and day 17 of treatment.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); p<0.05; p<0.01; * p<0.001.FIG.", "32: Effect of therapeutic APFO administration on tumour weight.", "Values are Mean±SD.", "Significantly different from respective control group (0 mg/kg); * p<0.05; p<0.01; * p<0.001.FIG.", "33: Effect of prophylactic APFO administration on nu/nu mouse body weight.", "FIG.", "34: Effect of therapeutic APFO administration on nu/nu mouse body weight.", "FIG.", "35: Activation of Mouse PPARα by APFO.", "FIG.", "36: Interaction of APFO with Ligand Binding Domain of Human PPARγ.", "EXAMPLE 1 Effect Of Perfluorinated Fatty Acid On Insulin, Glucose, Cholesterol And Triglyceride Levels, And On Body Weight METHODS Male Fisher 344 rats (initially 6 weeks old) were administered ammonium perfluorooctanoate (APFO, 300 ppm) in the diet for periods of time up to one year.", "Control rats received powdered diet that did not contain APFO.", "Body weights were initially determined daily and then weekly.", "Food consumption was determined weekly.", "Clinical observations were made daily.", "Rats were sacrificed at 1, 2, 7, 14, 28, 90, 182 and 365 days.", "There were 8 rats per group.", "At sacrifice blood was sampled by cardiac puncture and submitted for clinical chemistry.", "The following assay methods/kits were used: Assay Supplier Kit Number ALT Roche Diagnostics MPR 1087 568 AST Roche Diagnostics Unimate 3 0736414 Glucose Roche Diagnostics MPR2 1442 449 Trigylcerides Roche Diagnostics Peridochrom GPO-PAP 701 882 Cholesterol Roche Diagnostics CHOD-AP MPR3 236691 Insulin Amersham RNP 2567 RESULTS Administration of APFO to male Fischer 344 rats lead to marked reductions in bodyweight gain (FIG.", "1).", "Treated animals had body weights approximately 25-30% lower than concurrent controls.", "This weight change was not accompanied by any adverse clinical symptoms or changes in activity.", "Food consumption expressed per rat was markedly decreased (to approximately 50% of control consumption) during the first week of treatment.", "However after this time food consumption per rat increased to 80-90% of control values (FIG.", "2).", "When expressed as weight of food consumed per unit bodyweight, food consumption was decreased by approximately 30% during the first week of APFO administration.", "However, at later times APFO-treated animals consumed between 10 and 30% more food per unit bodyweight than controls (FIG.", "3).", "Plasma cholesterol, glucose and insulin concentrations were decreased at all time points examined (FIGS.", "4-6), while plasma triglycerides were decreased at 7 days and beyond (FIG.", "7).", "These data suggest that APFO and related compounds may be useful for treatment of obesity, diabetes, hypertriglycerideaemia and hypercholesterolaemia and diseases where alterations in lipid or eicosanoid status may be desirable, such as arthritis or cancer.", "EXAMPLE 2 Effect of APFO in Reversing Obesity and Diabetes The effect of APFO in animal models of obesity and diabetes was studied in order to establish the therapeutic potential of APFO to reverse obesity and diabetes.", "The studies reported in Example 1 involving the administration of APFO to Sprague Dawley rats for up to one year, demonstrated the compound's anti-diabetic and anti-obesity potential.", "Following an initial reduction in food consumption during the first week of the study, an increase in food consumption per unit body weight was coupled to a marked reduction in body weight gain in treated animals throughout the test period.", "Furthermore, plasma cholesterol and insulin concentrations were decreased at all time points examined, while plasma glucose and triglycerides were decreased at 7 days and beyond.", "In this Example, these observations in healthy rats are extended by investigations in two models of metabolic disease—the obese mouse (ob/ob) and the diabetic GK/Mol rat.", "1.1 Mouse Model (ob/ob) The C57BL/6J-ob/ob mouse is an obese, leptin-deficient animal that is widely accepted as a model of obesity and diabetes.", "Age-paired, disease-free (lean) animals (C57BL/6J-+/+) were also included in the study to observe the ‘normal’ response.", "1.1.1 Experimental Design and Methods Three groups (n=5) of ob/ob (C57BL/6J-ob/ob) mice were treated with 3 dose levels of APFO (5, 15 and 25 mg/kg/day).", "Animals were administered APFO, dissolved in water, by oral gavage, daily for 14 days.", "One group of 10 ob/ob mice was also treated with vehicle (water) alone.", "Additionally, to observe the ‘normal’ response, 5 age-paired, disease-free animals (C57BL/6J-+/+) were administered 25 mg/kg APFO and a similar disease-free control group was administered vehicle only.", "Twenty-four hours after the last dose the animals were killed by an increasing concentration of carbon dioxide.", "Blood was collected by cardiac puncture and plasma prepared and stored at −70° C. until analysed.", "Major tissues were weighed, sampled, flash frozen in liquid nitrogen and stored at −70° C. Plasma was analysed for triglycerides, cholesterol and glucose using kits purchased from Sigma (Poole, Dorset).", "Concentrations of plasma insulin and leptin were determined using commercially available enzymeimmunoassay-based kits from Amersham Life Sciences and Crystalchem Inc., (Chicago) respectively.", "All assays were carried out as specified by the manufacturer.", "1.1.2 Results and Discussion Both strains of mice treated at 25 mg/kg/day lost bodyweight over the treatment period.", "In +/+ mice this was only apparent after day 4; these animals also lost less weight, as a percentage of initial bodyweight, than the ob/ob mice (26% versus 33%).", "In ob/ob mice treated at 15 mg/kg/day 20% bodyweight loss was noted over the study period.", "Animals treated at 5 mg/kg/day were unaffected (FIG.", "9).", "No other adverse clinical observations were observed.", "The bodyweight losses were reflected in marked, APFO dose-related, decreases in food consumption (76%, 53% and 17% lower than control mice at the high, intermediate and low dose levels respectively) (FIG.", "10).", "In +/+ mice a decrease in food consumption was evident over the first nine days of treatment, following which there was a steep recovery towards control values without equalling them.", "The overall consumption was still 31% lower than in +/+ controls.", "When expressed in terms of food consumed per gram of bodyweight the pattern of effect was similar, although the recovery in values seen in +/+ mice after day 9 was greater and the subsequent values more nearly equal to those of their controls (FIG.", "11).", "There was a very marked reduction (greater than 90%) in plasma insulin concentrations in all treated ob/ob mice, which was broadly related to dose level (FIG.", "12).", "In +/+ mice, control insulin levels were notably lower than in the ob/ob mice.", "However, APFO-treatrnent still led to a marked reduction in plasma insulin concentrations.", "APFO-treated (15 or 25 mg/kg/day) ob/ob mice showed dose-related reductions in plasma glucose down to approximately 20% of control values, similarly plasma triglyceride concentrations were decreased to 40% of control values (FIGS.", "13 and 14).", "At 5 mg/kg/day administered to ob/ob mice, glucose was reduced by approximately 50% but there was no effect on triglyceride concentrations (FIG.", "14).", "In +/+ mice, APFO (25 mg/kg/day) decreased glucose and triglycerides to 55% and 35% of control plasma values respectively.", "At a dose of APFO of 25 mg/kg/day to both strains there was an approximate 30% reduction in plasma cholesterol concentrations.", "This was not evident at the low and mid dose levels in the ob/ob mice (FIG.", "15).", "Plasma leptin concentrations in the ob/ob mice were below the levels of quantitation; this was expected as there is an early stop codon within the leptin gene of this mouse strain.", "Treatment of the +/+ mouse, which possesses a normal leptin gene, with APFO (25 mg/kg/day) resulted in decreased plasma leptin concentrations to, or below the level of quantitation (FIG.", "16).", "Epididymal fat pad (white adipose tissue) weights were 7-fold higher in control ob/ob mice compared to control +/+ mice.", "APFO-treatment decreased the weight of the epididymal fat pads in a dose-related manner in ob/ob mice and +/+ mice (FIG.", "17).", "1.1.3 Conclusions In summary, there were a number of significant physiological effects that could be related to the administration of APFO.", "In lean controls, there was a slight reduction in body weight, and this loss reached a nadir after 10 days with no weight loss occurring after this time.", "Food consumption in this group remained constant.", "At the high and intermediate dose levels, ob/ob mice continued to lose body weight.", "At 25 mg/kg, ob/ob mice also displayed appetite loss (reflected in body weight changes).", "In this group there was also marked reduction in glucose levels.", "This appeared to suggest that the anti-obesity effects may have been due to reduced food consumption.", "However, in animals treated with 5 mg/kg APFO, a 17% reduction in food consumption was associated with a 50% reduction in plasma glucose levels, which suggested that the anti-obesity effects observed in ob/ob mice were due to metabolic changes caused by APFO, and not to a loss of appetite.", "These data suggest that APFO causes weight loss in obese animals, but not, significantly, in lean animals and so may be used as an anti-obesity agent.", "Additionally the APFO-induced decreases in plasma glucose and insulin suggest that this chemical may be of therapeutic use in Type II diabetes.", "1.2 Rat GK/Mol Model The GK/Mol rat is a non-obese, diabetic animal that is widely accepted as a model of Type II diabetes.", "In order to measure the ‘normal’ response, non-diabetic Wistar rats were also used in the study.", "1.2.1 Experimental Design and Methods Three groups (n=5) of GK/Mol rats were administered 3 dose levels of APFO (3, 10 and 30 mg/kg).", "Animals were administered APFO by oral gavage, daily for 14 days.", "One group of 10 GK/Mol rats was also treated with vehicle (water) alone.", "Additionally, to observe the ‘normal’ response, 5 age-paired, disease-free Wistar rats were administered 30 mg/kg APFO and a similar disease-free group was administered vehicle only.", "Twenty-four hours after the last dose the animals were killed by an increasing concentration of carbon dioxide.", "Blood was collected by cardiac puncture and plasma prepared and stored at −70° C. until analysed.", "Major tissues were weighed, sampled, flash frozen in liquid nitrogen and stored at −70° C. Plasma was analysed for, triglycerides, cholesterol, glucose, insulin and leptin as described in section 1.1.1.1.2.2 Results and Discussion APFO administration to GK/Mol rats resulted in a dose-dependent decrease in body weight gain to 90%, 71% and 44% of control values at the low, mid and high dose levels respectively (FIG.", "18).", "There was no effect on bodyweight gain in treated Wistar rats.", "Treated GK/Mol rats had slightly lower total food consumption (86-98% of control values), although this difference was not related to dose level (FIG.", "19).", "There was no difference in food consumption between treated Wistar rats and their controls.", "No pattern was discernible when the data were expressed as food eaten per gram of bodyweight (FIG.", "20).", "There was a marked dose-dependent reduction in the plasma concentration of insulin, reaching about 10% of control values in both strains of rat (FIG.", "21).", "Plasma glucose concentrations in GK/Mol rats were lowered by APFO to about 85% of control values at dose levels of 30 mg/kg/day (FIG.", "22).", "Plasma Triglycerides (FIG.", "23) and cholesterol (FIG.", "24) concentrations were lower by between 10 and 20% in treated GK/Mol rats.", "In Wistar rats, plasma cholesterol was reduced to 73% of control values.", "Group mean plasma concentrations of leptin (FIG.", "25) were slightly lower (by approximately 40%) in Wistar rats treated at 30 mg/kg/day than in their controls.", "There were no differences in leptin concentrations in the GK/Mol rats that could be indicative of a treatment-related effect.", "1.2.3 Conclusions The GK/mol study followed a similar pattern to the investigation in Sprague Dawley rats (Example 1).", "APFO caused a reduction in the levels of glucose, triglycerides and cholesterol coupled to reduced weight gain in treated animals; there was also a marked reduction in the level of plasma insulin.", "In conclusion APFO demonstrated anti-diabetic effects in a rat model for type II diabetes, further indicating it may be an effective agent for the treatment of this condition.", "2.Therapeutic Potential of APFO as an Anti-cancer Agent The effect of APFO in vitro against human tumour cell lines and in vivo in a human tumour xenografi model was examined.", "2.1 In vitro Anti-tumour Activity Three human cancer cell lines were exposed to APFO and cytotoxicity levels assessed in order to assess APFO's functions as an anti-cancer agent.", "2.1.1 Experimental Design and Methods HT-29 cells (human colon tumour-derived), MCF7 cells (human breast cancer-derived) and HepG2 cells (human liver cancer-derived) were cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% heat-inactivated foetal calf serum, 2 mM L-glutamine, penicillin (50 IU/ml), streptomycin (50 μg/ml) and I% non-essential amino acids.", "Cells were harvested by trypsinisation and diluted to 5×104 cells/ml, and 200 μl of cell suspension was plated into each well of a 96 well plate and allowed to attach overnight at 37° C. with 5% CO2.Cells were exposed to various concentrations of APFO in growth medium (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 and 1000 μM) for four hours.", "20 μl of a 5 mg/1 m MTT solution was added to each well and the cells were incubated for 4 hours at 37° C. The medium was removed and 200 μl DMSO was added to dissolve formazan crystals.", "Plates were read at 570 nm and background at 690 nm was subtracted.", "Results were displayed graphically as percentage cell survival versus APFO concentration.", "2.1.2 Results and Discussion APFO elicited a cytotoxic effect after 4 hours at concentrations exceeding 500 μM (FIG.", "26).", "2.1.3 Conclusions This study indicated that APFO was effective at killing a range of human cancers in vitro.", "2.2 HT-29 Xenograft Model in Nude Mice The objective of this study was to examine the anti-tumour capabilities of APFO in a xenografted animal model.", "Effects of APFO on tumour progression were tested on a human colon cancer-derived cell line xenografted into immune-deficient nu/nu mice.", "Prophylactic and therapeutic effects of APFO were assessed by measurement of tumour size at regular intervals during administration of APFO.", "2.2.1 Experimental Design and Methods Athymic nude (nu/nu) mice from ICRF stock (HsdOla:ICRF-nu) were obtained from Clare Hall (Pofters Bar, UK).", "All animals were female and approximately 9 weeks old.", "Animals, housed in isolator cages and handled under laminar flow, were divided into one control group and two treatment groups, with 5 female mice per group for both the prophylactic and therapeutic schedules HT-29 cells were cultured according to the conditions described in section 2.1.1.Cells were harvested, pooled by centrifugation and resuspended in 5 ml medium to which 5 ml Matrigel (basement membrane matrix) was added.", "100 μl of cell suspension was injected subcutaneously into each flank of the mice (1.75×106 cells per flank).", "In order to assess the prophylactic effect of APFO, half of the animals were administered the compound immediately following tumour cell implantation.", "For the therapeutic schedule, APFO was injected once tumours had developed.", "Animals were administered APFO, dissolved in water, by intra-peritoneal (ip) injection 3 times per week for one month.", "The doses of APFO were 15 mg/kg and 25 mg/kg bodyweight.", "The volume of the dosing solution was 10 ml/kg bodyweight.", "Control animals received an equivalent volume of water.", "Animal bodyweights were recorded throughout the study.", "Tumour growth was measured 3 times per week using digital calipers and the volume was calculated using the formula: 4 / 3 ⁢ π · { ( d 1 + d 2 ) 3 4 } Where d1=mean length (n=2) and d2=mean width (n=2).", "(NB, n=4 if tumour was an irregular shape).", "The maximum permitted tumour volume, according to the terms of the Home Office licence, was 1.44 cm3.Results were expressed graphically for each time point as mean tumour volumes.", "Tumour weights were recorded at the end of the study, and tumour samples were either snap frozen in liquid nitrogen or fixed in formal saline for further analysis.", "2.2.2 Results and Discussion HT-29-derived tumours developed approximately 14 days into the study.", "Tumour growth in both prophylactic and therapeutic groups proceeded at a much faster rate in control groups compared to tumours in APFO-treated animals (FIGS.", "27 and 30 respectively).", "Consequently, the therapeutic study was not completed because control animals were lost either because tumour volume exceeded the permitted size, or because the tumours were deemed ulcerated and again continuance was not permitted under the terms of the Project Licence.", "Hence, animals in the therapeutic study were injected at 8 time points compared to 13 time points in the prophylactic study.", "Tumour growth rate in animals treated prophylactically was markedly slower in APFO-treated animals, with a lag phase of 15 days for control groups compared to 22 days for APFO-treated mice (FIG.", "27).", "In animals administered 25 mg/kg APFO, tumour growth reached a plateau after 26 days, while in animals dosed at 15 mg/kg, tumour volume continued to increase (FIG.", "27).", "Tumour volume in the prophylactic groups increased 18 fold in controls, 8 fold at 15 mg/kg and 6 fold at 25 mg/kg between the start of tumour measurement (day 1) and the end of the study (day 15) (FIG.", "28).", "Upon necroscopy, in the 15 mg/kg and 25 mg/kg groups respectively, tumour weights were 22% and 58% smaller than in control animals (FIG.", "29).", "Tumour growth rate was also markedly slower in animals treated therapeutically with APFO, with a lag phase of 17 days in control tumours compared to 26 days in treated mice (FIG.", "30).", "No plateau was reached in the highest dose group, but the study was incomplete as animals were treated for a shorter period than intended.", "Tumour volume increased 15-fold in controls, 14-fold at 15 mg/kg, and 7-fold at 25 mg/kg between day 1 and day 17 (FIG.", "31).", "Tumour weights were 45% and 37% smaller in the 15 mg/kg and 25 mg/kg dose groups respectively (FIG.", "32).", "It should be noted that 5 animals (4 controls and 1 high dose animal) had been lost from the study, thus affecting mean values of final tumour weights.", "Tumours were removed from animals at the end of the study period and examined macroscopically.", "Control tumours from the prophylactic group were solid, while APFO-treated animals produced tumours that were fluid-filled, suggesting cell death in the centre of the tumours.", "Differences between control and treated groups were less obvious in animals treated therapeutically, but these animals were dosed for a shorter period.", "Samples of tumours were formalin fixed and also flash frozen in liquid nitrogen for histopathalogical examination.", "Animal body weights were monitored and recorded throughout the study.", "There was no significant difference between control and treated animals in either the prophylactic or therapeutic groups in animals implanted with HT-29 cells (FIGS.", "33 and 34).", "2.2.3 Conclusions In summary, APFO demonstrated anti-tumour capabilities in a human cancer cell line when either given concomitantly with the tumour cells or following tumour establishment.", "Additionally, body weight remained unaffected by the test agent, suggesting that treatment-associated weight loss would not occur, a major advantage for chemotherapeutic agent.", "It is probable that treatment-associated weight loss did not occur because APFO selectively targets obese subjects and not lean subjects (eg.", "nu/nu mice).", "Finally, APFO showed anti-tumour capabilities against HT-29 cells, a human colon cancer cell line, thus demonstrating that it is capable of inhibiting the growth of human tumour cells.", "3.The Potential Anti-inflammatory Properties of APFO 3.1 In vitro Studies The ability of APFO (or other test compound) to inhibit cyclooxygenase 1 (COX1) and cyclooxygenase 2 (COX-2) inhibition is examined using an EIA-based human COX inhibitor assay kit as described by the manufacturer (Cayman Chemical, Michigan).", "3.2 In vivo Studies The anti-inflammatory potential of APFO (or other test compound) is examined in a rat model.", "Animals are dosed with APFO or dexamethasone, after which the animal's immune system is challenged with lipopolysaccharide (LPS) and plasma cytokines are measured.", "The study may consist of one control group and three treatment groups, with 10 male CD rats (80-120 g) per group.", "The control group is administerd vehicle (water) only followed by LPS (30 μg per 100 g rat) 24 hrs later.", "Treatment group 1 animals receive APFO (or other test compound) at 30 mg.kg.", "Treatment group 2 animals receive APFO (or other test compound) at 30 mg.kg followed by LPS (30 μg per 100 g rat) 24hrs later.", "Treatment group 3 animals received dexamethasone (10 mg.ml in corn oil) followed by LPS (30μg per 10 g rat) 1 hour later.", "The plasma from 5 animals per group is harvested lhour or 2 hours post-treatment.", "Plasma cytokines (II-6, II-1β and TNF) are measured using commercially available kits as specified by the manufacturer (Endogen Inc., Massachusetts).", "4.Interaction of APFO with PPAR Isoforms Transactivation assays involving mouse PPAR alpha cDNA and ligand binding assays using human PPAR gamma were performed in order to demonstrate that APFO interacts with PPAR isoforms.", "4.1 Mouse PPAR Ti-ausactivation Assay 4.1.1 Experimental Design and Methods COS-1 cells (cultured in medium described in section 2.1.1 but without non-essential amino acids) were plated into 6 well tissue culture dishes at 3×105 cells per well and allowed to adhere overnight at 37° C. The next day the medium was aspirated and the cells washed with PBS, pH7.4, and 200 μl of a transient transfection cocktail was added to each well.", "The transfection cocktail was composed of 50 ng of vector DNA carrying mouse PPAR alpha, 500 ng of plasmid DNA containing the PPAR response element of liver fatty acid binding protein and, as a transfection control, 500 ng of a vector harbouring β-Galactosidase.", "DNA was dissolved in PBS containing 50 μg.ml DEAE-Dextran.", "Control cells were exposed to a transfection cocktail that contained no plasmid DNA.", "Cells were incubated at 37° C. for 30 minutes before 2 ml of medium containing 80 μM chloroquine was added and the cells incubated for a further 2.5 hours at 37° C. The medium was aspirated and the cells shocked with 10% DMSO in medium for 2.5 minutes at room temperature.", "Cells were washed with PBS then allowed to recover at 37° C. in growth medium for 24 hours.", "Transiently transfected cells were exposed to APFO (dissolved in water) in medium at 0, 3, 10, 30, 100, 300 and 1000 μM for 16 hours at 37° C. Cells were then washed, lysed, and luciferase and β-Galactosidase activities were measured using kits according to the methods specified by the manufacturer (Promega, Madison, USA) by flash luminescence and spectophotometry respectively.", "Luciferase expression was normalised by dividing by the flash luminescence reading with constitutive β-Galactosidase expression levels measured at 415 nm following a colourimetric assay.", "Data were graphed, fitted to non-linear regression curves and EC50 values calculated using GraphPad Prism software.", "4.1.2 Results and Discussion Activation of mouse PPAR alpha by APFO occurred, with an effective concentration (EC50) of 102 μM.", "(FIG.", "27).", "4.1.3 Conclusions The data presented here demonstrate that APFO is a mouse PPAR alpha activator at μM concentrations.", "4.2 PPAR Gamma Ligand Binding Studies His-tagged human PPARγ ligand binding domain was expressed in E. Coli as described previously [Palmer, CAN and Wolf, C R. FEBS Letts.", "431, 476-480, (1998)].", "The receptor protein was partially purified by nickel affinity chromatography.", "4.2.1 Ligand Binding Studies This recombinant receptor protein has been used previously to study interactions with the fluorescent fatty acid—cis-parinaric acid (CPA)[Palmer CAN and Wolf C R. FEBS Letts.", "431, 476-480, (1998); Causevic M, Wolf C R and Palmer CAN.", "FEBS Letts.", "463, 205-210, (1999)].", "On binding to the receptor, changes in the spectral properties of the fatty acid occurs.", "These are quantitatively related to the binding of the ligand to the receptor and can be used to calculate binding constants.", "A competitive displacement assay can be utilised to examine the binding characteristics of other compounds.", "APFO was assayed for its ability to displace cis-parinaric acid from PPARγ by this method.", "Data were analysed as described in section 4.1.4.2.2 Results and Discussion Competitive ligand binding assays using the ligand binding domain of human PPAR gamma showed that displacement of cis-parinaric acid occurred, with an EC50 of 355 μM (FIG.", "28).", "3.2.4 Conclusions These data indicate that APFO interacts with the ligand binding domain of human PPAR gamma." ] ]	Patent_10468331
[ [ "Catalysts", "A tethered ligand comprising the reaction product of an organofunctional silica and a ligand containing a functional group capable of reaction with said organofunctional silica, wherein the organofunctional silica is prepared from an alkyl silicate and an organofunctional silane is described.", "A supported catalyst is also described comprising additionally a source of catalytically-active metal.", "Methods for preparing the tethered ligand and supported catalyst are provided and uses of the supported catalyst for performing asymmetric reactions are claimed.", "The catalysts are readily separable from the reaction mixtures and may be re-used if desired." ], [ "1.A tethered ligand comprising the reaction product of a meso-porous organofunctional silica having an average pore width, as measured by BET porosimetry, of between 20 and 500 Angstroms and a ligand containing a functional group capable of reaction with said organofunctional silica, wherein the organofunctional silica is prepared from an alkyl silicate and an organofunctional silence.", "2.A tethered ligand according to claim 1, wherein the alkyl silicate has the general formula Si(OR)4 in which each R may be the same or different and is an alkyl group or substituted alkyl group having between 1 and 4 carbon atoms.", "3.A tethered ligand according to claim 1, wherein the organofunctional silane has the general formula (Y)aSi((Z)X)b in which; Y is a halogen or alkoxy group having 1 to 3 carbon atoms; Z is an alkyl, aryl or alkyl-aryl group, which optionally contains at least one heteroatom selected from oxygen, nitrogen, phosphorus or sulphur; X is a functional group selected from halide, hydroxyl, carbonyl, carboxyl, anhydride, carbene, methacryl, epoxide, vinyl, nitrile, mercapto, amine, imine,.", "amide and imide; a=3 or 2, b=1 or 2 and a+b=4.4.A tethered ligand according to claim 1, wherein the organofunctional silane is prepared by reaction of an organofunctional silane with a linker molecule that contains a functional group that is capable of reacting with the organofunctional silane and a functional group capable of reacting with a functional group-containing ligand, said linker molecule selected from the group comprising C1-C10 alkyl, alkoxy, alkyl-aryl, aryl, phenoxy or anilide compounds containing functional groups selected from halide, hydroxyl, carbonyl, carboxyl, anhydride, carbene, methacryl, epoxide, vinyl, nitrile, mercapto, isocyanate, amine, imine, amide and imide.", "5.A tethered ligand according to claim 1, wherein more than one organofunctional silane is used.", "6.A tethered ligand according to claim 1, wherein the organofunctional silica is prepared from an alkyl silicate and an organofunctional silane and a silane not having a functional group capable of reaction with a functional group-containing ligand.", "7.A tethered ligand according to claim 1, wherein the functional group on the organofunctional silica is changed by chemical conversion before reaction of the organofunctional silica with the functional group-containing ligand.", "8.A tethered ligand according to claim 1, wherein the organofunctional silica is reacted with a linker molecule that contains a functional group that is capable of reacting with the organofunctional silica and a functional group capable of reacting with a functional group-containing ligand.", "9.A tethered ligand according to claim 1, wherein the ligand containing a functional group is a chiral or non-chiral mono-, bi-, tri- or tetra-dentate ligand having a reactive group capable of reacting with the organofunctional silica.", "10.A tethered ligand according to claim 9, wherein the ligand containing a functional group is a racemic or non-racemic mixture or single enantiomer of a β-diketonate, β-ketoester, alkanolamine, Schiff base, aminoacid, peptide, phosphite, phosphate, alkyl- or aryl-phosphine, diamine, crown-ether and bis-oxazoline, each having a reactive group selected from the group consisting of halide, hydroxyl, carbonyl, carboxyl, anhydride, carbene, methacryl, epoxide, vinyl, nitrile, mercapto, amine, imine, amide and imide.", "11.A method for the preparation of a tethered ligand comprising the steps of; a) forming an organofunctional silica by the reaction of an alkyl silicate, an organofunctional silane and water, optionally in the presence of a template compound, b) removing the template compound if present, and c) reacting said organofunctional silica with a ligand containing a functional group capable of reaction with said organofunctional silica.", "12.A method according to claim 11, wherein the functional group present on the organofunctional silica formed in step (a) is changed by a chemical conversion before reaction of the organofunctional silica with the functional group-containing ligand.", "13.A method according to claim 11, wherein the organofunctional silica formed in step (a) is reacted with a linker molecule that contains a functional group that is capable of reacting with the organofunctional silica and a functional group capable of reaction with a function group-containing ligand.", "14.A method according to claim 11, wherein the molecular ratio of alkyl silicate to organofunctional silane is 1:1 to 99:1.15.A supported catalyst comprising the reaction product of a tethered ligand as claimed in claim 1 and a source of catalytically-active metal.", "16.A catalyst according to claim 15, wherein the catalytically-active metal is selected from the group comprising Sc, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Tc, Re, Fe, Ru, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, Al, Ge, Sb and Sn.", "17.A catalyst according to claim 15, wherein the source of catalytically-active metal is an organic complex of the metal or metal salt.", "18.A method for the preparation of a supported catalyst comprising the steps of; a) forming an organofunctional silica by reaction of an alkyl silicate, an organofunctional silane and water, optionally in the presence of a template compound, b) removing the template compound if present.", "c) reacting said organofunctional silica with a ligand containing a functional group capable of reaction with said organofunctional silica to produce a tethered ligand and d) carrying out a chemical reaction between a metal compound and said tethered ligand.", "19.The use of a supported catalyst according to claim 15 for hydrogenation reactions, dihydroxylation reactions, hydrolysis reactions, metathesis reactions, carbon-carbon bond formation reactions, hydroamination reactions, epoxidations, aziridinations, cycloadditions, hetero-Diels-Alder reactions, hetero-ene reactions, Claisen rearrangements, carbonyl reductions, sigmatropic rearrangements, additions of nucleophiles to π-bonds, addition of nucleophiles to carbonyl groups and ring-opening reactions.", "20.The use of a supported catalyst according to claim 19 for hydrogenation reactions, hydrolysis reactions and carbon-carbon bond formation reactions.", "21.The use of a supported catalyst according to claim 19, wherein the catalyst is separated from the reaction mixture, and re-used in subsequent reactions.", "22.The use of a supported catalyst according to claim 20, wherein the catalyst is separated from the reaction mixture, and re-used in subsequent reactions.", "23.The use of a supported catalyst according to claim 21, wherein the catalyst is separated from the reaction mixture, and re-used in subsequent reactions." ], [ "This invention relates to ligands tethered to solid supports and in particular to ligands tethered to silica that provide a means for supporting metal catalysts which are useful for directing chemical reactions.", "The products of such reactions are useful, for example, as chemical intermediates or reagents for use in the production of fine chemicals or pharmaceutical intermediates.", "The fixing of homogeneous catalysts to solid supports provides the potential for extending the benefits of heterogeneous catalysts to homogeneous systems.", "These benefits include easier separation of catalyst and reaction products leading to shorter work up times and improved process efficiency, the potential for re-activation and re-use of the supported catalysts which are often based on expensive metals and complex ligand geometry, and the possible adaptation of the immobilised catalyst to continuous flow fixed-bed processes.", "Strategies for homogeneous catalyst immobilisation have been based on absorption, ion exchange or tethering the catalysts to a support using covalent attachment.", "By covalent attachment we mean the formation of a covalent bond between support and catalyst.", "Covalent attachment is attractive for providing catalysts that may be more robust to catalyst leaching and hence retain higher activities upon re-use.", "Covalent attachment of the metal catalyst may be achieved by forming chemical bonds between a ligand and particles of a polymer, for example polystyrene, or oxide material, for example silica, that has been subjected to a surface functionalisation.", "For example, Jacobsen (J.", "Am.", "Chem.", "Soc.", "1999, 121, 4147-4154) has reported the covalent attachment of the ligand ‘salen’ to polystyrene and fine silica particles.", "The ligand was immobilised by functional group interconversion to add a linking molecule with a siloxy group at the end.", "The ligand was then grafted onto 10 μm spherical silica particles and used for the hydrolytic kinetic resolution of a variety of terminal epoxides.", "Kim et al (Catalysis Today.", "2000, 63, 537-547) described confining a chiral catalyst on an alkyl silicate-derived silica-MCM-41 by a multi-step process including surface treating the silica material with 3-aminopropyl trimethoxysilane to activate the silica prior to reaction with a precursor of the required chiral catalyst and then further treatment to produce the immobilised catalyst.", "These methods for providing covalent attachment of ligand and catalyst depend upon the surface activity of the silica material being subjected to treatment Variations in the silica material may result in catalysts with lower enantioselectivities and/or efficiencies than the homogeneous counterpart.", "Furthermore, the practical advantages gained by covalent attachment to a support are often outweighed by the added complexity associated with synthesising the appropriately modified support and/or chiral ligand.", "Improved consistency in functionalised silicas is required.", "Clarke and Macquarrie (Chem.", "Commun., 1998, 2707-2708) describe a method for generating a silica-supported peroxycarboxylic acid for the epoxidation of alkenes wherein the silica is prepared via a co-hydrolysis of tetraethylorthosilicate and 2-cyanoethyltriethoxysilane.", "However, the resulting peroxyacid is not suitable for tethering homogeneous catalysts.", "We have now found that homogeneous catalysts may be successfully immobilised using functionalised silicas prepared from alkyl silicates and organofunctional silanes.", "According to the invention we provide a tethered ligand comprising the reaction product of an organofunctional silica and a ligand containing a functional group capable of reaction with said organofunctional silica, wherein the organofunctional silica is prepared from an alkyl silicate and an organofunctional silane.", "According to a further aspect of the invention we provide a supported catalyst comprising the reaction product of an organofunctional silica, a ligand containing a functional group capable of reaction with said organofunctional silica and a source of catalytically-active metal, wherein the organofunctional silica is prepared from an alkyl silicate and an organofunctional silane.", "According to a further aspect of the invention, we also provide a method for the preparation a tethered ligand comprising the steps of; a) forming an organofunctional silica by the reaction of an alkyl silicate, an organofunctional silane and water, optionally in the presence of a template compound, b) removing the template compound if present, and c) reacting said organofunctional silica with a ligand containing a functional group capable of reaction with said organofunctional silica.", "According to a further aspect of the invention, we also provide a method for the preparation of a supported catalyst comprising the steps of; a) forming an organofunctional silica by reaction of an alkyl silicate, an organofunctional silane and water, optionally in the presence of a template compound, b) removing the template compound if present, c) reacting said organofunctional silica with a ligand containing a functional group capable of reaction with said organofunctional silica to produce a tethered ligand and d) carrying out a chemical reaction between a metal compound and said tethered ligand.", "According to a further aspect of the invention, we also provide the use of a supported catalyst comprising the reaction product of an organofunctional silica, a ligand containing a functional group capable of reaction with said organofunctional silica and a source of catalytically-active metal, wherein the organofunctional silica is prepared from an alkyl silicate and an organofunctional silane, for hydrogenation reactions, dihydroxylation reactions, hydrolysis reactions, metathesis reactions, carbon-carbon bond formation reactions, hydroamination reactions, epoxidations, aziridinations, cycloadditions, hetero-Diels-Alder reactions, hetero-ene reactions, Claisen rearrangements, carbonyl reductions, sigmatropic rearrangements, additions of nucleophiles to π-bonds, addition of nucleophiles to carbonyl groups and ring-opening reactions.", "The present invention relates to tethered ligands.", "By the term ‘tethered ligand’ we mean an organic ligand covalently bound to a solid silica support.", "By the term ‘ligand’ we mean any molecule capable of reacting with a metal compound to produce a catalyst Organofunctional silicas of the present invention are prepared from alkyl silicates and organofunctional silanes.", "The alkyl silicates are tetraalkylsilicates which have the general formula Si(OR)4 in which each R may be the same or different and is an alkyl group or substituted alkyl group having between 1 and 4 carbon atoms.", "Alkyl silicates useful for the present invention include tetramethylorthosilicate, tetraethylorthosilicate and tetrapropylorthosilicate or mixtures of these.", "Preferably, the alkyl silicate is tetraethylorthosilicate or tetramethylorthosilicate.", "The organofunctional silane of the present invention may be halo or alkoxy organofunctional silane according to the general formula (Y)aSi((Z)X)b in which; Y is a halogen or alkoxy group having 1 to 3 carbon atoms; Z is an alkyl, aryl or alkyl-aryl group which optionally contains at least one heteroatom selected from oxygen, nitrogen, phosphorus or sulphur; and X is a functional group selected from halide, hydroxyl, carbonyl, carboxyl, anhydride, carbene, methacryl, epoxide, vinyl, nitrile, mercapto, amine, imine, amide and imide; a=3 or 2, b=1 or 2 and a+b=4.In the above formula, if Z contains an alkyl group it preferably has between 1 and 16 carbon atoms, is branched or linear and saturated or unsaturated.", "If Z contains an aryl group it is preferably a substituted or unsubstituted phenyl, phenoxy or anilide moiety.", "Where X is bound to an alkyl group it may be bound on either a primary, secondary or tertiary carbon.", "The organofunctional silane may be selected from those commercially available or if desired may be prepared by reaction of an organofunctional silane with a linker molecule that provides a functional group capable of reaction with a functional group-containing ligand.", "The linker molecule may be any that contains a functional group that is capable of reacting with the organofunctional silane and provides a suitable functional group in the resultant silica material capable of reacting with a functional group-containing ligand.", "Suitable linker molecules include C1-C10 alkyl, alkoxy, alkyl-aryl, aryl, phenoxy or anilide compounds containing functional groups selected from halide, hydroxyl, carbonyl, carboxyl, anhydride, carbene, methacryl, epoxide, vinyl, nitrile, mercapto, isocyanate, amine, imine, amide and imide.", "Advantages of preparing the organofunctional silane in this way are that new functional groups may be introduced in a way that is not generally possible in commercially available silanes, functional groups may be introduced at a greater distance from the silicon atom than is possible with currently available silanes, and such modification may provide organofunctional silanes that provide improvements in the properties of the resulting organofunctional silica, e.g.", "porosity.", "Mixtures of organofunctional silanes having different functional groups may be used in the present invention.", "In such mixtures 2 or more silanes may be present.", "Optionally, a silane not having a functional group capable of reaction with a functional group-containing ligand may be used In combination with an organofunctional silane as described above to e.g.", "reduce the surface concentration of functional groups on the silica material and improve ligand attachment.", "Such non-functionalised silanes may also be used to improve other properties of the resulting silica material such as porosity or pore size.", "Typically silanes such as alkyl silanes can be used although other non-functionalised silanes may also be used.", "The molecular ratio of functionalised and non-functionalised silanes used may be any that provides a sufficient number of reactive sites for tethering the ligand containing a functional group to the resulting organofunctional silica to provide a useful level of catalytic activity in the final catalyst.", "Molecular ratios may be in the range 1:99 to 99:1 for any given pair of silanes, preferably 1:9 to 9:1.Organofunctional silanes useful for the present invention include vinyltrimethoxy silane, vinyltriethoxysilane, dichlorodivinylsilane, 3-(aminopropyl)trimethoxysilane, 3-(aminopropyl)triethoxysilane, [3-(methacryloyloxy)-propyl]trimethoxysilane, [3-[tri(ethoxy/methoxy)silyl]propyl]urea, 3-(glycidoxypropyl)trimethoxysilane, 4-(triethoxysilyl)butyronitrile, 3-(lodopropyl)trimethoxysilane, 3-(mercaptopropyl)-trimethoxysilane, 3-(triethoxysilyl)propionitrile, 4-(triethoxysilyl)butyronitrile, ((chloromethyl)-phenylethyl)trimethoxysilane, and ((chloromethyl)phenyl)trimethoxysilane and mixtures of these.", "Non-functionalised silanes useful for the present invention include alkyl silanes having 1 to 16 carbon atoms such as propyltrimethoxysilane, propyltriethoxysilane, butyltrimethoxysilane, hexylbutyltrichlorosilane, dodecyltrichlorosilane, octadecyltrimethoxysilane, octyltrimethoxysilane, octyltriethoxysilane and mixtures of these.", "The ligand containing a functional group suitable for reaction with the organofunctional silica may be a chiral or non-chiral mono-, bi-, tri- or tetra-dentate ligand.", "If a chiral ligand is used it may be a racemic or non-racemic mixture or single enantiomer having a reactive group capable of reacting with the organofunctional silica.", "Typically such ligands include β-diketonates, β-ketoesters, alkanolamines, Schiff bases, aminoacids, peptides, phosphates, phosphites, alkyl- or aryl-phosphines, diamines, crown-ethers and bis-oxazolines.", "Preferred ligands containing a functional group are chiral ligands and include, but are not limited to racemic and non-racemic mixtures or single enantiomers of bidentate ligands such as; The functional groups to be reacted with the organofunctional silica may conveniently be introduced into the ligand during its preparation.", "If a functional group available on the ligand is unsuitable for reaction with the organofunctional silica, it may be converted by chemical reaction or alternatively, the ligand may be reacted with a linker molecule that provides a suitable functional group capable of reaction with the organofunctional silica.", "Suitable functional groups on the ligand include halide, hydroxyl, carbonyl, carboxyl, anhydride, carbene, methacryl, epoxide, vinyl, nitrile, mercapto, amine, imine, amide and imide.", "The catalytically-active metal to be reacted with the ligand tethered to the organofunctional silica is selected from the group comprising Sc, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Tc, Re, Fe, Ru, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, Al, Ge, Sb and Sn.", "Preferably, for asymmetric hydrogenation, the metal is selected from Rh, Ir or Ru; preferably for hydrolytic kinetic resolution of epoxides the metal is selected from Co; preferably for ring-opening reactions the metal is selected from Cr and Al; and preferably for Heck reactions the metal is Pd.", "The source of the metal may be any suitable for reaction with the ligand tethered to the organofunctional silica.", "Preferably the source of the metal is an organic complex of the metal or a metal salt.", "For example rhodium may be reacted as a 1,5-cyclooctadiene complex and for manganese, palladium or cobalt the metal may be reacted as the di-acetate.", "A method for preparing an organfunctional silica by the co-hydrolysis of an alkyl silicate and an organofunctional silane is depicted below.", "The wavy line represents the silicon atom on or within the resulting silica material.", "In one embodiment, alkyl silicate and organofunctional silane are added to a mechanically stirred mixture of solvent and water.", "The alkyl silicate and organofunctional silane may be added together, sequentially in any order or in alternating portions.", "In an alternative embodiment the alkyl silicate, organofunctional silane and solvent are added to the mechanically stirred water and in yet a further embodiment the alkyl silicate and organofunctional silane without solvent are added to the mechanically stirred water.", "The alkyl silicate and organofunctional silane are added at a molecular ratio of greater than or equal to 1:1 (silicate:silane).", "Preferably the ratio of alkyl silicate to silane is between 1:1 and 99:1 and more preferably between 1:1 and 10:1.Typically the solvent, if used, is an alcohol but may be any other solvent suitable for performing the co-hydrolysis reaction.", "For example, the solvent may be methanol, ethanol or propanol.", "Water is present in sufficient quantity to cause complete hydrolysis of the alkoxide moieties on the alkyl silicate and is generally added in large excess.", "A template compound may be added to the hydrolysis mixture to influence the resulting pore structure and potentially the disposition of the organofunctionality within the pores of the resulting organofunctional silica.", "Depending upon the method used, the template may be added to for example, the solvent/water mixture, the alkyl silicate/organofunctional silane/solvent mixture or the alkyl silicate/organofunctional silane mixture.", "The templates function by becoming entrapped in the silica as it forms during the co-hydrolysis of the alkyl silicate and organofunctional silane.", "Once the co-hydrolysis is complete, the entrapped template may then be removed by, for example, solvent extraction to leave behind pores corresponding to the structure of the template molecule.", "Suitable solvents for solvent extraction include alcohols, e.g.", "ethanol.", "Template compounds may be used in the preparation of mico- and meso-porous silicas (where a micro-porous silica has an average pore width of less than 20 Å and a meso-porous silica has an average pore width of between 20 and 500 Å).", "Preferably the organofunctional silica of the present invention is a meso-porous silica having an average pore width, as measured by BET porosimetry, of between 20 and 500 Å. Template compounds include amines, quaternary ammonium salts and non-ionic poly(ethylene oxide)/(propylene oxide) surfactants such as amphilic block copolymers.", "Suitable amphilic block copolymers are tri-block copolymers, e.g.", "PLURONIC™ F127 (EO106PO70EO106) and PLURONIC™ 123 (EO20PO70EO20) and mixtures of these.", "The quaternary ammonium compounds are quaternary ammonium salts or hydroxides e.g.", "of general formula [R4N]+[Z]− in which R may be the same or different and is alkyl or substituted alkyl (C1-C30), and Z is preferably Cl, Br, or OH.", "Quaternary ammonium compounds wherein the nitrogen atom forms part of a ring structure may also be used.", "Suitable quaternary ammonium compounds are tetrapropylammonium hydroxide, tetrapropylammonium chloride, tetrabutylammonium hydroxide, benzyltrimethylammonium hydroxide, benzylcetyldimethylammonium chloride, benzyltrimethylammonium hydroxide, benzyltrimethylammonium chloride, cetyltrimethyl ammonium hydroxide, cetyltrimethylammonium bromide, cetyltrimethylammonium chloride and mixtures of these.", "Preferably the template molecule is an amine.", "Suitable amines are amines having 10 or more carbon atoms in the structure and preferably are C12 to C18 alkyl amines such as n-dodecylamine or n-octadecylamine or mixtures of these.", "The amount of template compound required will depend upon a number of factors including the amount of alkyl silicate used.", "In general the relative amounts of template molecule may vary in the range 1:10 to 50:1 and preferably in the range 1:1 to 10:1 parts by weight silicate:template compound.", "In addition to the template, a pore-enlarging additive may be included in the hydrolysis mixture.", "A known pore-enlarging additive is mesitylene (1,3,5-trimethylbenzene).", "Depending upon the method chosen for performing the co-hydrolysis reaction, the pore swelling additive may for example, be combined with the silicate and organofunctional silane or with the water or water/solvent mixture or may be added separately to the hydrolysis mixture.", "The amount of pore-enlarging additive that may be added will depend upon the properties of the additive, for example in the present invention 1-2 moles of mesitylene may be added per mole of alkyl silicate.", "The co-hydrolysis reaction may be performed at room temperature or if desired at elevated temperature depending on the physical properties of the solvent chosen.", "For example the co-hydrolysis reaction may be carried out at between 10-50° C. for periods between 1 and 36 hours.", "When the co-hydrolysis reaction is complete the organofunctional silica is recovered by, for example filtration and the template, if present, removed by solvent extraction.", "Solvent extraction may be effected for example by heating the re-suspended organofunctional silica in a suitable solvent such as ethanol.", "This may be repeated as necessary to remove all of the template prior to attachment of the functional group-containing ligand.", "It may be desirable to change the functional groups present on the organofunctional silica to, for example, provide a different functional group with which to react the functional group containing-ligand.", "For example, if the functional group containing-ligand has a pendant hydroxyl group capable of reaction with the organofunctional silica, it may be desirable to chemically convert the functional group on the silica from, for example a cyano-group to a carboxyl group or, a carboxyl group to an acid-chloride group, to facilitate a reaction between said silica and said ligand.", "Such conversions may also provide a means for providing functional groups on the silica not practical as a result of the method used for preparing the organofunctional silica.", "For example, isocyanate groups that would be unstable to the water used during the co-hydrolysis of alkyl silicate and organofunctional silane may be provided by inter-conversion of acid-chloride via a Curtis rearrangement Alternatively, a chemical conversion may be performed to reduce the surface concentration of functional groups capable of reaction with the functional group-containing ligand by, for example, converting the functional groups to unreactive species such as hydrogen or alkyl groups.", "Alternatively the organofunctional silica may be reacted with a linker molecule that provides a functional group capable of reaction with a functional group-containing ligand, to form a new organofunctional silica.", "This may be of particular use where chemical conversion of functional groups is difficult.", "The linker molecule may be any that is capable or reacting with the organofunctional silica and provides a suitable functional group for reacting with the ligand.", "Suitable linker molecules include C1-C10 alkyl, alkoxy, alkyl-aryl, aryl, phenoxy or anilide compounds containing functional groups selected from halide, hydroxyl, carbonyl, carboxyl, anhydride, carbene, methacryl, epoxide, vinyl, nitrile, mercapto, isocyanate, amine, imine, amide and imide.", "Suitable linker molecules include (3-Formylindol-1-yl)acetic acid, [3-({Ethyl-Fmoc-amino}-methyl)-indol-1-yl]-acetic acid, 2,4-Dimethoxy-4′-hydroxy-benzophenone, 3,5-Dimethoxy-4-formyl-phenol, 3-(4-Hydroxymethylphenoxy)propionic acid, 3-Carboxypropanesulfonamide, 3-Hydroxy-xanthen-9-one, 3-Methoxy-4-formylphenol, 4-(2-Bromopropionyl)phenoxyacetic acid, 4-(4-[Bis-(4chlorophenyl)hydroxymethyl]phenoxy)butyric acid dicyclohexylammonium salt, 4-(4-Formyl-3-methoxy-phenoxy)-butyric acid, 4-(4-Hydroxymethyl-3-methoxyphenoxy)-butyric acid, 4-[4-(1-(Fmocamino)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid, 4-[4-(1-Hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid, 4-[4-(2,4-Dimethoxybenzoyl)phenoxy]butyric acid, 4-[4-(Diphenylhydroxymethyl)phenoxy]butyric acid dicyclohexylammonium salt, 4-[4-Hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoic acid, 4-Hydroxy-2,6-dimethoxy-benzaldehyde, 4-Hydroxy-2-methoxy-benzaldehyde, 4-Hydroxymethylbenzoic acid, 4-Hydroxymethylphenoxyacetic acid, 4-Sulfamoyl-butyric acid and p-[(R,S)-α-[1-(9H-Fluoren-9-yl)-methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic acid A reaction of a ligand containing a functional group with an organofunctional silica to provide a tethered ligand is depicted below.", "Y represents the functional group on the ligand capable of reaction with the organofunctional silica and A represents the covalent bond between ligand and organofunctional silica; The reaction may be achieved by any effective chemical reaction between the functional groups of the organofunctional silica and the functional group containing-ligand.", "Typical reactions include for example, esterification reactions, amidation reactions, addition reactions, substitution reactions, insertion reactions and carbon-carbon coupling reactions and may be performed by any method known to those skilled in the art.", "For example, esterification reactions may be performed between ligand and silica either having carboxyl and hydroxyl groups, anhydride and hydroxyl groups or acid-chloride and hydroxyl groups in the presence of suitable catalysts or reagents.", "Amidation reactions may be performed between ligand and silica either having carboxyl groups and primary or secondary amine groups or anhydride groups and primary or secondary amine, again in the presence of suitable catalysts or reagents.", "A reaction of a metal with a tethered ligand to provide a supported catalyst is depicted below; The reaction may be achieved by methods known to those skilled in the art and is preferably effected by reaction of a metal compound with the tethered ligand.", "Such reactions include, for example, ligand substitution reactions and metal-insertion reactions.", "The metal may also, if desired, be subjected to steps of oxidation or reduction to provide the necessary catalytic activity.", "For example cobalt catalysts may be oxidised from Co(II) to Co(III) or rhodium catalysts may be reduced from Rh(III) to Rh(I).", "Preferably the tethered ligands of the present invention are chiral ligands providing supported chiral catalysts.", "The supported chiral catalysts of the present invention may be applied to a large number of asymmetric reactions used to produce chiral products.", "Such reactions include hydrogenation reactions, dihydroxylation reactions, hydrolysis reactions, metathesis reactions, carbon-carbon bond formation reactions such as Heck or Suzuki reactions, hydroamination reactions, epoxidations, aziridinations, cycloadditions, hetero-Diels-Alder reactions, hetero-ene reactions, Claisen rearrangements, carbonyl reductions, sigmatropic rearrangements, additions of nucleophiles to π-bonds, addition of nucleophiles to carbonyl groups and ring-opening reactions.", "Preferably the reactions are hydrogenation reactions, hydrolysis reactions and carbon-carbon bond formation reactions.", "The advantages of the catalysts of the present invention are that they are readily separated from the reaction products and may be re-used if so desired.", "The invention is illustrated by the following examples.", "EXAMPLE 1 Preparation of Catalyst A (a) Preparation of Mesoporous Carboxylic Acid-Functionalised Silica A mixture of ethanol (105 ml), de-ionised water (105 ml) and n-dodecylamine (10 g) was prepared and stirred vigorously at room temperature until homogeneous.", "To this mixture, tetraethylorthosilicate (20.4 g) and 3-(triethyoxysilyl)propionitrile (23.1 g) were added stepwise over 30 minutes with continued stirring for 24 hours.", "After this time the precipitate was filtered, washed with de-ionised water (500 ml) and ethanol (500 ml) then allowed to dry at room temperature for 24 hours.", "Extraction of n-dodecylamine was achieved by heating the solid at reflux in ethanol (200 ml) for 3 hours.", "This reflux was repeated three times.", "The solid was then filtered and dried in a vacuum oven at 80° C. overnight.", "Hydrolysis of the nitrile functionality was achieved by heating the solid to 120° C. in 50% v/v aqueous sulphuric acid (150 ml per 5 g) for 3 hours.", "The solid was filtered and washed with excess de-ionised water until natural pH was achieved before drying in a vacuum oven at 80° C. overnight.", "BET porosimetry analysis of the silica indicated that it had an average pore width of 40 Å.", "(b) Tethering of Ligand To a stirred suspension of the mesoporous carboxylic acid functionalised silica (0.1 g), bppm (0.05 g, 0.11 mmol) and 4-dimethylaminopyridine (DMAP) (3.0 mg,) in dichloromethane (2.5 ml) was added diisopropylcarbodiimide (DIC) (17 μl, 0.11 mmol).", "The resulting suspension was stirred overnight at room temperature.", "The mixture was filtered and the resulting solid washed with dichloromethane (5 ml), methanol (5 ml) and dichloromethane (5 ml).", "The solid was dried under vacuum to give the ligand supported Silica as a colourless solid.", "(c) Insertion of Metal A mixture of the ligand supported silica (0.1 g) and [Rh(cod)2]BF4 (44 mg, 0.1 mmol) in dichloromethane (2.5 ml) was stirred for 1 hr under nitrogen.", "The mixture was then filtered and the solid washed with dichloromethane (2.5 ml), methanol (5 ml) and dichloromethane (2.5 ml).", "The solid was dried under vacuum to give the Rh-ligand supported silica as a yellow solid.", "The tethering of ligand (I) and insertion of metal (II) may be depicted as follows; EXAMPLE 2 Hydrogenation of Dimethylitaconate Dimethylitaconate was hydrogenated according to the following scheme; Dimethylitaconate substrate and catalyst were weighed into a glass-liner that was placed inside a 50 ml autoclave to give a substrate:catalyst molar ratio of 100:1.The autoclave was sealed and flushed with nitrogen.", "The autoclave was then pressurised with hydrogen to 80 psi (506.6 kPa) and then released (cycle repeated 5 times).", "Sufficient methanol was added to the autoclave to give an approximately 1M solution and the 5 cycles of pressurising-releasing with hydrogen were repeated.", "Finally the autoclave was pressurised with H2 to 80 psi (506.6 kPa) and left to stir.", "After the desired time the stirring was stopped and the H2 released slowly.", "The autoclave was opened and the mixture filtered to recover the supported catalyst.", "Gas-chromatographic analysis of the filtrate was performed to determine conversion and enantiomeric excess (ee %).", "The results are given below.", "Catalyst Conversion (%) ee (%) Catalyst A 100 17 The result demonstrates that highly active catalysts can be prepared according to the present invention, that provide a means for producing chiral products and which are easily separated from the reaction mixture.", "EXAMPLE 3 Preparation of Tethered Ferrocenyl Ligand An organofunctional silica prepared according to the method of example 1 part (a) was reacted with a ferrocenyl-bis(phosphine) ligand according to the following scheme; An oven dried Schlenk tube was charged under nitrogen with carboxy-functional silica prepared according to the method of example 1 part (a) (0.213 g, 0.64 mmol; approx.", "3 mmol/g loading) and dry dichloromethane (3 ml).", "1,1′-Carbonyldiimidazole (0.104 g, 0.64 mmol) was added, and the mixture was stirred until the bubbling ceased (approx.", "15 min).", "The phosphine 1 (0.185 g, 0.32 mmol) was added followed by dry dichloromethane (1 ml).", "After stirring for 20 hours at room temperature under nitrogen, methanol (10 ml) was added, the mixture was filtered, and the solid was washed with dichloromethane, methanol, and once more with dichloromethane.", "The resulting beige solid was dried under high vacuum to give 0.202 g 2.ICP analysis: 0.52% Fe (w/w), 0.093 mmol/g loading; 0.60% P (w/w), 0.097 mmol/g loading; the average loading is therefore 0.095 mmol/g.", "EXAMPLE 4 Heck Reaction Using a Supported Catalyst A Heck reaction was performed using the tethered ligand of Example 3 reacted with palladium acetate according to the following scheme; An oven dried Schlenk flask was charged with Pd(OAc)2 (2.2 mg, 0.01 mmol, 1 mol %) and tethered ligand 2 (45.8 mg, 0.0044 mmol, 0.44 mol %).", "The flask was evacuated and backfilled with nitrogen and then capped with a rubber septum.", "Dry dimethylformamide (5 ml) was added and the mixture was stirred for 60 min at room temperature under nitrogen.", "4′-Bromoacetophenone (0.199 g, 1.0 mmol), styrene (0.137 ml, 1.2 mmol), and triethylamine (0.195 ml, 1.4 mmol) were added successively.", "The rubber septum was replaced with a glas stopper, the flask was sealed, and the mixture was stirred at 120° C. for 20 h. After cooling to room temperature, the mixture was filtered, and the solid was washed with methyl-t-butyl ether (MTBE), water, MeOH, acetone, and once more with MTBE.", "The phases of the filtrate were separated and the aqueous phase was extracted with MTBE (2×30 ml).", "The combined organic extracts were dried over MgSO4 and concentrated under reduced pressure.", "The crude product was purified by flash chromatography on silica gel (hexane/EtOAc, 10/1 to 1/1) to give E-4-acetylstilbene (0.192 g, 86%) as a white solid.", "EXAMPLE 5 Preparation of a Carboxylic Acid-Functionalised Silica Using a Mixture of Functionalised and Non-Functionalised Silanes A mixture of dodecylamine (10 g), deionised water (105 ml) and denatured (5% MeOH) ethanol (105 ml) was prepared and stirred for 30 minutes until homogeneous.", "To this, mesitylene (1 9.5 g) was added and the resultant cloudy emulsion was stirred for a further 30 minutes.", "After this time, a freshly prepared mixture of 4-triethoxysilylbutyronitrile (11.5 g) and propyltrimethoxysilane (8.5 g) was added with continual stirring.", "After a further 60 minutes tetraethylorthosilicate (20.8 g) was added to the mixture and the resultant slurry stirred for a period of 24 hours.", "After this time the precipitate was filtered, washed with de-ionised water (500 ml) and ethanol (500 ml) then allowed to dry at room temperature for 24 hours.", "Template extraction and hydrolysis of the nitrile functionality were performed as described in Example 1 part (a) to yield a white solid having a nitrogen content of 3.6% by weight compared with 7.2% by weight for the silica material prepared without the propyltrimethoxysilane." ] ]	Patent_10468340
[ [ "Compositions and methods to prevent metastasis from primary malignancies", "We have now discovered a new method for treating carcinoembriyonic antigen (CEA) associated cancers.", "This involves blocking a protein expressed by the hnRNP M4 gene (preferably the human hnRNP M4 gene).", "We identified and isolated a liver-derived recombinant cDNA clone, termed heterogeneous nuclear RNA binding protein M4 (hnRNP M4) SEQ ID NO: 1, from rat macrophages Kupffer cells (KC) that encodes a novel protein interacting with CEA molecules and is 91% homologous with the deletion mutant of the human hnRNP M4 gene (# U32577).", "The novel protein is, hereinafter, considered as one protein population with the human homologue and referred to herein as hnRNP M4 CEA receptor, or hnRNP M4." ], [ "1.A method of preventing tumor metastasis comprising administering to a host having a primary tumor a compound that inhibits binding of CEA to a hnRNP M4 receptor.", "2.The method of claim 1, wherein the compound is an antibody.", "3.The method of claim 1, wherein the compound is an antisense DNA.", "4.The method of claim 1, wherein the compound is a soluble receptor.", "5.An isolated DNA segment encoding a protein comprising the amino acids of SEQ ID NO: 2.6.A DNA segment encoding an hnRNP M4 receptor having the amino acid of SEQ ID NO: 2.7.An isolated DNA segment encoding a human hnRNP M4 receptor.", "8.A protein encoded by the DNA of claim 5, 6 or 7.9.An antibody directed to the protein of claim 8.10.An isolated DNA segment comprising the nucleotide sequence of SEQ ID NO: 1, or the complement thereof.", "11.A host cell containing the DNA of claim 5, 6 or 7.12.A bioassay for detecting hnRNP M4 mRNA in a biological sample comprising the steps of: i) contacting said biological sample with a DNA segment according to claims 5, 6 or 7 under conditions such that a DNA:RNA hybrid molecule containing said DNA segment and complementary RNA can be formed; and ii) determining the amount of said DNA segment present in said hybrid molecule.", "13.A bioassay for testing potential analogs of ligands of hnRNP M4 receptors for the ability to affect an activity mediated by said hnRNP M4 receptors, comprising the steps of: i) contacting a molecule suspected of being a ligand with hnRNP M4 receptors produced by a cell according to claim 11; and ii) determining the amount of a biological activity mediated by said hnRNP M4 receptors in said cells.", "14.An assay for detecting an hnRNP M4 in a biological sample comprising the steps of: i) contacting said sample with an antibody according to claim 9, under conditions such that specific complexes of said antibody and an antigen can be formed; and ii) determining the amount of said antibody present as said complexes.", "15.A method for targeting a therapeutic drug to cells having high levels of hnRNP M4 receptors, comprising the steps of: i) conjugating an antibody according to claim 9, or an active fragment thereof, to said drug; and ii) administering the resulting conjugate to an individual with cells having high levels of hnRNP M4 receptors in an effective amount and by an effective route such that said antibody is able to bind to said receptors on said cells.", "16.Use of antibody of claim 9, or an active fragment thereof, conjugated to a therapeutic drug to target said therapeutic drug to cells having high levels of hnRNP M4 receptors." ], [ "<SOH> BACKGROUND <EOH>1.Field of the Invention The present invention relates to carcinoembryonic antigen (CEA) molecules generally and metastasis of malignant cells.", "More specifically, the present invention relates to a method for treating CEA-associated cancers.", "2.Background of the Invention The liver is a common site for metastasis from various forms of primary malignancies.", "Both experimental and clinical results reveal that the presence of CEA enhances liver metastasis from colorectal carcinoma cells (1, 2).", "Increased amount of CEA in the serum correlates with the development of metastatic recurrence after the surgical removal of the primary tumor.", "In malignant conditions, the reported incidence of elevated serum CEA level ranges from 9% in testicular, ovary, lung, pancreatic and thyroid carcinomas to more than 50% in the metastatic colorectal carcinomas.", "CEA is an FDA approved tumor marker in the management of metastatic colon and breast cancers (3).", "It has been previously shown that CEA production of human colorectal cancer cell lines directly correlates with the metastatic potential (4, 5).", "Poorly metastatic colon cancer cell lines become highly metastatic when transfected with the cDNA coding for CEA (6).", "As a member of the immunoglobulin supergene family, CEA is involved in the intercellular recognition and may facilitate attachment of colorectal carcinoma cells to sites of metastasis.", "In an experimental metastasis model of colorectal carcinoma in athymic nude mice, systemic injection of CEA enhanced experimental liver metastasis and implantation in liver by weakly metastatic tumor cells (7).", "The mechanism by which CEA causes enhancement of metastasis is largely unknown.", "We have shown earlier that CEA is rapidly cleared from the circulation of experimental animals, accumulates in the liver and is endocytosed in vitro by KC (8).", "This uptake is independent of carbohydrates and is mediated by an 80 kD protein (9).", "The structure and the sequence of this protein is unknown.", "We have further shown that CEA is recognized by this binding protein through a five amino acid sequence, Pro-Glu-Leu-Pro-Lys (PELPK), located at the hinge region (amino-acids 108-112) between the N-terminal and the first immunoglobulin loop domain in the CEA sequence (10), and that the CEA binding to Kupffer cells initiates series of signaling events that lead to the tyrosine phosphorylation (23) and induction of IL-1α, IL-6, IL-10 and TNF-α cytokines (24).", "Molecular modeling studies have suggested that this region is exposed on the surface of the molecule (P. A. Bates, personal communication).", "In order to successfully treat a multitude of malignant conditions, it is necessary to prevent the development of metastasis from cancerous cells after the treatment or removal of the primary malignancy.", "Therefore, there is a need to elucidate the mechanism by which CEA initiates series of signaling events that lead to metastasis from cancerous cells to healthy tissues, e.g., liver." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>We have now discovered a new method for treating carcinoembryonic antigen (CEA) associated cancers.", "This involves blocking a protein expressed by the hnRNP M4 gene (preferably the human hnRNP M4 gene).", "We identified and isolated a liver-derived recombinant cDNA clone, termed heterogeneous nuclear RNA binding protein M4 (hnRNP M4) SEQ ID NO: 1, from rat macrophages Kupffer cells (KC) that encodes a novel protein interacting with CEA molecules and is 91% homologous with the deletion mutant of the human hnRNP M4 gene (# U32577).", "The novel protein is, hereinafter, considered as one protein population with the human homologue and referred to herein as hnRNP M4 CEA receptor, or hnRNP M4.We have surprisingly discovered that this gene product is the receptor for CEA.", "For example, transfection of rat hnRNP M4 cDNA into mouse macrophage cell line p388D1 resulted in CEA binding.", "To isolate the novel CEA receptor we used two approaches: screening of a KC cDNA library with the specific antibody and the yeast 2-hybrid system for the protein interaction using as a bait N-terminal part of the CEA encoding the binding sequence.", "Both techniques resulted in the isolation of a deletion mutant (amino acids 158-197) of the rat and human hnRNP M4 proteins.", "Thus, the new rodent clone is the rat homologue of the human hnRNP M4 (# U32577).", "The full-length cDNA is a 2351 bp complete ORF with the polyadenylation signal AATAAA (SEQ ID NO: 3) and a termination polyA tail.", "The mRNA shows ubiquitous tissue expression as a 2.4 kb transcript.", "The deduced amino acid sequence comprised a 78-kD membrane protein with 3 putative RNA-binding domains, arginine-methionine-glutamine rich C-terminus and 3 potential membrane spanning regions.", "Computer assisted evaluation of the hnRNP M4 sequence revealed a motif for tyrosine phosphorylation (KVGEVTY, SEQ ID NO: 4), 7 potential protein kinase C phosphorylation sites, 11 casein kinase 11 phosphorylation sites, two glucosaminoglycan attachment sites and 15 N-myristoylation sites.", "When hnRNP M4 protein is expressed in pGEX3T-4 vector system in E. coli it binds 125 I labeled CEA in a Ca 2+ -dependent fashion.", "These data provide an evidence for a new function of hnRNP M4 protein as a CEA binding protein.", "To further study the interaction between the peptide sequence PELPK (SEQ ID NO: 5) and rat Kupffer cells, the CEA binding protein was purified using a combination of gel filtration, preparative polyacrylamide gel electrophoresis and affinity chromatography on CEA sepharose (11).", "A polyclonal antibody to the rat 80-kD protein was produced in mice that blocks both CEA and PELPK-albumin uptake by isolated rat Kupffer cells and shows a high degree of specificity for the rat 80-kDa protein by FACS analysis and Western blotting (11).", "The present invention further provides DNA segments encoding the hnRNP M4 receptor proteins.", "The present invention also provides an isolated DNA encoding a protein comprising the amino acids as set forth in SEQ ID NO: 2, as well as an isolated protein comprising the amino acids as set forth in SEQ ID NO: 2.Antibodies directed to amino acids of SEQ ID NO: 2, or a protein comprising amino acids of SEQ ID NO: 2 are also included.", "A DNA segment comprising the nucleotides as set forth in SEQ ID NO: 1 is further provided.", "The present invention further provides assays for expression of the RNA and protein products of the DNA of the present invention to enable determining whether abnormal expression of such DNA is involved with a particular disease, e.g., cancer.", "The present invention also provides antibodies, either polyclonal or monoclonal, specific to a unique portion of the receptor protein; a method for detecting the presence of a receptor ligand that is capable of either activating or down-regulating, i.e., modulating, the receptor protein; a method of screening potential ligand analogs for their ability to modulate the receptor protein; and procedures for targeting a therapeutic drug to cells having a high level of the receptor protein.", "The present invention also provides binding assays that permit the ready screening for molecules that affect the binding of the receptor and its ligands.", "The present invention further provides use of the receptor for intracellular or extracellular targets to affect binding.", "Intracellular targeting can be accomplished through the use of intracellularly expressed antibodies referred to as intrabodies.", "Extracellular targeting can be accomplished through the use of receptor specific antibodies.", "Additionally, the soluble form of the receptor can be used as a receptor decoy or aptamer to inhibit binding.", "The present invention also provides use of antisense technology to affect binding of the CEA receptor and its ligands by designing an antisense nucleic acid molecule which is complementary to a nucleic acid molecule encoding the CEA receptor.", "The present invention also provides an assay to determine the presence or absence of the receptors that can be used as a diagnostic or prognostic tool to identify the presence or stage of differentiation of tissue, e.g., tumor tissue.", "Finally, a nucleic acid of the invention could be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.", "RNA-mediated interference (RNAi) may also be used." ], [ "GOVERNMENT FUNDING This invention was made with government support under CA 74941 awarded by the National Institutes of Health.", "The government has certain rights in the invention.", "BACKGROUND 1.Field of the Invention The present invention relates to carcinoembryonic antigen (CEA) molecules generally and metastasis of malignant cells.", "More specifically, the present invention relates to a method for treating CEA-associated cancers.", "2.Background of the Invention The liver is a common site for metastasis from various forms of primary malignancies.", "Both experimental and clinical results reveal that the presence of CEA enhances liver metastasis from colorectal carcinoma cells (1, 2).", "Increased amount of CEA in the serum correlates with the development of metastatic recurrence after the surgical removal of the primary tumor.", "In malignant conditions, the reported incidence of elevated serum CEA level ranges from 9% in testicular, ovary, lung, pancreatic and thyroid carcinomas to more than 50% in the metastatic colorectal carcinomas.", "CEA is an FDA approved tumor marker in the management of metastatic colon and breast cancers (3).", "It has been previously shown that CEA production of human colorectal cancer cell lines directly correlates with the metastatic potential (4, 5).", "Poorly metastatic colon cancer cell lines become highly metastatic when transfected with the cDNA coding for CEA (6).", "As a member of the immunoglobulin supergene family, CEA is involved in the intercellular recognition and may facilitate attachment of colorectal carcinoma cells to sites of metastasis.", "In an experimental metastasis model of colorectal carcinoma in athymic nude mice, systemic injection of CEA enhanced experimental liver metastasis and implantation in liver by weakly metastatic tumor cells (7).", "The mechanism by which CEA causes enhancement of metastasis is largely unknown.", "We have shown earlier that CEA is rapidly cleared from the circulation of experimental animals, accumulates in the liver and is endocytosed in vitro by KC (8).", "This uptake is independent of carbohydrates and is mediated by an 80 kD protein (9).", "The structure and the sequence of this protein is unknown.", "We have further shown that CEA is recognized by this binding protein through a five amino acid sequence, Pro-Glu-Leu-Pro-Lys (PELPK), located at the hinge region (amino-acids 108-112) between the N-terminal and the first immunoglobulin loop domain in the CEA sequence (10), and that the CEA binding to Kupffer cells initiates series of signaling events that lead to the tyrosine phosphorylation (23) and induction of IL-1α, IL-6, IL-10 and TNF-α cytokines (24).", "Molecular modeling studies have suggested that this region is exposed on the surface of the molecule (P. A. Bates, personal communication).", "In order to successfully treat a multitude of malignant conditions, it is necessary to prevent the development of metastasis from cancerous cells after the treatment or removal of the primary malignancy.", "Therefore, there is a need to elucidate the mechanism by which CEA initiates series of signaling events that lead to metastasis from cancerous cells to healthy tissues, e.g., liver.", "SUMMARY OF THE INVENTION We have now discovered a new method for treating carcinoembryonic antigen (CEA) associated cancers.", "This involves blocking a protein expressed by the hnRNP M4 gene (preferably the human hnRNP M4 gene).", "We identified and isolated a liver-derived recombinant cDNA clone, termed heterogeneous nuclear RNA binding protein M4 (hnRNP M4) SEQ ID NO: 1, from rat macrophages Kupffer cells (KC) that encodes a novel protein interacting with CEA molecules and is 91% homologous with the deletion mutant of the human hnRNP M4 gene (# U32577).", "The novel protein is, hereinafter, considered as one protein population with the human homologue and referred to herein as hnRNP M4 CEA receptor, or hnRNP M4.We have surprisingly discovered that this gene product is the receptor for CEA.", "For example, transfection of rat hnRNP M4 cDNA into mouse macrophage cell line p388D1 resulted in CEA binding.", "To isolate the novel CEA receptor we used two approaches: screening of a KC cDNA library with the specific antibody and the yeast 2-hybrid system for the protein interaction using as a bait N-terminal part of the CEA encoding the binding sequence.", "Both techniques resulted in the isolation of a deletion mutant (amino acids 158-197) of the rat and human hnRNP M4 proteins.", "Thus, the new rodent clone is the rat homologue of the human hnRNP M4 (# U32577).", "The full-length cDNA is a 2351 bp complete ORF with the polyadenylation signal AATAAA (SEQ ID NO: 3) and a termination polyA tail.", "The mRNA shows ubiquitous tissue expression as a 2.4 kb transcript.", "The deduced amino acid sequence comprised a 78-kD membrane protein with 3 putative RNA-binding domains, arginine-methionine-glutamine rich C-terminus and 3 potential membrane spanning regions.", "Computer assisted evaluation of the hnRNP M4 sequence revealed a motif for tyrosine phosphorylation (KVGEVTY, SEQ ID NO: 4), 7 potential protein kinase C phosphorylation sites, 11 casein kinase 11 phosphorylation sites, two glucosaminoglycan attachment sites and 15 N-myristoylation sites.", "When hnRNP M4 protein is expressed in pGEX3T-4 vector system in E. coli it binds 125I labeled CEA in a Ca2+-dependent fashion.", "These data provide an evidence for a new function of hnRNP M4 protein as a CEA binding protein.", "To further study the interaction between the peptide sequence PELPK (SEQ ID NO: 5) and rat Kupffer cells, the CEA binding protein was purified using a combination of gel filtration, preparative polyacrylamide gel electrophoresis and affinity chromatography on CEA sepharose (11).", "A polyclonal antibody to the rat 80-kD protein was produced in mice that blocks both CEA and PELPK-albumin uptake by isolated rat Kupffer cells and shows a high degree of specificity for the rat 80-kDa protein by FACS analysis and Western blotting (11).", "The present invention further provides DNA segments encoding the hnRNP M4 receptor proteins.", "The present invention also provides an isolated DNA encoding a protein comprising the amino acids as set forth in SEQ ID NO: 2, as well as an isolated protein comprising the amino acids as set forth in SEQ ID NO: 2.Antibodies directed to amino acids of SEQ ID NO: 2, or a protein comprising amino acids of SEQ ID NO: 2 are also included.", "A DNA segment comprising the nucleotides as set forth in SEQ ID NO: 1 is further provided.", "The present invention further provides assays for expression of the RNA and protein products of the DNA of the present invention to enable determining whether abnormal expression of such DNA is involved with a particular disease, e.g., cancer.", "The present invention also provides antibodies, either polyclonal or monoclonal, specific to a unique portion of the receptor protein; a method for detecting the presence of a receptor ligand that is capable of either activating or down-regulating, i.e., modulating, the receptor protein; a method of screening potential ligand analogs for their ability to modulate the receptor protein; and procedures for targeting a therapeutic drug to cells having a high level of the receptor protein.", "The present invention also provides binding assays that permit the ready screening for molecules that affect the binding of the receptor and its ligands.", "The present invention further provides use of the receptor for intracellular or extracellular targets to affect binding.", "Intracellular targeting can be accomplished through the use of intracellularly expressed antibodies referred to as intrabodies.", "Extracellular targeting can be accomplished through the use of receptor specific antibodies.", "Additionally, the soluble form of the receptor can be used as a receptor decoy or aptamer to inhibit binding.", "The present invention also provides use of antisense technology to affect binding of the CEA receptor and its ligands by designing an antisense nucleic acid molecule which is complementary to a nucleic acid molecule encoding the CEA receptor.", "The present invention also provides an assay to determine the presence or absence of the receptors that can be used as a diagnostic or prognostic tool to identify the presence or stage of differentiation of tissue, e.g., tumor tissue.", "Finally, a nucleic acid of the invention could be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.", "RNA-mediated interference (RNAi) may also be used.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 illustrates SEQ ID NO: 1 which is the nucleotide sequence of rat hnRNP M4 cDNA.", "This cDNA was isolated from rat KC as encoding the CEA binding protein.", "It is a full-length sequence that consists of a 5′ end nontranslated region of 21 bp and an open reading frame (ORF) that starts from the initiation sequence AAAATGG.", "The initiation, termination codons and a polyA tail shown in bold.", "The two N-terminal RNA-binding domains followed by methionine-arginine-glycine rich region and C-terminal RBD-3 shown as boxes.", "Inside the RBD-1 and RBD-2 the regions of homology with the Mycf-2 transcription factor are underlined.", "The linker sequence between the RBD-1 and RBD-2 containing 158-197 deletion shown in italic.", "The double underlined amino acids correspond the primer's sequences (5′-GGAAGGCCACTGAAAGTCAA (SEQ ID NO: 6), 3′-TCCACGACTTTTCCCATCTT (SEQ ID NO: 7)) employed for RT-PCR amplification.", "The GeneBank accession No.", "for the sequence is U32577.FIGS.", "2(a) and 2(b) show the predicted amino acid sequence of hnRNP M4 cDNA.", "FIG.", "2(a) shows the domain structure of rat hnRNP M4 protein.", "The protein composed of 2 N-terminal RNA-binding domains followed by methionine-arginine-glycine rich region and C-terminal RBD-3 shown as boxes.", "The potential transmembrane regions are shown as black boxes.", "The numbers above correspond to the amino acids.", "FIG.", "2(b) shows the posttranslational modification sites of rat hnRNP M4 protein.", "The predicted tyrosine kinase phosphorylation site, KVGEVTY (SEQ ID NO: 4), (aa 100-106) is shown between the arrows.", "The (#) symbols indicate 2 potential N-linked glycosylation sites (aa 36-39; 379-382).", "The (o) symbols indicate 7 potential protein kinase C phosphorylation sites (aa 1-3; 74-76; 92-94; 130-132; 144-146; 632-634; 685-687).", "The (*) symbols indicate 11 potential casein kinase II phosphorylation sites (aa 1-4; 50-53; 92-95; 105-108; 399-402; 419-422; 434-437; 448-451; 480-483; 520-523; 685-688).", "The (+) symbols indicate 15 potential casein kinase II phosphorylation sites (aa 11-16; 270-275; 290-295; 305-310; 322-327; 331-336; 37-362; 363-368; 387-392; 454-459; 503-508; 575-580; 595-600; 603-608; 681-686.).", "FIG.", "3 illustrates SEQ ID NO: 2 which is a comparison of published (Kafasla P., et al., Biochem J.; 2000, 350: 495-503) and deduced from cDNA amino acid sequences of rat hnRNP M4 protein.", "Amino acids are shown in single letter code.", "The sequences are indicated on the top and the amino acid numbers on the right and on the left sides.", "The consensus sequence shown in the middle represents the identical amino acids in both proteins.", "FIG.", "4 is a Western blot of rat hnRNP M4 protein expressed in E coli showing CEA binding.", "Rat cDNA corresponding to hnRNP M4 ORF was placed into a prokaryotic expression vector pGEX3T-4 allowing production of a GST-hnRNP M4 fusion protein.", "The Kupffer cell lysate and pGEX-4T-3/hnRNP M4 fusion proteins (2 clones) were subjected to SDS/PAGE and transferred into the PVDF membrane.", "This membrane was exposed to the soluble 1 μM CEA followed by the anti-CEA antibody.", "Similar to KC (line 1), hnRNP M4 protein (lines 2 and 3) binds to the soluble CEA and has a molecular weight 80 KD.", "Line 4 corresponds to a negative control that is a pGEX3T-4 vector without the hnRNP M4 cDNA.", "FIGS.", "5A-C show distribution of different splicing forms of hnRNP M4 protein in rat tissues.", "To determine whether the form with the deletion in a spacer region between the RBD-1 and RBD-2 is expressed in Kupffer cells we synthesized PCR primers that incorporated the deleted region as part of their product.", "Amplification will result in a larger PCR product for the full-length form (321 bp) and a shorter product (204 bp) for the deletion mutant.", "We examined a number of rat tissues.", "The results show that the deletion form encoding amino acids 158-197 of hnRNP M4 is distributed throughout all rat tissues examined (FIGS.", "5A, 5B and 5C).", "The larger form is generally also present but in what seems to be lower copy numbers.", "Comparison of the cDNA sequences indicates that there are multiple subclasses of mRNA that arise by alternative pre-mRNA splicing.", "This suggests that multiple forms of the hnRNP M4 protein may exist, possibly with different functions in vivo.", "Both mRNA forms (with and without the deletion) were characteristic to the rat KC.", "Only the short form was determined in human KC.", "FIG.", "6 shows that the expression of the 80 kD CEA receptor and the ability to uptake CEA are cell specific and perhaps tissue restricted to KC and alveolar macrophages.", "To elucidate whether the macrophage cell lines can uptake soluble CEA, in vitro experiments with 125I, labeled CEA were performed.", "Previously we have reported that LPS treatment can stimulate the CEA uptake (Toth CA et al, J. Leukocyte Biology.", "1989; 45: 370-376.).", "A very high level of CEA uptake characteristic to KC was used as a positive control.", "The macrophage cell lines pretreated with LPS (1 μg/ml, 1 hour) and without LPS exposure were incubated with the iodine labeled CEA (5 μg/ml) for 15, 30, 45 and 60 minutes.", "Comparatively, none of the macrophage cell lines was able to uptake CEA with or without LPS pretreatment.", "We have shown earlier that only freshly isolated lung alveolar macrophages express 80 kD protein and rapidly endocytosed CEA in a similar manner to KC.", "These data suggest that the expression of the 80 kD CEA receptor and the ability to uptake CEA are cell specific and perhaps are tissue restricted to KC and alveolar macrophages.", "FIG.", "7 illustrates that transfection of hnRNP M4 cDNA can initiate CEA binding in P388D1 macrophage cell line.", "To determine the mechanism underlying the intracellular signaling associated with KC activation and CEA binding, the hnRNP M4 cDNA in the pBK-CMV expression vector was introduced into the CEA non-responsive macrophage cell lines.", "The hnRNPM4 transcription in this system was driven by the CMV promoter.", "The two mouse (P388D1 and IC21) and a rat macrophage cell line (CRL2192) that did not bind CEA were transfected with the hnRNP M4/PBK-CMV expression vector.", "As shown in FIG.", "7, transient transfection of bnRNPM4 (48 hours) in P388D1 cells results in CEA binding.", "The uptake was increased with increasing time of exposure and concentration of the hnRNP M4 plasmid DNA.", "An approximately 5 times increase in CEA uptake was observed on transfecting 10 g of hnRNP M4/pBK-CMV plasmid.", "This fact implies that hnRNP M4 is involved in CEA metabolism in P388D1 macrophages.", "Interestingly, P388D1, but not IC21 or CRL2192 cells were able to uptake CEA.", "At present, the reasons for lack of response by IC21 and CRL2192 cells is not known.", "It is possible that these cells may not have the regulatory factors that can induce hnRNP M4 gene expression as a result of differentiation and tissue specificity.", "FIG.", "8 is a schematic of CEA internalization by Kupffer cells.", "DETAILED DESCRIPTION The present invention provides a method of inhibiting CEA-associated process of cancer metastasis by identifying and targeting the human hnRNP M4 receptor that mediates this process.", "The novel receptor for CEA is the hnRNP M4 protein which was isolated by means of expression cloning using a mouse polyclonal 80 kD protein specific antiserum.", "The novel receptor for CEA from the rat KC is homologous to the deletion mutant of the human hnRNP M4 gene.", "The cDNA displays features similar to that of 80 kD KC CEA receptor by four independent criteria: (1) By SDS/PAGE the molecular masses of both the recombinant and the cellular proteins were found to be similar corresponding 78-80 kD; (2) The rat hnRNP M4 fusion protein expressed in E. coli binds CEA and this interaction is a Ca++ dependent process; (3) Transfection of rat hnRNP M4 cDNA in P388D1 mouse macrophages results in CEA uptake; (4) The same gene was identified for the binding protein in both rat and human liver using two different approaches: screening with the anti-80 kD antibody and two-hybrid assay for the protein interactions.", "Earlier we have shown that the CEA binding to KC initiates series of signaling events that lead to the induction of IL-1α, IL-6, IL-10 and TNFE-α cytokines (24).", "Data presented in this study shows that the hnRNP M4 protein can bind CEA.", "Without wishing to be bound by theory, we predict that it is involved in a multi-protein complex that can recognize CEA on the surface of KC.", "Earlier it was shown that the increase in cytokines production is associated with tyrosine phosphorylation (23).", "The analysis of conserved domains of the encoded protein by Swiss-Prot database revealed that hnRNP M4 has a tyrosine internalization signal, KVGEVTY (SEQ ID NO: 4), aa 100-106.Enhancement in tyrosine phosphorylation after receptor activation is considered an important signaling event leading to cellular responses.", "For example, the hnRNP K protein is tyrosine phosphorylated in vitro by Src and Lck that regulates in vivo K-protein-protein and K-protein-RNA interactions by changing recruitment of signaling effectors (33).", "HnRNP K has a diverse repertoire of molecular partners including G coupled receptor protein, tyrosine and serine/threonine kinases as well as the proto-oncoprotein Vav (34).", "It was shown that of hnRNP A2 and hnRNP C1 proteins in the liver can be phosphorylated and this process is modulated by calmodulin (35).", "In addition to a tyrosine kinase phosphorylation site, the hnRNP M4 possesses 7 potential protein kinase C that can also play an important role in the signal transduction pathways.", "Similar to hnRNP K the cell distribution pattern of hnRNP M4 depends on the tissue examined.", "In Hela cells this protein is evenly present throughout the nucleus and the cytoplasm (19).", "In contrast, in mice's embryos the protein is localized predominantly in the nucleus (20).", "The hnRNP M4 protein can also form complexes with hnRNP K and hnRNP A1 shuttling proteins (34, 36).", "We therefore discovered a new physiological role for the hnRNP M4 protein as a CEA binding protein in Kupffer cells and also in lung alveolar macrophages (32).", "One of the mechanisms of how the hnRNPs can carry different functions is structural rearrangement and the appearance of different splicing forms (15).", "The present invention shows that at least 3 splicing forms of hnRNP M4 are ubiquitously expressed in rat and human tissues.", "Two low molecular weight mRNA forms (with and without the deletion of aa 158-197) were characteristic to rat KC.", "Only the short form is transcribed in human KC.", "When rat cDNA with the deletion was translated in the prokaryotic system it was able to bind with 125I labeled CEA or PELPK (SEQ ID NO: 5) albumin conjugate in the presence of 10 mM Ca++.", "This binding was abolished in the presence of 10 mM EDTA.", "This data shows that the low molecular weight form of the protein that has a deletion of aa 158-197 in the spacer region between RBD-1 and RBD-2 can function as a CEA receptor.", "The signaling pathway of KC activation by CEA leading to the enhancement of metastasis is not known.", "Based on the present invention, several mechanisms of cytokine regulation by CEA can be envisioned.", "First, structural findings reveal that hnRNP M4 receptor has a potential to be involved in signaling pathways and to be modified by a variety of enzymes.", "Second, high level of homology of N-terminal RBD-1 and RBD-2 domains with the Myef-2 transcription factor suggests a role of hnRNP M4 as a transcription factor.", "By participating in transcription and regulating the promoter function of the genes, hnRNP M4 could modulate the cytokine production.", "Third, the hnRNPs can also effect the cytokine production through a direct binding with nRNA and control of mRNA stability.", "For many protooncogenes, lymphokines, and cytokines, a common feature is the existence of A+U-rich elements (15).", "It was shown that all four hnRNP M proteins bind to poly (U) stretches in high salt conditions (1M NaCl) (17, 18, 19).", "Uridylate stretches also found in regulatory regions of RNAs such as the 3′ splice site of introns appear to be common targets for RNA-binding proteins (15).", "In support of our previous findings (32), the present invention teaches that the ability to take up CEA is tissue specific.", "Several macrophage cell lines: CRL2192, P388D, IC21, PH1 and raw 264.7 were analyzed and none of them were as effective in taking up CEA as KC.", "We transfected these cells with the expression pBK-CMV/hnRNP M4 vector to elucidate whether the hnRNP M4 protein expression can result in CEA uptake.", "Only one cell line, a mouse macrophage cell line p388D1, initiated CEA binding in transient transfection assay.", "This is an important evidence of the role of hnRNP M4 in CEA metabolism.", "A developed system represents an in vitro model to study and to reconstitute the signaling events that occur in vivo in KC.", "Further investigations by using deletions of specific domains within the gene and examining the effects on specific functions will result in the identification of CEA (PELPK (SEQ ID NO: 5)) binding domains and the structure-function relationships associated with the CEA binding.", "This model also allows examination of downstream signaling events and protein domains needed to initiate the receptor mediated endocytosis and to induce the cytokine secretion in macrophages by CEA.", "Identifying the nature of the biochemical differences between KC and macrophage cell lines, which allow hnRNP M4 function more efficiently in some cell lines than others, will help our understanding of the complex regulatory mechanisms involved in the CEA receptor expression and CEA binding.", "CEA is not only a serum marker to monitor metastasis but is also a target for therapeutic treatment.", "Conventional treatments of metastatic colon cancer such as chemo- and radiotherapy are lacking specificity and have dose limitations due to toxicity in normal tissues.", "Anti-CEA antibodies are widely used for localization of colorectal carcinoma in radioinimunoguided surgery (37), radioimmunotherapy (38) and anti-CEA antibody-directed cytokine targeting (39).", "CEA is also a potential T-cell target in antigen-specific vaccination-based cancer therapy (40).", "Development of specific antibodies and vaccines to the CEA receptor seems as a more straightforward approach to study and prevent metastasis, e.g., hepatic metastasis.", "The receptor of the present invention may also be used diagnostically.", "Determining the level of this receptor in individuals can be an important tool in determining whether an individual is at a greater risk for developing metastasis of cancerous cells post treatment or post surgery.", "This knowledge can be used in determining a more effective treatment plan for that individual.", "The determination of the number of receptors present on the cells of an individual can readily be accomplished by standard means, for example, using FACS analysis or analysis of RNA levels.", "The level can be compared to a reference level, which can be determined by standard means.", "These assays are further discussed below.", "Another preferred embodiment of this invention is in the diagnosis of diseases associated with this receptor.", "Using any suitable technique known in the art, such as Northern blotting, quantitative PCR, reverse transcriptase PCR, etc.", "the nucleotide sequences of the receptors or fragments thereof can be used to measure levels of receptor RNA expression.", "Alternatively, the antibodies of the invention can be used in standard techniques such as Western blotting to detect the presence of cells expressing receptors and using standard techniques, e.g.", "FACS or ELISA, to quantify the level of expression.", "One can treat diseases associated with the expression of the receptors of the present invention by blocking receptor/ligand interaction.", "This can be accomplished by a range of different approaches.", "For example, antibodies, aptamers, decoys, small molecules, antagonists, etc.", "One preferred approach is the use of antibodies to these receptors.", "Antibodies specifically binding to amino acids of SEQ ID NO: 2 are preferred.", "Antibodies to these receptors can be prepared by standard means.", "For example, one can use single chain antibodies to target these receptors.", "An alternative strategy is to use receptor decoys.", "For example, one could prepare a decoy comprising the portion of the receptor present on the exterior of the cell membrane.", "Another strategy is to prepare soluble forms of these receptors.", "This can be done by standard means including using PCR to clone a gene, site-directed mutagenesis to make changes in the structure, deletions to make fragments, etc.", "as discussed below.", "Compounds that affect this receptor/ligand interaction can be directly screened for example using a direct binding assay.", "For example, the compound of interest can be added before or after the addition of the labeled ligand and the effect of the compound on binding can be determined by comparing the degree of binding in that situation against a base line standard with that ligand, not in the presence of the compound.", "The binding assay can be adapted depending upon precisely what is being tested.", "The present invention also provides use of antisense technology to affect binding of the CEA receptor and its ligands.", "Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.", "An antisense nucleic acid molecule which is complementary to a nucleic acid molecule encoding the CEA receptor can be designed based upon the isolated nucleic acid molecules encoding the receptor provided by the invention.", "An antisense nucleic acid molecule can comprise a nucleotide sequence which is complementary to a coding strand of a nucleic acid, e.g.", "complementary to an mRNA sequence, constructed according to the rules of Watson and Crick base pairing, and can hydrogen-bond to the coding strand of the nucleic acid.", "The antisense sequence complementary to a sequence of an mRNA can be complementary to a sequence in the coding region of the mRNA or can be complementary to a 5′ or 3′ untranslated region of the mRNA.", "Furthermore, an antisense nucleic acid can be complementary in sequence to a regulatory region of the gene encoding the mRNA, for instance a transcription initiation sequence or regulatory element.", "Preferably, an antisense nucleic acid complementary to a region preceding or spanning the initiation codon or in the 3′ untranslated region of an mRNA is used.", "An antisense nucleic acid can be designed based upon the nucleotide sequence of SEQ ID NO: 1 (shown in FIG.", "1).", "A nucleic acid is designed which has a sequence complementary to a sequence of the coding or untranslated region of the shown nucleic acid.", "Alternatively, an antisense nucleic acid can be designed based upon sequences of the CEA receptor, which can be identified by screening a genomic DNA library with an isolated nucleic acid of the invention.", "For example, the sequence of an important regulatory element can be determined by standard techniques and a sequence which is antisense to the regulatory element can be designed.", "The antisense nucleic acids and oligonucleotides of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.", "The antisense nucleic acid or oligonucleotide can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids e.g.", "phosphorothioate derivatives and acridine substituted nucleotides can be used.", "Alternatively, the antisense nucleic acids and oligonucleotides can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e.", "nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).", "The antisense expression vector is introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced, For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol.", "1 (1) 1986.A nucleic acid of the invention could be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.", "See for example Bartel, D. and Szostak, J. W. Science 261: 1411-1418 (1993).", "RNA-mediated interference (RNAi) (Fire, et al., Nature 391: 806-811, 1998) may also be used.", "Aptamers can be produced using the methodology disclosed in a U.S. Pat.", "No.", "5,270,163 and WO 91/19813.The DNA segments according to this invention are useful for detection of expression of the receptors in tissues, as described in the Examples below.", "Therefore, in yet another aspect, the present invention relates to a bioassay for determining the amount of receptor mRNA in a biological sample comprising the steps of i) contacting that biological sample with a nucleic acid isolate consisting essentially of a nucleotide sequence that encodes the receptor or a unique portion thereof, e.g., such the nucleotide sequence of SEQ ID NO: 1 (shown in FIG.", "1), under conditions such that a nucleic acid:RNA hybrid molecule, such as a DNA:RNA hybrid molecule, can be formed; and ii) determining the amount of hybrid molecule present, the amount of hybrid molecule indicating the amount of receptor raRNA in the sample.", "Findings described in the Examples, below, indicate that increased expression of the receptor of the present invention, as detected by this method of this invention, plays a critical role in the success of treatment of some human malignancies, e.g., colon and breast cancer.", "Of course, it will be understood by one skilled in the art of genetic engineering that in relation to production of polypeptide products, the present invention also includes DNA segments having DNA sequences other than those in the present examples that also encode the amino acid sequence of the polypeptide product of the receptor gene.", "For example, it is known that by reference to the universal genetic code, standard genetic engineering methods can be used to produce synthetic DNA segments having various sequences that encode any given amino acid sequence.", "Such synthetic DNA segments encoding at least a portion of the amino acid sequence of the polypeptide product of the receptor gene also fall within the scope of the present invention.", "Further, it is known that different individuals may have slightly different DNA sequences for any given human gene and, in some cases, such mutant or variant genes encode polypeptide products having amino acid sequences which differ among individuals without affecting the essential function of the polypeptide product.", "Still further, it is also known that many amino acid substitutions can be made in a polypeptide product by genetic engineering methods without affecting the essential function of that polypeptide.", "Accordingly, the present invention further relates to a DNA segment having a nucleotide sequence that encodes an amino acid sequence differing in at least one amino acid from the amino acid sequence of receptor, or a unique portion thereof, and having greater overall similarity to the amino acid sequence of the receptor than to that of any other polypeptide.", "The amino acid sequence of this DNA segment includes at least about 4 to 6 amino acids which are sufficient to provide a binding site for an antibody specific for the portion of a polypeptide containing this sequence.", "The present invention further relates to a recombinant DNA molecule comprising a DNA segment of this invention and a vector.", "In yet another aspect, the present invention relates to a culture of cells transformed with a DNA segment according to this invention.", "These host cells transformed with DNAs of the invention include both higher eukaryotes, including animal, plant and insect cells, and lower eukaryotes, such as yeast cells, as well as prokaryotic hosts including bacterial cells such as those of E. coli and Bacillus subtills.", "Various standard recombinant systems, such as those cited above as well as others known in the art, are suitable as well for production of large amounts of the novel receptor proteins using methods of isolation for receptor proteins that are well known in the art.", "Therefore, the present invention also encompasses an isolated polypeptide having at least a portion of the amino acid sequence of SEQ ID NO: 2.The isolated nucleotide sequences and isolated polypeptides of the invention encoding receptors can be mutagenized by any of several standard methods including treatment with hydroxylamine, passage through mutagenic bacterial strains, etc.", "The mutagenized sequences can then be classified “wild type” or “non-wild type” depending whether it will still facilitate infectivity or not.", "Mutagenized sequences can contain point mutations, deletions, substitutions, rearrangements etc.", "Mutagenized sequences can be used to define the cellular function of different regions of the receptors they encode.", "This information can be used to assist in the design of small molecules or peptides mimicking the interactive part of the receptor.", "Another approach is to use small molecules that will selectively bind to one of the receptors.", "Such molecules and peptides can be synthesized by known techniques.", "Another strategy is to express antibodies to these receptors in individuals intracellularly.", "This can be done by the method of Marasco and Haseltine set forth in WO94-02610 (PCT/US93/06735 filed Jul.", "16, 1993) published Feb. 3, 1994.In addition, additional compounds that bind to these receptors can readily be screened for.", "For example, one can select cells expressing high numbers of these receptors, plate them; e.g.", "add labeled ligand and screen for compounds or combinations of compounds that will interact with, e.g.", "binding of, these receptors by standard techniques.", "Alternatively, one can use known techniques to prepare cells that will express these receptors and use those cells in drug screens.", "One can also prepare cell lines stably expressing the receptors.", "Such cells can be used for a variety of purposes including an excellent source of antigen for preparing a range of antibodies using techniques well known in the art.", "Therapeutic and Pharmaceutic Compositions.", "An exemplary pharmaceutical composition is a therapeutically effective amount of a decoy, antibody etc.", "that affects the ability of the receptor to bind ligand optionally included in a pharmaceutically-acceptable and compatible carrier.", "The term “pharmaceutically-acceptable and compatible carrier” as used herein, and described more fully below, includes (1) one or more compatible solid or liquid filler diluents or encapsulating substances that are suitable for administration to a human or other animal, and/or (ii) a system, such as a retroviral vector, capable of delivering the molecule to a target cell.", "In the present invention, the term “carrier” thus denotes an organic or inorganic ingredient, natural or synthetic, with which the molecules of the invention are combined to facilitate application.", "The term “therapeutically-effective amount” is that amount of the present pharmaceutical compositions which produces a desired result or exerts a desired influence on the particular condition being treated.", "Various concentrations may be used in preparing compositions incorporating the same ingredient to provide for variations in the age of the patient to be treated, the severity of the condition, the duration of the treatment and the mode of administration.", "The term “compatible”, as used herein, means that the components of the pharmaceutical compositions are capable of being commingled with a small molecule, nucleic acid and/or polypeptides of the present invention, and with each other, in a manner such that does not substantially impair the desired pharmaceutical efficacy.", "Dose of the pharmaceutical compositions of the invention will vary depending on the subject and upon particular route of administration used.", "Dosages can range from 0.1 to 100,000 μg/kg per day, more preferably 1 to 10,000 Ag/kg.", "By way of an example only, an overall dose range of from about, for example, 1 microgram to about 300 micrograms might be used for human use.", "This dose can be delivered at periodic intervals based upon the composition.", "For example on at least two separate occasions, preferably spaced apart by about 4 weeks.", "Other compounds might be administered daily.", "Pharmaceutical compositions of the present invention can also be administered to a subject according to a variety of other, well-characterized protocols.", "For example, certain currently accepted immunization regimens can include the following: (i) administration times are a first dose at elected date; a second dose at 1 month after first dose; and a third dose at 5 months after second dose.", "See Product Information, Physician's Desk Reference, Merck Sharp & Dohme (2002) (e.g., Hepatitis B Vaccine-type protocol); (ii) Recommended administration for children is first dose at elected date (at age 6 weeks old or older); a second dose at 4-8 weeks after first dose; a third dose at 4-8 weeks after second dose; a fourth dose at 6-12 months after third dose; a fifth dose at age 4-6 years old; and additional boosters every 10 years after last dose.", "See Product Information, Physician's Desk Reference, Merck Sharp & Dohme (2002) (e.g., Diptheria, Tetanus and Pertussis-type vaccine protocols).", "Desired time intervals for delivery of multiple doses of a particular composition can be determined by one of ordinary skill in the art employing no more than routine experimentation.", "The small molecules and polypeptides of the invention may also be administered per se or in the form of a pharmaceutically-acceptable salt.", "When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of this invention.", "Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene-sulfonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzenesulphonic.", "Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.", "Thus, the present invention also provides pharmaceutical compositions, for medical use, which comprise nucleic acid and/or polypeptides of the invention together with one or more pharmaceutically acceptable carriers thereof and optionally any other therapeutic ingredients.", "The compositions include those suitable for oral, rectal, intravaginal, topical, nasal, ophthalmic or parenteral administration, all of which may be used as routes of administration using the materials of the present invention.", "Other suitable routes of administration include intrathecal administration directly into spinal fluid (CSF), direct injection onto an arterial surface and intraparenchymal injection directly into targeted areas of an organ.", "Compositions suitable for parenteral administration are preferred.", "The term “parenteral” includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.", "The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.", "Methods typically include the step of bringing the active ingredients of the invention into association with a carrier which constitutes one or more accessory ingredients.", "Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the nucleic acid and/or polypeptide of the invention in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.", "Preferred compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the molecule of the invention which is preferably isotonic with the blood of the recipient.", "This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.", "The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.", "Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.", "In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.", "For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides.", "In addition, fatty acids such as oleic acid find use in the preparation of injectibles.", "Antibodies The term “antibodies” is meant to include monoclonal antibodies, polyclonal antibodies and antibodies prepared by recombinant nucleic acid techniques that are selectively reactive with polypeptides encoded by eukaryotic nucleotide sequences of the present invention.", "The term “selectively reactive” refers to those antibodies that react with one or more antigenic determinants of the receptors and do not react with other polypeptides.", "Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.", "Antibodies can be used for diagnostic applications or for research purposes.", "For example, antibodies may be raised against amino-terminal (N-terminal) or carboxyl-terminal (C-terminal) peptides of a polypeptide encoded by the receptors.", "One approach is to isolate a peptide sequence that contains an antigenic determinant for use as an immunogen.", "This peptide immunogen can be attached to a carrier to enhance the immunogenic response.", "Although the peptide immunogen can correspond to any portion of a polypeptide encoded by a eukaryotic nucleotide sequence of the invention, certain amino acid sequences are more likely than others to provoke an immediate response, for example, an amino acid sequence including the N- or C-terminus of a polypeptide encoded by a gene that contains nucleotide sequences of the invention.", "Preferably one can use a cell line expressing only the receptor, select those cells with the highest levels of expression and use the whole cell as an antigen.", "For example, cDNA clone encoding a receptor or a fragment thereof may be expressed in a host using standard techniques (see above; see Sambrook et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.: 1989) such that 5-20% of the total protein that can be recovered from the host is the desired protein.", "Recovered proteins can be electrophoresed using PAGE and the appropriate protein band can be cut out of the gel.", "The desired protein sample can then be eluted from the gel slice and prepared for immunization.", "Alternatively, a protein of interest can be purified by using conventional methods such as, for example, ion exchange hydrophobic, size exclusion, or affinity chromatography.", "Once the protein immunogen is prepared, mice can be immunized twice intraperitoneally with approximately 50 micrograms of protein immunogen per mouse.", "Sera from such immunized mice can be tested for antibody activity by immunohistology or immunocytology on any host system expressing such polypeptide and by ELISA with the expressed polypeptide.", "For immunohistology, active antibodies of the present invention can be identified using a biotin-conjugated anti-mouse immunoglobulin followed by avidin-peroxidase and a chromogenic peroxidase substrate.", "Preparations of such reagents are commercially available; for example, from Zymad Corp., San Francisco, Calif. Mice whose sera contain detectable active antibodies according to the invention can be sacrificed three days later and their spleens removed for fusion and hybridoma production.", "Positive supernatants of such hybridomas can be identified using the assays described above and by, for example, Western blot analysis.", "To further improve the likelihood of producing an antibody as provided by the invention, the amino acid sequence of polypeptides encoded by a eukaryotic nucleotide sequence of the present invention may be analyzed in order to identify portions of amino acid sequence which may be associated with increased immunogenicity.", "For example, polypeptide sequences may be subjected to computer analysis to identify potentially immunogenic surface epitopes.", "Such computer analysis can include generating plots of antigenic index, hydrophilicity, structural features such as amphophilic helices or amphophilic sheets and the like.", "For preparation of monoclonal antibodies directed toward polypeptides encoded by a eukaryotic nucleotide sequence of the invention, any technique that provides for the production of antibody molecules by continuous cell lines may be used.", "For example, the hybridoma technique originally developed by Kohler and Milstein (Nature, 256: 495-497, 1973), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies, and the like, are within the scope of the present invention.", "See, generally Larrick et al., U.S. Pat.", "No.", "5,001,065 and references cited therein.", "Further, single-chain antibody (SCA) methods are also available to produce antibodies against polypeptides encoded by a eukaryotic nucleotide sequence of the invention (Ladner et al.", "U.S. Pat.", "Nos.", "4,704,694 and 4,976,778).", "The monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.", "The present invention provides for antibody molecules as well as fragments of such antibody molecules.", "Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies or to other molecules of the invention.", "See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference.", "Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities.", "This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation.", "The preferred binding is, however, covalent binding.", "Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules.", "Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules.", "For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines.", "This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents.", "(See Killen and Lindstrom 1984, “Specific killing of lymphocytes that cause experimental Autoimmune Myasthenia Gravis by toxin-acetylcholine receptor conjugates.” Jour.", "Immun.", "133:1335-2549; Jansen, F. K., H. E. Blythman, D. Carriere, P. Casella, O. Gros, P. Gros, J. C. Laurent, F. Paolucci, B. Pau, P. Poncelet, G. Richer, H. Vidal, and G. A. Voisin.", "1982.“Immunotoxins: Hybrid molecules combining high specificity and potent cytotoxicity”.", "Immunological Reviews 62:185-216; and Vitetta et al., supra).", "Preferred linkers are described in the literature.", "See, for example, Ramakrishnan, S. et al., Cancer Res.", "44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester).", "See also, Umemoto et al.", "U.S. Pat.", "No.", "5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker.", "Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene (Pierce Chem.", "Co., Cat.", "(21558G); (ii.)", "SPDP (succinimidyl-6 [3(2-pyridyldithio) propionamido] hexanoate (Pierce Chem.", "Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)propianamide] hexanoate (Pierce Chem.", "Co. Cat.", "#2165-G); and (v) sulfo-NHS(N-hydroxysulfo-succinimide: Pierce Chem.", "Co., Cat.", "#24510) conjugated to EDC.", "The linkers described above contain components that have different attributes, thus leading to conjugates with differing physiochemical properties.", "For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates.", "NHS ester containing linkers are less soluble than sulfo-NHS esters.", "Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability.", "Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available.", "Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings.", "Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.", "These antibodies may also be used as carriers to form immunotoxins.", "As such, they may be used to deliver a desired chemical or cytotoxic moiety to cell expressing the receptor.", "The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial, fungal or plant origin, or an enzymatically active polypeptide chain or fragment (“A chain”) of such a toxin.", "Enzymatically active toxins and fragments thereof are preferred and are exemplified by diphtheria toxin A fragment, non-binding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aer-uginosa), ricin A chain, abrin A chain, modeccin A chain, alphasarcin, certain Aleurites fordii proteins, certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin, Ricin A chain, Pseudomonas aeruginosa exotoxin A and PAP are preferred.", "Conjugates of the monoclonal antibody and such cytotoxic moieties may be made using a variety of bifunctional protein coupling agents.", "Examples of such reagents are N-succinimidyl-3-(2pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters such as dimethyl adelpimidate HCl, active esters such as disuccininidyl suberate, aldehydes such as glutaradehyde, bis-azido compounds such as bis(p-diazoniumbenzoyl)ethylenediamine, diisocyanates such as tolylene 2,6-diisocyante, and bisactive fluorine compounds such as 1,5-difluoro-2,4-dinitrobenzene.", "The enzymatically active polypeptide of the immunotoxins according to the invention may be recombinantly produced.", "Recombinantly produced ricin toxin A chain (RRTA) may be produced in accordance with the methods disclosed in PCT WO85/03508 published Aug. 15, 1985.Recombinantly produced diphtheria toxin A chain and non-binding active fragments thereof are also described in PCT WO85/03508 published Aug. 15, 1985.Antibodies of the present invention can be detected by appropriate assays, e.g., conventional types of immunoassays.", "For example, a sandwich assay can be performed in which the receptor or fragment thereof is affixed to a solid phase.", "Incubation is maintained for a sufficient period of time to allow the antibody in the sample to bind to the immobilized polypeptide on the solid phase.", "After this first incubation, the solid phase is separated from the sample.", "The solid phase is washed to remove unbound materials and interfering substances such as nonspecific proteins which may also be present in the sample.", "The solid phase containing the antibody of interest bound to the immobilized polypeptide of the present invention is subsequently incubated with labeled antibody or antibody bound to a coupling agent such as biotin or avidin.", "Labels for antibodies are well-known in the art and include radionuclides, enzymes (e.g.", "maleate dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluors (fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescamine), biotin, and the like.", "The labeled antibodies are incubated with the solid and the label bound to the solid phase is measured, the amount of the label detected serving as a measure of the amount of anti-urea transporter antibody present in the sample.", "These and other immunoassays can be easily performed by those of ordinary skill in the art.", "The following Examples serve to illustrate the present invention, and are not intended to limit the invention in any manner.", "EXAMPLES Example #1 The cDNA Library Construction and Cloning of the CEA-Binding Protein To isolate the CEA receptor a rat KC cDNA library was constructed using the MAP Express vector system (Stratagene, La Jolla, Calif.).", "The library was screened with the mouse polyclonal anti-80 kD antibody (11).", "By screening of 1011 plaques one positive cDNA clone with an insert size of 2.4 kb was isolated.", "Sequencing and a Blast search of human genome data bases for close related family members revealed that this cDNA represents a novel rodent gene with 91% of homology to human hnRNP M4 protein.", "The hnRNPs are a large group of over 20 proteins (hnRNP A-hnRNP U) that associate with pre-mRNAs in eukaryotic cell (12).", "The majority of hnRNPs are extremely abundant nuclear components, their amounts approximately the same as those of the core histones (13).", "Most hnRNPs are expressed only in the nucleus.", "However, several, among them, hnRNP A, D, E, I, and K have been found to shuttle between the nucleus and the cytoplasm, and are believed to promote mRNA export by acting as adapters between mRNA and the transport machinery (14).", "In general, these proteins interact with RNA and are involved in mRNA processing and maturation (capping, splicing and polyadenilation.", "They can affect mRNA stability (15).", "Some of hnRNPs also bind single and double stranded DNA and act as transcription factors (16).", "The hnRNP M4 protein belongs to the family of hnRNP M proteins that consists of 4 splicing forms with the molecular weight 68-80 k) (17).", "The human hnRNP M4 protein has been shown to participate in RNA splicing and processing, as well as to changes related with heat shock (18, 19, 20).", "Evidence that this protein can be expressed on the cell surface comes from the fact that the close homologue of human hnRNP M4 was also described as a monomer of N-acetylglucosamine-specific receptor of the thyroid hormone NAGR1 (21).", "Later studies showed that this receptor identical to the hnRNP M4 (22).", "The differences between the isolated rat and full-length human hnRNP M4 cDNA (# NM 005968.1) are mostly single substitutions of nucleotides throughout the sequence.", "However, the significant difference between the isolated rat and full-length human hnRNP M4 cDNA (# NM 005968.1) is a deletion of 117 nucleotides in a spacer region between two N-terminal RNA-binding domains (RBD-I and RBD-2).", "Similar deletion is also characteristic to Homo sapiens hnRNP M4 protein deletion mutant cDNA (# AF 061832).", "The new clone has been referred to as a rat homologue of human hnRNP M4 protein.", "The cDNA sequence was submitted to the Gene Bank (# U32577).", "Since the anti-80 KD antiserum used to probe the rat KC library is polyclonal, there is potential to recognize the nonspecific protein epitopes.", "Independently, we screened for the CEA binding proteins using the yeast two-hybrid system (HybriZAP-2.1 Two-Hybrid Predigested Vector System, Stratagene, La Jolla, Calif.).", "As a bait, an engineered fragment of the full length CEA, containing a 146 amino acid (aa) fragment surrounding the PELPK binding site was used.", "In addition to rat Kupffer cell cDNA library, a commercial human liver cDNA library (Stratagene, La Jolla, Calif.) was acquired as a target library for the yeast two-hybrid system.", "As a result of interaction, a single cDNA clone was isolated that was identical in sequence to the human hnRNP M4 protein.", "Thus, both screening techniques, antibody probing and the yeast two-hybrid system, gave the same result indicative of a new functional role for the hnRNP M4 protein as the CEA-binding protein in KC.", "Example #2 Analysis of Rat hnRNP M4 Homologue cDNA The cloned rat cDNA is a 2351 nucleotides long full-length sequence with the polyadenylation signal AATAAA and a termination polyA tail.", "SEQ ID NO: 1 (shown in FIG.", "1) is the cDNA sequence of rat homologue of human hnRNP M4.The sequence consists of a 5′ end nontranslated region of 21 bp and an open reading frame (ORF) that starts from the initiation sequence AAAATGG.", "The cDNA contains 3 putative RNA-binding domains (RBD).", "It is an evolutionarily conserved domain present in pre-mRNA-, mRNA-, pre-rRNA-, and snRNA-binding proteins, including hnRNP proteins, splicing factors, and polyadenylation factors (12).", "The first and second domains (RBD-1 and RBD-2) are arranged in a tandem close to the N-terminus and RBD-3 locates near the C-terminus (FIGS.", "1 and 2a).", "It was shown that RBD motifs are characteristic of the RNA binding proteins and can provide RNA and DNA binding activity (16).", "N-terminal RBD-1 region (nucleotides 297-376) has.", "87% of homology with Homo sapiens myelin gene expression factor 2 (MyEF-2) cDNA (25).", "The RBD-2 region (nucleotides 634-740) has 82% of identity to MyEF-2.The MyEF-2 is a transcription factor that was isolated from mouse brain, maps to mouse chromosome 2 and represses transcription of myelin basic protein gene.", "The MyEF-2 protein contains two RNA-binding domains (RBD-1 and RBD-2), previously shown to be responsible for sequence specific binding to both RNA and single stranded DNA (25).", "RBD-1 and RBD-2 demonstrate a lesser degree of homology to RBDs found in a wide variety of RNA and single strand DNA binding proteins (26).", "Sequence analysis demonstrated that, hnRNP M4 contains no other commonly recognized DNA binding motifs, such as zinc finger, homeobox, POU, or helix-loop-helix domain.", "Presumably, similar to MyEF-2, RBDs can be responsible for the single strand DNA binding activity of hnRNP M4 and their presence suggests that this protein may also bind RNA.", "The rat M4 protein sequence has 90-92% identity to a Homo sapiens chromosome 19 clone CTD-3182G2, complete sequence.", "Human gene by in situ hybridization was previously assigned to subbands p13.3-p13.2 of chromosome 19 (18, 21).", "The localization of the hnRNP M4 in the rat genome is not known.", "Example #3 Analysis of Rat hnRNP M4 Protein Sequence analysis of rat hnRNP M4 indicates that it encodes a novel protein of 775 amino acids (aa) consistent with its apparent molecular mass in SDS-polyacrylamide gel electrophoresis of 78-kD-80 kD.", "The protein is composed of 3 putative RNA-binding domains (aa 77-155, 171-248, 620-696).", "It has 3 potential spanning regions (aa 263-282, 290-311 and 597-616) (FIG.", "2a).", "The intracellular domain contained what appeared to be a tyrosine phosphorylation motif (aa 100-106) and two glucosaminoglycan attachment sites (aa 36-39), (aa 379-382).", "These chains can serve to orient the receptor proteins on the cell surface by correct insertion into the plasma membrane (27) and to stabilize the protein (28).", "Based on the translation of the primary sequence rat hnRNP M4 is a multifunctional signaling protein that can be modified by variety of enzymes.", "Structural analysis of the protein using Swiss-Prot database revealed that the protein possesses 7 potential protein kinase C phosphorylation sites, 11 casein kinase II phosphorylation sites and 15 N-myristoylation sites throughout the sequence (FIG.", "2b).", "The C-terminus contains 17 repeats of glycine-, arginine-, methionine and glutamine.", "Arginine methylation modification is a part of the mechanism by which protein-RNA complexes are recognized for nuclear export (29).", "Arginine is the only known methylated amino acid residue in hnRNPs.", "In fact, hnRNPs contain about 65% of methylated arginines in the cell nucleus (30), indicating that the methylation of these proteins is likely to have an important effect on their functions.", "It can influence the interactions of hnRNP M4 with other proteins (19) and affect its RNA-binding activity (31).", "This data demonstrates that the hnRNP M4 protein contains several important structural motifs that are characteristic to receptors and molecules involved in the signaling transduction pathways.", "In support of this hypothesis is the partial protein sequence of the rat hnRNP M4 that has been recently published (17).", "We compared the two protein sequences and found 74% homology between them (FIG.", "3, SEQ ID NO: 2).", "In contrast to our data, the published rat hnRNP M4 protein represents a partial sequence that is missing the N-terminal part of the protein.", "It also represents a splicing form without the 158-197 deletion in a spacer region between RBD-1 and RBD-2.Example #4 In Vitro CEA Binding Studies The ability of hnRNP M4 to interact with CEA was examined by preparing membrane lifts from 7ZAP Express-hnRNP M4 phage plates and incubating them with 125I labeled CEA or PELPK albumin conjugate in the presence of 10 mM Ca++ or 10 mM EDTA.", "Both CEA and PELPK albumin bound strongly to the phages in the presence of Ca++ but there was no binding when EDTA was present.", "The data confirms our observations on the Ca++ requirements of the CEA binding protein using isolated Kupffer cells and adds further evidence that hnRNP M4 is the Kupffer cell CEA binding protein.", "To further substantiate this finding and to estimate the molecular mass of the encoded protein, the cDNA corresponding to hnRNP M4 ORF was placed into a prokaryotic expression pGEX system allowing production of a GST-hnRNP M4 fusion protein.", "A series of pGEX vectors were designed for inducible, high level intracellular expression of the cDNA as a fusion protein with Schistosoma japonicum GST protein (Amersham Pharmacia Biotech, Piscataway, N.J.).", "The hnRNP M4 cDNA was inserted into pGEX-4T-3 vector to maintain the proper reading frame.", "The fusion protein was obtained as described below.", "The Kupffer cell lysate and pGEX-4T-3/hnRNP M4 fusion proteins (2 clones) were subjected to SDS/PAGE and transferred to a PVDF membrane (FIG.", "4).", "This membrane was exposed to the soluble 1 μg/ml CEA followed by an anti-CEA antibody.", "KC lysate was used as a positive control (line 1) and the empty vector as a negative (line 4) control.", "A single 80 kD band was determined in Kupffer cells (line 1) and in the fusion proteins (lines 2, 3).", "This band was absent for the vector alone (line 4).", "This data indicated that rat hnRNP M4 protein encoded by the cDNA is able to bind soluble CEA similar to the 80 kD KC receptor.", "The molecular weights of the hnRNP M4 protein and an 80 KD receptor from KC were indistinguishable.", "Example #5 Tissue Distribution of hnRNP M4 Both rat and human tissues were examined for the presence of the binding protein mRNA using Northern blots.", "A nick translated probe with [32P] corresponding hnRNP M4 cDNA was used.", "The results from rat and human tissues were similar.", "Northern blot analysis revealed that the mRNA encoding hnRNP M4 is abundantly expressed as a single transcript of 2.4 kb in the liver, heart, lung, skeletal muscle, kidney, and stomach.", "The RNA seems to have a ubiquitous distribution but the Northern blot analysis was unable to distinguish between the full length and deletion mutant.", "To determine whether the form with the deletion in a spacer region between the RBD-1 and RBD-2 is expressed in Kupffer cells we synthesized PCR primers that incorporated the deleted region as part of their product.", "Amplification results in a larger PCR product for the full-length form (321 bp) and a shorter product (204 bp) for the deletion mutant, as illustrated in FIG.", "5.The results show that the deletion form encoding amino acids 158-197 of hnRNP M4 is distributed throughout all rat tissues examined.", "The larger form is generally also present but in what seems to be lower copy numbers.", "Comparison of the cDNA sequences indicates that there are multiple subclasses of mRNA that arise by alternative pre-mRNA splicing.", "Without wishing to be bound by theory, these results indicate that multiple forms of the hnRNP M4 protein may exist, possibly with different functions in vivo.", "Both mRNA forms (with and without the deletion) were characteristic to the rat KC.", "Only the short form was found in human KC.", "PCR results suggest that the short form of hnRNP M4 is exclusively expressed in human and is present in rat KC and can function as a CEA receptor.", "This data further supports our finding that the deleted forms of rat and human hnRNP M4 were isolated as CEA binding proteins.", "Example #6 Transfection of hnRNP M4 in Macrophage Cell Lines To elucidate whether the macrophage cell lines can uptake soluble CEA, in vitro experiments with 125I labeled CEA were performed.", "We examined several macrophage cell lines: CRL2192, P388D, IC21, PH1 and raw 264.7.CEA uptake by isolated rat KC was used as a positive control.", "Cells were incubated with the iodine labeled CEA (5 μg/ml) for 15, 30, 45 and 60 minutes.", "None of the macrophage cell lines was able to take up labeled CEA in vitro.", "We have shown earlier that freshly isolated lung alveolar macrophages express the 80 kD protein and rapidly endocytose CEA in a similar manner to KC (32).", "These data suggest that the expression of the 80 kD CEA receptor and the ability to uptake CEA are cell specific and perhaps are tissue restricted to KC and alveolar macrophages.", "To begin to elucidate the mechanism underlying the intracellular signaling associated with KC activation and CEA binding, the hnRNP M4 cDNA in the pBK-CMV expression vector was introduced into CEA non-responsive macrophage cell lines.", "The hnRNP M4 transcription in this system was driven by the CMV promoter.", "The mouse alveolar macrophage: P388D1 and mouse peritoneal macrophage: IC21 cell lines that do not bind CEA and do not express CEA receptor were transfected with the hnRNP M4/PBK-CMV expression vector.", "As shown in FIG.", "6, in transient transfection after 48 hours, the hnRNP M4 induced CEA binding in P388D1 cells was increased with increasing time of exposure and concentration of the hnRNP M4 plasmid DNA as compared with the co transfection of matching concentration of the vector.", "An approximately 5 times increase in CEA uptake was observed on transfecting 10 g of hnRNP M4/pBK-CMV plasmid.", "This fact implies that hnRNP M4 cDNA can initiate CEA binding in P388D1 alveolar macrophage cell line and can act as a receptor for CEA.", "Interestingly, only P388D1, but not IC21 cells were able to take up CEA (FIG.", "7).", "At present, the reasons for lack of response by IC21 cells are not known.", "Example #7 RNA Preparation and Analysis The total RNA from various tissues, KC and cell lines was isolated using RNAzol method (Biotex Laboratories, Inc., Houston, Tex.)", "and the mRNA using mRNA isolation kit (Qiagen Inc., GmbH, Germany).", "Approximately 20 μg of total RNA were size fractionated on a formaldehyde/agarose gel, stained with ethidium bromide, and then transferred into nylon membrane (Hybond N, Amersham).", "HnRNP M4 probes were labeled by nick translation with [32P]-dCTP and hybridized at a concentration of 2.10.6 cpm/ml at 65° C. in a solution containing 5×SSC, 2× Denhardt's solution, 0.1% SDS and 0.3 mg/ml salmon sperm DNA.", "The filters were washed under high stringency conditions (65° C. 0.1×SSC-0.1% SDS) prior to autoradiography.", "Example #8 Rat cDNA Library Construction and Screening Rat KC library was constructed on the basis of the ZAP Express vector system (Stratagene, La Jolla, Calif.) according to the manufacturer's instructions.", "In brief, bacterial strain XL1 was incubated with recombinant phages.", "Approximately 1.106 pfu, of the library were plated on 150 mm Luria-Bertini agar plates containing 10 mM MgSO4 and maintained at 42° C. for 2-3 hours.", "The PVDF filters pretreated in 10 mM IPTG were overlaid in the plates and incubated at 37° C. for 12 hours.", "Then the filters were dried and blocked in 3% milk prior to exposure to the anti-80 kD antibody (1:100 dilution).", "Enhanced chemiluminescence technique was used to detect positive cDNA clones (Amersham, Pharmacia).", "By screening 1011 phages one positive clone was identified, and after plaque purification, the insert was subcloned in pBK-CMV vector for DNA sequencing.", "Example #9 DNA Sequencing The sequencing was carried out using dideoxy terminator fluorescent DNA sequencing method with the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems).", "Reactions were purified by ethanol precipitation and separated on the ABI 377-96 slab gel automated DNA sequencer.", "The contingency sequence was assembled and analysed using the Vector NTI Suite program (Informax, Gathersburg, Mass.).", "The sequences were verified by sequencing of the opposite strand.", "Example #10 Isolation of Kupffer Cells Kupffer cells were isolated according to our standard laboratory protocol (11) from the livers of male Sprague Dawley rats or Balb/C mice.", "The rodents are starved overnight and the livers perfused through the portal vein with collagenase.", "Suspended cells are separated into parenchymal and non-parenchymal fractions by differential centrifugation.", "Kupffer cells are purified from the non-parenchymal cell fraction by using 17.5% solution of metrizamide in Gey's balanced salt solution for final separation.", "The interface layer containing Kupffer cells is isolated and washed in phosphate buffer.", "Further purification of the cells is achieved by attachment to plastic plates for 2 hr.", "Generally 3-5×108 cells are routinely isolated from one liver.", "Kupffer cells are identified by their ability to phagocytose latex particles, staining for endogenous peroxidase activity and by electron microscopy.", "Example #11 Western Blot Analysis Cells (1×107) were washed in PBS and then lysed in RIPA buffer (Santa Cruz, Calif.) with protease inhibitors (1 mM Sodium Orthovanadate, 2 mM Sodium Fluoride, 10 μg/ml leupeptin, and 0.5 mM PMSF).", "Cell lysates were centrifuged at 15,000 rpm for 10 minutes at 4° C. Supernatants were collected and protein concentration was measured by BCA kit (Pierce, Rockford, Ill.).", "Equal amount of protein was loaded on SDS-PAGE.", "After electrophoresis with Mini-Protein II apparatus at 200V for 50 min, samples were transferred into Sequi-Blot-PVDF nitrocellulose membrane (Bio-Rad Laboratories, Hercules, Calif.) for 1 hr at 100V.", "The membrane was incubated with the primary antibody (in concentration 1:250-1:500) dissolved in 3% milk/TBS blocking buffer overnight at 4° C. or for 1 hr at room temperature.", "Subsequently it was washed 3 times for 10 min with 1×TBS buffer and incubated with the corresponding secondary HRP antibody at 1:1-10,000 dilution for 1 hour at room temperature.", "After washing with 1×TBS buffer for 3 times (15 min each) at room temperature, detection of proteins of interest was carried out by ECL (Amersham, Life Science Inc, Cleveland, Ohio).", "Example #12 Prokaryotic Protein Expression For production of CEA binding protein in a bacterial system, the EcOR1/Xho1 fragment corresponding to hnRNP M4 cDNA from the recombinant phage was cloned into the EcOR1/Xho1 sites of pGEX-4T-3 vector (Amersham Pharmacia Biotech, Piscataway, N.J.).", "The pGEX-4T-3 backbone was chosen from the variety of pGEX-GST vectors to maintain the original reading frame.", "The plasmid was introduced into B21 E. coli host bacteria and the transformed cells were cultured in L-broth to OD600=0.5 after which IPTG (0.3 mM) were added and cultures were maintained at 37° C. for 2 hours.", "Total bacterial proteins were prepared and fusion proteins were purified using glutathione Sepharose 4B affinity chromatography, according the manufacturer's instructions (Amersham Pharmacia Biotech, Piscataway, N.J.).", "Example #13 RT-PCR Amplifications were performed as per recommended protocols by the suppliers of the reagents.", "Briefly, RNA was isolated from cell lines, embryos and tissues using a single step RNA isolation method as described above.", "Typically, 1 g of total RNA was used for reverse transcription reaction using M-MLV reverse transcriptase and random hexamer or oligo dT primers (Promega Corp., USA) in a 50 l reaction.", "One tenth of the RT-products were used for PCR amplifications using Taq polymerase (Promega Corp., USA; Life Technologies, USA) along with gene specific primer pairs employing various cycling parameters depending on the primer combinations (described in the relevant section).", "The PCR reaction products were analyzed on agarose gels by ethidium bromide staining and photographed using a gel documentation set up (Kodak digital science, USA).", "For isolation of the PCR products, the appropriate bands containing DNA were excised from the agarose gels and purified using gel extraction kits (Qiagen, GmbH, Germany).", "The purified DNA fragments were dissolved in TE (pH 7.6) and used for direct sequencing and/or subcloning into plasmid vectors.", "Example #14 In Vitro CEA Binding Assay and CEA 125I Labeling These procedures were performed as described previously (10).", "Example #15 Transient Transfection of Expression Plasmids into Macrophage Cell Lines Transient transfection of hnRNP M4 plasmid was carried out in P388D1 mouse lymphoid macrophage, IC21 mouse peritoneal and CRL2192 rat alveolar macrophage cell lines.", "As a positive control were used rat KC.", "The P388D1 cells were propagated in DMEM medium supplemented with 10% horse serum.", "The CRL 2192 cells were grown in F-12K medium (Kaighn's modification) supplemented with 15% FBS.", "The IC21 cells were grown in RPM1 1640 ATCC modified medium supplemented with 10% FBS.", "All cells were maintained at 37° C. in 5% CO2 atmosphere.", "All tissue culture products were obtained from Life Technologies, USA.", "Cells for transfection were plated at 2-2.5.105 cells per well in six well tissue culture clusters (Nunc products, Germany).", "Cells were allowed to grow for 24 h and plasmid construct was transfected in serum free medium using Gene Pulser II Electroporation system (250 μF-300V) (Bio-Rad, Richmond, Calif., USA).", "The cells were washed once with PBS and resuspended at a density of 1.10.7 cells/ml in RPM1 media without FBS.", "10 μg of pBK-CMV/hnRNP M4 expression vector DNA was transfected per sample.", "0.4 ml of the cell suspension was used per electroporation in 0.4 cm cuvettes.", "The cells were maintained at room temperature prior to and after electroporation.", "At 10 minutes post electroporation, the cells are placed into growth media.", "At 48 hours post electroporation, the cells are harvested and resuspended in a volume of 200 μl for measuring of CEA binding activity.", "The references cited herein and set forth below are incorporated herein in their entirety.", "REFERENCES 1.Kim J. 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[ [ "Method for producing gene libraries", "The presented invention refers to a method for the creation of gene libraries wherein a defined number of adjacent nucleotides is exchanged and gene libraries are produced which code for protein variants having more manifold amino acid exchanges and a more homogenous distribution of mutations than can be obtained using conventional methods.", "DNA-strands are incorporated at random positions into a gene of interest.", "Then parts of the donor strands and parts of the gene sequence that is flanking these strands are removed, however, a defined number (e.g.", "3) of nucleotides that originate from the donor strand remain in the gene at the place of a defined number (e.g.", "3) of nucleotides of the original gene having been removed from it.", "Combined with a selection step after the incorporation of the donor strand into the gene it can be ensured that the nucleotides to be exchanged/introduced are in a specific reading frame.", "When the nucleotides of the donor strand that remain in the genes are degenerate, gene libraries can be produced with variants that have any codon at any position." ], [ "1-44.", "(canceled) 45.A method for producing sequence variation in DNA, which comprises the steps of: a) incorporation of a transposon (DONOR) into said DNA (GEN) at different random positions and b) specific removal of DONOR from GEN and specific removal of a defined number of adjacent nucleotides of GEN from GEN, such that at exactly the position of these removed nucleotides within the sequence of GEN a defined number of nucleotides remain that originate from DONOR and which can be completely degenerate.", "46.A method according to claim 45, wherein step 1 (b) occurs by several cycles of the following steps (a) to (d), followed by the steps (e) to (g): a) restriction digestion of GEN and at least parts of DONOR containing DNA using a restriction endonuclease of type IIs b) by demand, treatment of the DNA-ends with enzymes that make the DNA-ends blunt and/or isolation of the GEN containing part of the restricted DNA c) intramolecular ligation of the free DNA ends of the GEN containing part of the restricted DNA by which a circular strand of DNA is formed, which such receives a new recognition site for a restriction enzyme of type IIs and d) by demand isolation and/or amplification of this circular DNA-strand e) restriction digestion using at least one restriction enzyme of type IIs of the products obtained from the last cycle in step (c) or, when necessary of the amplified and/or isolated products from the last cycle in step (d) f) treatment of the DNA-ends with enzymes that make DNA-ends blunt and/or isolation of the GEN containing part of the restricted DNA g) intramolecular ligation of the free DNA-ends of the GEN containing part of the restricted DNA by which a circular DNA strand (cGEN') is formed, which has the original sequence of GEN with the exception of few nucleotides of GEN that were replaced by degenerate nucleotides from DONOR.", "47.A repertoire of sequence variants of DNA that has been produced using a method according to claim 45.48.A kit for creating sequence variation in DNA based on a method according to claim 45.49.A method for producing sequence variation in DNA, comprising the steps: a) introduction of a double strand breakage in said DNA (GEN), b) ligation of a DNA Strand (DONOR) to both DNA-ends of GEN, formed by the double strand breakage of GEN, producing a ligation product (LP), c) removal of the major part of DONOR from LP apart from few nucleotides that can be degenerate and removal of a small part of GEN from LP d) intramolecular ligation of the free DNA-ends of the remaining part of LP such that a circular DNA strand (cGEN') is formed, which has the original sequence of GEN with the exception of few nucleotides of GEN that were replaced by degenerate nucleotides from DONOR.", "50.A repertoire of sequence variants of DNA that has been produced using a method according to claim 49.51.A kit for creating sequence variation in DNA based on a method according to claim 49.52.A repertoire of sequence variants of DNA that has been produced using a method according to claim 2.53.A kit for creating sequence variation in DNA based on a method according to claim 2." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Enzymes are increasingly applied in the biotechnological and chemical industry, as well as in the medical diagnostics and therapie 1 .", "They help to improve existing processes and make new applications possible.", "Therefore, there is an increasing demand of enzymes with new or improved properties.", "The improvement/optimization of enzymes by rational or computerized methods, however, has been mostly without success 2 .", "Directed evolution of proteins, on the contrary, has been quite successful in optimizing properties of enzymes 3 .", "Directed evolution consists of two steps.", "Firstly, a gene library is created by varying one or more DNA sequences encoding the according enzyme and secondly, using selection or screening methods, those variants of the genes are isolated that encode enzymes variants with the desired, optimized properties.", "The advantage of this approach is that it does not require any information on the structure of the enzyme in question, its dynamics or its interactions with different substrates.", "Therefore, methods of directed evolution are increasingly accepted and applied by many biotech companies worldwide 1,4 .", "Success and failure of directed evolution above all depend on the quality of the libraries and the efficiency of the selection or screening method.", "The quality of a library is determined by the available sequence variation and depends mostly on the method used to create the sequence variation.", "Nowadays mainly two approaches are used to create sequence variation: in-vitro DNA-recombination 5-7 and random mutagenesis 8-10 .", "In-vitro DNA-recombination is based on a small repertoire of DNA sequences that are similar but not identical to each other.", "The differences (mutations) between these sequences can originate from natural evolution (family shuffling) 11 or could have been introduced by a combination of random mutagenesis and selection 5 .", "They therefore represent a pre-selection and in general improve or at least are neutral to the properties of the enzyme encoded by the DNA sequence.", "In the in-vitro DNA-recombination a repertoire of DNA sequences is produced, the variants of which contain new combinations of the mutations that were present in the original repertoire.", "The advantage of this approach is that a high number of positions in a gene are varied at once and that mostly advantageous mutations are recombined.", "The disadvantage of this approach is that variation is confined to sequence positions that show variation in the original repertoire and additionally to the types of mutations present at these positions.", "Random mutagenesis, on the contrary, provides access to completely new mutations.", "It has the advantage that all positions of a gene can contain every possible variation.", "Random mutagenesis, therefore, is not limited to mutations that are already present somewhere.", "Nowadays there are two techniques that are almost exclusively used for introducing random mutations into genes: error prone PCR 8 and site specific (cassette-) mutagenesis using degenerated oligonucleotides 9 .", "Error Prone PCR The by far most dominant method of the site-nonspecific random mutagenesis is to use error prone PCR.", "Here the gene to be mutated is amplified by PCR using a thermostable polymerase (usually Taq-polymerase) that introduces wrong nucleotides in a rate that depends on the conditions of the PCR einbaut 12 .", "A common variant of this method applies Manganese(II)-Ions 8 or nucleotide analoga 13 to adjust the error rate to suitable levels.", "Error prone PCR is inexpensive and simple, but it has the following drastic disadvantages: 1.)", "Due to the construction of the genetic code—three nucleotides encode one amino acid, each amino acid is encoded by up to six different codons—a single nucleotide exchange can only lead to 9 new codons (three new nucleotides on three positions in the codons) which on average give rise to only 6 different amino acids.", "Many amino acid exchanges can only be achieved when two or even all three nucleotide in a codon are exchanged.", "For example, a codon for Isoleucine (ATT, ATA, ATC) can only be gained by two exchanges when starting from the codon CAA (Glutamine) or even three exchanges when starting from the codon CAG (also Glutamine).", "There are examples from applications of mutagenesis for enzyme improvement where the exchange of all three nucleotides of a codon was indeed necessary to achieve the required properties of the enzyme 14, 15 .", "A repertoire of variants of a protein that contains all single amino acid mutations (each amino acid is represented on each position of the protein) has to be very large when it is produced by single nucleotide exchanges (e.g.", "error prone PCR).", "An average protein of 300 amino acid length has exactly 300×19=5700 variants that differ to each other by exactly one amino acid.", "A repertoire of variants of the same protein, which was produced by on average three nucleotide exchanges contains (900×3)ˆ3=2×10 10 different variants.", "Repertoires of such sizes can only be handled by few selection methods 16 .", "Error prone PCR, therefore, can not be used to obtain complete high quality repertoires.", "2.)", "Error prone PCR does not exchange all nucleotides alike.", "Transversions and transitions occur with different rates 17 so that some mutations occur more frequently than others do.", "This effect leads to a further diminishing of the effective size of a repertoire produced by error prone PCR.", "3.)", "The high redundancy of the codons—several codons encode the same amino acid—leads to the phenomenon that on average 23% of all nucleotide exchanges result in synonymous mutations in which the mutated codon encodes the same amino acid as the original one.", "Such mutations might lead to desired changes in the expression rate of proteins 18 , important intrinsic properties of the a protein, like for example the enzymatic activity or stability, however, are unchanged.", "The effect of this phenomenon is that the effective size of a repertoire is diminished.", "4.)", "On average 4% of all nucleotide exchanges introduce new stop codons.", "By choosing a high mutation rate to achieve a maximum number of amino acid exchanges, many stop codons can be introduced which leads to shortened gene products.", "A repertoire produced with an average mutation rate of three exchanges per gene (in theory necessary to place at least all amino acids on one position) has already more than 10% prematurely terminated gene products [1−(1−0.04)ˆ3]=0.115.5.)", "The introduction of mutations is mostly a stochastic process in which the number of mutations per gene in each single variant in one repertoire follows a Poisson distribution.", "Depending on the average mutation rate a significant part of the repertoire can have no mutation at all while others contain a very high number of mutations.", "The result is a further diminishment of the effective size of the repertoire.", "6.)", "The introduction of non-natural amino acids in the biosynthesis of proteins can be achieved by using modified components of the complex biochemical apparatus for protein translation together with new codons that consist of four instead of three bases 19 .", "To generate protein variants that contain such non-natural amino acids at random positions complete codons, meaning three consecutive nucleotides have to be exchanged against four nucleotides.", "This is effectively impossible.", "Site Directed Random Mutagenesis An alternative for error prone PCR is to exchange several nucleotides at once at several defined positions in a gene by using degenerated oligonucleotides and PCR.", "When all three nucleotides of one codon are varied freely, any natural amino acid can be encoded at the position of this codon within the repertoire 20 .", "However, a repertoire obtained such a way is distributed rather unevenly.", "For example, Arginine and Serine are represented 6 times as often as Methionine or Tryptophane.", "Additionally, the introduction of stop codons can be problematic.", "The degree of degeneracy of the oligonucleotides can be adjusted by using combinations or mixtures of certain nucleotides at different positions and such that some of these problems are circumvented.", "So, for example, DNA sequence repertoires can be obtained that encode only a specific part of all amino acids or that encode all amino acids with the same frequency 21,22 .", "In theory a better alternative for nucleotide mixtures in the chemical synthesis of DNA strands is the usage of nucleotide triplets, entire codons so to say, instead of single nucleotides 23-27 .", "Unfortunately the different tri-nucleotides all show a different efficiency of the chemical coupling during DNA synthesis, leading again to unevenly distributed repertoires 27 .", "And even in the case that all these problems can be overcome, site directed random mutagenesis, as its name implies, is always limited to pre-selected, defined sites in a gene.", "All presented limitations of the currently applied methodologies for random mutagenesis clearly show, how important and advantageous it was, to have a method that could: 1.)", "exchange complete codons (independent on the number of nucleotides within a codon) instead of single nucleotides randomly along the entire strand of DNA, and 2.)", "introduce a defined number of mutations (i.e.", "codon mutations) per each strand of DNA.", "The invention presented here exactly describes such a method.", "Repertoires of gene variants prepared by the invented method have a better quality than repertoires that were prepared by conventional methods of error prone PCR.", "They contain a higher number of different variants while having the same number of repertoire members.", "When coupled to a selection or screening variants that encode proteins with new and desired properties can be isolated more efficiently from these repertoires.", "These proteins, for example, can be applied as enzymes in the biotechnological industry, in medical diagnostics or therapy.", "The above features and many other advantages of the invention will be become better understood by reference to the following detailed description when taking in conjunction with the accompanying drawings." ], [ "FIELD OF INVENTION The presented invention refers to a method for the creation of sequence variation in DNA.", "Especially, it can be used for the creation of libraries of genes of which the genes encode protein sequences and of which the genes in each library are distinguished to each other by the mutation (exchange) of complete codons.", "By using methods of selection or screening, gene variants can be isolated from these libraries that code for proteins with special properties, like for example, a change in activity or an increase in stability.", "BACKGROUND OF THE INVENTION Enzymes are increasingly applied in the biotechnological and chemical industry, as well as in the medical diagnostics and therapie1.They help to improve existing processes and make new applications possible.", "Therefore, there is an increasing demand of enzymes with new or improved properties.", "The improvement/optimization of enzymes by rational or computerized methods, however, has been mostly without success2.Directed evolution of proteins, on the contrary, has been quite successful in optimizing properties of enzymes3.Directed evolution consists of two steps.", "Firstly, a gene library is created by varying one or more DNA sequences encoding the according enzyme and secondly, using selection or screening methods, those variants of the genes are isolated that encode enzymes variants with the desired, optimized properties.", "The advantage of this approach is that it does not require any information on the structure of the enzyme in question, its dynamics or its interactions with different substrates.", "Therefore, methods of directed evolution are increasingly accepted and applied by many biotech companies worldwide1,4.Success and failure of directed evolution above all depend on the quality of the libraries and the efficiency of the selection or screening method.", "The quality of a library is determined by the available sequence variation and depends mostly on the method used to create the sequence variation.", "Nowadays mainly two approaches are used to create sequence variation: in-vitro DNA-recombination5-7 and random mutagenesis8-10.In-vitro DNA-recombination is based on a small repertoire of DNA sequences that are similar but not identical to each other.", "The differences (mutations) between these sequences can originate from natural evolution (family shuffling)11 or could have been introduced by a combination of random mutagenesis and selection5.They therefore represent a pre-selection and in general improve or at least are neutral to the properties of the enzyme encoded by the DNA sequence.", "In the in-vitro DNA-recombination a repertoire of DNA sequences is produced, the variants of which contain new combinations of the mutations that were present in the original repertoire.", "The advantage of this approach is that a high number of positions in a gene are varied at once and that mostly advantageous mutations are recombined.", "The disadvantage of this approach is that variation is confined to sequence positions that show variation in the original repertoire and additionally to the types of mutations present at these positions.", "Random mutagenesis, on the contrary, provides access to completely new mutations.", "It has the advantage that all positions of a gene can contain every possible variation.", "Random mutagenesis, therefore, is not limited to mutations that are already present somewhere.", "Nowadays there are two techniques that are almost exclusively used for introducing random mutations into genes: error prone PCR8 and site specific (cassette-) mutagenesis using degenerated oligonucleotides9.Error Prone PCR The by far most dominant method of the site-nonspecific random mutagenesis is to use error prone PCR.", "Here the gene to be mutated is amplified by PCR using a thermostable polymerase (usually Taq-polymerase) that introduces wrong nucleotides in a rate that depends on the conditions of the PCR einbaut12.A common variant of this method applies Manganese(II)-Ions8 or nucleotide analoga13 to adjust the error rate to suitable levels.", "Error prone PCR is inexpensive and simple, but it has the following drastic disadvantages: 1.)", "Due to the construction of the genetic code—three nucleotides encode one amino acid, each amino acid is encoded by up to six different codons—a single nucleotide exchange can only lead to 9 new codons (three new nucleotides on three positions in the codons) which on average give rise to only 6 different amino acids.", "Many amino acid exchanges can only be achieved when two or even all three nucleotide in a codon are exchanged.", "For example, a codon for Isoleucine (ATT, ATA, ATC) can only be gained by two exchanges when starting from the codon CAA (Glutamine) or even three exchanges when starting from the codon CAG (also Glutamine).", "There are examples from applications of mutagenesis for enzyme improvement where the exchange of all three nucleotides of a codon was indeed necessary to achieve the required properties of the enzyme14, 15.A repertoire of variants of a protein that contains all single amino acid mutations (each amino acid is represented on each position of the protein) has to be very large when it is produced by single nucleotide exchanges (e.g.", "error prone PCR).", "An average protein of 300 amino acid length has exactly 300×19=5700 variants that differ to each other by exactly one amino acid.", "A repertoire of variants of the same protein, which was produced by on average three nucleotide exchanges contains (900×3)ˆ3=2×1010 different variants.", "Repertoires of such sizes can only be handled by few selection methods16.Error prone PCR, therefore, can not be used to obtain complete high quality repertoires.", "2.)", "Error prone PCR does not exchange all nucleotides alike.", "Transversions and transitions occur with different rates17 so that some mutations occur more frequently than others do.", "This effect leads to a further diminishing of the effective size of a repertoire produced by error prone PCR.", "3.)", "The high redundancy of the codons—several codons encode the same amino acid—leads to the phenomenon that on average 23% of all nucleotide exchanges result in synonymous mutations in which the mutated codon encodes the same amino acid as the original one.", "Such mutations might lead to desired changes in the expression rate of proteins18, important intrinsic properties of the a protein, like for example the enzymatic activity or stability, however, are unchanged.", "The effect of this phenomenon is that the effective size of a repertoire is diminished.", "4.)", "On average 4% of all nucleotide exchanges introduce new stop codons.", "By choosing a high mutation rate to achieve a maximum number of amino acid exchanges, many stop codons can be introduced which leads to shortened gene products.", "A repertoire produced with an average mutation rate of three exchanges per gene (in theory necessary to place at least all amino acids on one position) has already more than 10% prematurely terminated gene products [1−(1−0.04)ˆ3]=0.115.5.)", "The introduction of mutations is mostly a stochastic process in which the number of mutations per gene in each single variant in one repertoire follows a Poisson distribution.", "Depending on the average mutation rate a significant part of the repertoire can have no mutation at all while others contain a very high number of mutations.", "The result is a further diminishment of the effective size of the repertoire.", "6.)", "The introduction of non-natural amino acids in the biosynthesis of proteins can be achieved by using modified components of the complex biochemical apparatus for protein translation together with new codons that consist of four instead of three bases19.To generate protein variants that contain such non-natural amino acids at random positions complete codons, meaning three consecutive nucleotides have to be exchanged against four nucleotides.", "This is effectively impossible.", "Site Directed Random Mutagenesis An alternative for error prone PCR is to exchange several nucleotides at once at several defined positions in a gene by using degenerated oligonucleotides and PCR.", "When all three nucleotides of one codon are varied freely, any natural amino acid can be encoded at the position of this codon within the repertoire20.However, a repertoire obtained such a way is distributed rather unevenly.", "For example, Arginine and Serine are represented 6 times as often as Methionine or Tryptophane.", "Additionally, the introduction of stop codons can be problematic.", "The degree of degeneracy of the oligonucleotides can be adjusted by using combinations or mixtures of certain nucleotides at different positions and such that some of these problems are circumvented.", "So, for example, DNA sequence repertoires can be obtained that encode only a specific part of all amino acids or that encode all amino acids with the same frequency21,22.In theory a better alternative for nucleotide mixtures in the chemical synthesis of DNA strands is the usage of nucleotide triplets, entire codons so to say, instead of single nucleotides23-27.Unfortunately the different tri-nucleotides all show a different efficiency of the chemical coupling during DNA synthesis, leading again to unevenly distributed repertoires27.And even in the case that all these problems can be overcome, site directed random mutagenesis, as its name implies, is always limited to pre-selected, defined sites in a gene.", "All presented limitations of the currently applied methodologies for random mutagenesis clearly show, how important and advantageous it was, to have a method that could: 1.)", "exchange complete codons (independent on the number of nucleotides within a codon) instead of single nucleotides randomly along the entire strand of DNA, and 2.)", "introduce a defined number of mutations (i.e.", "codon mutations) per each strand of DNA.", "The invention presented here exactly describes such a method.", "Repertoires of gene variants prepared by the invented method have a better quality than repertoires that were prepared by conventional methods of error prone PCR.", "They contain a higher number of different variants while having the same number of repertoire members.", "When coupled to a selection or screening variants that encode proteins with new and desired properties can be isolated more efficiently from these repertoires.", "These proteins, for example, can be applied as enzymes in the biotechnological industry, in medical diagnostics or therapy.", "The above features and many other advantages of the invention will be become better understood by reference to the following detailed description when taking in conjunction with the accompanying drawings.", "DESCRIPTION OF FIGURES FIG.", "1: (A) Schematic description of the insertion of a Donor-strand of DNA (dark box) into random positions of a gene (open box), which is in a circular form (e.g.", "Plasmid).", "The Donor-strand can carry the gene of a reporter protein.", "(B) The Donor-strand is removed in such a way that the original gene is recovered with the exception that one codon (shown as examples are CAG and CTG) is replaced by a different one (shown as examples are ATT and TAC.", "The numbers exemplary represent the numbering of amino acids.", "FIG.", "2: Detailed schematic description of the separate essential steps for the introduction of codon mutations into a gene.", "For detailed explanation the description of the invention below is referred.", "The dashed line symbolizes the circularity of the gene; it could show, for example, a plasmid.", "The numbers correspond to an exemplary numbering of the amino acids.", "The arrows above the genes mark restriction sites (not recognition sites) of restriction enzymes R1, R2, R3 and R4.FIG.", "3: Detailed course of the mutagenesis shown exemplary by the exchange of codon 86 from GFP (AGT, nt 256-258) against TGG (amino acid mutation S86W).", "(i) .", ".", ".", "Introduction of a double strand break, (ii) .", ".", ".", "Insertion of the Donor-strand, (iii) .", ".", ".", "Restriction digestion with BseRI, (iv) .", ".", ".", "Creating blunt DNA ends, (v) .", ".", ".", "Religation.", "On top of the DNA sequences nucleotide numbering is shown, below the DNA sequences the codon (amino acid) numbering is shown.", "The recognition sites for BseRI are in bold letters; the actual cutting sites are marked with dashed lines.", "FIG.", "4: Agarose gel electrophoreses of PCR products to analyze the position of the placement of the Donor-strand with the GFP gene.", "The ladder on the side shows the size of the fragments in bp.", "DEFINITIONS Before the detailed description of the invention several terms are defined: DNA (deoxyribonucleic acid), polynucleotide, nucleotide sequence or oligonucleotide means any chain or sequence of the chemical building blocks Adenine (A), Cytosine (C), Guanine (G) and Thymine (T) (nucleotide bases).", "Nucleotides and oligonucleotide are degenerate, when they can have more than one nucleotide base at on or more positions.", "DNA can consist of one strand of nucleotide bases (single strand) or two complementary strands (double strand), which form a double helical structure.", "The term single strand break refers to breakage of one strand in double strand DNA after which the double strand is maintained.", "The term double strand break refers to breakage of both strands of double strand DNA after which two new DNA ends arise.", "Blunt DNA ends means that the ends of both strands in double strand DNA are equally long, overhanging or sticky DNA ends means that one strand is longer than the other.", "A blunt-end-ligation means that two double strands with blunt DNA ends are covalently attached to each other.", "The terms library, repertoire or ensemble, as they are used here are identical and mean a collection of polynucleotides or polypeptides.", "A gene- or DNA-library or a repertoire of gene or DNA-sequences is a collection of polynucleotides or DNA-sequences, that are derivatives of on ore more polynucleotides or DNA-sequences and that are in most parts identical to each other.", "Each single polynucleotide or each single DNA-sequence of a library is referred to as a member of the library.", "Several members of a library that are identical to each other are referred to as one gene variant or as one variant of the library.", "The effective size of a library or of a repertoire is a measure of the number of different variants within this library.", "A large library has many members, but when it had only a small effective size it had only few different variants.", "Enzymes or terms of molecular biology as restriction enzyme, restriction enzyme of type IIs, DNase I, Nuclease, Exonuclease, DNA-Ligase, DNA-Polymerase, Transposase, Transposon, vector and plasmid are defined in their function as they are described in state of the art literature about molecular biology28, 29.DETAILED DESCRIPTION OF THE INVENTION The invention consists of (also refer to FIG.", "1): A.)", "The insertion of a piece of DNA into a gene at different, randomly positioned sites.", "B.)", "The directed removal of this piece of DNA and of a defined number of adjacent nucleotides of said gene, while instead at this position a defined number of nucleotides from said inserted piece of DNA is remaining.", "In detail the invention is marked by the following steps (also refer to FIG.", "2): 1.)", "Into molecules of a gene or of a DNA sequence, preferably as part of a vector or any other circular form, exactly one double strand breakage per molecule is performed by mans of molecular biological methods (FIG.", "2,i).", "This can, for example, be achieved by treating the DNA with a) an enzyme that site-nonspecifically introduces single strand breaks (e.g.", "DNase I) and a single strand specific nuclease (e.g.", "S1 nuclease), b) an enzyme that site-specifically introduces single strand breaks, a 5′-3′ exonuclease, DNA-polymerase and single strand specific nuclease (e.g.", "S1 nuclease), c) an enzyme that site-nonspecifically introduces double strand breaks (e.g.", "modified variants of restriction enzymes that have lost their sequence specificity), or d) a transposon and a transposase that does not have a sequence specificity.", "It is preferred that an ensemble of gene variants is produced in which the double strand breaks are located at different positions and (apart from case d) in which the double strand breaks lead to blunt DNA ends.", "In case d) the double strand breakage is achieved under simultaneous incorporation of a DNA strand (transposon) and possibly the doubling of several nucleotides from the gene.", "2.)", "Into the gene variants of this ensemble a DNA strand (Donor-strand) is incorporated by blunt-end-ligation (FIG.", "2,ii).", "In the case of the utilization of a transposase (1d) the Donor-strand, as a transposon, has been incorporated already during the previous step.", "It is preferred that the Donor-strand encodes a genetically selectable marker, e.g.", "a resistance against an antibiotic.", "It is further preferred that the expression of this marker is dependent on that the inserted Donor-strand is incorporated into the correct reading frame of the gene.", "Preferably, the Donor-strand contains recognition sites for restriction enzymes of type IIs.", "Such obtained DNA constructs can be completely or partially amplified by PCR30, 31 and/or can be amplified in and isolated from microorganisms after having transformed them.", "It is preferred that the growth of the microorganisms is performed in culture media containing antibiotics against which the microorganisms are resistant due to the gene product that is encoded on the Donor-strand.", "3.)", "By restriction digestion with said restriction enzymes of type IIs the Donor-strands are mostly removed from the amplified gene variants.", "The DNA ends are made blunt by treatment with a DNA Polymerase or with a single strand specific nuclease (FIG.", "2,iii).", "It is preferred that the positions of the recognition sites of said restriction enzymes of type IIs are chosen such that in addition to the removal of most of the Donor-strand a defined number of n nucleotides is removed from the original gene and that at their position a defined number of m nucleotides from the original Donor-strand is remaining.", "It is preferred that these remaining m nucleotides are degenerate, meaning that in the gene variants of the ensemble different nucleotide compositions are remaining.", "It is preferred that exactly three nucleotides are replaced (n=3, m=3).", "In case that the variability of the nucleotide composition in the close proximity (10 to 40 base pairs) of the ends of the Donor-strand is restricted, e.g.", "by conserved sequences of a transposon, it is preferred that the Donor-strand contains several recognition sites for restriction enzymes of type IIs.", "These are preferably positioned in a way that the Donor-strand is removed from the gene bit by bit in several cycles of a) restriction digestion with one or two of said enzymes, b) when necessary, treatment to create blunt DNA ends and c) followed by the fusion of the DNA ends by intramolecular ligation, until the entire Donor-strand apart from m nucleotides is removed together with a defined number n nucleotides from the original gene (FIG.", "2,iv and v).", "These remaining m nucleotides preferably are degenerate, it is preferred that exactly 3 nucleotides are replaced (n=3, m=3).", "4.)", "By intramolecular blunt-end-ligation the DNA-ends of the variants of the ensembles are closed and complete, continuous genes are obtained (FIG.", "2,vi).", "The genes can be subjected to a further round of introduction of mutations.", "Alternatively the genes can be expressed in vivo after transformation of an expression host or in vitro by using an in vitro—translation system to yield the protein variants that are encoded by the genes.", "The here disclosed method is so far the only method for mutagenesis that allows the random exchange of several adjacent nucleotides in a gene.", "So far several methods of molecular biology have been published that are based on random double strand breaks of DNA strands or on the insertion of DNA sequences, including transposons at random positions into DNA strands.", "These methods, however, are limited to the experiments to find new termini for proteins32, to randomly delete parts of protein sequences or insert additional sequences into proteins33.They have not been applied and, by themselves, they are not even applicable to exchange nucleotides at random positions in DNA sequences in such a way that single amino acids in the accordingly encoded proteins are exchanged to produce a repertoire of genes whose products are distinguished to each other in the type of a defined number of amino acids.", "What is Predicted: It is predicted that the fraction of any theoretically possible mixture of nucleotides within the degenerate part of the Donor DNA can be accurately adjusted, e.g.", "in such a way that in an area of 3 degenerate nucleotides each amino acid is represented by exactly one codon.", "It is predicted that the disclosed method allows incorporating mutations not only into the entire lengths of a gene but also into limited parts of genes.", "For example, after incorporation of the Donor-strands these parts can be amplified by PCR using flanking primers, the amplified products can be fused into the complete gene by the use of GenSOEing34 and the such modified gene can then be further subjected to the described protocol.", "It is further predicted that during the stepwise removal of the Donor-strands, required restriction sites of type IIs are only created in the process, e.g.", "by restriction and religation.", "It is predicted that Donor-strands that are incorporated into genes as transposons by the action of a transposase can be modified with mutations within the transposase recognition sequence that necessary for the transposition, such that new recognition sites for restriction enzymes of type IIs are created within the transposase recognition sequence.", "It is predicted that there are other techniques that can be applied to introduce double strand breaks into DNA than the ones exemplary indicated in the description of the invention.", "It is predicted that by applying immobilization techniques genes with incorporated Donor-strands can be physically separated from genes that do not contain Donor-strands or that by applying immobilization techniques Donor-strands that are incorporated into genes can be physically separated from Donor-strands not incorporated into genes.", "EXAMPLE The possibilities and the approach of the invention will become even clearer in the following example.", "The example of practicing the invention is understood to be exemplary only, and do not limit the scope of the invention or the appended claims.", "A person of ordinary skill in the art will appreciate that the invention can be practiced in many forms according to the claims and disclosure here.", "Example 1 Introduction of Codon Mutations into the Gene of the Green Fluorescent Protein (GFP) (Also Refer to FIG.", "3 and 4) 1.)", "Introduction of Randomly Positioned Double Strand Breaks in the Plasmid pGFP The plasmid pGFP (Clontech, Palo Alto, USA) contains the GFP-gene under the control of the lac-promoter.", "For the amplification in E. coli the plasmid contains the gene for the resistance against ampicillin.", "E. coli XL1-Blue cells were transformed with pGFP and from 200 ml of a culture of the transformed cells 300 μg pGFP DNA were prepared (Maxikit, Quiagen, Hilden, Germany).", "In 200 μl 33 mM Tris/HCl, pH 7,5, 10 mM MgCl2 and 50 μg/ml BSA 40 μg pGFP were incubated with 0.01 mu DNase I (Roche Diagnostik, Penzberg, Germany) for 5 min at 28° C. The reaction was stopped by addition of 20 mM EDTA (final conc.)", "and cooling on ice.", "The analysis of the reaction by agarose gel electrophoresis revealed that approx.", "40% of pGFP had been converted into the open-circular form.", "This open circular DNA was isolated using preparative agarose gel electrophoresis.", "In 100 μl 7.4× S1 Buffer (MBI Fermentas, St. Leon Roth, Germany) 5 μg of the open-circular form were incubated with 100 u S1 Nuclease (1 μl, MBI Fermentas, St. Leon Roth, Germany) for 2 h at 16° C. after which the reaction was stopped with 10 μl S1-Stop solution.", "The analysis of the DNA by agarose gel electrophoresis revealed that approx.", "50% of the open circular DNA was linearised.", "This linearised DNA was isolated by preparative agarose gel electrophoresis.", "2.)", "Preparation of the DNA Strand to be Inserted (Donor-Strand) The gene of chloramphenicol acetyltransferase (CAT) was amplified by PCR with the primers NNS GGG CCT GGG TCT CCT CCT GGC GAG AAA AAA ATC ACT GGA TAT ACC (SEQ.", "ID NO: 1) and GGC GTA GCT CCT CGC GTT TAA GGG (SEQ.", "ID NO: 2) and the Plasmid pACYC184 (NEB, Beverly, Mass., USA) as template.", "The PCR was performed following standard protocols (NEB, Beverly, Mass., USA), 30 cycles were performed applying an annealing temperature of 55° C. and an extension time of 45 sec.", "Vent-Polymerase was used (NEB, Beverly, Mass., USA).", "The PCR product was precipitated with EtOH and resuspended in a small volume TE to give a concentration of 150 ng/μl.", "3.)", "Insertion of Donor-Strand into the Plasmid and Transformation In 50 μl ligase buffer (Gibco BRL, Eggenstein, Germany) (final volume) 10 μl linearised plasmid (approx.", "300 ng, refer to 1.)", "and 14 μl PCR product (approx.", "2 μg, refer to 2.)", "were incubated with 5 u T4-DNA ligase (Gibco BRL, Eggenstein) for 20 h at 16° C. Subsequently the ligation mix was desalted by microdialysis and used to transform XL1-Blue cells by electroporation.", "Transformed cells were plated on dYT-Agar including 100 μg/ml Ampicillin, 8 μg/ml Chloramphenicol and 1 mM IPTG.", "Growth of transformed bacteria was basically limited to cells transformed with plasmids that contained the PCR fragment under the control of the lac-promoter in the correct reading frame after a start codon.", "Approx.", "10000 transformants were obtained.", "4.)", "Analysis of Library 95 colonies were analyzed by colony-PCR using the primers CCA TGA TTA CGC CAA GCT TGC (SEQ.", "ID NO: 3) (binds to the 5′-end of the GFP-gene and GTG CTT ATT TTT CTT TAC GGT C (SEQ.", "ID NO: 4) (binds within CAT-gene) for whether an insertion of the PCR-product into the plasmid had occurred within or outside the GFP gene and whether the insertion into the GFP gene had occurred in the correct direction of translation and at positions randomly distributed.", "From approx.", "80% of the transformants a fragment between ca.", "270 to ca.", "1000 bp in length could be amplified (see FIG.", "4a as an example.", "For all those variants the insertion of the PCR-product had occurred within the gene sequence of GFP in a way that the direction of translation of the gene for chloramphenicol acetyltransferase lies in the same as for the gene for GFP.", "5.)", "Donor-Removal All colonies of the transformed bacteria were collected from the agar plates, pooled and plasmid DNA was prepared (Mini kit, Quiagen, Hilden, Germany).", "2 μg of the plasmid DNA, which represents a repertoire of pGFP with randomly inserted PCR products, was completely digested with BseRI (NEB, Beverly, Mass., USA).", "This restriction enzyme of Type IIs cuts outside its recognition site CTCCTC (FIG.", "3,iii).", "The products of the restriction digestion were treated with Klenow fragment, subsequently separated by agarose gel electrophoresis and the DNA band that had a length of approx.", "3.4 kb was isolated from the agarose (QiaexII, Qiagen, Hilden, Germany).", "Ca.", "40 ng of the DNA were incubated in 50 μl ligation buffer with 1 u T4-DNA-Ligase for 20 h at 16° C. Subsequently the ligation mix was desalted by microdialysis and 5 μl were used to transform XL1-Blue cells by electroporation.", "Transformed cells were plated on dYT agar including 100 μg/ml Ampicillin.", "6.)", "Second Analysis of Library The transformants should contain the desired library of codon-mutated variants of GFP.", "For the analysis of this library 5 transformants were randomly selected and the entire sequence of GFP was determined to establish type and location of the mutation.", "The following mutations were found: S86W (AGT→TGG), G51H (GGA→CAC), N164R (AAC→CGC) and V219N (GTC-→AAG).", "One mutation was not in the correct reading frame and lead to the double mutation Q204L, S205A (CAATCT→CTCGCT).", "6.)", "Phenotypic Analysis The library of variants of GFP can be examined for variants that show a desired phenotypic change compared to wildtype GFP (e.g.", "increased expression of GFP, shift of excitation or emission wavelength, etc.).", "Desired variants can then be isolated and applied according to their properties.", "LIST OF REFERENCES 1.Rubingh, D. 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Arnold, Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair.", "Nucleic Acids Res., 1999.27(18): p. e18.7.Zhao, H., et al., Molecular evolution by staggered extension process (StEP) in vitro recombination.", "Nat.", "Biotechnol., 1998.16(3): p. 258-61.8.Fromant, M., S. Blanquet, and P. Plateau, Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction.", "Anal.", "Biochem., 1995.224(1): p. 347-53.9.Lahr, S. J., et al., Patterned library analysis: A method for the quantitative assessment of hypotheses concerning the determinants of protein structure.", "Proc.", "Nat.", "Acad.", "Sci.", "USA, 1999.96(26): p. 14860-14865.10.Greener, A., M. Callahan, and B. 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[ [ "Control of priority and instruction rates on a multithreaded processor", "A method and apparatus for controlling issue rate of instructions for an instruction thread to be executed by a processor is provided.", "The rate at which instructions are to be executed for an instruction thread are stored (46) and requests are issued (44) to cause instructions to execute in response to the stored rate.", "The rate at which instruction requests are issued is reduced in response to instruction executions and is increased in the absence of instruction executions.", "In a multi-threaded processor, instruction rate is controlled by storing the average rate at which each thread should execute instructions (48).", "A value representative of the number of instructions available and not yet issued is monitored and is decreased in response to instruction executions (42).", "Execution of instructions is prevented on a thread if the number of instructions available but not yet issued falls below a defined value.", "A ranking order is assigned to a plurality of instructions threads for execution on a multi-threaded processor.", "A plurality of metrics related to the threads and required for establishment of the rank order are provided.", "Each metric is assigned to a set of bits and these are assembled in a composite metric being assigned to the most significant bits and the least important metric being assigned to the least significant bits.", "A ranking order is then assigned to the composite metrics in dependence on their values." ], [ "1.A method for issue rate control of instructions for an instruction thread to be executed by a processor comprising the steps of: storing the rate at which instructions are to be executed for an instruction thread; issuing requests to cause instructions to execute in response to the stored rate; reducing the rate at which instruction requests are issued in response to instruction executions; increasing the rate at which instructions are to be executed in the absence of instruction executions.", "2.A method according to claim 1 including the step of accumulating a difference value between an intended rate of execution and the actual rate of execution of instructions; and and wherein the step of issuing requests to cause instructions to execute is dependent on the deficit between the actual rate and intended rate of execution.", "3.A method according to claim 2 including the step of stopping the executio of instructions in response to an accumulated excuses.", "4.A method according to claim 1 in which the processor is a multithreaded processor which handles a plurality of instructions threads.", "5.A method according to claim 4 including the step of assigning a priority to each thread and executing instructions on each thread in dependence on the priority of each thread.", "6.A method according to claim 5 in which the step of assigning a priority to the threads comprises: providing a plurality of metrics required for the establishment of rank order for each thread; assigning each metric to a set of bits; and assembling the sets of bits in a composite metric for each thread with the most important metric being assigned to the most significant bits and the least important metric being assigned to the least significant bits of the composite metric.", "7.A method according to claim 5 including the step of monitoring a real time deadline for execution of the thread and adjusting the priority of the thread in dependence on the time left to expiry of the deadline.", "8.Apparatus for issue rate control of instructions for an instruction thread to be executed by a processor comprising: means for storing the rate at which instructions are to be executed for an instruction thread; means for issuing requests to cause instructions to execute in response to the stored rate; means for reducing the rate at which requests are issued in response to executions; and means for increasing the rate at which instructions are to be executed in the absence of instruction executions.", "9.Apparatus according to claim 8 including: means for accumulating a difference in value between an intended rate of execution and the actual rate of execution; wherein the means for issuing requests to cause instructions to execute is dependent on to the deficit between the actual and intended rate of execution.", "10.Apparatus according to claim 8 including means to stop execution in response to an accumulated excess between the actual and intended rate of execution.", "11.Apparatus according to claim 8 in which the processor is a multithreaded processor which handles a plurality of instruction threads.", "12.Apparatus according to claim 11 including means to assign a priority to each thread and wherein the means for issuing requests does so in dependence on the priority of each thread.", "13.Apparatus according to claim 12 in which the means for assigning a priority to a thread comprises; means for providing a plurality of metrics required for establishment of rank order for each thread; means for assigning each metric to a set of bits; and means for assembling the sets of bits in a composite metric for each thread with the most important metric being assigned to the most significant bits and the least significant metric being assigned to the least significant bits of the composite metric 14.Apparatus according to claim 12 including means to monitor a real time deadline for execution of the thread and means for adjusting the priority of the thread in dependence on the time left to expiry of the deadline.", "15.A method for assigning a ranking order to a plurality of instruction threads to be executed on a multi-threaded processor, comprising the steps of: providing a plurality of metrics required for the establishment of rank order for each thread; assigning each metric to a set of bits; assembling the sets of bits in a composite metric with the most important metric being assigned to the most significant bits of the composite metric and the least important metric being assigned to the least significant bits of the composite metric; and assigning a ranking order to the composite metrics in dependence on their values.", "16.A method according to claim 15 in which the step of assigning a ranking order to the composite metrics comprises the steps of: comparing a pair of composite metrics and swapping their order if the lower positioned composite metric has a greater or equal value than the higher positioned composite metric.", "17.A method according to claim 16 in which the step of comparing a pair of composite metric proceeds from the lowest ranked pair to the highest ranked pair of composite metrics.", "18.A method according to claim 17 in which the process returns to the lowest ranked pair after comparison of the highest ranked pair.", "19.Apparatus for assigning a ranking order to a plurality of instruction threads to be executed on a multi-threaded processor comprising: means for providing a plurality of metrics required for establishment of rank order for a thread; means for assigning each metric to a set of bits; means for assembling the sets of bits in a composite metric with the most important metric being assigned to the most significant bits and the least important metric being assigned to the least significant bits; and assigning a ranking order to the composite metrics in dependence on their values.", "20.Apparatus according to claim 19 in which the means for assigning a ranking order to the composite metric comprises: means to compare a pair of composite metrics; and means to swap the ranking order of the pair of composite metrics if the lower positioned composite metric has a greater of equal value than the higher positioned composite metric.", "21.Apparatus according to claim 20 in which the means to compare composite metrics proceeds from the lowest ranked pair to the highest ranked pair.", "22.Apparatus according to claim 21 in which the comparison means returns to the lowest ranked pair after comparison of the highest ranked pair.", "23.A method for controlling the instruction issue rate of threads executing on a multithreaded processor comprising the steps of: storing the average rate at which each thread should execute instructions; monitoring a value representative of the number of instructions not yet issued for a thread, increasing the value in dependence on the stored average rate for each thread; and decreasing the value in response to instruction executions.", "24.A method according to claim 23 including the step of accumulating the value representative of the number of instructions not yet issued for a thread; 25.A method according to claim 24 including the step of setting a maximum value on the number of instructions that can be accumulated for a thread.", "26.A method according to claim 24 in which a thread which builds up an accumulation of instructions not yet issued may be allocated a burst of execution time in which the instructions execute at the higher rate, the burst being bounded by the accumulated value.", "27.Apparatus for controlling the instruction issue rate of threads executing on a multithreaded processor comprising: means for storing the average rate at which each thread should execute instructions; means for monitoring a value representative of the number of instructions available but not yet issued for a thread; means for increasing the value in dependence on the stored average rate for each thread; means for decreasing the value in response to instruction executions on a thread; and means for preventing execution of instructions on a thread if the number of instructions available but not yet issued falls below a defined value.", "28.Apparatus according to claim 27 including means for accumulating the average instruction issue rate into the value representative of the number of instructions available but no yet issued for a thread.", "29.Apparatus according to claim 28 including means for setting a maximum value on the number of instructions available but not yet issued for a thread.", "30.Apparatus according to claim 28 in which a thread which builds up an accumulation of instructions not yet issued may be allocated a burst of execution time in which instructions execute at a rate higher than the average rate, the burst being bounded by the accumulated value.", "31-36.", "(Cancelled)" ], [ "This invention relates to the control of priorities and instruction issue rates on a multithreaded processor which is configured to process at any one time one of a number of different instruction threads.", "This invention is particularly beneficial when used with a system such as that described in our international patent application no.", "WO97/38372 the contents of which are incorporated herein by reference.", "This document discloses a processing system which is able to manage the processing of a number of different instruction threads by one or more data processors.", "This architecture looks repeatedly to the resources available and the instructions which have to be executed to determine which instruction thread should be processed on a following clock cycle.", "Such an architecture has many advantages in real time systems where the processor interacts with external devices such as hardware peripherals or other processors.", "In a real time system it is crucially important to ensure that all processing associated with an event is completed within a defined time.", "This is easy to verify for a processor which only performs one task but becomes very complex when the processor has many tasks to perform.", "In a system in which a processor has multiple threads it is quite possible to disturb the operation of a program running on one thread by changing the behaviour of a program running on different thread.", "This lack of thread conformity makes it difficult to develop programs which can execute reliably without prior knowledge of what is running on other threads.", "A conventional processor uses a priority system which permits urgent tasks to be handled more quickly than normal or non-urgent tasks.", "However, a processor which also has multiple hardware threads such as that described in WO97/38372 gives more flexibility than conventional processors and therefore requires a more flexible priority control.", "Preferred embodiments of the present invention seek to provide an issue rate control scheme for a multi threaded processor system.", "In particular, preferred embodiments seek to permit a program executing on one thread to control its use of processor resources in such a way that the processing requirements of both that program and any programs executing on other threads are met.", "In order to do this the program must be able to: define the rate at which its instructions are issued regardless of the behaviour of programs executed on other threads; handle an urgent event quickly whilst controlling any disruption to programs executed on other threads; and, adapt to disruptions in its defined rate of instruction issue caused by the handling of urgent events on other threads.", "The preferred embodiment is sufficiently robust to enable a thread to recover from processor overload in a reliable way, and to ensure that deviation from the defined bounds of execution rate on a thread can be detected.", "Furthermore it seeks to provide a control scheme that can operate at a number of levels of complexity whilst allowing a programmer to ignore aspects of the scheme that he does not require.", "A further embodiment seeks to minimise processor power consumption by clocking the processor at the minimum rate required to complete all its tasks.", "Preferred embodiments seek to assign ranked priorities to instruction threads to ensure the most effective use of processor resources.", "The invention is defined in the appended claims to which reference should now be made.", "A preferred embodiment of the invention will now be described in detail by way of example with reference to the accompanying drawings in which: FIG.", "1 is a block diagram of the base architecture of a multithreaded processor system; FIG.", "2 is a block diagram of the thread scheduling portion of the Media Control Core of FIG.", "1; FIG.", "3 is a block diagram of the thread ranking circuitry of FIG.", "2; FIG.", "4 is a block diagram of the issue request control circuitry of FIG.", "2; FIG.", "5 shows the arrangement of the deadline counter.", "The base architecture of the embodiment of the invention described here is shown in FIG.", "1.The central part of this is a media control core (MCC) 2 which comprises a fine grained multithreaded processor.", "It has a plurality of inputs and outputs which can be coupled to real time data input and output devices 4 which may be, for example, video sources, audio sources, video outputs, audio outputs, data sources, storage devices, etc.", "In the most simple example only a single input and a single output will be provided.", "Also coupled to the MCC 2 are a plurality of data processing units 6.Each of these comprises a data processing core 8 which controls the processing of data via a data pipeline 10.The core 8 decodes and sequences micro instructions for the pipeline 10.Also coupled to the media control core 2 is a multibank cache memory 12 from which data may be retrieved by the MCC 2 and data processing unit 6 and into which data may be written by the MCC 2 and the data processing units 6.It includes temporary storage for data and instructions to be performed by the data processing cores on the input data and other internally generated data.", "These various sets of instructions will, when activated, comprise processing threads.", "The MCC 2 is a fine grained multithreading processing unit which directs data from inputs 4 to data processing cores 6 or to storage in cache 12 and provides data to outputs.", "It is arranged so that it may switch tasks on every clock cycle should this be required.", "To achieve this it checks on every clock cycle which possible operations it could perform by looking at the tasks to be performed and the resources available for those tasks to be performed.", "It also checks which of these tasks have the highest priority.", "More than one operation can commence on each clock cycle if sufficient processing power is provided.", "This resource checking ensures that everything required for a particular task to be performed is in place before an instruction is issued, including external resources such as data to an input port, or availability of data storage devices or outputs.", "It also includes the checking of internal resources such as registers for temporary storage, processing cores, or previously processed data required for a particular new processing operation.", "The MCC 2 operates to direct data from an input to an appropriate data processing unit 6 and for processing to take place routes appropriate instructions to the unit 6, and routes processed data to an output when required, making use of the cache as necessary.", "Once execution of a set of instructions is commenced on a processing unit the MCC 2 can look again at the various threads it can run and resources available for these whilst the program continues to run on the data processing unit.", "This resource and priority checking means that tasks which perform on real time data such as video input are able to be performed without the large memory buffers usually required for real time inputs.", "In e.g.", "video input, the MCC will look to see whether data is available at the IO port and if it is will receive that data and send it to either a portion of the multibanked cache or to data storage registers in preparation for processing by one of the data processing unit 6.Scheduling of the data processing unit 6 is under the control of MCC 2.For example, the data pipeline 10 of FIG.", "1 will be made up of a number of processing elements such as multipliers, adders, shifters, etc, under the control of an associated data processing core 8 which runs a sequence of instructions retrieved from the cache to perform a data processing algorithm.", "Each of these processing cores has its own micro instruction ROM and/RAM storing sequences of instruction to perform a particular data process.", "The MCC 2 invokes the data processing unit 6 to perform its particular operation sequence by e.g.", "passing an address offset into its micro instruction ROM and instructing it to commence execution.", "It will then perform a particular process on either data from the multibank cache or on data passed from one of the inputs to the MCC 2 until it has completed, at which point it will signal to the MCC 2 that its processing is complete.", "In this embodiment of the present invention, the thread scheduling performed by the MCC 2 has two main elements.", "These are thread instruction issue rate control and thread priority.", "Instruction issue rate control allows the number of million instructions per second (MIPS) requested by each thread to be defined as a burst rate Bn.", "This allows the processor loading to be balanced so that each thread can operate independently of activity on other threads.", "So long as the total loading never exceeds the processor capacity, issue rate control is the only mechanism required to ensure that all the threads receive the processor resources they require.", "If the total loading requested by all the threads exceeds the processor capacity, the thread priority mechanism allows processor resources to be applied to the highest priority threads first.", "The issue rate control system monitors the processing deficit for the lower priority threads which do not receive the processor resources they have requested, and ensures that the balance is restored once the processor overload has ended.", "The issue rate control mechanism and priority mechanism are described below.", "Each thread being monitored by the MCC 2 can be in one of two states.", "These are WAITING and RUNNING.", "In the WAITING state the thread is blocked on an external event such as a trigger and the instruction issue rate is zero.", "In the RUNNING state the thread is executing normally and the instruction issue rate is controlled by a value written to a burst rate register 46 (Bn) which is shown in the Schematic diagram of FIG.", "4.Generally speaking, an executing thread will have periods of WAITING for events which synchronise the operation of the program and periods of RUNNING when tasks are performed.", "In some cases a thread may execute continuously without WAITING on any synchronising event.", "Both patterns of execution are handled by an issue rate control scheme which will be described below.", "The total of all the burst rates requested by all the processor threads may exceed the processor capacity.", "Each thread being processed has an assigned priority which is used to determine the choice of thread for instruction issue.", "On each clock cycle the MCC identifies the threads which can accept a new instruction, and chooses the thread with the highest priority.", "This ensures that when the processor is overloaded for a period, it is the least important threads that are slowed down.", "The priority control mechanism interacts with thread scheduling logic in the MCC as does the issue rate control mechanism, which can optionally also interact with the clock generator.", "The signals which implement the interface between the priority and instruction issue rate control systems and the processor instruction scheduler are described in table 1 below for a processor with four instruction threads.", "This number is selected purely by way of example and any other number of threads can be used.", "I/O Signal Name Description Priority Signals TO RANK0id RANK0idD indicates the identity scheduler of the thread with the lowest ranking.", "TO RANK1id RANK1id indicates the identity of scheduler the thread with ranking 2 of 4.TO RANK2id RANK2id indicates the identity of scheduler the thread with ranking 3 of 4.TO RANK3id RANK3id indicates the identity of scheduler the thread with the highest ranking.", "NB for an N thread processor, there are N signals RANKxid.", "Issue Rate Control Signals TO FREEZEn (n = 0:3) Indicates that thread n wishes to scheduler halt instruction issue.", "FREEZEn acts as an interlock signal to the instruction scheduler.", "TO REQUESTn (n = 0:3) Indicates that thread n requires the scheduler issue of an instruction FROM GRANTn (n = 0:3) Indicates that an instruction has scheduler been issued to thread n. FROM BLOCKEDn(n = 0:3) Indicates that thread n is.", "blocked scheduler (waiting) on an external event such as a trigger.", "The behaviour of the instruction scheduler on each processor cycle is generally defined by the following rules: 1.Disregard all threads which are blocked (cannot run) and all threads which have asserted their FREEZE signals.", "2.Schedule the highest priority thread which is not blocked and does not have its FREEZE signal asserted and has asserted its REQUEST signal.", "3.If no thread has been scheduled at stage 2, schedule the highest numbered non-REQUEST-ing thread which is not blocked and does not have FREEZE asserted.", "4.If a thread is scheduled, assert the corresponding GRANT signal.", "This mechanism influences the thread instruction issue rate but does not directly control it on a cycle by cycle basis.", "An optional clock gating circuit in the clock generator ensures that processor clock pulses are deleted whenever all the signals REQUESTn are de-asserted.", "Thus the gated processor clock rate may be controlled to run at exactly the rate required to achieve the total instruction rates demanded by all the threads, and thus processing power consumption may be kept to a minimum.", "The MCC 2 of FIG.", "1 is scalable in all important respects.", "Because it is constructed from banks which localise storage (register files) and processing (ALU) additional banks can be added without creating any unmanageable routing and interconnection problems.", "The number of processing threads which can be supported can then be increased by adding registers to the program counter bank included in the multibank cache and modifying the control unit accordingly.", "The number of input/output streams which can be supported by the MCC can be increased by adding further I/O banks.", "A block diagram of the portion of the MCC 2 which deals with thread scheduling is shown in FIG.", "2.It comprises a resource checker 20 which receives resource status information relating to the status of data processing units, pipeline and memory bank status, and I/O port status along with connections to a FREEZEn signal output from each of a set of issue request control units 22.These issue request control units 22 each have a REQUESTn output and a DELAY_COUNTn output to a thread ranking unit 24.This also receives a PRIORITYn signal and a DEADLINE_COUNTn signal associated with each instruction thread.", "The sources of these two signals will be described with reference to FIGS.", "4 and 5.This thread ranking unit sends out ranking signals for each instruction thread to a priority selection unit 26.The priority selection unit 26 also receives interlock signals from a resource checker 20 which indicate which of the threads have the resources available to be executed.", "Thus, if the highest rank thread does not have all its resources available, the priority selection unit will select the next highest ranked thread which has resources available.", "The priority selection unit 26 has a GRANTn output which indicates which thread will execute an instruction on the next clock cycle.", "This GRANTn signal is also fed back to the issue rate request control units 22.Because of the indirect nature of the interaction with the scheduler shown in FIG.", "2 there may be some delay between the assertion of a REQUESTn signal and the issue of an instruction on an associated thread.", "This delay may be very long if the processor is overloaded, and therefore a buffer is required to hold instruction issue requests.", "The issue request buffer is implemented as a counter called the DELAY_COUNTn accumulator 42, which forms part of the issue request control unit 22.The issue request control unit is shown in detail in FIG.", "4.The unit comprises a POOL_COUNTn accumulator 40 and a DELAY_COUNTn accumulator 42.The POOL_COUNTn accumulator 40 receives a value from the MAX_POOL_COUNTn register 49 which represents the maximum allowable value in the POOL_COUNTn accumulator 40.It also receives a value from the average rate register 48 (An).", "The DELAY_COUNTn accumulator 42 receives a signal BLOCKEDn on one of its inputs and on its other input it receives the value in the burst rate register 46 (Bn).", "Both POOL_COUNTn accumulator 40 and DELAY_COUNTn accumulator 42 receive a signal GRANTn from the priority selector 26 of FIG.", "2.The DELAY_COUNTn accumulator 42 and the POOL_COUNTn accumulator 40 influence thread execution independently of each other and are therefore described separately.", "First we describe the operation of the DELAY_COUNTn accumulator 42.A thread defines the instruction issue rate required for a particular task by writing a value to its burst rate register 46.The value in the burst rate register 46 is repeatedly added into the DELAY_COUNTn accumulator 42 to represent the rate at which instruction issues are required by thread n. The DELAY_COUNTn accumulator 42 is decremented each time the signal GRANTn is asserted, so that if the processor executes instructions at the rate defined by the value in Bn then the value in the DELAY_COUNTn accumulator 42 will remain close to zero.", "If the processor is unable to execute at the rate defined by the value in Bn, there will be a residual positive value in the DELAY_COUNTn accumulator 42.The value in the DELAY_COUNTn accumulator 42 represents the number of instruction issues that thread n requires for its current task but has not yet received.", "The value in the DELAY_COUNTn accumulator 42 is used to generate the signals REQUESTn and FREEZEn for thread n so that the instruction issue rate on the thread is controlled.", "The value written to Bn to define the required instruction issue rate is calculated by the equation: Bn=required—issue—rate/delay—count—update—rate The DELAY_COUNTn accumulator 42 may be updated on every processor clock cycle, in which case Bn is calculated by: Bn=required_issue_rate/processor_clock_rate In order to save power, the DELAY_COUNTn accumulator 42 may be updated only once in each accumulation period of KR processor clock cycles, where KR is the rate control decimation factor.", "On each update the value in the burst rate register 46 is added to DELAY_COUNTn, and at the same time DELAY_COUNTn is decremented by the number of times that GRANTn was asserted during the accumulation period.", "The use of this mechanism ensures that the value in DELAY_COUNTn accumulator 42 at the end of each accumulation period matches the value it would have held if updates were applied on every processor clock cycle.", "At all other times the values may differ.", "Nevertheless, power consumption is reduced by a factor of KR, at the cost of coarser timing resolution.", "The value of KR must be chosen as a compromise between power consumption and granularity of instruction issue control.", "As a guide, it is expected that the value of KR in a range 4-16 should give acceptable timing granularity, whilst keeping power consumption to a reasonable level.", "If the DELAY_COUNTn accumulator 42 is updated once every KR processor clock cycles, then Bn is calculated by the equation: Bn=required—issue—rateKR/processor—clock—rate The program running on a thread may write to its burst rate register 46 at any time to define a new instruction issue rate.", "Typically this will be done in response to an event which requires processor activity, or in response to the completion of a task.", "The BLOCKEDn signal to the DELAY_COUNTn accumulator is generated by the resource checker 20 of FIG.", "2 and indicates that execution on the thread is blocked waiting for an external event.", "When BLOCKEDn is asserted, updates of the DELAY_COUNTn accumulator 42 are inhibited.", "When BLOCKEDn is deasserted indicating that an event has happened, the DELAY_COUNTn accumulator 42 is initially cleared and is subsequently updated once every accumulation period by the addition of the value Bn, and by the subtraction of one every time the signal GRANTn is asserted.", "The DELAY_COUNTn accumulator 42 value is limited to the largest 2's complement value it can represent so that it can avoid errors due to number wrapping.", "In operation, the DELAY_COUNTn accumulator 42 ensures that the instruction issue rate matches the rate defined by the burst rate register 46, provided that its value does not reach the maximum.", "The REQUESTn generation unit 44 of FIG.", "4 receives an output from the DELAY_COUNTn accumulator 42, and in response to this generates a REQUESTn signal to the thread ranking unit.", "The DELAY_COUNTn signal is also output directly to the thread ranking unit.", "The FREEZEn generation unit 45 of FIG.", "4 receives an outputs from the DELAY_COUNTn accumulator 42 and the POOL_COUNTn accumulator 40, and in response to these it generates a FREEZEn signal to the resource checking unit 20.The REQUESTn generation unit 44 determines the number of instructions to be requested during the next accumulation period by examining the value of DELAY_COUNTn at the start of that period, and asserting REQUESTn the appropriate number of times.", "REQUESTn will not be asserted if DELAY_COUNTn is less than or equal to zero.", "REQUESTn will be asserted KR times if the value of DELAY_COUNTn is greater than or equal to KR.", "REQUESTn will be asserted DELAY_COUNTn times if the value of DELAY_COUNTn is greater than zero and less than KR.", "The REQUESTn generation unit 44 ensures that the instruction requests are maximally distributed over the accumulation period by assertion of REQUESTn at the appropriate times.", "A succession of GRANTn assertions may lead to DELAY_COUNTn falling below zero, thus causing REQUESTn to be de-asserted and freeing up processor capacity for other threads to execute.", "Subsequent additions of the value in burst rate register 46 will cause DELAY_COUNTn to increase above zero thereby causing REQUESTn for that thread to be asserted.", "The output of the DELAY_COUNTn accumulator 42 is connected to a FREEZEn generation unit 45 which sends a FREEZE signal to the resource checker.", "The other input to this FREEZEn generation unit 45 is the output of the POOL_COUNTn accumulator 40.If the clock gating option referred to above is implemented, then the instruction issue rate on thread n is controlled by the signal REQUESTn, and if not implemented then the instruction issue rate is controlled by the signal FREEZEn.", "If the clock gating option described above is implemented then FREEZEn is asserted when DELAY_COUNTn is less than −15 and is de-asserted when DELAY_COUNTn is greater than zero.", "If clock gating is not implemented, FREEZEn is asserted when DELAY_COUNTn is less than zero and de-asserted when DELAY_COUNTn is greater than Bn.", "The operation of the DELAY_COUNTn accumulator 42 controls the rate at which instructions are issued so that it matches the rate defined in the burst rate register 46.This may be changed frequently as thread loading varies and thus it is difficult to ensure that the total processor loading due to one thread is bounded so that it does not exceed a defined value.", "A POOL_COUNTn accumulator 40, with value POOL_COUNTn is therefore provided to keep track of the average processor load and to limit the duration of processor overloads, and to ensure that no one thread can take over the processor.", "We now describe the operation of the POOL_COUNTn accumulator 40.A thread n which defines the average instruction issue rate that it requires does this by writing to its average rate register 48 (An).", "POOL_COUNTn accumulator 40 is incremented at a regular rate defined by the value in An and is decremented each time GRANTn is asserted for that thread.", "This is done using the same KR processor clock cycle accumulation period as is used to update the DELAY_COUNTn accumulator.", "If the POOL_COUNTn accumulator 40 is updated once every KR processor clock cycles, then An is calculated by the equation: An=average_issue_rateKR/processor_clock_rate The program running on a thread will typically write to its average rate register 48 only once, when the program is started.", "Having established the average instruction issue rate that it requires, the value in the average rate register 48 will usually not be altered.", "The value in the POOL_COUNTn accumulator 40 represents the number of instruction issues that a thread may require to complete its tasks but has not yet received.", "The value in the POOL_COUNTn accumulator 40 may be read by programs executing on other threads to assess the maximum possible duration of a processor overload.", "If POOL_COUNTn accumulator 40 becomes zero or negative then REQUESTn is de-asserted for that thread regardless of the condition of the DELAY_COUNTn accumulator 42, because the zero or negative value indicates that thread n has exhausted its allocation of instruction issues.", "The value in the POOL_COUNTn accumulator 40 is limited to a maximum value defined by the MAX_POOL_COUNTn register 49.This defines the number of instructions that a thread may build up “on account” to be executed later at a higher rate, and placing a limit on this value allows the duration of processor overloads to be bounded.", "When the POOL_COUNTn accumulator 40 reaches the value in the MAX_POOL_COUNTn register 49, further increments of the POOL_COUNTn accumulator 40 are inhibited.", "Thus, a thread which waits for an external event before executing at a high rate may build up a ‘reservoir’ of instructions in POOL_COUNTn accumulator 40 while it waits, and then executes at a higher rate defined by the burst rate register 46 for a limited time following the event.", "Any processing deficit in other threads which builds up during the high rate execution burst will be balanced later by the operation of their DELAY_COUNTn accumulators 42.It is possible to provide a facility which enables extra instructions to be issued to a thread such that the value in POOL_COUNTn accumulator 40 will go to a negative value.", "This can only happen if it is not preventing any other thread from receiving its required instruction rate.", "That is to say, this facility enables the allocation of spare instructions to other threads, thereby optimising processor usage.", "A default mode of operation is called ‘cycle strict’.", "In this mode, FREEZEn is asserted when the POOL_COUNTn accumulator 40 becomes zero or negative via the FREEZEn generator 44 of FIG.", "4 and the thread will not receive any more instruction issues until the POOL_COUNTn accumulator 40 is greater than zero again, as a result of increments by the value of the average rate register 48.Thus, a hard limit is enforced to the load that a thread can place on the processor.", "In the optional ‘cycle lenient’ mode, FREEZEn is not asserted when the POOL_COUNTn accumulator 40 is less than one.", "If this state arises and the thread with an empty instruction pool can run when no other threads is available to receive an instruction issue, the instruction scheduler may issue an instruction to the thread with an empty pool, causing the value of the POOL_COUNTn accumulator 40 to become more negative.", "It should be noted that REQUESTn is de-asserted when the POOL_COUNTn accumulator 40 is less than one regardless of the mode of operation.", "It should also be noted that a real time system will normally be designed such that the POOL_COUNTn accumulator 40 never empties.", "When the POOL_COUNTn accumulator 40 reaches the most negative value it can represent, further decrements are inhibited to ensure that the accumulator value does not wrap from a negative value to a positive one.", "In summary, therefore, the circuitry of FIG.", "4 will cause a REQUESTn signal to be generated in response to the DELAY_COUNTn accumulator 42, and a FREEZEn signal in response to the DELAY_COUNTn accumulator 42 or the POOL_COUNTn accumulator 40.If neither REQUESTn nor FREEZEn is asserted, an instruction will be issued to thread n if no other threads can run, but any other thread which can run will take priority over thread n. The instruction threads which run on the processor have a number of different possible states.", "These are as follows: RUNNING State In this state instructions are normally issued at the rate defined by the value in the burst rate register 46.The DELAY_COUNTn value will normally stay close to zero unless another thread has a burst of high execution rate.", "The value in the DELAY_COUNTn accumulator 42 represents the number of instructions by which the thread has been delayed from its intended pattern of execution, either by stalling or by the operation of other threads.", "POOL_EMPTYn State When the POOL_COUNTn accumulator 40 is zero or less than zero, for a particular thread then the thread is defined as being in the POOL_EMPTYn state.", "In this state the thread operates as it does in the running state but the issue rate may be limited to the value AN from its average rate register 48, if the thread is operating in cycle strict mode.", "Protection is provided to the other threads in the event that thread n attempts to run at an issue rate great than AN for a long time.", "In normal operation the POOL_EMPTYn state should never be entered.", "WAITING State When a thread is blocked on an external event such as a trigger, BLOCKEDn is asserted by the resource check unit 20 and the thread is defined as WAITING.", "If the clock gating option described above is implemented then no processor clock pulses are produced for the thread in the WAITING state.", "The priority control selection unit 26 of FIG.", "2 provides, to the instruction scheduler in the media control core of FIG.", "1, a rank order of preference for the threads.", "In the condition when the total of all the burst rates Bn for all the input threads is lower than the available processor instruction rate, the priority mechanism has no net effect.", "That is to say, the priority control unit is only relevant when the processor is overloaded.", "In the event of processor overload, the instruction scheduler issues instructions to the thread with the highest rank that is not interlocked.", "A combination of three metrics is used for establishing the rank order of threads, as illustrated in FIG.", "3.This processor is described below.", "The inputs to the thread ranking unit relating to each instruction thread are numbered 0, 1, 2, and 3.Only thread 0 is fully illustrated.", "The priority signal for a thread is input to a shift register from which it is supplied to an adder.", "DEADLINE_COUNT and DELAY_COUNTn each enter format conversion units 32 before passing to similar shift registers 30 to normalise them with the priority signal before all three are combined in an adder 34 thus producing a metric relating to that thread.", "Similar metrics are produced for each other thread and these are compared in a rank order comparator 36.This includes a gating unit 38 which then assigns a rank to each of the threads.", "These ranks are output in parallel.", "The metric with highest significance in establishing rank order is the thread priority.", "A thread defines its priority by writing an unsigned byte value to its priority register.", "The larger the number in this priority register, the higher its ranking.", "The second highest significance metric is the DEADLINE_COUNTn illustrated in FIG.", "5 and this provides a second input to the thread ranking unit 24.The DEADLINE_COUNTn mechanism allows a thread to define a deadline time within which it must complete a task.", "The DEADLINE_COUNTn register 50 is held static when the thread is in the WAITING state, and it is initialised to DEADLINE_DEFAULTn and subsequently decremented at a regular rate when the thread changes from the WAITING state to the RUNNING state in response to an external event.", "The inputs to the deadline counter 50 are the BLOCKEDn signal from the resource checker 20, the value held in the DEADLINE_DEFAULT register, representing the maximum deadline for that thread to execute, a DEADLINE_INCREMENTn value which is added to DEADLINE_COUNTn to adjust the deadline when a thread handles a number of different events each with different deadlines, and a DEADLINE_ENABLEn signal to enable the operation of the DEADLINE_COUNTn counter.", "The initialisation value represents the time from the event to the expiry of the shortest deadline.", "The DEADLINE_COUNTn register 50 then decrements at a regular rate, ensuring that its value represents the time remaining to expiry of the deadline.", "In order to save power, DEADLINE_COUNTn is updated only once in every period of KD clock cycles, where KD is the deadline counter decimation factor, in a similar manner to the updating of the DELAY_COUNTn accumulator 42 of FIG.", "4.As DEADLINE_COUNTn decreases, so the ranking of its thread should increase.", "If all threads are provided with a deadline priority mechanism then the processor resources are allocated in the best way to ensure that all deadlines are met.", "In a typical system there may be threads which do not have deadlines and therefore it is not absolutely necessary for all threads to use this mechanism.", "When a thread returns to WAITING state from the RUNNING state, the DEADLINE_COUNTn register stops decrementing, holding its final value until it leaves the WAITING state again, at which point it is re-initialised to a new value of DEADLINE_DEFAULTn.", "A thread may handle a number of different events, each with different deadlines.", "In this case, the DEADLINE_DEFAULTn register input to the DEADLINE_COUNTn 50 is programmed to represent the shortest deadline to be handled.", "If an event with a longer deadline occurs then its thread writes a deadline increment to a register DEADLINE_INCREMENTn (not illustrated) and this is then added to the value in the deadline counter 50.A DEADLINE_ENABLEn input to deadline counter 50 enables the deadline counter to be switched on or off with a control bit.", "When the deadline counter is disabled DEADLINE_COUNTn is set to its maximum value.", "A further input (not illustrated) can be used to halt the decrementing of DEADLINE_COUNTn during soft deadline scheduling.", "The deadline counter 50 only operates correctly if a thread n is in the WAITING state when an event with the deadline occurs.", "This can only be guaranteed where a thread handles only one event, and where event handling must complete before the next event.", "Where such restrictions are not enforced, deadline control must be handled by a separate processing thread dedicated to the task.", "Such a dedicated deadline control thread would perform the initial handling of all events, noting the deadline expiry time and then queuing the events to the appropriate threads.", "The work done by this thread is kept to a minimum so that the time taken to detect an event is as short as possible.", "When a thread picks up an event from its queue it adjusts DEADLINE_COUNTn for that thread by an amount recorded in the task queue entry via the deadline control thread.", "The adjustment value will be calculated by the deadline control thread at the time when the event occurs.", "The metric with the lowest significance used by the thread ranking unit is DELAY_COUNTn, described above.", "If this metric is used to control the thread ranking, then in the event of a processor overload, the instruction scheduler allocates processor resources so that all threads experience the same delay from their intended processing profile.", "For each thread, the three metrics are combined into a single composite metric by concatenation in the adder 34 of FIG.", "3.Priority is in the most significant position and DELAY_COUNTn in the least significant.", "The format converter 32 performs a bit wise inversion on the DEADLINE_COUNTn to ensure that a reducing count gives an increasing number.", "DELAY_COUNTn has its most significant bit inverted to convert it from 2's complement to offset-binary representation.", "This is to avoid misinterpretation of negative count values.", "The composite metrics are placed in rank order by the rank order comparator 36 which uses a sequential implementation of what is known as a bubble-sort algorithm.", "This is as follows: 1.On each clock cycle of the sorter, two composite metrics are compared and their rankings are swapped if the lower ranking value is greater than or equal to the higher ranking value.", "2.On successive clock cycles, the comparison proceeds from the lowest ranking pair of composite metrics towards the highest ranking pair.", "3.On the clock cycle following the comparison of the highest-ranking pair of composite metrics, the lowest ranking pair are compared.", "The effect of this algorithm is to place the largest composite metric in the highest ranking position in no more than N−1 clock cycles (where N is the number of threads), and can establish the correct order in a maximum of N2/2−N/2 clock cycles.", "If two or more composite metrics have identical values, their rank orders will cycle over time due to the swap taking place if values are equal.", "This behaviour emulates equal ranking by time slicing, therefore ensuring that the threads with equivalent composite metrics all receive equivalent priority from the instruction scheduler.", "The scheme described will control the instruction issue rates on all threads, provided that the processing limits of the processors are not exceeded.", "If they should be exceeded, it may be helpful to provide detection mechanisms to assist the debugging and characterisation.", "These are described below.", "To monitor and control instruction issue shortfall and excesses, two triggers are provided for each thread.", "The POOL_FILLEDn trigger is set when the POOL_COUNTn accumulator 40 value equals the value in the MAX_POOL_COUNTn register 49.The POOL_EMPTY trigger is set when the POOL_COUNTn accumulator 40 value is zero or negative.", "The states of POOL_FILLEDn and POOL_EMPTYn and the value of POOL_COUNTn accumulator 40 can then be read from a status register.", "The value of DELAY_COUNTn can be read from a status register.", "The deadline priority mechanism can be monitored by a trigger for each thread.", "This is called DEADLINE_MISSEDn and is set when DEADLINE_COUNTn falls to zero.", "The state of DEADLINE_MISSEDn and the value of DEADLINE_COUNTn can both be read from the status register.", "A summary table of the registers and triggers of the issue rate control system described here are listed below in table 2.Format IO Name N F T Description R/W An 8 −4 t (average instruction issue rate in MIPS) KR_/R+ R/W Bn 8 −4 t (Burst instruction issue rate in MIPS) KR/R R/W PRIORITYn 8 0 u Thread priority R/W DEADLINE_DEFAULTn 20 0 u Default deadline for thread (in units of KD/R) W DEADLINE_INCREMENTn 20 0 u Value by which to extend deadline counter (in units of KD/R) R/W MAX_POOL_COUNTn 22 0 U Maximum value of instruction pool counter R/W AMA_CONTROLn 3 0 u AMA control register R DELAY_COUNTn 27 −4 t Number of instructions that thread n has been delayed from its required burst rate.", "R/W DEADLINE_COUNTn 20 0 u Number of cycles remaining until event deadline expiry# R POOL_COUNTn 27 −4 t Number of potential instructions available to thread R DEADLINE_MISSEDn — — — Trigger indicating deadline has expired R POOL_FILLEDN — — — Triggger indicating instruction pool has filled (reached MAX_POOL_COUNTn) R POOL_EMPTYn — — — Trigger indicating instruction pool has emptied R is the processor clock rate in MHz.", "*The 20 bits of DEADLINE_COUNTn are read in positions D[23 .", ".", ".", "4], ensuring that the count value read by the MCC is the number of cycles remaining (truncated to the next lowest multiple of KD).", "Format: N—total number of bits F—number of fractional bits T—data storage type: u—unsigned data t—2's complement data.", "AMA Control Register: Bits are provided for: cycle strict operation deadline disable deadline halt A DELAY_COUNTn with 23 integer bits as given in Table 2, accumulates to a maximum of 222—approximately 4 million instructions.", "For a 50 MHz instruction issue rate, the counter can accumulate for about 80 ms.", "This is the same for the POOL_COUNTn.", "A 20 bit DEADLINE_COUNTn accumulates to a maximum of 220.Given a deadline decimation factor (KD) of 16, the DEADLINE_COUNTn can therefore represent a maximum of approximately 16.7 million cycles.", "For a 200 MHz processor, this represents a deadline of approximately 84 ms." ] ]	Patent_10468434
[ [ "Intraocular lens", "An intraocular lens system to be implanted in the posterior chamber of an eye, the system comprises a lens having an optical axis and at least two haptics extending from the circumference of the lens.", "The two haptics each includes one or more teeth located on their periphery." ], [ "1.An intraocular lens system to be implanted in the posterior chamber of an eye, the system comprising a lens having an optical axis and at least two haptics extending from the circumference of the lens, said two haptics each including one or more teeth located on their periphery.", "2.An intraocular lens system according to claim 1, wherein said teeth are capable of penetrating the ciliary sulcus of the scleral wall of the eye, to anchor the lens in place.", "3.An intraocular lens system according to claim 2, wherein at least one of said teeth is oriented to form an acute angle with the circumference of the haptic, so as to allow smooth rotation of the intraocular lens system in one direction relative to the optical axis and to enable penetration of the teeth into the ciliary sulcus when rotated in the other direction.", "4.An intraocular lens system according to claim 1, wherein at least one of said teeth has a harpoon-shaped outer surface.", "5.An intraocular lens system according to claim 1, wherein at least one of said teeth has a smooth outer surface.", "6.An intraocular lens system according to claim 1, wherein at least one of said teeth has a jagged outer surface.", "7.An intraocular lens system according to claim 1, wherein said lens and said haptics are produced as one body.", "8.And intraocular lens system according to claim 1, wherein said haptics are produced as separate bodies, attachable to said lens.", "9.An intraocular lens system according to claim 1, wherein said haptics and said teeth are produced as one body.", "10.An intraocular lens system according to claim 1, wherein at least one of said teeth is produced as a separate body, attachable to at least one of said haptics.", "11.An intraocular lens system according to claim 10, wherein said haptics are produced with teeth engaging means.", "12.An intraocular lens system according to claim 10, wherein at least one of said teeth is made of a biodegradable material.", "13.An intraocular lens system according to claim 10, wherein at least one of said teeth is made of a magnetic material.", "14.An intraocular lens system according to claim 1, further including longitudinal positioning holes to facilitate the manipulation of the haptics and the lens.", "15.An intraocular lens system according to claim 1, further including positioning holes at the haptic base and tip as to facilitate the manipulation of the haptics and the lens.", "16.An intraocular lens system according to claim 1, further including a rigid tool to support the lens system for directing it into its operative position.", "17.An intraocular lens system according to claim 16, wherein said tool is adapted to accommodate said lens system and to keep it secure by limiting its movement thereon.", "18.An intraocular lens system according to claim 17, wherein said tool has an operative end, which is soft and flexible to avoid damaging the ciliary sulcus.", "19.An intraocular lens system according to claim 1, further including a protective, possibly removable sleeve to be placed on at least one of said haptics for depressing said teeth thereon.", "20.Haptic to be attached to an intraocular lens in a lens system defined in claim 1.21.An intraocular lens system to be implanted in an eye, comprising a lens having an optical axis and at least two haptics extending from the periphery of the lens, said two haptics each being formed with teeth engaging means to enable the attachment thereto of one or more teeth.", "22.An intraocular lens system according to claim 21, adapted to be implanted in the posterior chamber of an eye, wherein said engaging means is so located on the haptics as to enable the teeth, when attached thereto, to penetrate the ciliary sulcus of the scleral wall of the eye, to anchor the lens in place." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Cataract is clouding of the natural lens of the eye or of its surrounding transparent membrane, which obstructs the passage of light causing various degrees of blindness.", "To correct this condition, a surgical procedure is known to be performed in which the opaque natural lens, or cataract, is extracted and replaced by an artificial intraocular lens.", "The natural lens, located behind the iris in the posterior chamber in front of the vitreous cavity of the eye, is composed of a capsular bag containing gelatinous material.", "If this bag, called the posterior capsule, is left intact during a cataract extraction procedure, it may serve as a stable support site for implanting an intraocular lens.", "However, in the course of surgery, the posterior capsule may be inadvertently damaged or removed along with the cataract, in which case it would no longer be able to provide a support base to keep the intraocular lens from floating back into the vitreous cavity.", "In his case, it is known to implant the lens in the anterior chamber in front of the iris, or in the posterior chamber behind the iris, wherein the iris serves as a carrier for the lens in both instances.", "In the latter case, it has also been known to fix the intraocular lens in place behind the iris by suturing it to the ciliary sulcus.", "In both of the above cases, to maintain the intraocular lens properly centered, it is normally equipped with extensions, called haptics, which may have positioning holes to facilitate the centering of the lens.", "U.S. Pat.", "No.", "4,750,904 discloses a method of implanting an intraocular lens in the posterior chamber by tying the haptics to the iris and using small, radially disposed loops formed on the lens to serve as suture sites for securing the implanted lens directly to the iris." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with the present invention, there is provided a novel solution for the self-fixation of an intraocular lens system in the posterior chamber of an eye.", "The lens system comprises a lens having an optical axis and at least two extending haptics attached to the circumference of the lens.", "These two haptics each have one or more teeth located on their periphery, which are particularly capable of penetrating the ciliary sulcus of the scleral wall of the eye, thereby anchoring the lens in place.", "The teeth may be oriented to form an acute angle with the circumference of the haptic, thereby allowing free rotation of the haptic in one direction relative to the optical axis and allowing penetration of the teeth into the ciliary sulcus, when rotated in the other direction.", "The teeth may be harpoon-shaped, smooth or jagged in order to further facilitate their penetration or grasping of the ciliary sulcus.", "The present invention provides for a secure self-attachment of the intraocular lens in the posterior chamber independently of the posterior capsule and without involving the iris." ], [ "FIELD OF THE INVENTION This invention relates to intraocular lens implantation and particularly to implantation in the posterior chamber of an eye.", "BACKGROUND OF THE INVENTION Cataract is clouding of the natural lens of the eye or of its surrounding transparent membrane, which obstructs the passage of light causing various degrees of blindness.", "To correct this condition, a surgical procedure is known to be performed in which the opaque natural lens, or cataract, is extracted and replaced by an artificial intraocular lens.", "The natural lens, located behind the iris in the posterior chamber in front of the vitreous cavity of the eye, is composed of a capsular bag containing gelatinous material.", "If this bag, called the posterior capsule, is left intact during a cataract extraction procedure, it may serve as a stable support site for implanting an intraocular lens.", "However, in the course of surgery, the posterior capsule may be inadvertently damaged or removed along with the cataract, in which case it would no longer be able to provide a support base to keep the intraocular lens from floating back into the vitreous cavity.", "In his case, it is known to implant the lens in the anterior chamber in front of the iris, or in the posterior chamber behind the iris, wherein the iris serves as a carrier for the lens in both instances.", "In the latter case, it has also been known to fix the intraocular lens in place behind the iris by suturing it to the ciliary sulcus.", "In both of the above cases, to maintain the intraocular lens properly centered, it is normally equipped with extensions, called haptics, which may have positioning holes to facilitate the centering of the lens.", "U.S. Pat.", "No.", "4,750,904 discloses a method of implanting an intraocular lens in the posterior chamber by tying the haptics to the iris and using small, radially disposed loops formed on the lens to serve as suture sites for securing the implanted lens directly to the iris.", "SUMMARY OF THE INVENTION In accordance with the present invention, there is provided a novel solution for the self-fixation of an intraocular lens system in the posterior chamber of an eye.", "The lens system comprises a lens having an optical axis and at least two extending haptics attached to the circumference of the lens.", "These two haptics each have one or more teeth located on their periphery, which are particularly capable of penetrating the ciliary sulcus of the scleral wall of the eye, thereby anchoring the lens in place.", "The teeth may be oriented to form an acute angle with the circumference of the haptic, thereby allowing free rotation of the haptic in one direction relative to the optical axis and allowing penetration of the teeth into the ciliary sulcus, when rotated in the other direction.", "The teeth may be harpoon-shaped, smooth or jagged in order to further facilitate their penetration or grasping of the ciliary sulcus.", "The present invention provides for a secure self-attachment of the intraocular lens in the posterior chamber independently of the posterior capsule and without involving the iris.", "BRIEF DESCRIPTION OF THE DRAWINGS In order to understand the invention and to see how it may be carried out in practice, different embodiments will now be described, by way of non-limiting examples only, with reference to the accompanying drawings, in which: FIG.", "1 is a schematic perspective view of an intraocular lens system according to the present invention; FIG.", "2 is a schematic view of the intraocular system shown in FIG.", "1, when implanted in the posterior chamber of an eye; FIGS.", "3A, 3B, and 3C illustrate haptics of the lens system of FIG.", "1, in accordance with three alternative embodiments of the present invention; FIG.", "4 is a schematic perspective view of an intraocular lens system according to another embodiment of the present invention; FIG.", "5 shows an optional supporting tool and a lens system according to the present invention, when supported by this tool for implantation; FIG.", "6 shows an optional protective sleeve for use with a lens system according to the present invention.", "DETAILED DESCRIPTION OF THE INVENTION An intraocular lens system 1 in accordance with one embodiment of the present invention is shown in FIG.", "1.The lens system 1 consists of a lens 2 of any known type having an optical axis 3 and two flexible haptics 4 extending away from the circumference of the lens 2.The haptics 4 include longitudinal positioning holes 5 near their base 8 and near their tip 9.These holes 5 aid in the positioning of the lens system 1 and facilitate manipulations thereof.", "The holes 5 may have any appropriate shape and each haptic 4 may have any number of them at any appropriate location.", "With reference to FIG.", "2, the lens system 1 is adapted to be implanted in an eye 30 to replace a cataract.", "FIG.", "2 shows that, when implanted, the lens system 1 is located in the posterior chamber 32 between the iris 34 and the posterior capsule 36, with its haptics 4 bearing against the ciliary sulcus 38 of the scleral wall 40 of the eye 30.In accordance with the present invention, and as seen in FIG.", "1, each haptic 4 is provided with teeth 6 located at its periphery in the region designed to contact with the ciliary sulcus 38, when the lens system 1 is implanted in an eye.", "Thus, upon insertion of the lens system 1 into the eye 30 and manipulation of the haptics 4, the teeth 6 are made to penetrate and embed themselves in the ciliary sulcus 38 of the scleral wall 40, thereby securely anchoring the intraocular lens system 1 in the posterior chamber 32.Reverting to FIG.", "1, the teeth 6 may be harpoon-shaped with one smooth side 12 and one indented side 14, which is oriented to form an acute angle a with the circumference of the haptic 4.Such shape and orientation of the teeth 6 enable free rotation of the haptic 4 and the system 1 in one direction relative to the optical axis 3 and penetration and embedding of the teeth 6 into the ciliary sulcus 38, when rotation is attempted in the other direction.", "FIGS.", "3A, 3B, and 3C show alternative designs for the teeth 6.In FIG.", "3A, the teeth 6a are smooth on both sides and are acutely angled to allow rotation of the haptic 4 in only one direction.", "In FIG.", "3B, the teeth 6b are similar to the teeth 6a but are oriented perpendicular to the circumference of the haptic 4 so as to prevent rotation in either direction.", "As shown in FIG.", "3C, the teeth 6c are jagged to ensure an extremely firm anchoring of the haptics 4 in the eye 30.To better secure the attachment of the lens system 1 and to center the lens 2, manipulation of the haptics 4 and the lens 2 can be performed by using the longitudinal positioning holes 5.It has been found that the longitudinal design of the positioning holes 5 is particularly useful as it allows for greater facility in the manipulation of the intraocular lens system 1 than conventional circular positioning holes.", "While oval positioning holes 5 are shown here, the longitudinal positioning holes 5 may be of various oblong shapes and sizes, with their length extending in the longitudinal direction of the haptic 4.Thus, rectangular or slit-like positioning holes may also be used.", "The oblong designs allow a surgeon to know when the intraocular lens system 1 has abutted the ciliary sulcus 38 during implantation.", "In each haptic 4, 4′, any number of teeth 6 may be used and they may be placed anywhere on the far outer periphery of the haptics and at various distances from each other.", "The arrangement of the teeth on the two haptics of the lens system may be similar or completely different.", "The lens 2 and haptics 4, 4′ may be produced as one body or alternatively, they may be produced as separate bodies, attachable to each other.", "The teeth 6 may also be produced as one body along with the haptics 4, 4′ or rather they may be produced separately from the haptics, for example, to be attached thereto prior to implantation.", "In the latter case, it may be especially advantageous to produce a lens system to which teeth can be attached, when needed, and which can also be used without such teeth.", "For this purpose, the haptics should be formed with suitable teeth engagement means and/or the teeth may be formed with corresponding haptic engagement means.", "The lens system of the present invention may be composed of various different substances.", "One example is making the teeth 6 of a biodegradable material, because it is beneficial to have the teeth 6 that penetrate the ciliary sulcus 38 completely dissolve over time.", "Another example is making the teeth 6 of a magnetic material so that they can be made to penetrate the sulcus 38 of the eye 30 by the use of an external magnet, after the haptic 4, 4′ has been positioned at the penetration site.", "FIG.", "5 illustrates an auxiliary tool 50 that may be used with an intraocular lens system of the present invention, for example, to facilitate its introduction into the eye 30.The auxiliary tool 50 may consist of a base 52 having a shape and dimensions to fully accommodate the lens system and a handle 54 attached to the base 52 at its end opposite its operative end 56.The tool 50 also has a covering member 58 to keep the lens system 1′ securely on the base 52 thereby restricting the motion of the lens system 1′ and reducing the likelihood of damaging the eye during implantation.", "While the auxiliary tool 50 should be rigid in order to direct the intraocular lens system 1′ into its operative position, its operative end 56 should preferably be soft and flexible to prevent damaging the eye.", "As an example of how this tool 50 can be used, an asymmetric intraocular lens system 1′ is shown thereon, having jagged teeth 6c on one haptic 4′ and harpoon-shaped teeth 6 on the other haptic 4.During its implantation, the lens system 1′ is brought to the ciliary sulcus 38 using the tool 50, and is pushed towards it, where the jagged teeth 6c are made to embed themselves firmly.", "The positioning holes 5 on the other haptic 4 are then used to embed the harpoon shaped teeth 6 at another desired site in the sulcus 38.FIG.", "6 shows an additional embodiment of the present invention, in which a thin protective sleeve 60 is placed over the teeth 6 so as to press them down to the haptic 4, thereby ensuring that they do not contact and damage the eye.", "The sleeve 60 should have a means for its removal from the haptic 4, such as an engaging loop 62, which can be pulled in the direction of the arrow to withdraw the sleeve 60 when the intraocular lens system 1 reaches the appropriate site of the ciliary sulcus 38, thereby exposing the teeth 6 and allowing them to penetrate the sulcus 38.It should be understood that any permutation and/or combination of different features of the above-disclosed embodiments is also possible.", "It should further be understood that the above described embodiments constitute only examples of an intraocular lens system and a manner of its implantation according to the present invention, and that the scope of the present invention fully encompasses other embodiments which may become obvious to those skilled in the art." ] ]	Patent_10468465
[ [ "Method of treating human-immunodeficiency virus (hiv) disease infection", "Human Immunodeficiency virus causes depletion of CD4 cells.", "The depletion of CD4 cells results in decrease in immunity of an infected individual.", "Due to decrease immunity various opportunistic infections occur.", "These infections are cause for morbidity and mortality in HIV infected individuals.", "The treatment of HIV these includes antiretroviral drugs.", "These drugs have their own side effects and immune reconstitution achieved is delayed and slow.", "Various attempts have been made to improve CD4 count, use of IL-2 is one of them.", "It is associated with systemic side effects during the period of its administration.", "The present invention provides method of using mycobacterium w for the management of HIV.", "According to present invention mycobacterium w when given intradermally is effective in prophylaxis and treatment of AIDS or AIDS related complex (ARC).", "It is found to improve immunity as well as CD4 count.", "It is found to eliminate symptoms like fever, diarrhea.", "The effect is seen even when no antiretrovirals are used." ], [ "1-21.", "(canceled) 22.A method of managing an HIV infection comprising administering to a patient a medicament comprising mycobacterium w or its constituents.", "23.The method of claim 22, wherein the method is for improving the immune function of HIV positive subjects.", "24.The method of claim 22, wherein the method is for managing opportunistic infections associated with an HIV infection.", "25.The method of claim 22, wherein the method is for amelioration of symptoms associated with HIV.", "26.The method of claim 22, wherein the method is for the prophylaxis or treatment of AIDS or AIDS related complex (ARC).", "27.The method of claim 22, wherein the method is for delaying development of AIDS or AIDS related complex (ARC) in patients infected by HIV.", "28.The method of claim 22, wherein the method is for regression or removal, of overt symptoms of AIDS even in patients where the disease is far advanced.", "29.The method of claim 22, wherein the method is for treating patients who are suffering from HIV infection with or without AIDS and with or without tuberculosis.", "30.The method of claim 22, wherein the medicament improves a CD4+T cell count in patients who are suffering from HIV.", "31.The method of claim 22, wherein the medicament improves a CD4+T cell count in patients who are suffering from HIV in the absence of as well as in the presence of antiretroviral therapy.", "32.The method of claim 22, wherein the mycobacterium w or its constituents are used alone or in combination with each other.", "33.The method of claim 32, wherein the medicament further comprises adjuvants, excipients, diluents, suspending agents or preservatives.", "34.The method of claim 22, wherein the mycobacterium w is dead mycobacterium w. 35.The method of claim 34, wherein the mycobacterium w is killed by physical methods like heat or radiation by heat in the form of autoclaving.", "36.The method of claim 34, wherein the mycobacterium w is killed by heat in the form of autoclaving.", "37.The method of claim 22, wherein the medicament comprises mycobacterium w constituents obtained by sonication.", "38.The method of claim 22, wherein the medicament comprises mycobacterium w constituents obtained by a high pressure cell fractionator.", "39.The method of claim 22, wherein the medicament comprises mycobacterium w constituents obtained by osmatic pressure ingredient.", "40.The method of claim 22, wherein the medicament comprises mycobacterium w constituents obtained by extraction.", "41.The method of claim 40, wherein the mycobacterium w constituents are extracted by organic solvents.", "42.The method of claim 41, wherein the organic solvents are selected from the group consisting of chloroform, ethanol, methanol, acetone, phenol, isopropyl alcohol, acetic acid, urea and hexane.", "43.The method of claim 22, wherein the medicament comprises mycobacterium w constituents obtained by enzymatic treatment.", "44.The method of claim 22, wherein the mycobacterium w is obtained by use of enzyme lyticase and/or pronase.", "45.The method of claim 22, wherein the medicament comprises mycobacterium w constituents having a molecular weight of more than 12,000 daltons.", "46.The method of claim 22, wherein the medicament comprises mycobacterium w constituents that are water insoluble.", "47.The method of claim 22, wherein the medicament is in a unit dosage form comprising equal to or more than 1×105 mycobacterium w. 48.The method of claim 22, wherein the medicament is in a unit dosage form comprising equal to or more than 1×107 mycobacterium w. 49.The method of claim 22, wherein the medicament is in a unit dosage form comprising between 1×108 and 1×1010 mycobacterium w. 50.The method of claim 22, wherein the mycobacterium w is urease negative, does not hydrolyse polyoxyethylene sorbitan monooleate, does not produce niacin and provides a strong positive response to nitrate reduction test." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Human immunodeficiency virus (HIV) was first isolated in 1983.The causative agent for AIDS is known to be a virus of the retrovirus family called HIV (human immunodeficiency virus).", "Infection with HIV does not, however, immediately give rise to overt symptoms of AIDS.", "Three to six weeks following primary HIV infection more than 50% of individuals develop acute HIV syndrome, which is self limiting.", "Clinical findings seen during this period include fever, pharyngitis, lymphadenopathy, headache, arthralgia, myalgia, malaise, lethargy, nausea, vomiting, diarrhoea, skin rash, mucocutaneous ulceration, meningitis, encephalitis, neuropathy etc.", "The only indication of exposure to the virus may be the presence of antibodies thereto in the blood of an infected subject who is then described as ‘HIV positive’.", "The infection may lie dormant; giving rise to no obvious symptoms, and the incubation period prior to development of AIDS may vary from several months to decades.", "Development of AIDS itself may be preceded by the AIDS-related complex (ARC), which is characterized by unexplained fever, weight loss, chronic cough or diarrhea.", "The development of AIDS and/or ARC is dependent on breakdown of immune system.", "The reasons for the variable period between infection with the virus and breakdown of the immune system in an infected individual are poorly understood.", "Factors at present unknown may trigger proliferation of the virus with consequential disruption of the immune system.", "The victims of the disease are then subject to various infections and malignancies, which, unchecked by the disabled immune system, lead to death.", "Thus HIV is characterized by the “acute HIV syndrome” followed by “asymptomatic stage” with clinical latency.", "Symptomatic stage sets in later with breakdown of immune system, which ultimately leads to the death of the individual infected with HIV.", "Though the disease is caused by virus, the morbidity and mortality associated with disease is due to breakdown of immune system.", "The breakdown of immune system is characterized by decreased CD4 + T lympocyte count.", "Because of this reason 1993 revised classification system for HIV infection is based on CD4 + T lymphocyte counts.", "HIV disease is empirically divided based on CD 4 count which is a measure of immunodeficiency.", "a) Early stage CD 4 +T cell count more than 500 b) Intermediate stage CD 4 +T cell count 200 to 500.c) Advanced stage CD 4 +T cell count less than 200.Individuals with nonprogressive HIV disease are found to have steady CD 4 counts.", "They are also observed to have strong immune response against the virus.", "There is evidence that in HIV infection, there is a dramatic loss of CD4 T-cells, which results in very rapid development of overt symptoms of AIDS.", "Most AIDs defining opportunistic infections and true malignancies occur in advanced stage of disease where in CD 4 count is less the 200 cells/mL.", "CD4 Count and HIV Though HIV is a viral infection, viral load can be determined by reasonable accuracy, CD4 count (a measure of immune status) plays major role in management of HIV due to following reasons.", "1.Morbidity and Mortality in HIV infected individuals is due to opportunistic infections.", "These opportunistic infections define onset of AIDS in HIV +ve individuals.", "The risk of opportunistic disease increases markedly when CD4+ cell count declines to less than 200 cells/mm 3 .", "2.CD4 count provides estimate of degree of existing immunodeficiency.", "Immune deficiency is responsible for HIV +ve individuals getting converted to AIDS.", "3.The initiation of antiretroviral therapy is also dependent on CD4 count.", "4.Outcome of antiretroviral therapy is also dependent on CD4 count.", "Higher survival are associated with higher initial CD4 count.", "5.Risk of progression to AIDs defining illness is associated with declining CD4 count.", "The risk is lower with higher CD4 count.", "6.Likelyhood of developing AIDs within 3 years is significantly higher when CD4 count is low (less than 200 CD4+ T cells) compared to high CD4+ T cell count.", "For viral load of greater than 55 k as per RT-PCR the risk is 32.6% if CD4+ T cell count is more than 500 cells/mm3 compared to 85.5% for individuals with CD4 count of less than 200 cells/mm 3 .", "7.Similarly for viral load of 20 k-55 k (RT-PCR) the risk of developing AIDs is 9.5% when CD4 count is more than 750 cells/mm3 compared to 40.1% when CD4 count is less than 350 cells/mm3.Goals of Therapy Maximal and durable suppression of viral load.", "Restoration and/or preservation of immunologic function.", "Improvement of quality of life.", "Reduction in HIV related morbidity and mortality.", "The method to treat HIV includes various therapeutic options.", "The options include management of symptoms and infections manifesting in HIV infected individuals at various stages, of the disease.", "The antiretroviral drugs are used to keep the HIV infection (viral load) in control.", "They keep the viral load in control.", "The early antiretroviral drugs like azothymidine delayed progression of disease and had no significant effect on CD 4 count.", "Protease inhibitors like indinavir, ritonavir which are introduced recently do improve CD 4 count while reducing viral, load.", "All the drugs (antiretroviral) have their own side effects.", "The resistance to drugs is also noted.", "Thus there is need to provide alternate mechanism of treating HIV.", "Since CD 4 count is important in maintaining immunity of individual and decreased CD 4 counts are associated with morbidity and mortality in HIV infection attempts are made to improve immunity for management of HIV.", "Various efforts have been done towards this end.", "This has resulted in introduction of immune modifying therapies with or without antiretroviral drugs.", "They comprise of antigens, cytokines organisms etc.", "It has surprisingly been found during the course of research by us that formulations of ‘ Mycobacterium W’ (M w ) with or without antigenic and/or immunomodulatory material derived from (M w ) is effective for management of Human Immunodeficiency Virus (HIV) disease/infection.", "Prior Art M. vaccae is apparently unique among known mycobacterial species in that heat-killed preparations retain its properties for the use as vaccine and immunotherapeutic.", "For example, M. bovis -BCG vaccines, used for vaccination against tuberculosis, employ live strains.", "Heat-killed M. bovis BCG and M. tuberculosis have no protective properties when employed in vaccines.", "A number of compounds have been isolated from a range of mycobacterial species, which have adjuvant properties.", "The effect of such adjuvants is essentially to stimulate a particular immune response mechanism against an antigen from another species.", "In U.S. Pat.", "No.", "6,001,361 the invention is related to compounds and methods for the treatment of mycobacterial infections including Mycobacterium tuberculosis and Mycobacterium avium .", "The invention is further related to compounds that function as non-specific immune response amplifiers, and the use of such non-specific immune response amplifiers as adjuvants in vaccination or immunotherapy against infectious disease, and in certain treatments for immune disorders and cancer.", "U.S. Pat.", "No.", "4,716,038 discloses diagnosis of, vaccination against and treatment of autoimmune diseases of various types, including arthritic diseases, by administering mycobacteria, including M. vaccae.", "U.S. Pat.", "No.", "6,210,684 describes method for delaying the onset of AIDS using killed M. Vaccae .", "Onset of AIDS is related to decrease in CD 4 count is not disclosed in the patent.", "Published studies shows that killed M. Vaccae has no effect on CD 4 count in HIV positive individuals.", "International Patent Publication WO 91/01751 discloses the use of antigenic and/or immunoregulatory material from M. vaccae as an immunoprophylactic to delay and/or prevent the onset of AIDS.", "International Patent Publication WO 94/06466 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for therapy of HIV infection, with or without AIDS and with or without associated tuberculosis.", "U.S. Pat.", "No.", "6,001,361 discloses an invention that relates generally to the treatment by vaccination or immunotherapy of skin disorders such as psoriasis, atopic dermatis, allergic contact dermatitis, alopecia areata, and the skin cancers basal cell carcinoma, squamous cell carcinoma and melanoma.", "In particular, the invention is related to the use of compounds, which are present in or have been derived from Macobacterium vaccae ( M. vaccae ) or from the culture filtrate of M. vaccae.", "U.S. Pat.", "No.", "5,599,545 discloses the use of mycobacteria, especially whole, inactivated M. vaccae , as an adjuvant for administration with antigens, which are not endogenous to M. vaccae .", "This publication theories that the beneficial effect as an adjuvant may be due to heat shock protein 65 (hsp 65).", "International Patent Publication WO 92/08484 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of uveitis.", "International Patent Publication WO 93/16727 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of mental diseases associated with an autoimmune reaction initiated by an infection.", "International Patent Publication WO 95/26742 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for delaying or preventing the growth or spread of tumors.", "Vacce is not associated with change in CD 4 count or viral load in HIV positive individuals.", "It does not provide any relief in HIV +ve individual.", "However in spite of various patents issued in relation to mycobacterium vaccae , it fails to provide any significant change in CD 4 count as well as viral load in individuals who are HIV positive.", "Similarly attempts have been made to improve CD 4 count using immunomodulator from various sources.", "Remune is a HIV-1 specific immunogen in incomplete Freund's adjuvant.", "It includes inactivated HIV-1 from which gp 120 is depleted.", "It is found to be safe with immunogenic potential in persons infected with HIV.", "In initial studies it was found to improve CD4-cell count in asymptomatic HIV cohort not taking antiretroviral agents.", "It is given intramuscularly as an injection into the triceps muscles.", "The recent study by Sukeepaisamcharoen w et at suggests that remune therapy is associated with stabilization of CD 4-cell counts.", "It is also suggests that it may be of value in participants with higher CD4+ T cell count.", "One such immunomodulator consists of mixture of antigens of inactivated bacteria with antigens of influenza virus (poly antigenic immunomodulator).", "It has not been possible to achieve improvement in CD 4 count using it.", "SB-73 is another immunomodulator made up of substance produced by pencillium P(PB-73 strain).", "In a small study it is found to improve CD 4 count in majority (10/14) of individuals, infected with HIV when given intramuscularly in a dose of 5 mgm.", "Reticulose, a peptide-nucleic acid is another immunomodulator found to be useful in improving CD 4 count in HIV infected individuals when given subcutaneously.", "It was given as two 1 ml subcutaneous (SC) injections per day for two weeks followed by 1 ml SC per day every other week for a total 60 days (30 days total treatment).", "It resulted in a significant improvement, in CD 4 count in absence of any other antiretroviral therapy.", "Thymosin α 1 is a 28-aminoacid peptide.", "It was evaluated for its efficacy to improve CD 4 along with interleukin-2.It was found to have no significant effect.", "Grenutocyte colong stimulating factor (Filgrastim) has also been evaluated to improve immune response in HIV without much success.", "OKT3, a CD 3 monoclonal antibody, has been successfully used in management of HIV along with antiretroviral and Interleukin-2 in three patients.", "Of various cytokines used in management of HIV, Interleukin-2 (IL-2) is extensively studied and used.", "It is used as injection to be administered intravenously or subcutaneously.", "It is found to improve CD 4 count significantly when used alone or along with antiretroviral drugs.", "It is given as intermittent therapy the side effects seen are sometimes intolerable.", "They are seen only during period of active administration.", "The improvement seen in CD 4 count is found to be stable.", "Other cytokines used in management of HIV includes IL-12 and IL-15.U.S.", "Pat.", "No.", "5,759,992 provides low molecular weight glycopeptide with a molecular weight of 919.2 dalton which is derived from supernatant of disrupted cells maintaining temprature of 4° C. through out the process.", "This is obtained from bacteria which includes E coli and Mycobacterium .", "It is found to improve CD 4 count in normal mice.", "It is not evaluated in HIV+ve animals.", "U.S. Pat.", "No.", "5,871,732 describes methods for preventing or treating AIDS, AIDS related complex and human immunodeficiency virus infection by anti-CD 4 antibody homologs to DNA sequences of encoding such homologs.", "Mycobacterium w is a non-pathogenic, cultivable, atypical mycobacterium , with biochemical properties and fast growth characteristics resembling those belonging to Runyons group IV class of Mycobacteria in its metabolic and growth properties but is not indentical to those strains currently listed in this group.", "It is therefore thought that (M w ) is an entirely new strain.", "The species identity of M w has been defined by polymerase chain reaction DNA sequence determination.", "It has been found to share antigens with Mycobacterium leprae and Mycobacterium tuberculosis .", "It is found to provide prophylaxis against leprosy in humans by converting lepromin negative individuals to lepromin positivity.", "It is also found to provide prophylaxis against tuberculosis in animals.", "In leprosy it is also found to reduce duration of therapy for bacterial killing, clearance as well as clinical cure when used along with multi drug therapy." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>According to present invention, immunomodulator made from ‘ Mycobacterium w’ (M w ) is found to be useful in the management of HIV infection.", "We have now found that the same therapeutic agent not only delays development of AIDS in patients infected by HIV, but also is capable of causing regression, or even removal, of overt symptoms of AIDS even in patients where the disease is far advanced.", "These effects have been found in patients suffering also from tuberculosis.", "These effects are also seen in patients who are suffering from HIV infection with or without AIDS and without associated tuberculosis.", "The immunomodulator as per present invention is also found useful in relieving symptoms of HIV.", "Therapeutic agent which may be used in the present invention resembles Mw a non-pathogenic, cultivable, atypical mycobacterium , with biochemical properties and fast growth characteristics resembling those belonging to Runyons group IV class of Mycobacteria in its metabolic and growth properties but is not indentical to those strains currently listed in this group.", "It is therefore thought that (M w ) is an entirely new strain.", "The species identity of Mw has been defined by polymerase chain reaction DNA sequence determination and differentiated from thirty other species of mycobacteria.", "It however differs from those presently listed in this group in on respect or the other.", "By base sequence analysis of a polymorphic region of pattern analysis, it has been established that M w is a unique species distinct from many other known mycobacterial species examined which are: M. avium, M. intracellulare, M. scrofulaceum, M. kansasii, M. gastri, M. gordonae, M. shimoidei, M. malmoense, M. haemophilum, M. terrae, M. nonchromogenicum, M. triviale, M. marinum, M. flavescens, M. simian, M. szulgai, M. xenopi, M. asciaticum, M. aurum, M. smegmatis, M. vaccae, M. fortuitum subsp fortuitum, M. fortuitum subsp.", "Peregrinum, M. chelonae subsp.", "Chelonae, M. chelonae subsp.", "Abscessus, M. genavense, M. tuberculosis, M. tuberculosis H 37 R v , M. paratuberculosis.", "The object of the present invention is to provide an immunomodulator Mycobacterium w containing ‘ Mycobacterium w’ (Mw) with or without obtained from Mw for the prophylaxis or therapy of AIDS or AIDS related complex, to a subject exposed to HIV infection or is HIV positive with or without, overt symptoms of AIDS.", "Yet another object of the invention is to provide immunomodulator derived from mycobacterium w that are useful for the management of HIV infection.", "Yet another object of the invention is to provide immunomodulator derived from Mycobacterium w to improve immune function of HIV +ve subjects.", "Yet another object of present invention is to provide immunomodulator to improve CD 4 count in HIV infected individuals.", "Yet another object of present invention is to provide immunomodulator effective in ameliorating symptoms associated with HIV infection.", "Yet another object of present invention is to provide an immunomodulator effective in management of opportunistic infections of associated with HIV infection.", "Yet another object to present invention is to provide an immunomodulator useful in improving immune function of HIV +ve subjects in presence or absence of antiretroviral drugs.", "Yet another object of the invention is to provide methods for the treatment of HIV, which results in the amelioration of symptoms of symptomatic stage of the disease.", "It is yet another object of the invention to provide for a method of treatment of HIV infection that results in improved CD4 + T cell count.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "This invention relates to the management of Human Immunodeficiency virus (HIV) Disease/Infection.", "BACKGROUND OF THE INVENTION Human immunodeficiency virus (HIV) was first isolated in 1983.The causative agent for AIDS is known to be a virus of the retrovirus family called HIV (human immunodeficiency virus).", "Infection with HIV does not, however, immediately give rise to overt symptoms of AIDS.", "Three to six weeks following primary HIV infection more than 50% of individuals develop acute HIV syndrome, which is self limiting.", "Clinical findings seen during this period include fever, pharyngitis, lymphadenopathy, headache, arthralgia, myalgia, malaise, lethargy, nausea, vomiting, diarrhoea, skin rash, mucocutaneous ulceration, meningitis, encephalitis, neuropathy etc.", "The only indication of exposure to the virus may be the presence of antibodies thereto in the blood of an infected subject who is then described as ‘HIV positive’.", "The infection may lie dormant; giving rise to no obvious symptoms, and the incubation period prior to development of AIDS may vary from several months to decades.", "Development of AIDS itself may be preceded by the AIDS-related complex (ARC), which is characterized by unexplained fever, weight loss, chronic cough or diarrhea.", "The development of AIDS and/or ARC is dependent on breakdown of immune system.", "The reasons for the variable period between infection with the virus and breakdown of the immune system in an infected individual are poorly understood.", "Factors at present unknown may trigger proliferation of the virus with consequential disruption of the immune system.", "The victims of the disease are then subject to various infections and malignancies, which, unchecked by the disabled immune system, lead to death.", "Thus HIV is characterized by the “acute HIV syndrome” followed by “asymptomatic stage” with clinical latency.", "Symptomatic stage sets in later with breakdown of immune system, which ultimately leads to the death of the individual infected with HIV.", "Though the disease is caused by virus, the morbidity and mortality associated with disease is due to breakdown of immune system.", "The breakdown of immune system is characterized by decreased CD4+ T lympocyte count.", "Because of this reason 1993 revised classification system for HIV infection is based on CD4+ T lymphocyte counts.", "HIV disease is empirically divided based on CD4 count which is a measure of immunodeficiency.", "a) Early stage CD4+T cell count more than 500 b) Intermediate stage CD4+T cell count 200 to 500.c) Advanced stage CD4+T cell count less than 200.Individuals with nonprogressive HIV disease are found to have steady CD4 counts.", "They are also observed to have strong immune response against the virus.", "There is evidence that in HIV infection, there is a dramatic loss of CD4 T-cells, which results in very rapid development of overt symptoms of AIDS.", "Most AIDs defining opportunistic infections and true malignancies occur in advanced stage of disease where in CD4 count is less the 200 cells/mL.", "CD4 Count and HIV Though HIV is a viral infection, viral load can be determined by reasonable accuracy, CD4 count (a measure of immune status) plays major role in management of HIV due to following reasons.", "1.Morbidity and Mortality in HIV infected individuals is due to opportunistic infections.", "These opportunistic infections define onset of AIDS in HIV +ve individuals.", "The risk of opportunistic disease increases markedly when CD4+ cell count declines to less than 200 cells/mm3.2.CD4 count provides estimate of degree of existing immunodeficiency.", "Immune deficiency is responsible for HIV +ve individuals getting converted to AIDS.", "3.The initiation of antiretroviral therapy is also dependent on CD4 count.", "4.Outcome of antiretroviral therapy is also dependent on CD4 count.", "Higher survival are associated with higher initial CD4 count.", "5.Risk of progression to AIDs defining illness is associated with declining CD4 count.", "The risk is lower with higher CD4 count.", "6.Likelyhood of developing AIDs within 3 years is significantly higher when CD4 count is low (less than 200 CD4+ T cells) compared to high CD4+ T cell count.", "For viral load of greater than 55 k as per RT-PCR the risk is 32.6% if CD4+ T cell count is more than 500 cells/mm3 compared to 85.5% for individuals with CD4 count of less than 200 cells/mm3.7.Similarly for viral load of 20 k-55 k (RT-PCR) the risk of developing AIDs is 9.5% when CD4 count is more than 750 cells/mm3 compared to 40.1% when CD4 count is less than 350 cells/mm3.Goals of Therapy Maximal and durable suppression of viral load.", "Restoration and/or preservation of immunologic function.", "Improvement of quality of life.", "Reduction in HIV related morbidity and mortality.", "The method to treat HIV includes various therapeutic options.", "The options include management of symptoms and infections manifesting in HIV infected individuals at various stages, of the disease.", "The antiretroviral drugs are used to keep the HIV infection (viral load) in control.", "They keep the viral load in control.", "The early antiretroviral drugs like azothymidine delayed progression of disease and had no significant effect on CD4 count.", "Protease inhibitors like indinavir, ritonavir which are introduced recently do improve CD4 count while reducing viral, load.", "All the drugs (antiretroviral) have their own side effects.", "The resistance to drugs is also noted.", "Thus there is need to provide alternate mechanism of treating HIV.", "Since CD4 count is important in maintaining immunity of individual and decreased CD4 counts are associated with morbidity and mortality in HIV infection attempts are made to improve immunity for management of HIV.", "Various efforts have been done towards this end.", "This has resulted in introduction of immune modifying therapies with or without antiretroviral drugs.", "They comprise of antigens, cytokines organisms etc.", "It has surprisingly been found during the course of research by us that formulations of ‘Mycobacterium W’ (Mw) with or without antigenic and/or immunomodulatory material derived from (Mw) is effective for management of Human Immunodeficiency Virus (HIV) disease/infection.", "Prior Art M. vaccae is apparently unique among known mycobacterial species in that heat-killed preparations retain its properties for the use as vaccine and immunotherapeutic.", "For example, M. bovis-BCG vaccines, used for vaccination against tuberculosis, employ live strains.", "Heat-killed M. bovis BCG and M. tuberculosis have no protective properties when employed in vaccines.", "A number of compounds have been isolated from a range of mycobacterial species, which have adjuvant properties.", "The effect of such adjuvants is essentially to stimulate a particular immune response mechanism against an antigen from another species.", "In U.S. Pat.", "No.", "6,001,361 the invention is related to compounds and methods for the treatment of mycobacterial infections including Mycobacterium tuberculosis and Mycobacterium avium.", "The invention is further related to compounds that function as non-specific immune response amplifiers, and the use of such non-specific immune response amplifiers as adjuvants in vaccination or immunotherapy against infectious disease, and in certain treatments for immune disorders and cancer.", "U.S. Pat.", "No.", "4,716,038 discloses diagnosis of, vaccination against and treatment of autoimmune diseases of various types, including arthritic diseases, by administering mycobacteria, including M. vaccae.", "U.S. Pat.", "No.", "6,210,684 describes method for delaying the onset of AIDS using killed M. Vaccae.", "Onset of AIDS is related to decrease in CD4 count is not disclosed in the patent.", "Published studies shows that killed M. Vaccae has no effect on CD4 count in HIV positive individuals.", "International Patent Publication WO 91/01751 discloses the use of antigenic and/or immunoregulatory material from M. vaccae as an immunoprophylactic to delay and/or prevent the onset of AIDS.", "International Patent Publication WO 94/06466 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for therapy of HIV infection, with or without AIDS and with or without associated tuberculosis.", "U.S. Pat.", "No.", "6,001,361 discloses an invention that relates generally to the treatment by vaccination or immunotherapy of skin disorders such as psoriasis, atopic dermatis, allergic contact dermatitis, alopecia areata, and the skin cancers basal cell carcinoma, squamous cell carcinoma and melanoma.", "In particular, the invention is related to the use of compounds, which are present in or have been derived from Macobacterium vaccae (M. vaccae) or from the culture filtrate of M. vaccae.", "U.S. Pat.", "No.", "5,599,545 discloses the use of mycobacteria, especially whole, inactivated M. vaccae, as an adjuvant for administration with antigens, which are not endogenous to M. vaccae.", "This publication theories that the beneficial effect as an adjuvant may be due to heat shock protein 65 (hsp 65).", "International Patent Publication WO 92/08484 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of uveitis.", "International Patent Publication WO 93/16727 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of mental diseases associated with an autoimmune reaction initiated by an infection.", "International Patent Publication WO 95/26742 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for delaying or preventing the growth or spread of tumors.", "Vacce is not associated with change in CD4 count or viral load in HIV positive individuals.", "It does not provide any relief in HIV +ve individual.", "However in spite of various patents issued in relation to mycobacterium vaccae, it fails to provide any significant change in CD4 count as well as viral load in individuals who are HIV positive.", "Similarly attempts have been made to improve CD4 count using immunomodulator from various sources.", "Remune is a HIV-1 specific immunogen in incomplete Freund's adjuvant.", "It includes inactivated HIV-1 from which gp 120 is depleted.", "It is found to be safe with immunogenic potential in persons infected with HIV.", "In initial studies it was found to improve CD4-cell count in asymptomatic HIV cohort not taking antiretroviral agents.", "It is given intramuscularly as an injection into the triceps muscles.", "The recent study by Sukeepaisamcharoen w et at suggests that remune therapy is associated with stabilization of CD 4-cell counts.", "It is also suggests that it may be of value in participants with higher CD4+ T cell count.", "One such immunomodulator consists of mixture of antigens of inactivated bacteria with antigens of influenza virus (poly antigenic immunomodulator).", "It has not been possible to achieve improvement in CD4 count using it.", "SB-73 is another immunomodulator made up of substance produced by pencillium P(PB-73 strain).", "In a small study it is found to improve CD4 count in majority (10/14) of individuals, infected with HIV when given intramuscularly in a dose of 5 mgm.", "Reticulose, a peptide-nucleic acid is another immunomodulator found to be useful in improving CD4 count in HIV infected individuals when given subcutaneously.", "It was given as two 1 ml subcutaneous (SC) injections per day for two weeks followed by 1 ml SC per day every other week for a total 60 days (30 days total treatment).", "It resulted in a significant improvement, in CD4 count in absence of any other antiretroviral therapy.", "Thymosin α1 is a 28-aminoacid peptide.", "It was evaluated for its efficacy to improve CD4 along with interleukin-2.It was found to have no significant effect.", "Grenutocyte colong stimulating factor (Filgrastim) has also been evaluated to improve immune response in HIV without much success.", "OKT3, a CD3 monoclonal antibody, has been successfully used in management of HIV along with antiretroviral and Interleukin-2 in three patients.", "Of various cytokines used in management of HIV, Interleukin-2 (IL-2) is extensively studied and used.", "It is used as injection to be administered intravenously or subcutaneously.", "It is found to improve CD4 count significantly when used alone or along with antiretroviral drugs.", "It is given as intermittent therapy the side effects seen are sometimes intolerable.", "They are seen only during period of active administration.", "The improvement seen in CD4 count is found to be stable.", "Other cytokines used in management of HIV includes IL-12 and IL-15.U.S.", "Pat.", "No.", "5,759,992 provides low molecular weight glycopeptide with a molecular weight of 919.2 dalton which is derived from supernatant of disrupted cells maintaining temprature of 4° C. through out the process.", "This is obtained from bacteria which includes E coli and Mycobacterium.", "It is found to improve CD4 count in normal mice.", "It is not evaluated in HIV+ve animals.", "U.S. Pat.", "No.", "5,871,732 describes methods for preventing or treating AIDS, AIDS related complex and human immunodeficiency virus infection by anti-CD4 antibody homologs to DNA sequences of encoding such homologs.", "Mycobacterium w is a non-pathogenic, cultivable, atypical mycobacterium, with biochemical properties and fast growth characteristics resembling those belonging to Runyons group IV class of Mycobacteria in its metabolic and growth properties but is not indentical to those strains currently listed in this group.", "It is therefore thought that (Mw) is an entirely new strain.", "The species identity of Mw has been defined by polymerase chain reaction DNA sequence determination.", "It has been found to share antigens with Mycobacterium leprae and Mycobacterium tuberculosis.", "It is found to provide prophylaxis against leprosy in humans by converting lepromin negative individuals to lepromin positivity.", "It is also found to provide prophylaxis against tuberculosis in animals.", "In leprosy it is also found to reduce duration of therapy for bacterial killing, clearance as well as clinical cure when used along with multi drug therapy.", "REFERENCE 1.Immunological parameters modified in HIV disease by the macrophage activity immunomodulator WF 10 (a phase II pathogenesis study) Herndier B; Lull R Ah Ching O; Broz M; Kuehne F W; Kahn J Int Conf AIDS.", "1998;12:347-8 2.Changes in viral expression and cytokine profile induced by a polyantigenic immunomodulator in HIV-infected peripheral blood mononuclear cells.", "Rios Z; Rios E O; Garcia M I; De Leon C; Guzman L M; Rodriguez W; Romero D; Hunter R. Cell Mol Biol (Noisy-le-grand).", "1995;41 Suppl 1:s93-101.3.Controlled clinical trial of reticulose, a peptide-nucleic acid with Immunomodulator activity, in patients with HIV infection.", "Levett P N; Roach T C; Hirschman S Z; Broome H; Fraser H S Abstr Gen Meet Am Soc Microbiol.", "1998 May 17-21; 98:53 4.Survival and quality of life in HIV-positive patients treated with poly antigenic immunomodulator.", "Colon J I; Encarnacion G; Ortiz Santini M R; Rodriguez Malave M T; Santiago Delpin E A; Marchand A M P R Health Sci J.", "1997 March; 16(1):9-14.5.SB-73 immunomodulator: phase II clinical trial on HIV infection.", "Bellucci S; Abreu W; Bertazzoli R; Tomita F; Lourenco C; Machodo L; Silva O Int Conf AIDS.", "1992 Jul.", "19-24;8(3):54 3.Interleukin-15 Triggers Activation and Growth of the CD8 T-Cell Pool in Extravaxcular Tissues of Patients With Acquired Immunodificiency Syndrome.", "Agostini C, Trentin L, Sancetta R, Facco M, Tassinari C, Cerutti A, Bortolin M, Milani A, Siviero M, Zambello R, Semenzato G. Blood, Vol 90, No 3 (August 1), 1997:pp 1115-1123 4.Ethylene Glycol-Modified Interleukin-2 and Thymosin α1 in Human immunodefiency Virus Type I Infection Journal of Infecious Disease, 1996; 173; 1005-81 5.OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T cell depletion.", "van Praag R M, Prins J M, Roos M T, Schellekens P T, Ten Berge I J, Yong S L Schuitemaker H, Eerenberg A J, Jurriaans S, de Wolf F, Fox C H, Goudsmit J. Miedema F, Lange J M J Clin Immunol 2001 May;21(3):218-26 6.How effective are complementary therapies for HIV and AIDs?—A systemic review.", "Ozsoy M, Ernst E. Int J STD AIDS 1999 October; 10 (10): 629-35 7.Mechanism of HIV persistence: implications for vaccines and therapy.", "Bremermann H J J Acquir Immune Defic Syndr Hum Retrovirol 1995 Aug. 15;9(5):459-83.8.Both serum HIV type 1 RNA levels and CD4 lymphocyte counts predict clinical outcome in HIV type 1— infected subjects with 200 to 500 CD4+cells per cubic millimeter.", "AIDs clinical trials group study 175 virology study team.", "Kim S, Hughes M D, Hammer S M, Jackson J B, DeGruttola V, Katzenstein D A.", "AIDs Res Hum Retroviruses 2000 May 1;16(7):645-53.9.Do HIV type 1 RNA levels provide additional prognostic value to CD4 (+) T lymphocyte counts in patients with advanced HIV type 1 infection?", "AIDs Res Hum Retroviruses 2001 Aug. 10;17(12):1099-105.10.Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolesccnts.", "Aug. 13, 2001(This guidelines were developed by the Panel on Clinical Practices for Treatment of HIV Infection convened by the Department of Health and Human Services (DHHS) and the Henry J. Kaiser Family Foundation.", "Leadership of the Panel consists of Anthony S. Fauci, National Institutes of Health, Bethesda, Md.", "(co-chair); Eric P. Goosby, DHHS, Washington, D.C., (co-convener); and Jennifer Kates, Henry J. Kaiser Foundatin, San Francisco, Calif., (co-convener).", "11.A controlled Trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less.", "Arduino J M, Fischl M A, Stanley K, Collier A C, Spiegelman D. The New Eng.", "Journal of Med.", "1997 Sep. 337(11):725-733.12.Treatment with Indinavir, Zidovudine, and Lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy.", "scott m. hammer, m.d., kathleen e. squires, m.d., michael d. hughes, ph.d., janet m. grimes, m.s.", "lisa m. emeter, m.d., judith s. currier, m.d., joseph j. eron, jr., m.d., judith e. feinberg, m.d., henry h. balfour, jr., m.d., lawrence r. eyton, m.d., jeffrey a. chodakewitz, m.d., et al.", "The New Eng.", "Journal of Med.", "1997 September 337(11):734-739.13.Ramdomised placebo-controlled trial of ritonavir in advanced HIV-1 disease Yazdanpanah Y, Chene G, Losina E, Goldie S J, Merchadou L D, Alfandari S, Seage G R 3rd, Sullivan L, Marimoutou C, Paltiel A D, Salamon R, Mouton Y, Freedberg K A. Int J Epidemiol.", "2001 August;30(4):864-71.22.Randomised trial of intradermal Mycobacterium vaccae or intradermal hepatitis 13 immunisation in children with HIV infection.", "Johnson D, Waddell R D, Pelton S I, Jaeger A S, Modlin J F, Yogev R, Morin P, Arbeit R D, von Reyn C F. Vaccine.", "1999 Jun.", "4; 17(20-21):2583-7.23.Immunization of HIV-infected adults with a three-dose series of inactivated Mycobacterium vaccae.", "Marsh B J, Fordlani von Reyn C, Arbeit R D, Morin P. Am J Med Sci.", "1997 June;313(6):377-83.24.Safety and immunogenicity of a five-dose series of inactivated Mycobacterium vaccae vaccination for the prevention of HIV-associated tuberculosis.", "Waddell R D, Chintu C, Lein A D, Zumla A, Karagas M R, Baboo K S, Habbema J D, Tosteson A N, Morin P, Tvaroha S, Arbeit R D, Mwinga A, von Reyn C F. Clin Infect Dis.", "2000 June;30 Suppl 3:S309-15.SUMMARY OF THE INVENTION According to present invention, immunomodulator made from ‘Mycobacterium w’ (Mw) is found to be useful in the management of HIV infection.", "We have now found that the same therapeutic agent not only delays development of AIDS in patients infected by HIV, but also is capable of causing regression, or even removal, of overt symptoms of AIDS even in patients where the disease is far advanced.", "These effects have been found in patients suffering also from tuberculosis.", "These effects are also seen in patients who are suffering from HIV infection with or without AIDS and without associated tuberculosis.", "The immunomodulator as per present invention is also found useful in relieving symptoms of HIV.", "Therapeutic agent which may be used in the present invention resembles Mw a non-pathogenic, cultivable, atypical mycobacterium, with biochemical properties and fast growth characteristics resembling those belonging to Runyons group IV class of Mycobacteria in its metabolic and growth properties but is not indentical to those strains currently listed in this group.", "It is therefore thought that (Mw) is an entirely new strain.", "The species identity of Mw has been defined by polymerase chain reaction DNA sequence determination and differentiated from thirty other species of mycobacteria.", "It however differs from those presently listed in this group in on respect or the other.", "By base sequence analysis of a polymorphic region of pattern analysis, it has been established that Mw is a unique species distinct from many other known mycobacterial species examined which are: M. avium, M. intracellulare, M. scrofulaceum, M. kansasii, M. gastri, M. gordonae, M. shimoidei, M. malmoense, M. haemophilum, M. terrae, M. nonchromogenicum, M. triviale, M. marinum, M. flavescens, M. simian, M. szulgai, M. xenopi, M. asciaticum, M. aurum, M. smegmatis, M. vaccae, M. fortuitum subsp fortuitum, M. fortuitum subsp.", "Peregrinum, M. chelonae subsp.", "Chelonae, M. chelonae subsp.", "Abscessus, M. genavense, M. tuberculosis, M. tuberculosis H37Rv, M. paratuberculosis.", "The object of the present invention is to provide an immunomodulator Mycobacterium w containing ‘Mycobacterium w’ (Mw) with or without obtained from Mw for the prophylaxis or therapy of AIDS or AIDS related complex, to a subject exposed to HIV infection or is HIV positive with or without, overt symptoms of AIDS.", "Yet another object of the invention is to provide immunomodulator derived from mycobacterium w that are useful for the management of HIV infection.", "Yet another object of the invention is to provide immunomodulator derived from Mycobacterium w to improve immune function of HIV +ve subjects.", "Yet another object of present invention is to provide immunomodulator to improve CD4 count in HIV infected individuals.", "Yet another object of present invention is to provide immunomodulator effective in ameliorating symptoms associated with HIV infection.", "Yet another object of present invention is to provide an immunomodulator effective in management of opportunistic infections of associated with HIV infection.", "Yet another object to present invention is to provide an immunomodulator useful in improving immune function of HIV +ve subjects in presence or absence of antiretroviral drugs.", "Yet another object of the invention is to provide methods for the treatment of HIV, which results in the amelioration of symptoms of symptomatic stage of the disease.", "It is yet another object of the invention to provide for a method of treatment of HIV infection that results in improved CD4+ T cell count.", "DETAILED DESCRIPTION OF THE INVENTION In accordance with the invention the composition of immunomodulator the method of preparation, HPLC charachteristic its safety and tolerability, methods of use and outcome of treatments are described in following examples.", "The following are illustrative examples of the present invention and scope of the present invention should not be limited by them.", "EXAMPLE 1 The Immunomodulator Compositions A.", "Each dose of 0.1 ml of therapeutic agent contains: Mycobacterium w., (heat killed) 0.50 × 109 Sodium Chloride I.P.", "0.90% w/v Thiomerosal I.P.", "0.01% w/v (As a Preservative) Water for injection I.P.", "q.s.", "to 0.1 ml B.", "Each dose of 0.1 ml of therapeutic agent contains Extract of Mycobacterium w after sonication from 1 × 1010 Mycobacterium w Sodium Chloride I.P.", "0.90% w/v Thiomerosal I.P.", "0.01% w/v (As a Preservative) Water for injection I.P.", "q.s.", "to 0.1 ml C. Each dose of 0.1 ml of therapeutic agent contains Methanol Extract of 1 × 1010 Mycobacterium w Sodium Chloride I.P.", "0.90% w/v Thiomerosal I.P.", "0.01% w/v (As a Preservative) Water for injection I.P.", "q.s.", "to 0.1 ml D. Each dose of 0.1 ml of therapeutic agent contains Chloroform Extract of 1 × 1010 Mycobacterium w Sodium Chloride I.P.", "0.90% w/v Thiomerosal I.P.", "0.01% w/v (As a Preservative) Water for injection I.P.", "q.s.", "to 0.1 ml E. Each dose of 0.1 ml of therapeutic agent contains Acetone Extract of 1 × 1010 Mycobacterium w Sodium Chloride I.P.", "0.90% w/v Thiomerosal I.P.", "0.01% w/v (As a Preservative) Water for injection I.P.", "q.s.", "to 0.1 ml F. Each dose of 0.1 ml of therapeutic agent contains Ethanol Extract of 1 × 1010 Mycobacterium w Sodium Chloride I.P.", "0.90% w/v Thiomerosal I.P.", "0.01% w/v (As a Preservative) Water for injection I.P.", "q.s.", "to 0.1 ml G. Each dose of 0.1 ml of therapeutic agent contains Liticase Extract of 1 × 1010Mycobacterium w Sodium Chloride I.P.", "0.90% w/v Thiomerosal I.P.", "0.01% w/v (As a Preservative) Water for injection I.P.", "q.s.", "to 0.1 ml H. Each dose of 0.1 ml of therapeutic agent contains Mycobacterium w (heat killed) 0.5 × 107 Extract of mycobacterium w obtained 1 × 103 Mycobacterium w by disruption, solvent extraction or enzymatic extraction.", "Sodium Chloride I.P.", "0.90% w/v Thiomerosal I.P.", "0.01% w/v (As a Preservative) Water for injection I.P.", "q.s.", "to 0.1 ml EXAMPLE 2 The Process of Preparing Immunomodulator A. Culturing using of Mycobacterium w. i) Preparation of calture medium.", "Mycobacterium w is cultured on solid medium like L J medium or liquid medium like middle brook medium or sauton's liquid medium.", "For better yield middle brook medium is enriched.", "It can be preferably enriched by addition of glucose, bactotryptone, and BSA.", "They are used in ratio of 20:30:2 preferably.", "The enrichment medium is added to middle brook medium.", "It is done preferably in ratio of 15:1 to 25:1 more preperably in ratio of 20:1.ii) Bioreactor operation a) Preparation of vessel: The inner contact parts of the vessel (Joints, mechanical seals, o-ring/gasket grooves, etc.)", "should be properly cleaned to avoid any contamination.", "Fill up the vessel with 0.1 N NaOH and leave as such for 24H to remove pyrogenic materials and other contaminants.", "The vessel is then cleaned first with acidified water, then wit ordinary water.", "Finally, the vessel is rinsed with distilled water (3 times) before preparing medium.", "b) Sterilization of bioreactor The bioreactor containing 9 L distilled water is sterilized with live steam (indirect).", "Similarly the bioreactor is sterilized once more with Middlebrook medium.", "The other addition bottles, inlet/outlet air filters etc.", "are autoclaved (twice) at 121° C. for 15 minutes.", "Before use, these are dried at 50° C. oven.", "c) Environmental parameter i. Temprature: 37±0.5° C. ii.", "pH: 6.7 to 6.8 initially.", "B.", "Harvesting and concentrating It is typically done at the end of 6th day after culturing under aseptic condition.", "The concentration of cells (palletisation) is done by centrifugation.", "C. Washing of cells The pallet so obtained is washed minimum three times with normal saline.", "It can be washed with any other fluid which is preferably isotonic.", "D. Adding pharmaceutically acceptable carrier.", "Pyrogen free normal saline is added to pallet.", "Any other pyrogen free isotonic fluid can be used as a pharmaceutical carrier.", "The carrier is added in amount so as get to desired concentration of active in final form.", "E. Adding preservative To keep the product free from other contaminating bacteria for its self life preservative is added.", "Preferred preservative is thiomesol which is used in final concentration of 0.01% w/v.", "F. Terminal Sterilization Terminal sterilization can done by various physical methods like application of heat or ionizing radiation or sterile filtration.", "Heat can be in the form of dry heat or moist heat.", "It can also be in the form of boiling or pasturisation.", "Ionizing radiation can be ultraviolet or gamma rays or mircrowave or any other form of ionizing radiation.", "It is preferable to autoclave the final product.", "This can be done before after filling in a final packaging.", "G. Quality Control i.", "The material is evaluated for purity, sterility.", "ii.", "The organisms are checked for acid fastness after gram staining.", "iii.", "Inactivation test: This is done by culturing the product on L J medium to find out any living organism.", "iv.", "Pathogenicity and/or contamination with pathogen.", "The cultured organisms are infected to Balb/c mice.", "None of the mice should die and all should remain healthy and gain weight.", "There should not be any macroscopic or microscopic lesions seen in liver, lung spleen or any other organs when animals are killed up to 8 weeks following treatment.", "v. Biochemical Test: The organism is subjected to following biochemical tests: a) Urease b) Tween 80 hydrolysis c) Niacin test d) Nitrate reduction test The organism gives negative results in urease, tween 80 hydrolysis and niacin test.", "It is positive by nitrate reduction test.", "H. Preparation of constituents of Mycobacterium w. The constituents of Mycobacterium w can be prepared for the purpose of invention by: I.", "Cell disruption II.", "Solvent extration III.", "Enzymatic extraction.", "The cell disruption can be done by way of sonication or use of high pressure fractionometer or by application of osmotic pressure ingredient.", "The solvent extraction can be done by any organic solvent like chloroform, ethanol, methanol, acetone, phenol, isopropyl alcohol, acetic acid, urea, hexane etc.", "The enzymatic extraction can be done by enzymes which can digest cell wall/membranes.", "They are typically proteolytic in nature.", "Enzyme liticase and pronase are the preferred enzymes.", "For the purpose of invention cell constituents of Mycobacterium w can be used alone in place of mycobacterium w organisms or it can be added to the product containing mycobacterium w. Addition cell constituents results in improved efficacy of the product.", "EXAMPLE 3 Characteristics of Constituents of Mycobacterium w by HPLC Analysis The constituents of mycobacterium w. used for the purpose of invention when subjected to HPLC analysis gives a single peak at 11 minutes.", "No other significant peaks are found beyond.", "The peak is homogenous and devoid of any notch suggesting homogeneity of material obtained HPLC analysis was done using a waters system high performance liquid chromatography apparatus Column: Novapak c1860A, 4 μm, 3.9×150 mm.", "The guard column: Novapak c 18 Column Temperature: 30° C. Flow rate: 2.5 ml/min Injection volume: 25 μL.", "Mobile phase: Solvent A: HPLC grade methanol.", "Solvent B: HPLC grade methylene chloride Binary gradient: The HPLb gradient initially comprised 98% (v/v) methanol (solvent B).", "The gradient was increased linearly to 80%.", "A and 20% B at one minute; 35% A and 65% B at 10 minutes, held for 5 seconds and then decreased over 10 seconds back to 98% A and 2% B.", "EXAMPLE 4 Safety of Immunomodulator Mycobacterium w when used in healthy animals or humans is found to be safe well tolerated and has no effect on any organ system, biochemistry or hematology including various blood cells.", "It is found not to cause lymphocytosis and nor change ratio of CD4:CD8 cells as seen with various other nonspecific immune stimulation.", "The only effect seen is at injection site.", "It includes morphologically formation of erythema, induration ulceration and scar formation.", "Histologically the injection site is found to have infiltration of various kinds of lymptocytes, plasma cells, giant cells giving a histological picture of epitheloid cell granulomas.", "EXAMPLE 5 Effect of Immunomodulator on Symptomatic HIV +ve Patients; 11 subjects who were HIV +ve and getting recurrent attacks of fever, upper respiratory tract infection, and diarrhea were given Mycobacterium w (5×108) intradermally.", "All improved and showed no recurrence of symptoms after 2nd month of treatment.", "EXAMPLE 6 Effect of Immunomodulator on CD4 Count in HIV 1 Infected Adult Patients a) When Immunomodulator Alone is Used: In 17 HIV positive individuals who had symptoms attributed to HIV and seeking help for the same Mycobacterium w was used as a sole therapy.", "Mycobacterium w was administered intradermally over a deltoid region.", "The amount of Mycobacterium injected was 5×108 in a single injection.", "At the time of inclusion in study Mycobacterium w was given as intradermal injection over both the deltoids making a total dose of 1×109 Mycobacterium w subsequently at the interval of a month a single intradermal injection was given over a deltoid region which included 5×108 organisms.", "There was no mortality or morbidity seen during trial.", "All subjects tolerated the therapy well and completed the trial.", "Symptomatic relief was seen within two months of initiation of therapy.", "All subjects were evaluated for their CD4 count at the beginning of therapy and 5 months later.", "The mean pretreatment CD4 count was 204.70 (range 430-6).", "All subjects showed improvement in CD4 count.", "At the end of 5 months mean change in CD4 count was 163.17 (range 8-628).", "In seven (41.2%) individuals increase in CD4 count was more than 80%.", "Improvement in CD4 count was less than 40% in 3 individuals (17.6%) only.", "The therapy was not associated with any side effects systemically.", "These were a local reaction seen at the site of injection.", "It was in the form of erythematous reaction which was associated with induration.", "It progressed to ulceration at the site of injection in few which healed spontaneously leaving behind a this scar.", "None of the subjects participating in a trial received any antiretroviral therapy.", "Summary of results A No.", "of HIV +ve subjects 17 B Mean base line CD4 count 204.70 C Mean post treatment CD4 count 368.93 Range 850 to 32 D Change in CD4 count 163.17 (104.43%) Range 628 to 8 (433.33 to 4.08%) Details of change in CD4 count in each individuals.", "(Table 1) TABLE 1 PRETREATMENT POST- CD4 TREATMENT CHANGE CHANGE NO.", "COUNT CD4 (NO.)", "(%) 1 168 270 102 60.7 2 302 948 628 196 3 298 453 155 52 4 280 496 216 77 5 120 182 62 51.6 6 330 620 290 88 7 430 850 420 97.6 8 226 338 112 49.5 9 294 519 225 76.7 10 47 61 14 29.78 11 112 224 112 100 12 196 204 8 4.08 13 186 384 198 106.5 14 182 270 88 48.35 15 42 110 68 161.90 16 6 32 26 433.33 17 261 311 50 19.15 MEAN 204.70 368.93 163.17 104.43 the result is much better than what is achieved with use of interleukin-2 when LISCLI along with two antiretroviral drugs.", "It is also better than what is achieved with HAART (Highly Active AntiRetroviral Therapy) alone for the same period.", "It is also worth noting, that all patients showed improvement of CD4 count.", "Natural course of disease in abasence of antiretroviral therapy is associated with decline in CD4 count month by month.", "On average 12 cells are lost per month as per ziduvadine study for symptomatic individuals published in New Eng.", "J. Med.", "In a large cohort of 2664 HIV+ve asymptomatic persons CD4 count decline is 4.6 cells/month.", "Irrespective of no.", "of CD4 count at the beginning of therapy improvement in CD4 count was seen in all individuals.", "The pretreatment CD4 count ranged from 6 to 430.The regression analysis of improvement (FIG.", "5) suggests that improvement seen over five month period is proportionate to initial CD4 count.", "Higher the count better is improvement.", "b) Two Antiretroviral Drugs (NRTI)+Immunomodulator In an another set of subjects who were HIV+ve and had symptoms related to HIV.", "Mycobacterium w was used along with two antiretroviral drugs (both NRTI).", "None of them had received any anti-retroviral prior to these.", "Mycobacterium w was administered intradermally over a deltoid region.", "The amount of Mycobacterium w injected was 5×108 in a single injection.", "At the time of inclusion in study Mycobacterium w was given as intradermal injection over both the deltoids making a total dose of 1×108 Mycobacterium w subsequently at the interval of a month a single intradermal injection was given over a deltoid region which included 5×108 organisms.", "There was no mortality or morbidity seen during trial.", "All subjects tolerated the therapy well and completed the trial.", "Symptomatric relief was seen within two months of initiation of therapy.", "All subjects were evaluated for their CD4 count at the beginning of therapy and 5 months later.", "The mean pretreatment CD4 count was 200.99 (286 TO 96).", "All subjects showed improvement in CD4 count.", "At the end of 5 months mean change in CD4 count was 137.37(range 24-588) The therapy was not associated with any side effects systemically.", "These were a local reaction seen at the site of injection.", "It was in the form of erythematous reaction which) was associated with induration.", "It progressed to ulceration at the site of injection in few which healed spontaneously leaving behind a this scar.", "None of the subjects participating in a trial received any antiretroviral therapy.", "Summary of Results A No.", "of HIV +ve subjects 16 B Mean base line CD4 count 200.99 C Mean post treatment CD4 count 338.37 Range 860 to 199 D Change in CD4 count 137.37 (68.44%) Range 58 to 24 (294.15 to 16%) Details of change in CD4 count in each individuals.", "(Table 2) TABLE 2 PRETREATMENT POST CD4 TREATMENT CHANGE NO.", "COUNT CD4 COUNT (NO.)", "CHANGE(%) 1 162 238 76 47 2 192 240 48 25 3 286 860 574 200 4 142 199 57 40 5 250 290 40 16 6 196 230 34 17.3 7 238 504 266 111.76 8 216 328 112 51.85 9 236 824 588 249.15 10 194 236 42 21.64 11 96 210 114 118.75 12 262 319 57 21.75 13 244 310 66 27.04 14 160 210 50 31.25 15 230 280 50 21.73 16 112 136 24 21.42 Mean 200.99 338.37 137.37 68.44 All subjects were evaluated for their CD4 count at the beginning of therapy and 5 months later.", "None of the subjects showed deterioration in CD4 count.", "All irrespective of pretreatment CD4 count (Range 96 to 286) showed improvement in CD4 count.", "Natural course of disease suggest minimal or no change in CD4 when two antiretroviral drugs are used as used in this study.", "Thus improvement seen in the steady is significantly much more and can not be attributed to antiretroviral therapy used in the study.", "Regression analysis (FIG.", "6) shows that improvement seen in CD4 count is proportionate to the initial CD4 count.", "Higher the initial count better is improvement following therapy comparison of improvement between patients receiving antiretroviral therapy (two drugs) and those not receiving therapy shows that rate of improvement is better without use of two antiretroviral drugs when initial CD4 count is low.", "However when initial CD4 count is high the rate of improvement is better when two anti-retroviral drugs are used.", "c) HAART Theray+Immunomodulator In an another set of subjects who were HIV+ve and had symptoms related to HIV.", "Mycobacterium w was administered along with HAART therapy (three drugs).", "None of them had received any anti-retroviral prior to this.", "Mycobacterium w was administered intradermally over a deltoid region.", "The amount of Mycobacterium w injected was 5×108 in a single injection.", "At the time of inclusion in study Mycobacterium w was given as intradermal injection over both the deltoids making a total dose of 1×108 Mycobacterium w subsequently at the interval of a month a single intradermal injection was given over a deltoid region which included 5×108 organisms.", "There was no mortality or morbidity seen during trial.", "All subjects tolerated the therapy well and completed the trial.", "Symptomatic relief was seen within two months of initiation of therapy.", "All subjects were evaluated for their CD4 count at the beginning of therapy and 5 months later.", "The mean pretreatment CD4 count was 213.23 (range 536-40).", "All subjects showed improvement in CD4 count.", "At the end of 5 months mean change in CD4 count was 258.79 (range 40-887).", "The therapy was not associated with any side effects systemically.", "These were a local reaction seen at the site of injection.", "It was in the form of erythematous reaction which was associated with induration.", "It progressed to ulceration at the site of injection in few which healed spontaneously leaving behind a this scar.", "None of the subjects participating in a trial received any antiretroviral therapy.", "Summary of Results A No.", "of HIV +ve subjects 17 B Mean base line CD4 count 213.23 C Mean post treatment CD4 count 445.58 Range 1423 to 130 D Change in CD4 count 258.79 (155.85%) Range 887 to 40 (338.88 to 17.4%) Details of change in CD4 count in each individuals.", "(Table 3) TABLE 3 PRETREATMENT POST CHANGE CHANGE NO CD4 COUNT TREATMENT (NO.)", "(%) 1 72 220 148 205 2 130 230 100 77 3 40 130 90 225 4 230 270 40 17.4 5 127 276 149 117 6 148 336 188 127 7 230 490 260 113.04 8 356 950 594 166.85 9 199 432 233 117.08 10 236 539 303 128.38 11 204 660 456 223.5 12 536 1423 887 165.48 13 108 269 161 149.07 14 92 198 106 115.21 15 203 582 379 186.69 MEAN 194.06 466.99 266.90 137.06 16 72 316 244 338.88 17 60 212 152 253.33 MEAN 66 264 198 296.10 MEAN 213.23 445.58 258.79 155.85 All subjects were evaluated for change in CD4 count at the beginning of therapy and end of therapy.", "All patients showed significant improvement in CD4 count.", "The patients no.", "1 to 15 had NNRTI as third drug.", "The patient No.", "16 and 17 had protease inhibitor used as third drug.", "The improvement was significantly more than even reported in literature.", "The improvement in CD4 count was significantly more when protease inhibitor is used compared to when NNRTI is used as part of HAART therapy.", "Regression analysis of results (FIG.", "7) suggests that rate of improvement seen is more or less identical irrespective of initial CD4 count.", "It was little lower when initial CD4 count was higher compared to when it was lower.", "EXAMPLE 7 Effect of Immunomodulator on CD4 Count in HIV-1 Infected Children Effect of Immunomodulator in children is also evaluated.", "Immunomodulator was given as intradermal injection of 0.1 ml every month over a deltoid region for five months.", "Of the five children treated with Immunomodulator alone.", "All showed improvement.", "Children TABLE PRE AND POST Treatment CD4 COUNT POST CHANGE CHANGE No PRETREATMENT TREATEMENT (NO.)", "(%) 28 506 1100 594 117.39 29 246 720 474 192.68 30 398 562 164 41.21 31 720 1230 510 70.83 32 1120 1460 340 30.36 MEAN 598 1014.4 416.4 90.49 Thus effect of Immunomodulator is not restricted to age of HIV positive patients.", "EXAMPLE 8 Effect of Immunomodulator in HIV-2 Infected Individuals In another set of three subjects (HIV II positive), Immunomodulator alone was given as ii therapy.", "It was given intradermally over a deltoid region.", "The amount of Immunomodulator injected was 0.1 ml in a single injection.", "At the time of inclusion in study Immunomodulator was given as intradermal injection over both the deltoids making a total dose of 0.2 ml subsequently at the interval of a month a single intradermal injection was given over a deltoid region.", "All the three subjects showed improvement in CD4 count.", "No.", "of Pre-treatement Post Treatment Change in subjects Gender CD4 count CD4 count CD4 count % 46 Female 268 312 54 17.30 32 Female 324 402 78 19.40 49 Male 363 427 64 14.98 Thus effect of Immunomodulator is not found to be limited to HIV 1 only.", "EXAMPLE 9 Effect of Immunomodulator in HIV +ve Subjects with Tuberculosis Cervical Lymphadenopathy Not Responding to Five Anti Tuberculosis Drugs Seven HIV +ve subjects with tuberculosis cervical lymphadenopathy not responding to five anti tuberculosis drugs were given Immunomodulator intradermally.", "All had initial increase in size of cervical lymph node, which became erythematous.", "Within 3 weeks the size of lymph nodes decreased in size and over two months lymphodenopathy healed completely.", "Mycobacterium w has been used in leprosy for faster clearance of M. Leprae from lesions, and improved clinical out come making it possible to release the patients from therapy (MDT, multi drug therapy) at an earlier date.", "It has also been found to convert lepromin negative persons to lepromin positive and there by provide immunity against leprosy.", "According to present invention it is found useful in management of HIV.", "It is seen that HIV related symptoms disappear quickly when Mycobacterium w is administered.", "It is also found to improve immunity in the form of CD4 count.", "It does all this in absence of any anti retroviral therapy.", "However when anti-retroviral are included along with Mycobacterium w in the form of HAART therapy, response is augmented.", "The lack of systemic side effects as seen with all other therapies makes it even more suitable.", "FIG.", "1.HPLC analysis of crude extract obtained after disruption of Mycobacterium w cell by sonication.", "FIG.", "2 HPLC analysis of methanol extract of Mycobacterium w. FIG.", "3 HPLC analysis of chloroform extract of Mycobacterium w. FIG.", "4 HPLC analysis of acetone extract of Mycobacterium w. FIG.", "5 Regression analysis of Pre & Post treatmen change in CD4 count when immunomodulator is used alone FIG.", "6 Regression analysis of Pre & Post treatmen change in CD4 count when immunomodulator is used with two antiretroviral drugs (NRTI) FIG.", "7 Regression analysis of Pre & Post treatmen change in CD4 count when immunomodulator is used along with HAART therapy" ] ]	Patent_10468467
[ [ "Method for secure storing of personal data and for consulting same, chip card, terminal and server used to carry out said method", "A method for the secure storing of personal data and for consulting same is carried out in a terminal that is connected to a chip card reader and fitted with a man-machine interface.", "A browser executing on the terminal conducts a dialogue with a remote server by way of a communication network.", "Pages of data are viewed on a display device of the interface.", "Personal data is input by a user in response to the pages displayed, and the data is stored locally for consultation and remotely on the server for saving." ], [ "1.A method for the secure storage of personal data and for consultation, comprising the following steps: using a terminal connected to a chip-card reader and provided with a man-machine interface comprising a display and data input means, using a browser capable of dialoguing with a remote server through a communication network, from the said terminal, displaying pages of data with the said display means, inputting personal data of a user in response to the pages displayed and storing them locally for consultation and remotely on the server for saving.", "2.A storage method according to claim 1, wherein the data pages are supplied by the server.", "3.A storage method according to claim 1, wherein the data pages are supplied during a communication and entry.", "4.A storage method according to claim 1, wherein the data entry is carried out on line.", "5.A storage method according to claim 1, wherein the personal data are recorded locally on a chip card connected to said reader and a copy is saved on the server.", "6.A storage method according to claim 3, wherein the saving of the copy remotely is carried out substantially simultaneously with the recording locally.", "7.A storage method according to claim 1, wherein the personal data are encrypted by a card connected to said reader before being saved.", "8.A storage method according to claim 7, wherein the personal data are encrypted by means of an enciphering algorithm using one or more keys saved in the card.", "9.A storage method according to claim 8, wherein the enciphering key or keys are also saved by an entrusted entity.", "10.A storage method according to claim 1, wherein the browser comprises the functions of a browser of the type defined by the S@T standard (SIM Alliance Toolbox).", "11.A storage method according to claim 10, wherein pages supplied by the server are pages of the type defined by the S@TML language.", "12.A chip card comprising a processing unit and one or more program memories comprising programs including the operating system of the card, and further including a browser program capable of dialoguing with a distant server through a terminal connected to a chip card reader, provided with a man-machine interface, and wherein the browser permits the entry of personal data by a user of the terminal on pages of data and their storage locally in the card for consultation and remotely on the server.", "13.A chip card according to claim 12, wherein said card is a SIM card.", "14.A chip card according to claim 12, wherein the browser comprises the functions of a browser of the type defined by the S@T (SIM Alliance Toolbox) standard.", "15.A communication terminal for implementing the method according to claim 1, said terminal being provided with a man-machine interface comprising display and inputting means able to establish communication through a network with a remote server, and including a browser able to display personal data entry pages and to store data entered both locally at the terminal and remotely on the server.", "16.A terminal according to claim 15, wherein the terminal is a mobile telephone.", "17.A terminal according to claim 15, wherein the terminal is of the microcomputer type, and a chip card is inserted in the terminal by a user at each use.", "18.A server for implementing the method according to claim 1, comprising an application able to supply to a distant browser, via a communication terminal, pages which can be interpreted and/or executed by the browser, the pages comprising at least requests for the input of personal information and requests for the local storage of this information, requests to return this information to the server, said application executing a step of storing information received." ], [ "The invention concerns a method for the secure storage of personal data and for consultation.", "The invention also concerns chip cards intended to dialogue with a remote server through a communication network via a terminal connected to a chip card reader.", "It also concerns the telecommunication terminals and in particular the mobile telephones equipped with a subscriber identification chip card provided, one or the other, with a browser affording dialogue with a server.", "At the present time an adult person has dozens of items of information which are personal or even confidential to him and which he may need at any time during the day.", "The number of these items of information is continually increasing with the appearance of new services or new industrial products.", "All this personal information includes bank account numbers, service subscription numbers, secret codes or passwords and of course telephone numbers, the volume of which is increasing simply because of the fact that it is more and more usual to add a mobile telephone number to a fixed telephone network number.", "The mass of confidential information which a person has to manage today is continually increasing.", "A large number of persons still use paper diaries for noting all this information, including the secret codes.", "It will easily be understood that this solution is no longer at all suitable for storing confidential information and, even less, secret information.", "Paper diaries may remain an advantageous solution, naturally in so far as the personal information is only telephone numbers and such information does not require frequent changes (updates).", "However, for the past few years, portable electronic devices called Personal Digital Assistants (PDAs) have appeared, which make it possible to store a large amount of information, in particular personal information.", "These devices enable a person to create a private file in which he will be able to store all the personal information that he wishes.", "A password is required to activate and decipher the data stored.", "Unfortunately these devices do not afford sufficient security since their operating system is an open operating system, that is to say one which is accessible and into which it is consequently possible to introduce spy programs (Trojan Horses) or viruses, even in the case where the data exchanged with the outside are enciphered.", "The present invention aims to resolve this problem.", "To this end, the invention proposes to enable a user to input personal data from a terminal connected to the chip card reader by means of a browser which may be present in the chip card and/or in the terminal, capable of controlling the display by the terminal of pages supplied by a server through a communication network and to demand a saving on the card of any data encrypted and input.", "In addition, the method makes it possible to make a copy of this back-up on the server in a way which is very simple and secure for the user.", "The user can thus, in the event or loss or theft of his terminal, request the server to upload his personal data onto the new equipment.", "The chip cards comprising application programs developed at the present time are similar to a computer in so far as they possess an operating system and one or more application programs which can be loaded or downloaded and whose execution is initiated by the operating system.", "The operating system is protected by the very fact that it is stored in read only memory (ROM memory).", "The operating system is because of this not modifiable within the card.", "Amongst the application programs executed (or interpreted) by the operating system, there is provided according to the invention a browser program able to dialogue with the server and able to supply pages for entering personal data of the user.", "Advantageously the data inputting is carried out on line.", "The object of the present invention is therefore a method for the secure storage of personal data and for consultation, principally characterised in that it comprises the following steps: using a terminal connected to a chip-card reader and provided with a man-machine interface comprising a display and data input means, using a browser capable of dialoguing with a remote server through a communication network, from the said terminal, displaying pages of data with the said display means, inputting personal data of a user in response to the pages displayed and storing them locally for consultation and remotely on the server for saving.", "According to another characteristic, the data pages are supplied by the server.", "According to another characteristic, the data pages are supplied during a communication and during inputting.", "The data inputting is carried out on line, the session remaining open throughout the duration of the inputting.", "The personal data are recorded locally on the chip card and a copy is saved on the server.", "The saving of the copy remotely is carried out substantially simultaneously with the recording locally.", "The personal data are preferably encrypted by the card before being saved and can be decrypted only by the card.", "The personal data are preferably encrypted by means of an enciphering algorithm using one or more keys saved in the card.", "The enciphering keys are also saved by an entrusted entity.", "The browser comprises the functions of a browser of the type defined by the S@T (SIM Alliance Toolbox) standard.", "The pages supplied by the server are pages of the type defined by the S@TML language.", "Another object of the invention is a chip card comprising a processing unit and one or more program memories comprising programs including the operating system of the card, principally characterised in that it also comprises a browser program capable of dialoguing with a remote server through a terminal connected to a chip card reader, provided with a man-machine interface, and in that the browser permits the entry of personal data by a user of the terminal on pages of data and their storage locally in the card for consultation and remotely on the server.", "The card preferably comprises a program for protecting the saved data.", "The program for protecting the data uses an encrypting algorithm utilising one or more keys stored in the card in order to encrypt the personal data entered before saving, and a decrypting algorithm for any consultation of these data by the user.", "This encrypting program can be integrated in the browser program.", "According to one example the card is a SIM card.", "Advantageously, the browser comprises the function of a browser of the type defined by the SOT (SIM Alliance Toolbox) standard.", "Another object of the invention is a communication terminal provided with a man-machine interface comprising display and inputting means able to establish communication through a network with a remote server, principally characterised in that it comprises a browser able to supply for display personal data entry pages and the storage locally of the data entered and remotely on the server.", "According to one example the terminal is a mobile telephone.", "In this example the said chip card is inserted in the terminal by a user and resides therein.", "According to another example the terminal is of the microcomputer type and the chip card is inserted by the user at each use.", "Another object of the invention is a server, principally characterised in that it comprises an application able to supply to a distant browser via a communication terminal pages which can be interpreted and/or executed by the browser, the pages comprising at least requests for the inputting of personal information, requests for the local storage of this information and requests to return this information to the server, the said application comprising a step of storing the said information received.", "Other particularities and advantages of the invention will emerge clearly from a reading of the following description which is given by way of non-limiting example and with regard to the figures, in which: FIG.", "1 illustrates the diagram of an example of a system for implementing the method according to the invention, FIG.", "2 illustrates an example of the inputting on several pages, FIG.", "3 illustrates a functional diagram of a chip card, FIG.", "4 illustrates a terminal provided with a chip card.", "The invention applies to terminals reading chip cards or connected to a chip card reader.", "Hereinafter a terminal reading chip cards or connected to a chip card reader will be spoken of in general terms.", "The invention therefore applies to any electronic device equipped with means of communication with a chip card.", "It is a case for example of mobile telephones, microcomputers, personal electronic diaries (PDAs) or banking terminals, including chip cards themselves in so far as there exist chip cards “reading” another chip card.", "The chip card is either resident in the terminal or inserted in the terminal, or connected to the terminal through a reader connected to the terminal.", "This connection can be cabled or infrared or radio for example or of the BlueTooth type.", "The chip cards which are intended to communicate with the terminal have a program for communication with the terminal.", "The terminal or the card comprises a browser for connecting to and exchanging with a remote server.", "In fact, in the present invention, the concern is with a chip card provided with a browser program, also referred to as a navigator or browser in English terminology.", "This browser makes it possible to dialogue via the terminal with a server through a communication network (for example GSM, UMTS or other).", "The application programs are in general stored in an electrically programmable memory.", "These programs can thus be updated and some downloaded by means of the terminal.", "An example will be described hereinafter in the case where the terminal is a telecommunication terminal such as a mobile telephone in which, it will be recalled, the chip card resides.", "There is therefore next chosen, by way of example, the case of SIM cards, which are subscriber identification cards.", "There is also chosen by way of example a browser as defined by the S@T standard of the SIM Alliance organisation (SIM Alliance Toolbox).", "FIG.", "1 gives an outline diagram in the case of this particular example.", "A mobile telephone T equipped with a chip card C (SIM) can communicate through a communication network R and a gateway P with a server S dedicated to this application and in which the user is listed, for example by a customer number.", "There is also shown in this FIG.", "1 the entity A which represents an entrusted third party with whom the secret keys of the user customers of the server S can be stored.", "Reference will now be made, for a better understanding of the invention, to the following tables given by way of example in order to illustrate the various exchanges between card, terminal and server during an inputting operation and during a consultation operation: An inputting operation is illustrated by the steps detailed 1 to 20 in the following table.", "Inputting of Personal Data: Dedicated servers Network (supporting an application (BTS, generating the STKML pages .", ".", ".", ", and updating the personal Step User Card (browser S@T) Mobile gateway database at the operator) 1 Selection of data entry mode 2 Sending of a request to server (STKML page) for loading a form or “template” to be completed 3 Send page 4 Send page 5 Preparation of form Sending of page containing form 6 Send page 7 Receive page 8 Reception of page Request mobile for display 9 Display page 10 Entry of information requested on the page: personal data (name, driving licence number) 11 Encrypting of data with encrypting algorithm (of the card or browser) and with application key stored in the card 12 Preparation of page with encrypted data for server 13 Local saving of encrypted data 14 Send page 15 Send page 16 Send page 17 Storage of encrypted private data in the personal database 18 Preparation of following form Sending of page containing the form 19 Send page 20 Etc, for all information requested Thus, as can be seen in this example, at any time, the database of the the server S and the updating data in the card C are consistent.", "In the case where the data are not stored on the server, for example through absence of coverage in the network, a recall function can be provided for automatically effecting or proposing this saving as soon as coverage on the network is detected.", "The application program can also comprise functions which give choices to the user: for saving the data both in the card and/or on the server; only one saving can be effected either in the card or on the server or in the terminal; the data to be stored in the terminal are preferably encrypted or may not be so according to the choice of the user.", "In addition, this program can make provision for the data pages to be generated by the card or by the terminal.", "The application can propose to carry out a storage at two points or not (on the SIM card, on the server, on another card, on the terminal, on a computer).", "A consultation operation is illustrated by steps 1 to 9 in the following table: Consulation of Personal Data: Dedicated servers Network (supporting an application (BTS, generating the STKML pages .", ".", ".", ", and updating the personal Step User Card (browser S@T) Mobile gateway database at the operator) 1 Selection of data consultation mode 2 Request dealt with locally (the data are stored up to date in the card) 3 Preparation of data read in the card and decrypted by means of the decrypting algorithm of the card or of the navigator and the application key stored in the card 4 Preparation of the page containing the form requested and the data 5 Request display of the page 6 Display page 7 Reading of information 8 Selection of following data type to be consulted 9 Request dealt with locally (the data are stored up to date in the card) .", ".", ".", "c.f.", "above .", ".", ".", "The updating mode (data entry) preferably takes place on line and the consultation mode takes place when not connected.", "Provision can also be made for the pages supplied by the server to be stored blank to enable the user to close the session and to input the data on the pages when disconnected (closed session).", "Naturally the session is opened in a secure and known manner, that is to say after the user has been identified (entry of an identification code for example).", "In addition, in the case of a mobile telephone, the PIN code (Personal Identification Number) can also be required.", "The browser provides the transmission/reception and interpretation of a page containing executable and/or interpretable commands.", "Amongst the commands presented in the executable and/or interpretable pages there are: the sending of a request to the server for the supply of an STKML page; the request to the terminal for display of wording or data contained in the pages; the invocation of the encrypting function or direct triggering of its execution if this function is integrated in the browser; the local saving of the data (in particular in an electrically programmable memory of the card); the request to send each page with the personal data entered, from the mobile for saving on the server.", "The display commands can be print commands or equivalent intended for the user.", "The man-machine interface then comprises a display screen or a printer.", "As stated above, the personal data entered and saved on the card are protected by the intrinsic security of the card (TPR hardware resisting intrusion attacks).", "This security is advantageously reinforced by encrypting of these data by means of a known algorithm, using a secret key reserved for this application and contained in the card.", "The encrypting/decrypting algorithm can be integrated in the browser or be in the form of a separate program which can be invoked by the browser.", "The data saved on the server are preferably also protected, that is to say encrypted by the card before sending.", "Only the card can decrypt them.", "This is because the data can be stored enciphered or encrypted before saving on the server.", "This encrypting is carried out on the basis of a key dedicated to this application stored on the card and a duplicate of which can be stored by an entrusted third party A.", "Before the display, the data are deciphered by the card.", "The invention thus makes it possible to effect a saving of personal data of a user on the card and on the server.", "Thus, in the event of loss or theft of the chip-card reading terminal and more precisely of the chip card, the user can recover his data and/or his key or keys in particular by requesting the loading of the saved copy of his personal data onto his new card.", "FIG.", "2 illustrates the example of the display of three successive pages and personal data able to be entered.", "FIG.", "3 illustrates the functional diagram of a chip card CP.", "The chip card or object with integrated circuit or equivalent comprises a central unit U connected to a non-volatile program memory M1 and at least one electrically programmable program memory M2.The memory M1 comprises the operating system of the card CP and possibly the browser program.", "The chip card can thus dialogue with the remote server via the telecommunication terminal.", "FIG.", "4 illustrates the diagram of a terminal T. This terminal possesses the functions of chip card reader which enable it to communicate with the chip card CP.", "It has a screen E and a keypad C. According to an example embodiment the terminal T is a mobile telephone, the card is a SIM card and the browser meets the S@T standard of SIM Alliance.", "According to another example the terminal can be a terminal in which the chip card does not reside but must be inserted in an associated reader by a user of the terminal.", "It may for example be a banking terminal or a microcomputer or a PDA." ] ]	Patent_10468480
[ [ "Method for production of an optical disc with a detachable module", "Method for production of an optical disc with a detachable module, where a standard mould is provided with an insert insertable into said standard mould.", "The insert restricting the internal dimensions of the mould cavity for shaping the optical disc into dimensions different from a standard optical disc.", "The insert comprises a line restrictor for providing a groove along at least one breaking line between the optical disc and the detachable module." ], [ "1-19.", "(canceled) 20.An optical disc with a detachable module, said optical disc with detachable module having a length of approximately 85.6 mm and a width of approximately 54 mm, the SIM plug being placed on a position corresponding to ISO 7810 standard a diagonal of the optical disc being at least 80 mm, and a skew edge of the SIM plug facing away from the optical disk.", "21.An optical disc with a detachable module according to claim 20, where the optical disc and the detachable module are divided by a groove along a breaking line, and where said groove along the breaking line is tapered from being less steep to being more steep seen in a direction from the optical disc towards the detachable module.", "22.An optical disc with a detachable module according to claim 20, where a frame is provided in connection with the optical disc and the detachable module, where the frame and the detachable module are divided by grooves along breaking lines, and where said grooves along breaking lines are tapered from being less steep to being more steep seen in a direction from the frame towards the detachable module.", "23.An optical disc with a detachable module according to claim 20, where a frame is provided in connection with the optical disc and the detachable module, where the frame and the optical disc are divided by grooves along breaking lines, and where said grooves along breaking lines are tapered from being less steep to being more steep seen in a direction from the frame towards the optical disc.", "24.An optical disc with a detachable module, said optical disc with detachable module having a length of approximately 85.6 mm and a width of approximately 54 mm, the SIM plug being placed on a position differing from ISO 7810 standard a diagonal of the optical disc being at least 80 mm, and a skew edge of the SIM plug facing towards the optical disk.", "25.An optical disc with a detachable module according to claim 24, where the optical disc and the detachable module are divided by a groove along a breaking line, and where said groove along the breaking line is tapered from being less steep to being more steep seen in a direction from the optical disc towards the detachable module.", "26.An optical disc with a detachable module according to claim 24, where a frame is provided in connection with the optical disc and the detachable module, where the frame and the detachable module are divided by grooves along breaking lines, and where said grooves along breaking lines are tapered from being less steep to being more steep seen in a direction from the frame towards the detachable module.", "27.An optical disc with a detachable module according to 24, where a frame is provided in connection with the optical disc and the detachable module, where the frame and the optical disc are divided by grooves along breaking lines, and where said grooves along breaking lines are tapered from being less steep to being more steep seen in a direction from the frame towards the optical disc.", "28.Method for production of an optical disc with a detachable module comprising providing a mould with mould cavity having internal dimensions corresponding to the dimensions of the optical disc with the detachable module, said mould having supplier means for supplying molten polymer into the mould cavity, providing a stamper in the mould cavity, said stamper having a surface towards the interior of the mould cavity, said surface having a surface structure being the counterpart for the corresponding surface structure of the moulded optical disc, supplying molten polymer into the mould cavity, said molten polymer filling the remaining space inside the mould cavity, cooling the polymer to a temperature where the polymer is hardened, and removing the moulded optical disc, providing at least one line restrictor, for restricting the thickness of the optical disc with detachable module along at least one breaking line between the optical disc and the detachable module, and providing the at least one line restrictor in the mould at a position between the optical disc and the detachable module for expanding a longitudinal dimension of the optical disc to a dimension of at least 59,1 mm.", "29.Method according to claim 28, wherein said method also comprises providing at least one module thickness restrictor for restricting the thickness, preferably between 0.8 mm and 0.85 mm, of the detachable module over at least an area that is intended to include an electronic circuit.", "30.Method according to claim 28, wherein said at least one line restrictor is comprised by at least one movable plunger and that the method comprises moving said at least one plunger into the mould cavity after having supplied said molten polymer and before said polymer hardens.", "31.Method according to claim 28, wherein said method comprises providing a mould with internal dimensions corresponding to the dimensions of the optical disc with the detachable module comprises providing a standard mould for a standard optical disc and providing an insert insertable into said standard mould, said insert inside said standard mould restricting the internal dimensions of the mould cavity for shaping the optical disc into dimensions different from a standard optical disc.", "32.Method according to claims 28, wherein said method further comprises providing said detachable module with a hollow for an electronic circuit during moulding of said optical disc, and after removal of said disc, providing a standard production machine for inserting an electronic circuit into said hollow.", "33.Method according to claim 28, wherein the optical disc after moulding is provided with a reflective layer and covered by spray coating or silk-screen printing.", "34.Method according to claim 28, wherein said method also comprises providing at least one mould cavity restricting the dimensions between a length of approximately 85.6 mm and a width of approximately 54 mm, of the optical disc with the detachable module.", "35.An injection moulding device for manufacturing an optical disc with a detachable module from a polymer material, said injection moulding device comprising a mould having a first and second mould section which are movable relative to one another between an open and a closed position, and between which in the closed position a mould cavity is defined into which a molten polymer is injectable to form the disc with detachable module, a stamper in the mould cavity having a surface towards the interior of the cavity, said surface having a surface structure being the counterpart for the corresponding surface structure of the moulded optical disc, and said injection moulding device also comprising at least one line restrictor, for restricting the thickness of the optical disc with detachable module along at least one breaking line between the optical disc and the detachable module, and wherein said line restrictor is provided with a tapering form with a taper angle facing towards the module of less than 15 degrees, preferably less than 12 degrees.", "36.An injection moulding device according to claim 35, wherein said device also comprises a module thickness restrictor for restricting the thickness, preferably between 0.8 mm and 0.85 mm, of the detachable module, said module being optionally intended for receiving an electronic circuit.", "37.An injection moulding device according to claim 35, wherein said device also comprises a mould cavity restricting the dimensions between a length of approximately 85.6 mm and a width of approximately 54 mm, of the optical disc with the detachable module.", "38.An injection moulding device according to claim 35, wherein said mould is a standard mould for standard optical discs, and wherein said injection moulding device also comprises an insert insertable into said standard mould, said insert inside said standard mould restricting the internal dimensions of the cavity for shaping the optical disc with detachable module differently from a standard optical disc.", "39.An injection moulding device according to claim 35, wherein said at least one line restrictor is comprised by at least one plunger movable into the cavity of the mould, preferably in a direction approximately orthogonal to said optical disc, said line restrictor being intended to be moved into the cavity of the mould after having supplied molten polymer into the cavity and before said polymer hardens.", "40.An injection moulding device according to claim 35, wherein said taper angle facing away from said module is larger than said taper angle towards said module.", "41.An injection moulding device for manufacturing an optical disc with a detachable module from a polymer material, said injection moulding device comprising a mould having a first and second mould section which are movable relative to one another between an open and a closed position, and between which in the closed position a mould cavity is defined into which a molten polymer is injectable to form the disc with detachable module, a stamper in the mould cavity having a surface towards the interior of the cavity, said surface having a surface structure being the counterpart for the corresponding surface structure of the moulded optical disc, and said injection moulding device also comprising at least one line restrictor, for restricting the thickness of the optical disc with detachable module along at least one breaking line between the optical disc and the detachable module, and wherein said line restrictor is provided with a tapering form with a taper angle facing away from the module of more than 8 degrees, preferably more than 10 degrees and most preferably more than 12 degrees.", "42.An injection moulding device according to claim 41, wherein said device also comprises a module thickness restrictor for restricting the thickness, preferably between 0.8 mm and 0.85 mm, of the detachable module, said module being optionally intended for receiving an electronic circuit.", "43.An injection moulding device according to claim 41, wherein said device also comprises a mould cavity restricting the dimensions between a length of approximately 85.6 mm and a width of approximately 54 mm, of the optical disc with the detachable module.", "44.An injection moulding device according claim 41, wherein said mould is a standard mould for standard optical discs, and wherein said injection moulding device also comprises an insert insertable into said standard mould, said insert inside said standard mould restricting the internal dimensions of the cavity for shaping the optical disc with detachable module differently from a standard optical disc.", "45.An injection moulding device according to claim 41, wherein said at least one line restrictor is comprised by at least one plunger movable into the cavity of the mould, preferably in a direction approximately orthogonal to said optical disc, said line restrictor being intended to be moved into the cavity of the mould after having supplied molten polymer into the cavity and before said polymer hardens.", "46.An injection moulding device according to claim 41, wherein said taper angle facing away from said module is larger than said taper angle towards said module.", "47.An insert insertable into a standard mould with cavity for moulding of standard optical discs, said insert inside said standard mould restricting the internal dimensions of the cavity for shaping an optical disc with detachable module differently from a standard optical disc, and said insert being fittingly inserted into the cavity of the standard mould." ], [ "<SOH> FIELD OF THE INVENTION <EOH>The present invention relates to the production of an optical disc with a detachable module.", "The invention also relates to an embodiment of such an optical disc with a detachable module." ], [ "<SOH> DESCRIPTION/SUMMARY OF THE INVENTION <EOH>This purpose is achieved with a method according to the invention as described in the following.", "A method is herewith disclosed for production of an optical disc with a detachable module, the method comprising the provision of a mould with mould cavity having internal dimensions corresponding to the dimensions of an optical disc with the detachable module, said mould having supplier means for supplying molten polymer into the mould.", "A stamper is provided in the mould cavity, said stamper having a surface towards the interior of the mould cavity, said surface having a surface structure being the counterpart for the corresponding surface structure of the moulded optical disc.", "Molten polymer is supplied into the mould cavity, said molten polymer filling the remaining space inside the mould cavity, after which the polymer is cooled to a temperature where the polymer is hardened.", "Finally the moulded optical disc is removed.", "In order to establish a smooth, preferably linear, groove for a breaking line between the optical disc and the detachable module, a line restrictor is provided restricting the thickness of the optical disc with detachable module along at least one breaking line between the optical disc and the detachable module.", "By providing a line restrictor according to the invention, it is not necessary to perform any milling action along this line for establishing a linear groove, which facilitates the production process tremendously as compared to known processes.", "An optical disc with detachable module is in this way according to the invention produced in one step with a precise shaping during the moulding process.", "The method according to the invention may as well comprise the provision of a thickness restrictor for restricting the thickness of the detachable module, for example to 0.85 mm, in an area that is intended to include an electronic circuit.", "In as far as the detachable module is or contains a SIM plug, the thickness of the SIM plug will be between 0.8 mm and 0.85 mm in order to fulfill thickness requirements, whereas the thickness of the optical disc is typically 1.2 mm.", "Optionally, already during the moulding, the detachable module is produced thinner than the optical disc.", "As explained in the introduction with reference to European patent EP 296677 and references therein, there exist standard moulds for standard optical discs.", "In order to use these standard moulds, it has turned out that it is very advantageous to provide an insert which is insertable into such standard moulds, where the insert restricts the internal dimensions of the mould in order to shape the optical disc differently from a standard optical disc.", "This insert may be shaped such that the optical disc is not circular but has other shapes, for example quadratic or rectangular.", "The insert may also comprise a module restrictor for restricting the thickness of the detachable module.", "As far as the detachable module comprises a SIM plug, the SIM plug itself may have a thickness of 0.8 mm and comprises in addition a hollow in the SIM plug into which the electronic circuit is replaced.", "The module restrictor according to the invention may take account for the simultaneous production of this hollow in the SIM plug.", "The line restrictor inside the mould reduces the speed at which the polymer may flow across the line restrictor.", "Because the distance between the edge of the restrictor and the opposite internal side of the mould is only spaced a short distance, the polymer is prevented from flowing across the line restrictor as freely as in the rest of the mould cavity.", "Therefore, temperature differences may occur across the hardening optical disc with detachable module which again may result in strain inside the hardened optical disc.", "This may have the consequence that the optical disc with the detachable module bends, when removed from the mould resulting in a non-satisfactory product.", "In order to minimize such temperature differences when the polymer is injected into the mould and flowing inside the mould to fill the cavity completely, the invention has foreseen a further development, where the line restrictor is comprised by a movable plunger in order to establish the breaking line.", "Such a plunger is preferably moved in a direction approximately orthogonal to the optical disc into the mould cavity.", "This action is performed after having supplied polymer into the cavity and before this polymer hardens.", "As the line restrictor is not inside the cavity during the filling of polymer into the mould, the polymer can flow freely inside the remaining cavity such that temperature differences across the polymer in the cavity is avoided.", "Still being a fluid, the plunger in the form of a line restrictor is inserted and forms the groove for the breaking line between the detachable module and the optical disc.", "Though only one line restrictor and one plunger is mentioned in the foregoing and the following, this has to be understood also such that more than one line restrictor and plunger may be provided and used.", "The detachable module that may be used for a SIM plug as mentioned earlier which has to be provided with a hollow for insertion of an electronic circuit.", "Also such a hollow can be produced by a plunger that is inserted into the cavity.", "Typically, a SIM plug has one hollow for the electronic circuit and inside this hollow a second, deeper hollow for attaching the electronic circuit to the SIM plate.", "This means that the material thickness of the polymer of the SIM plug at the thinnest is 0.2 mm.", "This implies in practice, if the mould form is solid without a plunger, that polymer has to flow between two faces of 0.2 mm distance.", "In such a narrow region, the polymer may cool faster than in the rest of the cavity, which may induce stress in the optical disc with a detachable module as describe earlier.", "By using a plunger to form this deep second hollow in the SIM plug such a too-fast-cooling is avoided.", "For the amount of polymer to be injected into the mould, the volume of the plungers is taken into account.", "By using plungers to provide grooves for breaking lines and, optionally, a hollow in the detachable module, the tolerances for injection speed of the polymer, for the time for the moulding process, for the pressure on the polymer and for the cooling time are much less strict than in corresponding processes without such plungers.", "Once the optical disc has been removed from the mould, a reflective layer, typically a nickel layer, is provided on that side of the optical disc which has been facing the stamper.", "On top of this reflecting layer, a coating is applied.", "On standard optical discs such coating is spun on the surface after which a print is provided on the surface coating.", "For a disc with detachable module according to the invention, such a spinning method is not optimal, because the coating may accumulate in the groove for the breaking line.", "Therefore, in a further development of the invention, it is foreseen that the surface coating is applied by spraying or by silk screen printing.", "It has turned out that spray-coating has another advantage as compared to spun coating as will be apparent in the following.", "A spun coating has typically a layer thickness 50 μm.", "Because a SIM plug has a very well-defined thickness, a 50 μm spun coating on the surface would imply that the polymer thickness between the glue in the hollow of the electronic module and the spun coating is only 0.15 mm, which is very thin and which easily may lead to deformation of the SIM plug during moulding and also during hardening of the spun coating and the applied glue inside the hollow.", "By applying spray coating, the coating on the SIM plug can be made thinner or be completely avoided, such that the wall thickness is not 0.15 mm but 0.2 mm, which results in a higher stability against bending of this thin wall.", "The spray-coating that is applied to the reflective side of the optical disc may be an ultra violet hardening coating or varnish.", "It should be non-corrosive for the reflective layer, for example nickel coating or aluminum coating.", "The injection moulding device for manufacturing optical discs with a detachable module from a polymer material according to the invention comprises a mould with a first and second mould section which are movable relative to another between an open and closed position.", "Between the first and second mould section in a closed position, a mould cavity is defined into which a moulding polymer is injectable to form the disc with the detachable module.", "The injection moulding device comprises a stamper in the mould cavity having a surface towards the interior of the cavity.", "The surface of the stamper has a surface structure being the counterpart for the corresponding surface structure of the moulded optical disc.", "The injection moulding device furthermore comprises at least one line restrictor for restricting the thickness of the optical disc with the detachable module along at least one breaking line between the optical disc and the detachable module.", "The injection moulding device in a further development of the invention also comprises a module restrictor for restricting the thickness of the detachable module.", "The module being optionally intended for receiving an electronic circuit.", "In an even further embodiment of the invention the mould is a standard mould for standard optical discs and the injection moulding device comprises in addition an insert insertable into the standard mould.", "The insert inside the standard mould restricts the internal dimensions of the cavity of the mould in order to shape the optical disc with detachable module differently from standard optical discs.", "Preferably, the at least one line restrictor is comprised by a plunger moveable into the cavity of the mould, preferably in a direction approximately orthogonal to the optical disc.", "The at least one line restrictor is intended to be moved into the cavity of the mould, after having supplied moulding polymer into the cavity and before this polymer hardens.", "A line restrictor is preferably provided with a tapering form having a taper angle of less than 15 degrees, preferably less than 12 degrees and most preferably less than 10 degrees on the side that is facing the module.", "The taper angle facing the module should be rather small if the module is desired with very well defined dimensions and steep side edges.", "The latter is especially important for modules like SIM plugs.", "The taper angle facing away from the detachable module is preferably larger than the taper angle facing the module and should be more than 8 degrees, preferably more than 10 degrees and most preferably more than 12 degrees.", "Generally, the taper angles should not be chosen too small.", "The reason is that for very small taper angles, the polymer that forms the optical disc with detachable module after hardening may have contracted so much around the line restrictor during the hardening process that the optical disc with detachable module cannot be removed from the mould without receiving gratings in the groove for the breaking line.", "Because the taper angle towards the module, especially when this module is a SIM plug has to be rather small, it is preferred that the taper angle facing away from the module is rather large, for example 20-30 degrees.", "This way, while being steep towards the detachable module, for example a SIM plug, the line restrictor may be so blunt that the groove for the breaking may not fasten on the line restrictor.", "In case that the line restrictor is not comprised by a plunger and the line restrictor instead is a non-movable feature of the mould form or of the insert in the mould form, the line restrictor should be blunt for another reason.", "In case that the taper angles towards the module and facing away from the module are small, the line restrictor will be sharp and thin as a knife edge.", "This again implies that the thin line restrictor may more easily be deformed when polymer under high pressure is passing the narrow passage between the line restrictor and the opposite side of the mould.", "Partly, this can be prevented by using very hard materials, as for instance tungsten carbide, however, steel may be desired as a material for the line restrictor by other reasons.", "Generally, the shape of the line restrictor may be designed in dependence of the viscosity of the polymer and the distance between the line restrictor and the opposite side of the mould cavity.", "The method of production of an optical disc with detachable module may result in a product being an optical disc with a detachable SIM plug, and said optical disc with SIM plug having a length of approximately 85.6 mm and a width of approximately 54 mm, the SIM plug being placed on a position corresponding to ISO 7810 standard, a diagonal of the optical disc being at least 80 mm, and a skew edge of the SIM plug facing away from the optical disk.", "The detachable module may be a SIM plug for telephones as described above, for example with GSM, GPRS or UMTS standard, but may also be a pay card to be installed into a mobile phone in order to be able to pay for goods or services with the mobile phone.", "Applications in connection with road pricing or intelligent surveillance of buildings or working machines are other possibilities.", "Alternatively, the detachable module is a general computer component, for example a memory type or processing unit type as ROM, RAM, or CPU.", "The user may, thus access the data information of the optical disc in order to learn about the module and its installation.", "The data information on the optical disc may also comprise a company profile of the producer or distributor, software drivers necessary for the installation of the microelectronics module, computer programs, software packages necessary for Internet access from a mobile or stationary telephone, purchase contracts, licenses, television receiver codes, or any other relevant information for the user.", "The invention will be explained in more detail in the following with reference to the drawings." ], [ "FIELD OF THE INVENTION The present invention relates to the production of an optical disc with a detachable module.", "The invention also relates to an embodiment of such an optical disc with a detachable module.", "DESCRIPTION OF PRIOR ART Optical discs (CD, MiniDisc, CD-ROM, Digital Video Disc) are typically produced by injection moulding with a so-called stamper in a mould form.", "Such a process is described in European patent 296 677 by Cools and assigned to Philips.", "For the production of the stamper, a glass plate is covered with a resin that is laser illuminated and developed.", "Subsequently, the resin is covered with a metallic layer, typically Nickel, by sputtering and galvanic processes.", "The Nickel stamper is then removed from the glass and can after removal of the resin be used as a mould form in mould processes for optical disc production.", "Typically, the stamper is adapted to the diameter of the mould form which normally is slightly larger in diameter than the final optical disc.", "In the mould form, the stamper is centered and fixed to ensure high accuracy during the moulding procedure.", "The mould is then closed and polymer, typically polycarbonate or PMMA (polymethylmethacrylate), is injected into the form at a temperature substantially over the melting temperature of the polymer, typically 340° C. for polycarbonate.", "The temperature has to be on the one hand lower than the temperature where the polymer start disintegrating and on the other hand so high that the polymer flows quickly in the mould form in order to avoid too large strain in the optical disc when it is removed from the mould.", "Electronic modules like SIM plugs may be integrated in such optical discs.", "Such combinations are known from German patent application DE 199 05 588 by Bierlich assigned to Deutsche Telekom AG.", "In order to be able to remove the electronic module easily from the remaining carrier card with optical disc, a number of solutions have been proposed, for example in German Utility Model DE 201 02 719U by C.U.B.A.", "and in German patent application DE 199 43 092 by Lüke assigned to Orga.", "Neither of these two documents disclose a possible method for production of the disclosed optical disc with electronic module.", "The term optical disc has to be understood in a general sense as a digital data carrier with optically readable data tracks, even though the shape of the digital data carrier may have shapes that differ from a circular form.", "In German Utility Model DE 201 02 719U by C.U.B.A., a SIM plug can be removed from the optical disc by breaking two connecting bridges.", "However after breaking, the remaining SIM plug will have remains from the bridges such that the edge with the broken bridges is not smooth.", "The tolerances for the size of SIM plugs to be used in telephones are narrow such that the SIM plug as described in this document is not suited for ordinary usage.", "In German patent application DE 199 43 092 by Lüke, a separation line can be broken to separate an electronic module from an optical disc.", "This optical disc with the module is produced by injection moulding, where the thickness of the electronic module is different from the thickness of the optical disc.", "However no clear advice is given for how to produce such an optical disc.", "In fact, a number of precautions have to be taken for such a process as will be apparent in the following, which is a reason why, so far, no such products are commercially available.", "A general aim for a product where an electronic module like a SIM plug is connected to an optical disc, which then functions as a carrier, is to observe the ISO 7810 standard.", "The ISO 7810 standard governs the position for the placement of the SIM plug on such a carrier, where the carrier according to this standard has a length of 85.6 mm and a width of 53.9 mm—which is the standard size of a credit card.", "Electronic modules on cards of ISO 7810 standard, for example as described In European Patent EP 495 216 by Blome and Freise assigned to Orga, are well known to produce because such cards are used in telephones, especially elder telephones, in parallel to SIM plugs.", "Normally, the cards are prefabricated, after which a SIM electronic circuit is placed on the card in other existing machines.", "The configuration of the electronic circuit is governed by another standard, namely the ISO 7816.Such other existing machines producing electronic circuits on such prefabricated ISO 7810 carriers are not suited for the production of most known optical discs with SIM plugs, because the fixed placement according to ISO 7810 of the SIM plug on the carrier card and the size of the SIM plug itself leaves not enough space for the optical disc tracks to contain data.", "This is a problem faced by the shown embodiments in German patent application DE 199 43 092 by Lüke and assigned to Orga.", "A way to overcome this difficulty is a complete new construction of production machines for placement of the electronic module.", "The difficulty with the placement of the SIM module on an ISO 7810 standard card with an optical disc Is one of the problems to be solved.", "In the aforementioned German patent application DE 199 43 092 by Lüke, there is no stated restriction on the actual size and shape of the optical disc with the SIM plug.", "However, the disclosure should be seen in the light of existing standards, for example the ISO 7810 standard that is mentioned in this German patent application.", "Also, though not restricted to the shape of the card in this document, only quadratic optical disc's are shown to which a SIM plug is attached.", "It is also mentioned in this document, that the quadratic form is preferred with an edge of 54 mm.", "This combination of features leads to another problem, namely that the diagonal length of the carrier card with the optical disk is 76 mm, which is 4 mm shorter than the minimum length required for a CD to be played in a large number of optical CD drive unit.", "For example, a CD of less than 80 mm will only be read properly in 60-70% of existing CD drive units.", "Thus, also in this respect, German patent application DE 199 43 092 gives no clear advice for how to construct a satisfactory CD with SIM plug.", "This is another problem to be solved.", "It is the purpose of the invention to provide a method for production of an optical disc with a detachable module, for example an electronic module and preferably a SIM plug, where the module after detachment has a smooth edge.", "Especially, it is the further purpose of the Invention to provide a product that functions properly.", "DESCRIPTION/SUMMARY OF THE INVENTION This purpose is achieved with a method according to the invention as described in the following.", "A method is herewith disclosed for production of an optical disc with a detachable module, the method comprising the provision of a mould with mould cavity having internal dimensions corresponding to the dimensions of an optical disc with the detachable module, said mould having supplier means for supplying molten polymer into the mould.", "A stamper is provided in the mould cavity, said stamper having a surface towards the interior of the mould cavity, said surface having a surface structure being the counterpart for the corresponding surface structure of the moulded optical disc.", "Molten polymer is supplied into the mould cavity, said molten polymer filling the remaining space inside the mould cavity, after which the polymer is cooled to a temperature where the polymer is hardened.", "Finally the moulded optical disc is removed.", "In order to establish a smooth, preferably linear, groove for a breaking line between the optical disc and the detachable module, a line restrictor is provided restricting the thickness of the optical disc with detachable module along at least one breaking line between the optical disc and the detachable module.", "By providing a line restrictor according to the invention, it is not necessary to perform any milling action along this line for establishing a linear groove, which facilitates the production process tremendously as compared to known processes.", "An optical disc with detachable module is in this way according to the invention produced in one step with a precise shaping during the moulding process.", "The method according to the invention may as well comprise the provision of a thickness restrictor for restricting the thickness of the detachable module, for example to 0.85 mm, in an area that is intended to include an electronic circuit.", "In as far as the detachable module is or contains a SIM plug, the thickness of the SIM plug will be between 0.8 mm and 0.85 mm in order to fulfill thickness requirements, whereas the thickness of the optical disc is typically 1.2 mm.", "Optionally, already during the moulding, the detachable module is produced thinner than the optical disc.", "As explained in the introduction with reference to European patent EP 296677 and references therein, there exist standard moulds for standard optical discs.", "In order to use these standard moulds, it has turned out that it is very advantageous to provide an insert which is insertable into such standard moulds, where the insert restricts the internal dimensions of the mould in order to shape the optical disc differently from a standard optical disc.", "This insert may be shaped such that the optical disc is not circular but has other shapes, for example quadratic or rectangular.", "The insert may also comprise a module restrictor for restricting the thickness of the detachable module.", "As far as the detachable module comprises a SIM plug, the SIM plug itself may have a thickness of 0.8 mm and comprises in addition a hollow in the SIM plug into which the electronic circuit is replaced.", "The module restrictor according to the invention may take account for the simultaneous production of this hollow in the SIM plug.", "The line restrictor inside the mould reduces the speed at which the polymer may flow across the line restrictor.", "Because the distance between the edge of the restrictor and the opposite internal side of the mould is only spaced a short distance, the polymer is prevented from flowing across the line restrictor as freely as in the rest of the mould cavity.", "Therefore, temperature differences may occur across the hardening optical disc with detachable module which again may result in strain inside the hardened optical disc.", "This may have the consequence that the optical disc with the detachable module bends, when removed from the mould resulting in a non-satisfactory product.", "In order to minimize such temperature differences when the polymer is injected into the mould and flowing inside the mould to fill the cavity completely, the invention has foreseen a further development, where the line restrictor is comprised by a movable plunger in order to establish the breaking line.", "Such a plunger is preferably moved in a direction approximately orthogonal to the optical disc into the mould cavity.", "This action is performed after having supplied polymer into the cavity and before this polymer hardens.", "As the line restrictor is not inside the cavity during the filling of polymer into the mould, the polymer can flow freely inside the remaining cavity such that temperature differences across the polymer in the cavity is avoided.", "Still being a fluid, the plunger in the form of a line restrictor is inserted and forms the groove for the breaking line between the detachable module and the optical disc.", "Though only one line restrictor and one plunger is mentioned in the foregoing and the following, this has to be understood also such that more than one line restrictor and plunger may be provided and used.", "The detachable module that may be used for a SIM plug as mentioned earlier which has to be provided with a hollow for insertion of an electronic circuit.", "Also such a hollow can be produced by a plunger that is inserted into the cavity.", "Typically, a SIM plug has one hollow for the electronic circuit and inside this hollow a second, deeper hollow for attaching the electronic circuit to the SIM plate.", "This means that the material thickness of the polymer of the SIM plug at the thinnest is 0.2 mm.", "This implies in practice, if the mould form is solid without a plunger, that polymer has to flow between two faces of 0.2 mm distance.", "In such a narrow region, the polymer may cool faster than in the rest of the cavity, which may induce stress in the optical disc with a detachable module as describe earlier.", "By using a plunger to form this deep second hollow in the SIM plug such a too-fast-cooling is avoided.", "For the amount of polymer to be injected into the mould, the volume of the plungers is taken into account.", "By using plungers to provide grooves for breaking lines and, optionally, a hollow in the detachable module, the tolerances for injection speed of the polymer, for the time for the moulding process, for the pressure on the polymer and for the cooling time are much less strict than in corresponding processes without such plungers.", "Once the optical disc has been removed from the mould, a reflective layer, typically a nickel layer, is provided on that side of the optical disc which has been facing the stamper.", "On top of this reflecting layer, a coating is applied.", "On standard optical discs such coating is spun on the surface after which a print is provided on the surface coating.", "For a disc with detachable module according to the invention, such a spinning method is not optimal, because the coating may accumulate in the groove for the breaking line.", "Therefore, in a further development of the invention, it is foreseen that the surface coating is applied by spraying or by silk screen printing.", "It has turned out that spray-coating has another advantage as compared to spun coating as will be apparent in the following.", "A spun coating has typically a layer thickness 50 μm.", "Because a SIM plug has a very well-defined thickness, a 50 μm spun coating on the surface would imply that the polymer thickness between the glue in the hollow of the electronic module and the spun coating is only 0.15 mm, which is very thin and which easily may lead to deformation of the SIM plug during moulding and also during hardening of the spun coating and the applied glue inside the hollow.", "By applying spray coating, the coating on the SIM plug can be made thinner or be completely avoided, such that the wall thickness is not 0.15 mm but 0.2 mm, which results in a higher stability against bending of this thin wall.", "The spray-coating that is applied to the reflective side of the optical disc may be an ultra violet hardening coating or varnish.", "It should be non-corrosive for the reflective layer, for example nickel coating or aluminum coating.", "The injection moulding device for manufacturing optical discs with a detachable module from a polymer material according to the invention comprises a mould with a first and second mould section which are movable relative to another between an open and closed position.", "Between the first and second mould section in a closed position, a mould cavity is defined into which a moulding polymer is injectable to form the disc with the detachable module.", "The injection moulding device comprises a stamper in the mould cavity having a surface towards the interior of the cavity.", "The surface of the stamper has a surface structure being the counterpart for the corresponding surface structure of the moulded optical disc.", "The injection moulding device furthermore comprises at least one line restrictor for restricting the thickness of the optical disc with the detachable module along at least one breaking line between the optical disc and the detachable module.", "The injection moulding device in a further development of the invention also comprises a module restrictor for restricting the thickness of the detachable module.", "The module being optionally intended for receiving an electronic circuit.", "In an even further embodiment of the invention the mould is a standard mould for standard optical discs and the injection moulding device comprises in addition an insert insertable into the standard mould.", "The insert inside the standard mould restricts the internal dimensions of the cavity of the mould in order to shape the optical disc with detachable module differently from standard optical discs.", "Preferably, the at least one line restrictor is comprised by a plunger moveable into the cavity of the mould, preferably in a direction approximately orthogonal to the optical disc.", "The at least one line restrictor is intended to be moved into the cavity of the mould, after having supplied moulding polymer into the cavity and before this polymer hardens.", "A line restrictor is preferably provided with a tapering form having a taper angle of less than 15 degrees, preferably less than 12 degrees and most preferably less than 10 degrees on the side that is facing the module.", "The taper angle facing the module should be rather small if the module is desired with very well defined dimensions and steep side edges.", "The latter is especially important for modules like SIM plugs.", "The taper angle facing away from the detachable module is preferably larger than the taper angle facing the module and should be more than 8 degrees, preferably more than 10 degrees and most preferably more than 12 degrees.", "Generally, the taper angles should not be chosen too small.", "The reason is that for very small taper angles, the polymer that forms the optical disc with detachable module after hardening may have contracted so much around the line restrictor during the hardening process that the optical disc with detachable module cannot be removed from the mould without receiving gratings in the groove for the breaking line.", "Because the taper angle towards the module, especially when this module is a SIM plug has to be rather small, it is preferred that the taper angle facing away from the module is rather large, for example 20-30 degrees.", "This way, while being steep towards the detachable module, for example a SIM plug, the line restrictor may be so blunt that the groove for the breaking may not fasten on the line restrictor.", "In case that the line restrictor is not comprised by a plunger and the line restrictor instead is a non-movable feature of the mould form or of the insert in the mould form, the line restrictor should be blunt for another reason.", "In case that the taper angles towards the module and facing away from the module are small, the line restrictor will be sharp and thin as a knife edge.", "This again implies that the thin line restrictor may more easily be deformed when polymer under high pressure is passing the narrow passage between the line restrictor and the opposite side of the mould.", "Partly, this can be prevented by using very hard materials, as for instance tungsten carbide, however, steel may be desired as a material for the line restrictor by other reasons.", "Generally, the shape of the line restrictor may be designed in dependence of the viscosity of the polymer and the distance between the line restrictor and the opposite side of the mould cavity.", "The method of production of an optical disc with detachable module may result in a product being an optical disc with a detachable SIM plug, and said optical disc with SIM plug having a length of approximately 85.6 mm and a width of approximately 54 mm, the SIM plug being placed on a position corresponding to ISO 7810 standard, a diagonal of the optical disc being at least 80 mm, and a skew edge of the SIM plug facing away from the optical disk.", "The detachable module may be a SIM plug for telephones as described above, for example with GSM, GPRS or UMTS standard, but may also be a pay card to be installed into a mobile phone in order to be able to pay for goods or services with the mobile phone.", "Applications in connection with road pricing or intelligent surveillance of buildings or working machines are other possibilities.", "Alternatively, the detachable module is a general computer component, for example a memory type or processing unit type as ROM, RAM, or CPU.", "The user may, thus access the data information of the optical disc in order to learn about the module and its installation.", "The data information on the optical disc may also comprise a company profile of the producer or distributor, software drivers necessary for the installation of the microelectronics module, computer programs, software packages necessary for Internet access from a mobile or stationary telephone, purchase contracts, licenses, television receiver codes, or any other relevant information for the user.", "The invention will be explained in more detail in the following with reference to the drawings.", "SHORT DESCRIPTION OF THE DRAWINGS FIG.", "1 shows a standard mould form and an insert, FIG.", "2 shows one embodiment of the optical disc and detachable module, FIG.", "3 shows alternative embodiments, FIG.", "4 shows possible embodiments of the line restrictor in cross section, FIG.", "5 shows a cross-sectional view of the insert inside the mould, FIG.", "6 shows a further embodiment of the Invention with two modules, FIG.", "7 shows an embodiment with a SIM plug, FIG.", "8 shows an embodiment with a SIM plug In a 7810 standard, FIG.", "9 shows an even further embodiment with two modules.", "DETAILED DESCRIPTION OF THE INVENTION FIG.", "1a shows a standard mould form 100 with a first 101 and second 102 mould section that can be moved—as indicated by arrows 103, 103′—relative to one another between an open and a closed position.", "In the closed position, as illustrated in FIG.", "1b, a mould cavity 104 is defined into which a molten polymer is injectable to form the disc with detachable module.", "A stamper 105 in the mould cavity 104 has a surface 106 towards the interior of the cavity 104, said surface 106 having a surface structure being the counterpart for the corresponding surface structure of the moulded optical disc.", "In the closed situation as illustrated in FIG.", "1b, the stamper 105 rests on a central disc 112 defining the center of rotation for the optical disc and on a ring 107, which defines the outer periphery of the final optical disc moulded in the cavity 104.The first section 101 of the mould is shown in a head-on perspective In FIG.", "1c.", "In order to produce optical disks with detachable modules according to the invention, an insert 108, as shown in FIG.", "1d, may be fittingly inserted into the cavity 104.The remaining internal volume 109 defines the dimensions of the optical disk with detachable module.", "Such an optical disc 200 with detachable module 201 is shown in a possible embodiment in FIG.", "2.Now, referring to FIG.", "1d, the insert 108 may be provided with a module restrictor 111 that defines the space 109″ for the detachable module 201 thinner, for example 0.8 mm or 0.85 mm, than the space for the optical disk 109′ with a larger thickness, for example 1.2 mm.", "The thickness of the module 201 may be restricted from either side, eventually from both sides.", "However, it is preferred that the thickness is restricted from the side opposite to the stamper such that the module 201 rests against the stamper surface.", "The insert 108 may comprise a line restrictor 110 for restricting the thickness of the optical disc 200 with detachable module 201 along at least one breaking line 202 between the optical disc 200 and the detachable module 201 as shown in FIG.", "2.For protection of the edge of the detachable module 201, a frame 203 may be connected to the optical disc 200.This frame 203 need not to be detachable from the disc 200, but this is preferred due to reasons of weight symmetry when reading the optical disk 200 in an appropriated reading unit.", "In FIG.", "5, the line restrictor 110 is shown in a cross section perspective together with part of the stamper 105 and the opposite side 113 of the cavity 104.The line restrictor 110 is optionally a part of the insert 108.Alternatively, the line restrictor 110 is a plunger 110′ as it is indicated with hatched outlines, such that the line restrictor 110 can be moved at least partly but preferably totally in and out of the cavity 104.As shown in FIG.", "2, a space between the frame 203 and the detachable module 201 may also be provided by corresponding structures on the insert 108.Alternatively, this space 204 or these spaces may be produced by one or more plungers inserted into the cavity 104 after having filled polymer into the cavity 104.A further, however not preferred, possibility, is to stamp or mill these spaces out of the moulded disc 200 with detachable module 201.Smaller sections of grooves for breaking lines may be provided, for example, as shown in FIG.", "3, where the groove for the breaking line 301 between the optical disk 200 and the detachable module 201 may actually be provided between the frame 203 and the detachable module 201.The detachable module 201 may in this case be detached from the frame 203, which, in principle could remain as part of the optical disk 200.The frame 203 can be attached to the optical disk 200 with a number of bridges 302 as shown in FIG.", "3a or, alternatively, with grooves 303 along breaking lines as shown in FIG.", "3b which take the place of the bridges.", "The line restrictor 110 may be provided as a wedge, as shown in FIG.", "4, where one side 401 of the line restrictor 110′ has a smaller taper angle 402 than other side 403.The side 401 with the smaller taper angle is usually used for edges that have to be steep, like it is preferred for SIM plugs.", "The less steep side on the wedge is used in order to provide a restrictor which is blunt enough so that the optical disk will not fasten to the line restrictor.", "Usually the edge of the optical disk is not necessarily steep.", "If the detachable module 201 and the optical disk 200 both are desired with steep edges, the embodiment of FIG.", "3b is preferred.", "The groove 301 for the breaking line between the detachable module 201 and the frame 203 may be constructed to be steep on the edge of the detachable module 301 and less steep on the frame.", "On the other hand, the grooves 303 for the breaking lines between the frame 203 and the optical disk 200 may be constructed such that the optical disc 200 has a steep edge.", "Other examples for possible shapes for line restrictors are shown in FIGS.", "4b and 4c.", "In FIG.", "6, an alternative approach is shown.", "In this case, the optical disk 200 has a circular dimension 601 and comprises two detachable modules 201, 201′.", "The detachable modules 201, 201′ are connected to the optical disc 200 by grooves 301, 301′ along breaking lines and are otherwise surrounded by open spaces 204.Limited by the placement of the modules 201, 201′ on FIG.", "6 are the tracks 602 of the optical disk 200.In contrast, for the embodiments shown in FIG.", "2 and FIG.", "3, the tracks 602 are limited by the outer dimension of the optical disk 200.In FIG.", "7a an embodiment is shown, where the position of the detachable module 201 with respect to the tracks 603 of the optical disc 200 is analogous to one of the shown embodiments as disclosed In International patent application WO 01/18750 by Lüke assigned to Orga.", "If the position of the SIM module is as required by ISO 7810 standard, the tracks 602 of the optical disk 200 are limited by the outer diameter 703 that is defined by the SIM module.", "For standard disk drives for reading CD-ROMs, where also a minimum diameter 704 is given for the reading the tracks 603, the space between the inner diameter 704 and the outer diameter 703 is almost negligible, leaving practically no space for data storage.", "Thus, this embodiment is not suited for standard CD-ROM drives or standard music CD drives, if the ISO standard 7810 is to be followed.", "However, the shown embodiment on FIG.", "7a has an advantage over the disclosure in International patent application WO 01/18750 by Lüke assigned to Orga, because the diameter 702 of the optical disc 200 is at least 80 mm, such that the diameter of the optical disc does not prevent readability in standard disc reading units.", "If the placement of the SIM plug should not exactly correspond to the ISO 7810 standard, the SIM plug 201 may be placed at the edge as shown in FIG.", "7b.", "This way, the SIM plug 201 does not limit the tracks 602 of the optical disc 200.Visible in this drawing is also the hollow 706 for the electronic circuit and the even deeper hollow 707 for the glue.", "In case that the optical disc together with the detachable module shall correspond to the ISO 7810 standard with a length of 85.6 mm and a width of 53.9 mm, a certain embodiment as shown on FIG.", "8 has turned out to be of great advantage.", "The detachable module 201 as shown is a SIM plug with an electronic circuit 701 with a position that fulfills the ISO 7816 standard.", "Thus, the SIM module 201 is connected such to the optical disk 200 that existing standard machines can be used for placing the electronic circuit 701 into the hollow in the SIM module 201.This reduces the production costs for the optical disk 200 with SIM module 201 according to the invention.", "However, though the position of the electronic circuit is correct, the programming of the connectors are changed, because the DIM plug 201 is turned 180 degrees as compared to standard configurations, where the standard configuration is shown in FIG.", "7, that is to say with the skew edge 801 of the SIM plug is facing away from the optical disk.", "This different programming by existing standard machines can be achieved by only minor modifications to these machines, the modifications being far less extensive than changing the placement of the module 201 itself.", "The placement of the SIM module 201 as shown in FIG.", "8 still leaves space enough for the tracks 602 of the optical disk to contain sufficient data for the user of the optical disk.", "The embodiment on FIG.", "8 furthermore Implies that the diagonal 702 of the optical disk 200 is at least 80 mm such that it is assured that the optical disk 200 is playable in all corresponding standard reading units.", "In the shown embodiments in FIG.", "2, 3 6, 7, and FIG.", "8, the location of the microelectronics module 200 is within the periphery 601 of typical circular CD-ROMs and, therefore, inside the maximum diameter, 12 cm, acceptable by a CD-ROM reading unit.", "Such a CD-ROM may be read before or after detachment of the microelectronics module.", "For instance, a user may read the information of the optical disk in his CD-reading unit prior to detaching of the SIM plug 201 for installation in a mobile phone.", "In this case, the user may study the information on the optical disk 200, before the user actually decides to use the SIM plug 201 in the user's telephone.", "After detachment of the SIM plug 2, the user may still read the information which may be related to the installation and the set-up of the telephone, once the SIM plug 201 is inserted into the mobile phone.", "The possibility to read the information on the optical disk 201 before detachment of the microelectronics module 201, for example a SIM plug or another computer component, may be important for the user in case he wants to read the information in order to find out, whether he actually wishes to purchase the module 201.On the other hand, the possibility to read the information after detachment may be important for the user in case that the information is Important during the user's installation of the module 201, where the Installation can be a number of difficult steps.", "FIG.", "9 shows a further embodiment of an optical disk that is a carrier card with optical disk 200 and with two detachable modules 201 and 201′, which, for example, may be microelectronics modules like SIM plugs.", "One of the modules 201 is inside the periphery 601 typical for circular CD-ROMs and therefore inside the maximum diameter acceptable by a CD-ROM reading unit, while the other microelectronics module 201′ is partly outside periphery 601 typical for circular CD-ROMs and may be outside the maximum diameter acceptable by a CD-ROM reading unit.", "In order to read the optical disk 200 in a CD-ROM drive, it is necessary to remove the second microelectronics module 201′.", "In case that the user after purchase of the optical disk 200 wants to return the optical disk 200 to the seller, this may be used as a safe and easy control for the seller whether the CD-ROM 200 has been read.", "Because the embodiment as shown on FIG.", "8 has outlines that exceed the normal standard for optical disks, the production of such an embodiment may require modification of existing mould devices.", "It is possible to form optical disks 200 with a variety of different shapes, for example polygonal, star-shaped, or oval, which may be used to catch the attention of the user when the user is visiting a seller's place and has to chose among a number of offers.", "A frame 203 as shown in FIGS.", "2 and 7 may function as a carrier component that Is provided with a shape appropriate for the device of interest into which the module 201 has to be installed.", "Thus, the frame 203 may function as an adapter in an analogue way as telephone SIM cards, where the carrier SIM card-fulfilling the ISO 7810 standard with the SIM plug 201 may be installed in some cellular telephones, while the SIM plug 201 itself may be detached from the carrier SIM card for installation into other cellular telephones.", "In FIG.", "9, one detachable module 201′ is attached to the carrier card with optical disk 200 with bridges 301.Alternatively, this detachable module 201′ may be attached to the optical disk along one or several perforated lines.", "Such bridges 301 or perforated lines may be produces by the above described plunger principle, though also the insert 105 in the mould cavity 104 may be constructed such that plungers may be avoided." ] ]	Patent_10468481
[ [ "Method for producing freshly catched seafood", "A process for the production of ready-to-prepare portions of freshly caught fish such as cod, saithe, salmon, trout etc., comprising the steps of: catch and quality control; 2) gutting and filleting; 3) deep-freezing; 3) acceptance at processing facility and quality control; 4) defrosting; 5) portioning and placing in transport or retail packaging; 6) cooling/freezing; and 8) packing, wherein a) the deep-freezing in step 3) is carried out as an “interleave” freezing of the individual fillets with interleaving to about −20° C.; b) the defrosting in step 5) is carried out in a brine solution containing 0.5-5% NaCl and 0.5-10% commercially available 60% K-Lac; c) the cooling/freezing in step 7) is carried out as a shell-freezing with CO2 saturation and cooling with CO2 in CO2 cabinets to as close to the freezing point of the product as possible, adjusted according to the intended distance of transport; d) packing in an open system where air is replaced by a mixture of N2+CO2 as a transport and packaging atmosphere." ], [ "1.A process for the production of ready-to-prepare portions of freshly caught fish such as cod, saithe, salmon, trout, etc., comprising the steps of: 1) catch and quality control; 2) gutting and filleting; 3) deep-freezing; 3) acceptance at processing facility and quality control; 4) defrosting; 5) portioning and placing in transport or retail packaging; 6) cooling/freezing; and 8) packing, characterised in that a) the deep freezing in step 3) is carried out as an “interleave” freezing of the individual fillets with interleaving to about −20° C.; b) the defrosting in step 5) is carried out in a brine solution containing 0.5-5% NaCl and 0.5-10% commercially available 60% K-Lac; c) the cooling/freezing in step 7) is carried out as a shell-freezing with CO2 saturation and cooling with CO2 in CO2 cabinets to as close to the freezing point of the product as possible, adjusted according to the intended distance of transport; d) packing in an open system where air is replaced by a mixture of N2+CO2 as a transport and packaging atmosphere.", "2.A process according to claim 1, characterised in that the defrosting in step 5) is carried out in a brine solution comprising 2% NaCl and 5% commercially available 60% K-Lac." ], [ "The present invention relates to a process for the production of ready-to-prepare portions of freshly caught seafood.", "Specifically, the invention relates to the production of such portions of fish such as cod, saithe, salmon, trout etc.", "Fish is found in today's foodstuff market in numerous deep-frozen forms such as breaded fish products, stuffed fillets and the like, where fish products of this type are intended for frying or heating through, for example, in a pan in an oven.", "Also available are non-processed deep-frozen products of the “natural” fish type, suitable for poaching.", "However, the consumers' desired access to fresh fish has to date basically been limited to regular fishmonger's shops or chain stores with a separate fish counter.", "The object of the present invention is to provide the market with a supply of a product that is as close to a fresh product as it is possible to get, taking into account the need for transport time.", "The object of the present invention is to be able to deliver portions of fish of a quality that is not inferior to the quality of true fresh fish, and which may even be better than true fresh fish and where the product is ready to prepare upon arrival at the retailer.", "The requirements made by today's society with respect to foodstuffs that are to be marketed as fresh are stringent, and of these requirements special mention should be made of traceability of the product, an extensive quality assurance requirement, and the requirement of documented shelf life.", "The object of the present invention is to provide products that also meet these stringent requirements.", "Accordingly, the present invention relates to a method for the production of ready-to-prepare portions of freshly caught fish such as cod, saithe; salmon, trout, etc., comprising the steps of: 1) catch and quality control; 2) gutting and filleting; 3) deep-freezing; 4) acceptance at processing facility and quality control; 5) defrosting; 6) portioning and placing in transport/retail packaging; 7) cooling/freezing; and 8) packing; and this process is characterised in that a) the deep freezing in step 3) is carried out as an “interleave” freezing of the individual fillets with interleaving to about −20° C.; b) the defrosting in step 5) is carried out in a brine solution containing 0.5-5% NaCl and is 0.5-10% commercially available 60% K-Lac; c) the cooling/freezing in step 7) is carried out as shell-freezing with CO2 saturation, and cooling with CO2 in CO2 cabinets to as close to the freezing point of the product as possible, adjusted according to the intended distance of transport; d) packing in an open system where air is replaced by a mixture of N2+CO2 as a transport and packaging atmosphere.", "K-Lac in the present context is meant to include lactates of sodium, potassium and calcium.", "The preferred values in step b) are 2% NaCl and 5% K-Lac.", "Products treated in this way have a shelf life of a minimum of 12 days when stored at +4° C. without any deterioration of quality.", "To the different steps in the process, the following may be added: Immediately after the fish has been caught, the raw material is frozen on board the fishing vessels, usually within six hours.", "Because quality assurance is an important feature in order to obtain the desired quality of the product described herein, the applicant has found that seiners give the best quality, although trawlers with good production and freezing routines may achieve a microbiological level that is particularly low compared with ordinary fresh fish.", "Similar results may be achieved for farmed fish if they are processed or filleted no later than 2 days after slaughtering, and where the slaughtering and filleting is carried out in a controlled manner under stringent hygienic conditions.", "The freezing after gutting and filleting is very important as this freezing contributes to the elimination of “smell-generating” bacteria, specifically Photobacterium phosphoreum which causes H2S and TMA production.", "The freezing in step 3) is carried out as a so-called “interleave” freezing, i.e., the individual fillet is frozen with interleaving in order to maintain the integrity of the fillet and to inhibit and prevent oxidation and dehydration On acceptance at the final production facility, another quality control takes place before the crucial defrosting and brine treatment is carried out.", "In order to maintain the quality throughout the defrosting process, the applicant has developed a method of carrying out the defrosting at the same time as the brine solution treatment.", "Thus, the defrosting in step 5) is carried out in a controlled manner in a brine solution with a careful supply of power in order to maintain the temperature close to 0° C., and with simultaneous gentle handling of the individually deep-frozen fillets.", "It would not be a departure from the scope of this invention if the defrosting were started in air but finished in brine.", "The brine solution used in this invention is a combination of water, cooking salt, and lactate, i.e., NaCl and K-Lac, in a mixture as defined above.", "This mixture provides a unique preservation of quality, especially the sensory quality, whilst the defined content of the said salts contribute to the desired retention of water and prevent water loss from the product after it has been packed.", "At the same time these salts prevent the growth of bacteria in general, and in particular dangerous bacteria such as Listeria.", "It should perhaps be mentioned here that the authorities have approved the use of lactate, calcium lactate and cooking salt, NaCl, and that they have been used in some cases, but not in a process and in the manner described herein.", "After the defrosting and the brine treatment, the pieces of fish are portioned and placed in transport or retail containers, and in step 7) a cooling/freezing of the fish using CO2 as freezing medium is carried out when the fish is to be packed in a controlled atmosphere.", "The applicant has developed a method for cooling/freezing fish which is to be packed in a modified atmosphere of CO2 and N2.Of course, the scope and degree of this freezing is dependent on the fish type and the effect the process has been found to have on the sensory quality of the fish.", "Thus, the degree of freezing may be from a pure shell-freezing of a few millimetres of the surface with a core temperature of about −1° C., to a deeper through freezing of the whole product to a lower temperature.", "The shell-freezing is preferably carried out to a temperature as close to the freezing temperature of the product as possible in CO2 cabinets having a CO2 atmosphere, and the degree of freezing depends not least on the distance of transport.", "The temperature in the CO2 atmosphere is preferably kept close to −60° C. The purpose is that the product should be defrosted to a fresh state when it reaches the retail outlet.", "In addition, this freezing in a partial CO2 atmosphere results in the fish portions being saturated with CO2, which means that CO2 is not absorbed by the fish during transport, which in turn means that the fish maintains its transport atmosphere and thus also a tasteful wrapping.", "A further purpose of this CO2 freezing treatment is to utilise the general inhibitory effects that CO2 has on microbial growth.", "In conclusion, the special combination of steps as described according to this application allows a unique fresh food product of a high and assured quality close to the real fresh product to be obtained." ] ]	Patent_10468504
[ [ "DIESEL ENGINE EXHAUST PURIFIER", "An exhaust emission control apparatus for diesel engines is offered which is capable of achieving better power performance and exhaust emission purification efficiency in an optimally reconciled manner, and from which torque deficiency during hill climbing on a steep slope is eliminated.", "Emulsified fuel including light oil, water and emulsifier supplied respectively from a light oil tank 4, a water tank 5 and an emulsifier cartridge 6, of which the water ratio is adjusted by flow variable control valves 11-13, is supplied to a combustion chamber 4.", "The water ratio is normally set to a basic water ratio corresponding to the signal of an accelerator opening sensor 39 (Step S32).", "However, when a difference between target acceleration and actual acceleration of a vehicle corresponding to the accelerator pedal opening exceeds a predetermined value (Step S36), the basic water ratio is corrected so as to reduce the water ratio (Step S37)." ], [ "1.An exhaust emission control apparatus for a diesel engine comprising: a fuel injection valve for injecting fuel into a combustion chamber of a diesel engine; a fuel supply system for supplying fuel; a water supply feed unit for supplying water; an emulsifier feed unit for supplying emulsifier; a load detection means for detecting an accelerator pedal depression amount; a water ratio adjusting device for adjusting a water ratio from said water supply feed unit in relation to fuel from said fuel supply system; a static mixer for blending emulsified fuel from said emulsifier, said adjusted fuel and water and for supplying this emulsified fuel to said fuel injection valve; a torque deficiency judgment means for judging whether or not a torque deficiency exists relative to said accelerator pedal depression amount; and a water ratio determination means for setting a basic water ratio in which the water ratio of a larger accelerator depression amount in an engine operating range increases more than a smaller accelerator pedal depression amount in an engine operating range corresponding to accelerator pedal depression amount detected by said load detection means and for controlling said water ratio adjusting device to regulate a basic ratio when said torque deficiency judgment means judges a torque deficiency does not exist and a lower water ratio when said torque deficiency judgment means judges a torque deficiency does exist.", "2.The exhaust emission control apparatus for a diesel engine according to claim 1, wherein said torque deficiency judgment means judges a torque deficiency exists when said accelerator pedal depression amount is an amount of the actual maximum depression of an accelerator pedal.", "3.The exhaust emission control apparatus for a diesel engine according to claim 1, wherein said torque deficiency judgment means judges a torque deficiency exists when an acceleration difference is acquired and this acceleration difference becomes higher than a predetermined value by subtracting the target acceleration of a vehicle or a wheel made to respond to said accelerator pedal depression amount from the actual acceleration of said vehicle or wheel.", "4.The exhaust emission control apparatus for a diesel engine according to claim 3, wherein a reduction of said water ratio while judging said torque deficiency is set so that as said acceleration difference becomes higher said water ratio becomes lower.", "5.The exhaust emission control apparatus for a diesel engine according to claim 3 or 4, wherein said water ratio determination means cancels reduction of said water ratio when said acceleration difference becomes lower than a preset value less than said predetermined value.", "6.The exhaust emission control apparatus for a diesel engine according to any one of claims 3 through 5, wherein said predetermined value has a plurality of different values corresponding to the magnitude of said accelerator pedal depression amount.", "7.The exhaust emission control apparatus for a diesel engine according to any one of claims 3 through 6, wherein said predetermined value increases as said accelerator pedal depression amount becomes higher.", "8.The exhaust emission control apparatus for a diesel engine according to claim 5, wherein said preset value is set to become lower as said accelerator pedal depression amount becomes higher.", "9.The exhaust emission control apparatus for a diesel engine according to any one of claims 3 through 8, wherein said torque deficiency judgment means judges torque deficiency when said acceleration difference becomes higher than a predetermined value even though said accelerator pedal depression amount increases more than a predetermined amount within a predetermined time.", "10.The exhaust emission control apparatus for a diesel engine according to any one of claims 3 through 8, wherein said torque deficiency judgment means judges torque deficiency when vehicle speed decelerates more than predetermined within a predetermined time even though said accelerator pedal depression amount is more than a predetermined amount and said accelerator pedal depression amount does not change substantially within a predetermined time.", "11.The exhaust emission control apparatus for a diesel engine according to any one of claims 1 through 10, wherein said accelerator pedal depression amount is a value corresponding to the fuel flow rate supplied to said fuel injection valve or a value corresponding to the flow rate difference of this flow rate and the return fuel flow rate discharged from said fuel injection valve.", "12.The exhaust emission control apparatus for a diesel engine according to any one of claims 1 through 11, wherein said water ratio determination means sets the water ratio to substantially zero for a period of a predetermined time from diesel engine start-up time until any one of the engine coolant temperature, the temperature of said return fuel in the return passage or the transmission temperature reaches a predetermined temperature.", "13.The exhaust emission control apparatus for a diesel engine according to any one of claims 1 through 12, wherein said water ratio determination means sets the water ratio to substantially zero until a predetermined time elapses after detecting a diesel engine stop signal.", "14.The exhaust emission control apparatus for a diesel engine according to any one of claims 1 through 13, wherein said water ratio is controlled corresponding to the exhaust gas temperature within a diesel particulate filter." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates to an exhaust emission control apparatus for diesel engines adapted to suppress particularly the generation of nitrogen oxides in the exhaust gas.", "As for a conventional exhaust emission control apparatus for diesel engines, a technique in which water and emulsifier are added to fuel and supplied to a combustion chamber as emulsified fuel to lower the highest combustion temperature in the combustion chamber whereby generation of nitrogen oxides are suppressed is known from the description in Japanese Laid-Open Patent Application (Kokai) (A) No.", "Heisei 7-166962 (1995) titled “ENGINE EMULSIFIED FUEL FEEDER UNIT” and Japanese Laid-Open Patent Application (Kokai) (A) No.", "Heisei 7-166963 (1995) titled “ENGINE EMULSIFIED FUEL FEEDER UNIT.” In the above official gazettes, it is surely described that the ratio of adding water (water ratio) to the fuel is decided depending on the state of load on the engine.", "However, they do not give any particular description about the above-mentioned water ratio, which is the most indispensable to make the technique practicable in the case of vehicles equipped with an engine that is operated in a broad load range corresponding to the running conditions, unlike the case of ships where the technique has already been put into practical use.", "According to research and the experiment of the inventors, it was found that engine power performance and exhaust emission purification efficiency cannot be optimally reconciled in a broad engine operational range by fixing the water ratio to the load at a specific level or by simply increasing the water ratio in a proportional manner corresponding to the increase of the load.", "The object of the present invention is to solve the above described problem and to provide an exhaust emission control apparatus for diesel engines capable of achieving better power performance and exhaust emission purification efficiency in a compatible and enhanced manner even in a broad engine operational range." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention of an exhaust emission control apparatus for a diesel engine comprises a fuel injection valve for injecting fuel into a combustion chamber of a diesel engine; a fuel supply system for supplying fuel; a water supply feed unit for supplying water; an emulsifier feed unit for supplying emulsifier; a load detection means for detecting an accelerator pedal depression amount; a water ratio adjusting device for adjusting a water ratio from the water supply feed unit in relation to fuel from the fuel supply system; a static mixer for blending emulsified fuel from the emulsifier, the adjusted fuel and water and for supplying this emulsified fuel to the fuel injection valve; a torque deficiency judgment means for judging whether or not a torque deficiency exists relative to the accelerator pedal depression amount; and a water ratio determination means for setting a basic water ratio in which the water ratio of a larger accelerator depression amount in an engine operating range increases more than a smaller accelerator pedal depression amount in an engine operating range corresponding to accelerator pedal depression amount detected by the load detection means and for controlling the water ratio adjusting device to regulate a basic ratio when the torque deficiency judgment means judges a torque deficiency does not exist and a lower water ratio when the torque deficiency judgment means judges a torque deficiency does exist.", "In the above constitution, fuel, water and an emulsifier are supplied respectively from a fuel supply system, a water supply feed unit and an emulsifier feed unit.", "Then, after optimum water ratio is decided corresponding to the accelerator pedal depression amount by the water ratio determination means, amount of the fuel and amount of the water are adjusted in the water ratio by the water ratio adjusting device to produce an emulsified fuel with the emulsifier.", "The emulsified fuel is preferably a water-in-oil type (W/O type).", "In the control of the water ratio, the basic water ratio is set up such that, during normal driving, the water ratio in the engine operational range having a large acceleration pedal depression amount is increased compared with that having a small accelerator pedal depression amount.", "Accordingly, since the combustion temperature is lowered due to the existence of the water, it is possible to reduce the generation of NOx.", "In this case, since the water ratio is controlled as described above, it is always possible to allow the exhaust emission control efficiency and engine power performance to be consistent with each other even in a broad engine operational range like the case of vehicles.", "Further, since it is determined by the torque deficiency judgment means whether it is the state of torque deficiency based on accelerator pedal depression amount, even when hill climbing on a steep slope or sudden acceleration with a heavy load, the water ratio is reduced smaller than the basic water ratio thereby a larger torque can be obtained.", "Accordingly, it is possible to ensure the power performance that enables the above-mentioned hill climbing and sudden acceleration.", "In this case, the generation of the NOx increases.", "However, since the above period of the operation is short and the engine speed decreases because of the large load, the amount of the generated NOx becomes less than that generated during normal driving.", "Although soot and the like increases, these can be removed by using an oxidation catalyst, heater or the like provided in the exhaust system.", "As described above, during normal driving even with a large load, it is possible to make engine power performance and exhaust emission purification efficiency optimally compatible.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, the torque deficiency judgment means judges a torque deficiency exists when the accelerator pedal depression amount is an amount of the actual maximum depression of an accelerator pedal.", "Since the judgment of the torque deficiency is made corresponding to the magnitude of the accelerator pedal depression amount, if the driver feels that torque is deficient and depresses the accelerator pedal to the maximum amount level, torque deficiency can be judged easily and the intention of the driver certainly detected.", "The maximum pedal depression of the accelerator pedal may be determined based on the magnitude of output voltage using a potentiometer which detects the pedal depression amount of the accelerator pedal, or may be constituted with a kick down switch so as to turn on at the maximum pedal depression of the accelerator pedal.", "Accordingly, the maximum pedal depression of the accelerator can be detected using a simple and inexpensive device.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, the torque deficiency judgment means judges a torque deficiency exists when an acceleration difference is acquired and this acceleration difference becomes higher than a predetermined value by subtracting the target acceleration of a vehicle or a wheel made to respond to the accelerator pedal depression amount from the actual acceleration of the vehicle or wheel.", "After deciding the target acceleration of the vehicle or wheel corresponding to the detected accelerator pedal depression amount, the actual acceleration is subtracted from the target acceleration.", "The acceleration difference is compared with the predetermined value by the torque deficiency judgment means, and when the acceleration difference is more than a predetermined value, it is judged as torque deficiency.", "If judged as torque deficiency, the water ratio determination means controls the water ratio adjustment device so that the water ratio becomes smaller than the basic water ratio.", "Accordingly, when hill climbing on a steep slope or a sudden acceleration with a heavy load, the water ratio is decreased so as to be a value smaller than the water ratio during normal driving such as on a flat road with a light load.", "As a result, the output torque of the engine is increased and the required power performance is ensured.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein a reduction of the water ratio while judging the torque deficiency is set so that as the acceleration difference becomes higher the water ratio becomes lower.", "As described above, by adapting the reduction of the water ratio so that a larger acceleration difference results in a smaller water ratio, it becomes possible to reconcile power efficiency and exhaust gas purification with more sufficient accuracy according to the actual running conditions.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the water ratio determination means cancels reduction of the water ratio when the acceleration difference becomes lower than a preset value less than the predetermined value.", "At the torque deficiency, as a result of the correction of the basic water ratio so that the water ratio is reduced to increase the output torque of the engine, the acceleration becomes larger and the difference between the target and actual acceleration becomes less than a set value that has been set to a value smaller than the predetermined value.", "Then, the control of water ratio reduction will be stopped and returns to the basic water ratio.", "Thereby, the rise of engine power is suppressed to the required torque and becomes possible to control exhaust gas purification.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the predetermined value has a plurality of different values corresponding to the magnitude of the accelerator pedal depression amount.", "By setting the predetermined value to two or more different values in accordance with the accelerator pedal depression amount, it becomes possible to control the water ratio more finely corresponding to the running situations and the engine operational conditions.", "Accordingly, power performance and exhaust emission purification efficiency can be optimally reconciled.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the predetermined value increases as the accelerator pedal depression amount becomes higher.", "In this case, by adapting so that the predetermined value increases as the accelerator pedal depression amount becomes larger, it becomes possible to obtain the acceleration closer to the acceleration requested by the driver.", "According to the present invention for the exhaust emission control apparatus for a diesel engine, wherein the preset value is set to become lower as the accelerator pedal depression amount becomes higher.", "By adapting the set value so as to decrease as the accelerator pedal depression amount becomes larger, it becomes possible to obtain the acceleration closer to the acceleration requested by the driver like above.", "This setting is suitable for vehicles which do not have much margin in maximum engine power.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the torque deficiency judgment means judges torque deficiency when the acceleration difference becomes higher than a predetermined value even though the accelerator pedal depression amount increases more than a predetermined amount within a predetermined time.", "The torque deficiency judgment means judges that it is the state of torque deficiency when the acceleration difference becomes more than a predetermined value even when the driver depresses the accelerator pedal sharply demanding acceleration.", "Accordingly, it is possible to reliably detect the will of the driver to accelerate positively by the sudden depression of the accelerator pedal by the driver.", "The deficiency of torque (accordingly, acceleration) in this case can be detected easily.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the torque deficiency judgment means judges torque deficiency when vehicle speed decelerates more than predetermined within a predetermined time even though the accelerator pedal depression amount is more than a predetermined amount and the accelerator pedal depression amount does not change substantially within a predetermined time.", "In this case, before a driver notices, it is easily detectable the vehicle's sudden slowdown and becomes torque deficient.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the accelerator pedal depression amount is a value corresponding to the fuel flow rate supplied to the fuel injection valve or a value corresponding to the flow rate difference of this flow rate and the return fuel flow rate discharged from the fuel injection valve.", "As described above, if the accelerator pedal depression amount, as a result of a water ratio are determined with a flow rate or flow rate difference, it will become unnecessary to receive an information signal from the engine control unit side.", "Thus, the exhaust emission control apparatus of the present invention can be applied to a variety of types of vehicles, which have already been put on the market from various makers.", "Notably, it is possible to apply the same concept to many different vehicles without taking into consideration the signal processing of the engine control unit and the transfer method of the signal, which are different with each maker and each vehicle.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the water ratio determination means sets the water ratio to substantially zero for a period of a predetermined time from diesel engine start-up time until any one of the engine coolant temperature, the temperature of the return fuel in the return passage or the transmission temperature reaches a predetermined temperature.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the water ratio determination means sets the water ratio to substantially zero until a predetermined time elapses after detecting a diesel engine stop signal.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the water ratio is controlled corresponding to the exhaust gas temperature within a diesel particulate filter.", "In the case when there is a possibility that the catalyst or filter may be damaged due to extraordinary high temperatures of the exhaust gas in a diesel particulate filter, it is possible to reduce the temperature of the exhaust gas by increasing the water ratio to prevent the above filter from overheating.", "Conversely, to accelerate the combustion of the soot or the like while the temperature in the filter is low, it is possible to raise the temperature in the filter easily and swiftly by reducing the water ratio to raise the temperature of the exhaust gas.", "The above and further objects and novel features of the present invention will more fully appear from the following detailed description when the same is read in conjunction with the accompanying drawings.", "It is to be expressly understood, however, that the drawings are for the purpose of illustration only and are not intended as a definition of the limits of the invention." ], [ "BACKGROUND OF THE INVENTION The present invention relates to an exhaust emission control apparatus for diesel engines adapted to suppress particularly the generation of nitrogen oxides in the exhaust gas.", "As for a conventional exhaust emission control apparatus for diesel engines, a technique in which water and emulsifier are added to fuel and supplied to a combustion chamber as emulsified fuel to lower the highest combustion temperature in the combustion chamber whereby generation of nitrogen oxides are suppressed is known from the description in Japanese Laid-Open Patent Application (Kokai) (A) No.", "Heisei 7-166962 (1995) titled “ENGINE EMULSIFIED FUEL FEEDER UNIT” and Japanese Laid-Open Patent Application (Kokai) (A) No.", "Heisei 7-166963 (1995) titled “ENGINE EMULSIFIED FUEL FEEDER UNIT.” In the above official gazettes, it is surely described that the ratio of adding water (water ratio) to the fuel is decided depending on the state of load on the engine.", "However, they do not give any particular description about the above-mentioned water ratio, which is the most indispensable to make the technique practicable in the case of vehicles equipped with an engine that is operated in a broad load range corresponding to the running conditions, unlike the case of ships where the technique has already been put into practical use.", "According to research and the experiment of the inventors, it was found that engine power performance and exhaust emission purification efficiency cannot be optimally reconciled in a broad engine operational range by fixing the water ratio to the load at a specific level or by simply increasing the water ratio in a proportional manner corresponding to the increase of the load.", "The object of the present invention is to solve the above described problem and to provide an exhaust emission control apparatus for diesel engines capable of achieving better power performance and exhaust emission purification efficiency in a compatible and enhanced manner even in a broad engine operational range.", "SUMMARY OF THE INVENTION The present invention of an exhaust emission control apparatus for a diesel engine comprises a fuel injection valve for injecting fuel into a combustion chamber of a diesel engine; a fuel supply system for supplying fuel; a water supply feed unit for supplying water; an emulsifier feed unit for supplying emulsifier; a load detection means for detecting an accelerator pedal depression amount; a water ratio adjusting device for adjusting a water ratio from the water supply feed unit in relation to fuel from the fuel supply system; a static mixer for blending emulsified fuel from the emulsifier, the adjusted fuel and water and for supplying this emulsified fuel to the fuel injection valve; a torque deficiency judgment means for judging whether or not a torque deficiency exists relative to the accelerator pedal depression amount; and a water ratio determination means for setting a basic water ratio in which the water ratio of a larger accelerator depression amount in an engine operating range increases more than a smaller accelerator pedal depression amount in an engine operating range corresponding to accelerator pedal depression amount detected by the load detection means and for controlling the water ratio adjusting device to regulate a basic ratio when the torque deficiency judgment means judges a torque deficiency does not exist and a lower water ratio when the torque deficiency judgment means judges a torque deficiency does exist.", "In the above constitution, fuel, water and an emulsifier are supplied respectively from a fuel supply system, a water supply feed unit and an emulsifier feed unit.", "Then, after optimum water ratio is decided corresponding to the accelerator pedal depression amount by the water ratio determination means, amount of the fuel and amount of the water are adjusted in the water ratio by the water ratio adjusting device to produce an emulsified fuel with the emulsifier.", "The emulsified fuel is preferably a water-in-oil type (W/O type).", "In the control of the water ratio, the basic water ratio is set up such that, during normal driving, the water ratio in the engine operational range having a large acceleration pedal depression amount is increased compared with that having a small accelerator pedal depression amount.", "Accordingly, since the combustion temperature is lowered due to the existence of the water, it is possible to reduce the generation of NOx.", "In this case, since the water ratio is controlled as described above, it is always possible to allow the exhaust emission control efficiency and engine power performance to be consistent with each other even in a broad engine operational range like the case of vehicles.", "Further, since it is determined by the torque deficiency judgment means whether it is the state of torque deficiency based on accelerator pedal depression amount, even when hill climbing on a steep slope or sudden acceleration with a heavy load, the water ratio is reduced smaller than the basic water ratio thereby a larger torque can be obtained.", "Accordingly, it is possible to ensure the power performance that enables the above-mentioned hill climbing and sudden acceleration.", "In this case, the generation of the NOx increases.", "However, since the above period of the operation is short and the engine speed decreases because of the large load, the amount of the generated NOx becomes less than that generated during normal driving.", "Although soot and the like increases, these can be removed by using an oxidation catalyst, heater or the like provided in the exhaust system.", "As described above, during normal driving even with a large load, it is possible to make engine power performance and exhaust emission purification efficiency optimally compatible.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, the torque deficiency judgment means judges a torque deficiency exists when the accelerator pedal depression amount is an amount of the actual maximum depression of an accelerator pedal.", "Since the judgment of the torque deficiency is made corresponding to the magnitude of the accelerator pedal depression amount, if the driver feels that torque is deficient and depresses the accelerator pedal to the maximum amount level, torque deficiency can be judged easily and the intention of the driver certainly detected.", "The maximum pedal depression of the accelerator pedal may be determined based on the magnitude of output voltage using a potentiometer which detects the pedal depression amount of the accelerator pedal, or may be constituted with a kick down switch so as to turn on at the maximum pedal depression of the accelerator pedal.", "Accordingly, the maximum pedal depression of the accelerator can be detected using a simple and inexpensive device.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, the torque deficiency judgment means judges a torque deficiency exists when an acceleration difference is acquired and this acceleration difference becomes higher than a predetermined value by subtracting the target acceleration of a vehicle or a wheel made to respond to the accelerator pedal depression amount from the actual acceleration of the vehicle or wheel.", "After deciding the target acceleration of the vehicle or wheel corresponding to the detected accelerator pedal depression amount, the actual acceleration is subtracted from the target acceleration.", "The acceleration difference is compared with the predetermined value by the torque deficiency judgment means, and when the acceleration difference is more than a predetermined value, it is judged as torque deficiency.", "If judged as torque deficiency, the water ratio determination means controls the water ratio adjustment device so that the water ratio becomes smaller than the basic water ratio.", "Accordingly, when hill climbing on a steep slope or a sudden acceleration with a heavy load, the water ratio is decreased so as to be a value smaller than the water ratio during normal driving such as on a flat road with a light load.", "As a result, the output torque of the engine is increased and the required power performance is ensured.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein a reduction of the water ratio while judging the torque deficiency is set so that as the acceleration difference becomes higher the water ratio becomes lower.", "As described above, by adapting the reduction of the water ratio so that a larger acceleration difference results in a smaller water ratio, it becomes possible to reconcile power efficiency and exhaust gas purification with more sufficient accuracy according to the actual running conditions.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the water ratio determination means cancels reduction of the water ratio when the acceleration difference becomes lower than a preset value less than the predetermined value.", "At the torque deficiency, as a result of the correction of the basic water ratio so that the water ratio is reduced to increase the output torque of the engine, the acceleration becomes larger and the difference between the target and actual acceleration becomes less than a set value that has been set to a value smaller than the predetermined value.", "Then, the control of water ratio reduction will be stopped and returns to the basic water ratio.", "Thereby, the rise of engine power is suppressed to the required torque and becomes possible to control exhaust gas purification.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the predetermined value has a plurality of different values corresponding to the magnitude of the accelerator pedal depression amount.", "By setting the predetermined value to two or more different values in accordance with the accelerator pedal depression amount, it becomes possible to control the water ratio more finely corresponding to the running situations and the engine operational conditions.", "Accordingly, power performance and exhaust emission purification efficiency can be optimally reconciled.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the predetermined value increases as the accelerator pedal depression amount becomes higher.", "In this case, by adapting so that the predetermined value increases as the accelerator pedal depression amount becomes larger, it becomes possible to obtain the acceleration closer to the acceleration requested by the driver.", "According to the present invention for the exhaust emission control apparatus for a diesel engine, wherein the preset value is set to become lower as the accelerator pedal depression amount becomes higher.", "By adapting the set value so as to decrease as the accelerator pedal depression amount becomes larger, it becomes possible to obtain the acceleration closer to the acceleration requested by the driver like above.", "This setting is suitable for vehicles which do not have much margin in maximum engine power.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the torque deficiency judgment means judges torque deficiency when the acceleration difference becomes higher than a predetermined value even though the accelerator pedal depression amount increases more than a predetermined amount within a predetermined time.", "The torque deficiency judgment means judges that it is the state of torque deficiency when the acceleration difference becomes more than a predetermined value even when the driver depresses the accelerator pedal sharply demanding acceleration.", "Accordingly, it is possible to reliably detect the will of the driver to accelerate positively by the sudden depression of the accelerator pedal by the driver.", "The deficiency of torque (accordingly, acceleration) in this case can be detected easily.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the torque deficiency judgment means judges torque deficiency when vehicle speed decelerates more than predetermined within a predetermined time even though the accelerator pedal depression amount is more than a predetermined amount and the accelerator pedal depression amount does not change substantially within a predetermined time.", "In this case, before a driver notices, it is easily detectable the vehicle's sudden slowdown and becomes torque deficient.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the accelerator pedal depression amount is a value corresponding to the fuel flow rate supplied to the fuel injection valve or a value corresponding to the flow rate difference of this flow rate and the return fuel flow rate discharged from the fuel injection valve.", "As described above, if the accelerator pedal depression amount, as a result of a water ratio are determined with a flow rate or flow rate difference, it will become unnecessary to receive an information signal from the engine control unit side.", "Thus, the exhaust emission control apparatus of the present invention can be applied to a variety of types of vehicles, which have already been put on the market from various makers.", "Notably, it is possible to apply the same concept to many different vehicles without taking into consideration the signal processing of the engine control unit and the transfer method of the signal, which are different with each maker and each vehicle.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the water ratio determination means sets the water ratio to substantially zero for a period of a predetermined time from diesel engine start-up time until any one of the engine coolant temperature, the temperature of the return fuel in the return passage or the transmission temperature reaches a predetermined temperature.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the water ratio determination means sets the water ratio to substantially zero until a predetermined time elapses after detecting a diesel engine stop signal.", "According to the present invention of the exhaust emission control apparatus for a diesel engine, wherein the water ratio is controlled corresponding to the exhaust gas temperature within a diesel particulate filter.", "In the case when there is a possibility that the catalyst or filter may be damaged due to extraordinary high temperatures of the exhaust gas in a diesel particulate filter, it is possible to reduce the temperature of the exhaust gas by increasing the water ratio to prevent the above filter from overheating.", "Conversely, to accelerate the combustion of the soot or the like while the temperature in the filter is low, it is possible to raise the temperature in the filter easily and swiftly by reducing the water ratio to raise the temperature of the exhaust gas.", "The above and further objects and novel features of the present invention will more fully appear from the following detailed description when the same is read in conjunction with the accompanying drawings.", "It is to be expressly understood, however, that the drawings are for the purpose of illustration only and are not intended as a definition of the limits of the invention.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a diagram showing the entire power train containing the exhaust emission control apparatus for diesel engines relating to the first embodiment of this invention; FIG.", "2 is a control flow chart for controlling water ratio adjustment in the exhaust emission control apparatus of the diesel engine shown in FIG.", "1; FIG.", "3 is a diagram showing the entire power train containing the exhaust emission control apparatus for diesel engines according to the second embodiment of this invention; and FIGS.", "4 and 5 are control flowcharts for controlling water ratio adjustment in the exhaust emission control apparatus of the diesel engine shown in FIG.", "3, wherein FIG.", "4 shows the first half thereof and FIG.", "5 shows the second half thereof.", "DETAILED DESCRIPTION OF THE INVENTION The present invention will hereinafter be described in detail with reference to the accompanying drawings.", "As shown in FIG.", "1, a power train equipped with an exhaust emission control apparatus for diesel engines according to this embodiment includes a fuel supply system 1 that generates an emulsified fuel by mixing fuel, water and emulsifier; a diesel engine 2 that burns the emulsified fuel supplied from the fuel supply system 1 to obtain driving output; and a rear exhaust emission control apparatus 3 that cleans, particularly, soot from exhaust gas emitted from the diesel engine 2.Hereafter, a detailed description will be given as to the above components.", "The fuel supply system 1 includes a light oil tank 4 for storing light oil as the fuel, a water tank 5 (synonymous with water supply feed unit) for storing water to be added to the fuel, an emulsifier cartridge 6 (synonymous with emulsifier feed unit) for storing an emulsifier for emulsifying the fuel and water.", "The light oil tank 4, the water tank 5 and the emulsifier cartridge 6 are connected with a fuel pump 7, a water pump 8 and an emulsifier pump 9 that are driven by an electric motor respectively, and each of them can suction the light oil, water and emulsifier respectively.", "An emulsion controller 16 controls the operation of these pumps 7-9 separately.", "The discharge opening of each pump is connected to the inlet of each of flow variable control valves (ratio adjustment units) 11-13.As described later, these flow variable control valves 11-13 control independently the size of the respective dimensions of the flow path by means of emulsion controller 16 according to the running conditions.", "The fuel, water and emulsifier of which the flow rate has been adjusted respectively by these flow variable control valves 11-13 flows into a static mixer 15, segmented and mixed into the emulsified fuel therein.", "The supply flow rate of the emulsified fuel to a fuel injection pump 23 in the above process is monitored by a supply flow rate sensor 20 and is fed back to the emulsion controller 16 to generate optimum emulsified fuel.", "The static mixer 15 generates water-in-oil type (W/O type) emulsified fuel.", "Accordingly, Hydrophile and Lipophile Balance (hereinafter referred to as HLB) makes the above-mentioned emulsifier about HLB range six, and the amount of supply is made into about a 1.2% mass of fuel and water (volume about 1.5%).", "Fundamentally, the water ratio is decided on the basis of the engine load, i.e., the amount of accelerator pedal depression.", "That is to say, the water ratio is controlled so that the basic ratio is resulted in such that the larger load on the engine the higher water ratio as follow, that is, in the case of normal driving when running on a flat road with a light load, the light oil:water is preferably set to, for example, 95:5 within a range where the accelerator pedal depression amount is lower; and the light oil:water is preferably set to, for example, 80:20 within a range where the accelerator pedal depression amount is higher.", "Depending on the engine or combustion conditions, it is possible that the water may be practically set not to 80:20 but, for example, to 70:30.However, in the case of vehicles, it is not practical that the water ratio is increased to a level as 50:50 like the case of ships or a boiler.", "In the above-described constitution, the light oil tank 4 and the fuel pump 7 constitute the fuel supply system; the water tank 5 and the water pump 8 constitute a water supply feed unit; and the emulsifier cartridge 6 and the emulsifier pump 9 constitute emulsifier supply equipment.", "However, the water pump 8 and/or the emulsifier pump 9 are not indispensable components.", "It is possible to adapt so that the water and/or emulsifier are suctioned into the fuel that flows into the static mixer 15.On the other hand, the flow variable control valves 11 and 12 for fuel and water constitute a water ratio adjusting device.", "The outlet of the static mixer 15 is connected to the inlet of the fuel injection pump 23 via a fuel supply path 45.In the fuel supply path 45, the supply flow rate sensor 20 is provided.", "The sensor always detects the flow rate of the emulsified fuel supplied to the fuel injection pump 23 from the static mixer 15.The flow rate signal is sent to the emulsion controller 16.The fuel injection pump 23, whose outlet is connected to the supply port of the fuel injection valve 22, is controlled by an engine control unit 38 to decide the injection amount and the injection timing of the emulsified fuel from the fuel injection valve 22 to a combustion chamber 41.The discharge port of the fuel injection valve 22 is connected to a return reservoir 31 via a return path 46.In the return path 46, a return flow rate sensor 21 for detecting the flow rate of the returned fuel is provided, which sends flow rate signal about the returned fuel to the emulsion controller 16.The return reservoir 31 stores the emulsified fuel drained from the discharge port of the fuel injection valve 22 so that the returned fuel can be supplied again to the fuel injection valve 22 via the static mixer 15 and the like.", "That is, connected to the return reservoir 31 is a suction port of a return pump 10 driven by an electric motor so that the return pump 10 can suction the returned fuel within the return reservoir 31.The discharge opening of the return pump 10 is connected to the inlet of the static mixer 15 via a flow variable control valve 14.In this case, the returned fuel in the return reservoir 31 is returned to the inlet of the static mixer 15 again.", "Here, it is adapted so that at least any one of the light oil from the light oil tank 4, the water from the water tank 5 and the emulsifier from the emulsifier cartridge 6 can be supplied to the static mixer 15 along with the returned fuel; thereby, the water ratio (ratio of water to the light oil) at the outlet of the static mixer 15 is controlled to a desired value.", "Basically, it is adapted so that the reuse of the returned fuel has the priority to the supply of the new fuel, water and emulsifier.", "As a result, when the water ratio does not substantially change in such a case of continuous high speed driving, there may be a case where only the returned fuel is to be supplied to the static mixer 15.For the purpose of the reuse as described above, provided to the return reservoir 31 is a water ratio detection sensor 32 for measuring the water ratio of the return fuel stored therein, and detection signal is input to the emulsion controller 16.Here, the water ratio detection sensor 32 measures the water ratio by using a liquid gravity measurement or an optical measurement of liquid visual density or the like.", "In this case, the emulsion controller 16 determines the amount light oil, water and emulsifier supplied respectively from the amount of returned fuel and the amount of the light oil tank 4, the water tank 5 and the emulsifier cartridge 6 while taking into consideration the information about the water ratio of the returned fuel and the re-supplied amount of the returned fuel, and controls the pumps 7-10 and the flow variable control valves 11-14 to be capable of generating an emulsified fuel with an optimum water ratio.", "At the same time, an agitator 44 capable of agitating the returned fuel therein is attached to the return reservoir 31.The agitator 44 is driven under predetermined conditions (for example, at every predetermined point of time, or at a point of engine start or the like), and the emulsion controller 16 controls to prevent the emulsified fuel from separating into fuel and water.", "Here, at the engine start or the like, the return pump 10 is not activated and the flow variable control valve 14 is left closed until the returned fuel in the return reservoir 31 is sufficiently agitated and the emulsified fuel is stabilized so that the new emulsified fuel is generated being supplied from the light oil tank 4, the water tank 5 and the emulsifier cartridge 6.The light oil tank 4, the water tank 5, the emulsifier cartridge 6 and the return reservoir 31 are provided with a residual sensors 17, 18, 19 and 33 respectively to send the associated residual signals to the emulsion controller 16; thereby the emulsion controller 16 emits residual alarm by means of alarm lamp, or changes the target water ratio of the emulsified fuel to be supplied to the fuel injection valve 22 after the residual amount has reduced to a predetermined amount or less.", "The fuel injection valve 22 is attached to a cylinder head 42, and the injection port thereof is located in the combustion chamber 41 formed by a cylinder 25 and a piston 24 of the diesel engine 2 to be capable of injecting the emulsified fuel into the combustion chamber.", "Formed in the cylinder head 42 are an intake port 27 for guiding intake air and an exhaust port 28 for discharging exhaust gas; and an intake valve 29 and an exhaust valve 30 opens and closes the portions between the combustion chamber 41 and the intake port 27 and the exhaust port 28, respectively.", "Provided to the cooling water path formed in the cylinder 25 is a cooling water temperature sensor 26 for detecting the water temperature to send the cooling water temperature signal detected therein to the engine control unit 38.The engine control unit 38 is input with accelerator opening signal, engine revolution signal and the like from an accelerator opening sensor (load detection means) 39, an engine revolution sensor 40 or the like to decide the injection amount, injection timing or the like necessary for operating the diesel engine 2.From the engine control unit 38 to the emulsion controller 16, accelerator opening signal, required fuel information signals (signals about injection amount, injection timing and the like) are input.", "The rear exhaust emission control apparatus 3 is constituted mainly of a Diesel Particulate Filter (DPF) 34, the DPF 34 being connected to the exhaust port 28 of the diesel engine 2 via an exhaust pipe.", "Provided to the exhaust port 28 is a combustion temperature sensor 35 for detecting the temperature of the exhaust gas discharged from the diesel engine 2 to send the temperature signal to the emulsion controller 16.The above-mentioned DPF 34 includes an oxidation catalyst reaction chamber 34a (oxidation chamber) at the upstream side and an ash accumulation chamber 34b at the downstream side.", "The oxidation catalyst reaction chamber 34a is provided with an oxidation catalyst for oxidizing particulate matter (mainly, soot such as carbon or the like) in the exhaust gas using nickel or the like, and the inside of which can be heated with an electric heater 43 disposed therein.", "A pre-heater controller 37 controls the electric heater 43.Further, the oxidation catalyst reaction chamber 34a has a reaction chamber temperature sensor 36 for detecting the temperature inside the chamber and sends the reaction chamber temperature signal detected by the reaction chamber temperature sensor 36 to the pre-heater controller 37 and the emulsion controller 16.Next, the working of the exhaust emission control apparatus for diesel engines according to the first embodiment, which has the above-described constitution, will be described.", "First of all, to start the engine, the starter switch is turned ON to rotate the starter (not shown) to give turning force to the diesel engine 2.Accompanying the engine start, the emulsion controller 16 judges the engine is the starting by detecting the ON-actuation of the starter switch.", "The emulsion controller 16 receives oxidation catalyst reaction temperature signal from the reaction chamber temperature sensor 36, exhaust gas temperature signal from the combustion temperature sensor 35, flow rate signal of the emulsified fuel from the supply flow rate sensor 20, flow rate signal of the returned fuel from the return flow rate sensor 21, residual signal and the like from each residual sensors 17, 18, 19 and 33 and starts to generate and supply the emulsified fuel having an optimum water ratio.", "As shown in a control flow chart in FIG.", "2, when the power supply to the emulsion controller 16 is turned on accompanying the ON-actuation of the starter switch, at Step S1, the emulsion controller 16 initializes the internal memory; at Step S2, reads the engine start signal.", "When the engine has started, the emulsion controller 16 proceeds to Step S3 and begins to measure the time from the point of the engine start with a timer.", "Then, at Step S4, the emulsion controller 16 reads pedal depression amount of the accelerator pedal from the accelerator opening sensor 39, and at Step S5, reads the coolant temperature from the cooling water temperature sensor 26.Then, at Step S6, the emulsion controller 16 reads a predetermined elapsed time corresponding to the read cooling water temperature from a map of cooling water temperature-predetermined elapsed time, which has been previously organized based on experiments and the like.", "At Step S7, starting control of the water ratio is started.", "That is, at the starting up, the emulsion controller 16 closes the flow variable control valves 12, 13 and 14 respectively for the water tank 5, the emulsifier cartridge 6, and the return reservoir 31, and opens only the flow variable control valve 11 for the light oil tank 4 up to the flow path dimensions corresponding to the accelerator pedal depression amount (ordinarily, initially, idling operation amount).", "The emulsion controller 16 drives only the fuel pump 7 and leaves the other pumps 8-10 stopped.", "Accordingly, only the fuel of 100% light oil (therefore, fuel of water ratio 0%), which is supplied from the flow variable control valve 11 to the static mixer 15, is sent to the fuel injection pump 23.Being controlled by the engine control unit 38, the fuel injection pump 23 injects the above-described light oil of optimum injection amount from the fuel injection valve 22 into the combustion chamber 41, in which the air is previously inhaled from the intake port 27, at optimum injection timing for optimum period of injection.", "The piston 24 rises in the situation that the intake valve 29 and the exhaust valve 30 close the intake port 27 and the exhaust port 28 to ignite the light oil by the heat of compression, then the piston 24 is lowered by the gas expansion due to the combustion so as to drive the engine to turn.", "As a result, in the above starting up, since the fuel is the 100% light oil including no water, the engine can be started easily same as the case of an ordinary diesel engine.", "As for the nitrogen oxides (NOx) generated at the starting up, although it is impossible to take positive measures, the amount of the NOx generated in the exhaust gas discharged from the engine is small since the engine is still cool and is in an idling operation state.", "Although Particulate Matter (PM) increases, a large amount of the generated PM such as soot can be captured and burnt in the exhaust emission control apparatus.", "That is to say, since the temperature of the DPF 34 detected by the reaction chamber temperature sensor 36 is lower than the oxidation catalyst activation temperature, the pre-heater controller 37 swiftly heats up the electric heater 43.In addition to that, since the combustion of the light oil 100% increases the temperature of the exhaust gas, the DPF 34 is heated as soon as possible to oxidize the PM discharged from the engine in the oxidation catalyst reaction chamber 34a of the DPF 34 to clean the exhaust gas.", "Also, accompanying the engine start, since the agitator 44 provided in the return reservoir 31 is agitated.", "The emulsification of the fuel is further advanced by being agitated again even when the emulsified fuel in the return reservoir 31 has separated into fuel and water due to the stoppage of the engine for a certain period of time.", "At Step S8, it is determined whether the measured time has reached the above-described predetermined elapsed time.", "When the predetermined elapsed time is not reached yet, the control returns to Step S3.When it has reached the predetermined elapsed time, the control proceeds to Step S9.At Step S9, no-water control is stopped and ordinary water ratio control is started.", "At Step S10, accelerator pedal depression amount signal is read out.", "At Step S11, it is determined whether the accelerator pedal depression amount is equal to or exceeds the predetermined pedal depression amount (here, set to the kick down equivalent opening).", "When it is smaller than the predetermined pedal depression amount, it is judged that it is not the torque deficiency, and the control proceeds to Step S12.Here, while referring to the map of accelerator pedal depression amount-water ratio, it is controlled corresponding to the accelerator pedal depression amount, in accordance with the basic water ratio such that the water ratio increases as the accelerator pedal depression amount becomes higher.", "Accordingly, the emulsion controller 16 drives each of the pumps 7, 8, 9 and 10 for the light oil tank 4, the water tank 5, the emulsifier cartridge 6, and the return reservoir 31, and opens the flow path dimensions of the flow variable control valves 11, 12, 13 and 14 to the ratio respectively so that the optimum water ratio is obtained corresponding to the accelerator pedal depression amount to supply them to the static mixer 15.As a result, the emulsified fuel of an optimum water ratio corresponding to the accelerator pedal depression amount is supplied to the fuel injection valve 22, and power performance and exhaust emission purification efficiency can be optimally reconciled.", "As for the map of the above accelerator pedal depression amount-water ratio, for example, in a range that the accelerator pedal depression amount is low, the light oil:water is set to 95:5; in a range that accelerator pedal depression amount is medium level, the same is set to 85:15; and in the range that the accelerator pedal depression amount is high, the same is set to 80:20.These serve as the basic water ratio.", "When Step S12 has completed, the control returns to Step S10.On the other hand, when the predetermined pedal depression amount is reached or exceeded, it is determined as torque deficiency, then the control proceeds to Step S13, and a control is made in which the water ratio is set to a ratio (here, water ratio is set to, for example, 0%) lower than the basic water ratio during the above-described normal driving.", "As a result, power performance (in this case, the magnitude of the torque is important) is increased, even when hill climbing on a steep slope or passing with a large load, hill climbing or sudden acceleration can be achieved without getting into the torque deficiency.", "During the water ratio reduction control, since the load is large, the engine speed is also prevented from rising.", "Thus, compared to the normal driving, the NOx reduces although the PM increases.", "The increased PM is captured by the DPF 34 and combusted therein to remove it.", "Also, since the period of the torque deficiency is short in almost all cases, from this viewpoint also, the decrease of the exhaust emission control performance is small as a whole.", "After performing the control at the torque deficiency, the control returns to Step S10.Although not described in the above control flow chart, it is arranged such that the emulsion controller 16, when receiving engine stop signal, switches to the fuel of no-water, i.e., light oil 100% and operates the engine for a predetermined period of time from that point to prevent water from remaining in the engine and the parts in the exhaust system to avoid corrosion.", "On the other hand, the PM in the exhaust gas enters the DPF 34 and is oxidized into CO2 by the oxidation catalyst in the oxidation catalyst reaction chamber 34a.", "Likewise, the carbohydrate (HC) in the exhaust gas is also changed into CO2 and H2O in the oxidation catalyst reaction chamber 34a of the DPF 34.The incombustible ash is captured in the ash accumulation chamber at the downstream of the oxidation catalyst reaction chamber 34a to prevent it from being discharged into the external.", "However, the amount of the ash is small.", "When information signal about the oxidation reaction chamber temperature detected by the reaction chamber temperature sensor 36 is input to the emulsion controller 16, and in the case where the oxidation reaction chamber temperature has extraordinary risen and the filter, catalyst or the like therein may be destroyed, the emulsion controller 16 increases the water ratio exceeding the basic water ratio to lower the exhaust gas temperature.", "To the contrary, to sharply increase the temperature in the DPF 34 to accelerate the oxidization, it is adapted so as to reduce the water ratio to be smaller than the basic water ratio.", "As described above, in the exhaust emission control apparatus for diesel engines according to the first embodiment, the water ratio is controlled in accordance with the magnitude of the accelerator pedal depression amount; thereby it is always possible to ensure required power performance in a broad engine operational range, while enhancing the exhaust emission control performance and reducing the generation of the NOx.", "When hill climbing on a steep slope or a sudden acceleration with a heavy load is required, it is arranged so that the water ratio is reduced smaller than the basic water ratio at the normal driving when it is detected that the pedal is depressed up to an accelerator pedal depression amount equal to or over a predetermined value such as a kick down point, therefore it is possible to obtain a large torque allowing the hill climbing on a steep slope and the sudden acceleration.", "In this case, the NOx increases slightly, however since the engine revolution is reduced, the NOx decreases to a lower level than that during normal driving and the period thereof is short, the NOx does not reach a level that causes a problem.", "Further, the PC such as soot or the like that is generated a lot at that time can be removed by the DPF 34.Furthermore, since it is arranged so that no-water control is made at the engine start and that this period of the control is set based on the cooling water temperature, it becomes possible to reliably start up the engine and to operate it stably until it is warmed up.", "And when the engine is stopped, likewise no-water control is made so as not to allow any water to remain in the engine or parts of the exhaust system.", "Accordingly, it is possible to prevent any rust from gathering.", "Then, an exhaust emission control apparatus for diesel engines according to a second embodiment of the invention will be described below.", "FIG.", "3 shows a constitution of a power train in the second embodiment.", "In FIG.", "3, the substantially same items as those in FIG.", "1 will be given with the same reference symbols as those in FIG.", "1; and the descriptions thereof will be omitted.", "A non-driving wheel (not shown) is provided with a wheel speed sensor 48 for detecting wheel speed to send a signal about the wheel speed to the emulsion controller 16.And in the return path 46 connecting between the fuel injection valve 22 and the return reservoir 31, a return temperature sensor 47 is provided to send a signal about the temperature of the returned fuel to the emulsion controller 16.The other constitution is substantially the same as that in FIG.", "1.The exhaust emission control apparatus for diesel engines having the above constitution is controlled in accordance with a control flow chart as described below.", "That is, in FIGS.", "4 and 5 showing the control flow chart, when the power supply to the emulsion controller 16 is turned on, control operation starts in accordance with the control flow chart.", "At Step S21, the memory in the emulsion controller 16 is initialized.", "At Step S22, engine start signal is read.", "When the engine has started, the control proceeds to Step S23, and time measurement is started from the point of engine start with the timer.", "Then, at Step S24, pedal depression amount of the accelerator pedal is read from the accelerator opening sensor 39; at Step S25, the returned fuel temperature is read from the return temperature sensor 47.At Step S26, starting control, in which only the light oil of water ratio 0%, i.e., 100% light oil is supplied, is made.", "Then, at Step S27, it is determined whether a predetermined period of time (for example, 2 minutes) has elapsed from the engine start.", "When the predetermined period of time has not reached yet, the control returns to Step S23.When it is determined that the predetermined period of time has elapsed, the control proceeds to Step S28, and determines whether the temperature of the returned fuel detected by the return temperature sensor 47 is higher than a predetermined temperature (for example, approximately 5° C.-15° C., more particularly, approximately 10° C. or so, i.e., approximately 8° C.-12° C. is the best).", "When it is not higher than the predetermined temperature, it is determined whether the measured time has reached a set period of time (it is preferably set to be a period of time longer than the above predetermined period of time, which is estimated the engine would be reliably warmed up).", "When it is shorter than the set period of time, the control returns to Step S23.On the other hand, when the set period of time has elapsed, or when the returned fuel temperature is higher than the predetermined temperature at Step S28, the control proceeds to Step S29 to stop the starting control and start the water ratio control.", "At Step S31, pedal depression amount of the accelerator pedal is read from the accelerator opening sensor 39.At Step S32, a water ratio corresponding to the pedal depression amount of the accelerator pedal is decided.", "The water ratio is set as the basic water ratio based on the same map as that of the first embodiment.", "Further, at Step S33, a target acceleration corresponding to the pedal depression amount of the accelerator pedal at the point of time is read out based on a map of accelerator pedal depression amount-target acceleration (acceleration equivalent to vehicle speed obtained during flat road driving with a light load), which has been previously set based on research and experiments.", "In the second embodiment, the acceleration equivalent to the vehicle speed is defined as the acceleration of the non-driving wheel.", "Step S33 constitutes a target acceleration setting means.", "Then, at Step S34, the wheel speed is read out from the wheel speed sensor 48 and the actual acceleration is calculated based on the above wheel speed at Step S35.These Steps S34 and S35 constitute actual acceleration detection means.", "At the next Step S36, it is determined whether the difference between the target acceleration and the actual acceleration is equal to a predetermined value or more.", "When the difference is smaller than the predetermined value, it is determined as state of non torque deficiency, and the control proceeds to Step S38.When the difference is equal to the predetermined value or more, it is determined as torque deficiency, and the control proceeds to Step S37.The above-predetermined value is decided based on data collected from experiments about the acceleration difference in the case where an ordinary driver feels as torque deficiency.", "Further, it is set so that, for example, when the accelerator opening is large, the predetermined value is larger than that when the accelerator opening is small.", "The Step S36 constitutes torque deficiency judgment means.", "That is to say, when the acceleration difference is equal to the predetermined value or more, the control proceeds to Step S37 to correct so as to reduce the basic water ratio decided at Step S32.In this case, it is adapted so that the correction amount of water reduction is changed corresponding to the magnitude of the above acceleration difference (reduction value).", "In this case, it is preferred to set so that the larger water reduction amount (or water reduction ratio) is set for the larger acceleration difference.", "After correcting the water ratio, a new acceleration difference is detected again at Step S39, and it is determined whether the acceleration difference (reduction value) is the set value or less.", "When the difference is the set value or less, the control proceeds to Step S40, and when the difference exceeds the set value, the control proceeds to Step S41.Here, the set value is set to a value smaller than the predetermined value at above Step S36 (minus value is also possible), which is a value that it is not felt as torque deficiency even when the water ratio is increased.", "As described above, by giving hysteresis between the predetermined value and the set value, the control of the water ratio is prevented from hunting while ensuring an optimum water ratio.", "When it is determined at Step S39 that the acceleration difference is the set value or less due to moderate slope inclination or the like, it is determined that the torque deficiency has been cleared.", "Then the water ratio reduction control is stopped at Step S40, the water is increased up to the basic water ratio, and the control returns to Step S31.On the other hand, when the acceleration difference exceeds the set value, it is decided at Step S41 that the water ratio is left being reduced because the torque deficiency is still continuing; and the process returns to Step S31.The above-described Step S32 and Steps S36-S41 constitute water ratio determination means.", "As describe above, in the exhaust emission control apparatus for diesel engines according to the second embodiment, the exhaust emission control performance and the power performance are allowed to be consistent with each other in a broad engine operational range during the normal driving, same as the first embodiment.", "In addition, the torque deficiency is determined in accordance with the acceleration difference between the target acceleration and the actual acceleration corresponding to the accelerator pedal depression amount to control the water ratio.", "Also, in this case the torque deficiency is easily detected and determined to allow the torque to be increased at a hill climbing or sudden acceleration with a heavy load resulting in ensuring the power performance.", "In this case, although the exhaust emission control performance decreases, the influence thereof is small.", "Further, since the torque deficiency is determined using the acceleration difference, it is possible to produce various modifications and corrections to obtain a control with higher preciseness as described later.", "Here, the invention is not limited to the above-described embodiments, but the following modification or correction may be used.", "In the above-described embodiments, the accelerator pedal depression amount is detected based on the pedal depression amount of the accelerator pedal.", "However, in place of this, supply flow rate of the emulsified fuel detected by the supply flow rate sensor 20, or the flow rate difference in which the flow rate of the returned fuel detected by the return flow rate sensor 21 is subtracted from the supply flow rate may be used to determine the torque deficiency or to decide the water ratio.", "In this case, since it is not necessary to receive any information signal from the engine control unit 38 side, there is a merit such that, even when the engine control unit or communication system of the information signal may differ depending on the maker or vehicle, they can be applied without knowing them.", "Further, in the above-described embodiment, the actual acceleration of the vehicle is calculated from the wheel speed.", "However, a speed change per time may be detected from a speed meter, or an acceleration meter, which detects positional change of mass corresponding to the acceleration, may be used.", "Furthermore, as for the judgment of the torque deficiency, it may be determined as the torque deficiency when the acceleration difference is equal to a predetermined value or more although the accelerator pedal depression amount has increased by a predetermined amount or more within a predetermined period of time; or when a vehicle speed decelerates at or exceeding a predetermined value within a predetermined period of time although depression amount of the accelerator pedal is equal to a predetermined amount or more, and accelerator pedal depression amount does not substantially change within a predetermined period of time.", "By adapting as described above, it is possible to reliably detect the driver's will to increase the speed based on the sharp depression of the accelerator pedal.", "In the second embodiment, the torque deficiency is determined based on whether the magnitude of the acceleration difference is equal to a predetermined value or more.", "In this case, it may be adapted so that the larger acceleration difference results in the smaller water ratio; two or more predetermined values are set corresponding to the magnitude of the accelerator pedal depression amount; or the value increases as the depression amount of the accelerator pedal increases, or the like.", "By adapting as described above, it is possible to obtain a fine control corresponding to the actual driving conditions.", "Likewise, by setting so that the set value becomes smaller as the accelerator pedal depression amount becomes higher, it is possible to obtain a fine control corresponding to the actual driving conditions.", "When the accelerator pedal depression amount becomes substantially zero and acceleration of a vehicle detected based on the wheel speed or the like exceeds a permissible value, in such a case as descending on a steep slope, it is the state that the engine is driven by the driving wheel-side, therefore the basic water ratio may be corrected so that the water ratio is increased.", "Owing to this arrangement, water can be increased without problem of engine stop resulting in an enhanced purification of the exhaust gas.", "When the above acceleration decreases to a value equal to the above permissible value or less, the control to correct to increase the water ratio is stopped and returns to the basic water ratio.", "Furthermore, unlike the above-described embodiments in which the emulsifier is stored in an emulsifier cartridge separated from the fuel tank, the emulsifier may be previously mixed with the fuel in the fuel tank.", "In this case, the fuel supply system substantially substitutes for the emulsifier supply equipment.", "INDUSTRIAL APPLICABILITY As described above, the exhaust emission control apparatus for diesel engines of the invention is constituted so that the water ratio is controlled to a basic water ratio corresponding to the accelerator pedal depression amount so that, during normal driving, in an engine operational range in which the accelerator pedal depression amount is larger, the water ratio increases from that in the engine operational range in which the accelerator pedal depression amount is smaller; and that the torque deficiency is determined based on the accelerator pedal depression amount and the water ratio decreases to a value lower than the basic water ratio in the case of torque deficiency.", "Therefore, it is possible to ensure the power performance while increasing the cleaning performance by suppressing, particularly, generation of the nitrogen oxides in the exhaust gas in a broad engine operational range.", "Accordingly, the invention is the most suitable to be applied to vehicles mounted with a diesel engine.", "While the present invention has been described with reference to the preferred embodiments, it is our intention that the invention be not limited by any of the details of the description thereof.", "As this invention may be embodied in several forms without departing from the spirit of the essential characteristics thereof, the present embodiments are therefore illustrative and not restrictive, since the scope of the invention is defined by the appended claims rather than by the description preceding them, and all changes that fall within meets and bounds of the claims, or equivalence of such meets and bounds thereof are intended to be embraced by the claims." ] ]	Patent_10468507
[ [ "Novel substituted imidazotriazinones", "The invention relates to novel substituted imidazotriazinones, to a method for their production and to the use thereof for producing medicaments, in particular to improve perception, powers of concentration, learning capacity and/or memory retentiveness." ], [ "1.Compounds of the general formula (I), in which R1 denotes phenyl which can be substituted up to three times identically or differently by radicals selected from the group consisting of (C1-C4)-alkyl, (C1-C4)-alkoxy, halogen, cyano, —NHCOR8, —NHSO2R9, —SO2NR10R11, —SO2R12, and —NR13R14, in which R8, R10, R11, R13 and R14 independently of one another are hydrogen or (C1-C4)-alkyl, and R9 and R12 independently of one another are (C1-C4)-alkyl, or R10 and R11 together with the adjacent nitrogen atom form an azetidin-1-yl, pyrrol-1-yl, piperid-1-yl, azepin-1-yl, 4-methyl-piperazin-1-yl or morpholin-1-yl radical, or R13 and R14 together with the adjacent nitrogen atom form an azetidin-1-yl, pyrrol-1-yl, piperid-1-yl, azepin-1-yl, 4-methyl-piperazin-1-yl or morpholin-1-yl radical, R2 and R3 independently of one another denote hydrogen or fluorine, R4 denotes (C1-C4)-alkyl, R5 denotes (C1-C3)-alkyl, R6 denotes hydrogen or methyl, R7 denotes (C1-C10)-alkyl, (C2-C10)-alkenyl or (C2-C10)-alkinyl, and L denotes carbonyl or hydroxymethanediyl, and their physiologically tolerable salts, hydrates and/or solvates.", "2.Compounds according to claim 1, where R1 denotes phenyl, whose meta and/or para positions are substituted up to three times identically or differently by radicals selected from the group consisting of (C1-C4)-alkyl, (C1-C4)-alkoxy and —SO2NR10R11, and R10 and R11 have the meaning indicated in claim 1.3.Compounds according to claim 1 or 2, where R7 denotes (C4-C7)-alkyl or (C4-C7)-alkenyl.", "4.Compounds according to claim 1, where R1 denotes phenyl whose meta and/or para positions are substituted up to three times identically or differently by radicals selected from the group consisting of (C1-C4)-alkyl, (C1-C4)-alkoxy and —SO2NR10R11, in which R10 and R11 independently of one another are hydrogen or (C1-C4)-alkyl, R2 and R3 denote hydrogen, R4 denotes methyl or ethyl, R5 denotes methyl, R6 denotes hydrogen or methyl, L denotes carbonyl or hydroxymethanediyl, and R7 denotes n-butyl, n-pentyl, n-hexyl or n-pent-4-en-1-yl.", "5.Process for the preparation of compounds of the general formula (I) according to claim 1, where [A] a compound of the general formula (IIa), in which R1, R2, R3, R4, R5, R6 and R7 have the meaning indicated in claim 1, is reacted under suitable condensation conditions to give a compound of the general formula (Ia), in which R1, R2, R3, R4, R5, R6 and R7 have the meaning indicated in claim 1, and then, if appropriate, [B] is reduced under suitable conditions to give a compound of the general formula (Ib) in which R1, R2, R3, R4, R5, R6 and R7 have the meaning indicated in claim 1.6.Compounds of the general formula (II), in which R1, R2, R3, R4, R5, R6, R7 and L have the meaning indicated in claim 1, and their salts.", "7.Compounds according to one of claims 1 to 4 for the treatment and/or prophylaxis of illnesses.", "8.Medicaments comprising a compound according to one of claims 1 to 4.9.Compounds according to one of claims 1 to 4 for improving perception, concentration power, learning power and/or memory power.", "10.Compounds according to one of claims 1 to 4 for the treatment and/or prophylaxis of disorders of perception, concentration power, learning power and/or memory power.", "11.Use of compounds according to one of claims 1 to 4 for the production of a medicament for improving perception, concentration power, learning power and/or memory power.", "12.Use of compounds according to one of claims 1 to 4 for the production of a medicament for the treatment and/or prophylaxis of disorders of perception, concentration power, learning power and/or memory power.", "13.Use according to claim 12, the disorder being a result of dementia.", "14.Use of compounds according to one of claims 1 to 4 for the production of a medicament for the treatment and/or prophylaxis of dementia." ], [ "The present invention relates to new substituted imidazotriazinones, processes for their preparation, and their use for the production of medicaments, in particular for improving perception, concentration power, learning power and/or memory power.", "Phosphodiesterases (PDEs) play an essential role in the regulation of the intracellular cGMP and cAMP levels Of the previously described phosphodiesterase isoenzyme groups PDE 1 to PDE 10 (Beavo and Reifsnyder Trends in Pharmacol.", "Sci.", "1990, 11, 150-155; Sonderling and Beavo Curr.", "Opin.", "Cell Biol.", "2000, 12, 174-179), the PDEs 1, 2, 5, 9 and 10 are mainly responsible for the metabolism of cGMP.", "On account of the varying distribution of these cGMP-metabolizing PDEs in the tissue, selective inhibitors should raise the cGMP levels in the corresponding tissue, depending on the tissue distribution of the appropriate isoenzyme.", "The particular feature of PDE 2 lies in its positive cooperative kinetics with respect to the substrate cGMP.", "It was postulated that small amounts of cGMP bind to the so-called cGMP-binding domain and thereby bring about activation of the enzyme.", "By this means, the affinity of the catalytic domain to cGMP and cAMP is also increased (Martins et al.", "J. Biol.", "Chem.", "1982, 257, 1973-1979).", "Therefore PDE 2 can hydrolyse and thereby also control both second messenger systems by means of small amounts of cGMP.", "PDE 2 has been isolated from various tissues, for example from heart, adrenal gland, liver, platelets and in particular brain.", "In the brain, PDE 2 mRNA is expressed strongly in the cortex, the basal ganglia and in the hippocampus (Sonnenburg et al.", "Biol.", "Chem.", "1991, 266, 17655-17661).", "The sequence of the human isoform PDE 2A3 was reported by Rosman et al.", "Gene 1997, 191, 89-95.Of the tissues investigated, the expression of PDE 2A was demonstrated strongly therein in the brain and heart and more weakly in liver, skeletal muscle, kidney and pancreas.", "U.S. Pat.", "No.", "4,278,673 discloses imidazopyrimidinones having cAMP PDE-inhibitory action for the treatment of asthma and bronchitis.", "WO-A-99/67244 and WO-A-99/24433 disclose 7-alkyl-2-phenyl-imidazotriazinones having PDE 5-inhibiting action for the treatment of vascular diseases.", "EP-A-0 771 799, WO-A-98/40384 and WO-A-00/12504 describe purinone, allopurinol and triazolopyrimidinone derivatives, their inhibitory action on cGMP-metabolizing PDEs and their suitability for the treatment of vascular diseases.", "The present invention relates to compounds of the general formula (I), in which R1 denotes phenyl which can be substituted up to three times identically or differently by radicals selected from the group consisting of (C1-C4)-alkyl, (C1-C4)-alkoxy, halogen, cyano, —NHCOR8, —NHSO2R9, —SO2NR10R11, —SO2R12, and —NR13R14, in which R8, R10, R11, R13 and R14 independently of one another are hydrogen or (C1-C4)-alkyl, and R9 and R12 independently of one another are (C1-C4)-alkyl, or R10 and R11 together with the adjacent nitrogen atom form an azetidin-1-yl, pyrrol-1-yl, piperid-1-yl, azepin-1-yl, 4-methyl-piperazin-1-yl or morpholin-1-yl radical, or R13 and R14 together with the adjacent nitrogen atom form an azetidin-1-yl, pyrrol-1-yl, piperid-1-yl, azepin-1-yl, 4-methyl-piperazin-1-yl or morpholin-1-yl radical, R2 and R3 independently of one another denote hydrogen or fluorine, R4 denotes (C1-C4)-alkyl, R5 denotes (C1-C3)-alkyl, R6 denotes hydrogen or methyl, R7 denotes (C1-C10)-alkyl, (C2-C10)-alkenyl or (C2-C10)-alkinyl, and L denotes carbonyl or hydroxymethanediyl, and their physiologically tolerable salts, hydrates and/or solvates.", "(C1-C10)-Alkyl, (C1-C4)-alkyl, (C1-C3)-alkyl and (C4-C7)-alkyl in the context of the invention represent a straight-chain or branched alkyl radical having 1 to 10, 1 to 4, 1 to 3 and 4 to 7 carbon atoms, respectively.", "Examples which may be mentioned are: methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, n-hexyl and n-heptyl.", "(C2-C10)-Alkenyl and (C4-C7)-alkenyl in the context of the invention represent a straight-chain or branched alkenyl radical having 2 to 10 carbon atoms and 4 to 7 carbon atoms, respectively.", "Examples which may be mentioned are: vinyl, allyl, isopropenyl, n-but-2-en-1-yl, n-pent-4-en-1-yl and n-hex-5-en-1-yl.", "(C2-C10)-Alkinyl in the context of the invention represents a straight-chain or branched alkinyl radical having 2 to 10 carbon atoms.", "Examples which may be mentioned are: ethinyl, n-prop-2-in-1-yl, n-but-2-in-1-yl, n-pent-4-in-1-yl and n-hex-5-in-1-yl.", "(C1-C4)-Alkoxy in the context of the invention represents a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms.", "Examples which may be mentioned are: methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-pentoxy and n-hexoxy.", "Methoxy and ethoxy are preferred.", "(C5-C8)-Cycloalkyl in the context of the invention represents cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.", "The following may preferably be mentioned are: cyclopentyl, cyclohexyl or cycloheptyl.", "Halogen in the context of the invention in general represents fluorine, chlorine, bromine and iodine.", "Fluorine, chlorine and bromine are preferred.", "Fluorine and chlorine are particularly preferred.", "Preferred salts in the context of the invention are physiologically acceptable salts of the compounds according to the invention.", "Physiologically acceptable salts of the compounds according to the invention can be acid addition salts of the substances according to the invention with mineral acids, carboxylic acids or sulphonic acids.", "Particularly preferred salts are, for example, those with hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic acid, naphthalenedisulphonic acid, acetic acid, propionic acid; lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.", "Salts which may be mentioned are, however, also salts with customary bases, such as, for example, alkali metal salts (e.g.", "sodium or potassium salts), alkaline earth metal salts (e.g.", "calcium or magnesium salts) or ammonium salts derived from ammonia or organic amines such as, for example, diethylamine, triethylamine, ethyl-diisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabietylamine, 1-ephenamine or methyl-piperidine.", "Hydrates of the compounds according to the invention are stoichiometric compositions of the compounds or salts thereof with water.", "Solvates of the compounds according to the invention are stoichiometric compositions of the compounds or salts thereof with solvent.", "The compounds according to the invention can exist in stereoisomeric forms, which either behave as image and mirror image (enantiomers), or which do not behave as image and mirror image (diastereomers).", "The invention relates both to the enantiomers and diastereomers or their respective mixtures.", "Like the diastereomers, the racemic forms can be separated into the stereoisomerically uniform constituents in a known manner.", "Preferred compounds of the general formula (I) are those where R1 denotes phenyl whose meta and/or para positions are substituted up to three times identically or differently by radicals selected from the group consisting of (C1-C4)-alkyl, (C1-C4)-alkoxy and —SO2NR10R11, and R2, R3, R4, R5, R6, R7, R10, R11 and L have the meaning indicated above.", "The meta and para positions of the phenyl ring are understood as meaning those positions which are meta or para to the CR2R3 group.", "These positions can be illustrated by the following structural formula (Ic): Particularly preferred compounds of the general formula (Ic) are those in which the para and one meta position of the phenyl radical are substituted, and the second meta position is unsubstituted.", "Likewise, preferred compounds of the general formula (I) are those where R7 denotes (C4-C7)-alkyl or (C4-C7)-alkenyl and R1, R2, R3, R4, R5, R6 and L have the meaning indicated above.", "Very particularly preferred are compounds of the general formula (I), where R1 denotes phenyl whose meta and/or para positions are substituted up to three times identically or differently by radicals selected from the group consisting of (C1-C4)-alkyl, (C1-C4)-alkoxy and —SO2NR10R11, in which R10 and R11 independently of one another are hydrogen or (C1-C4)-alkyl, R2 and R3 denote hydrogen, R4 denotes methyl or ethyl, R5 denotes methyl, R6 denotes hydrogen or methyl, L denotes carbonyl or hydroxymethanediyl, and R7 denotes n-butyl, n-pentyl, n-hexyl or n-pent-4-en-1-yl.", "A further aspect of the invention relates to a new preparation process for the compounds of the general formula (I), in which R1, R2, R3, R4, R5, R6, R7 and L have the meaning indicated above, where [A] a compound of the general formula (IIa), in which R1, R2, R3, R4, R5, R6 and R7 have the meaning indicated in claim 1, is reacted under suitable condensation conditions to give a compound of the general formula (Ia), in which R1, R2, R3, R4, R5, R6 and R7 have the meaning indicated in claim 1, and then, if appropriate, [B] is reduced under suitable conditions to give a compound of the general formula (Ib) in which R1, R2, R3, R4, R5, R6 and R7 have the meaning indicated in claim 1.The condensation according to reaction step [A] can be carried out by heating the compounds of the general formula (IIa) in the absence of a solvent or in the presence of an inert solvent, in particular of a solvent of the type which forms an azeotropic mixture with water, such as, for example, toluene or xylene, if appropriate in the presence of an acid catalyst and/or of a dehydrating agent.", "A suitable acid catalyst is, for example, hydrogen chloride and a dehydrating agent which can be used is, for example, acetyl chloride, phosphorus pentoxide or phosphorus oxychloride.", "The condensation is preferably carried out in an inert solvent in the presence of 1-10, preferably 3-7, equivalents of phosphorus oxychloride (cf.", "Chem.", "Ind.", "1983, 331-335).", "Suitable inert solvents for the condensation are the customary organic solvents which do not change under the reaction conditions.", "These include, for example, ethers such as diethyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether, or hydrocarbons such as benzene, toluene, xylene, hexane, cyclohexane or petroleum fractions, or halogenohydrocarbons such as dichloromethane, trichloromethane, tetrachloromethane, dichloroethane, trichloroethylene or chlorobenzene, or ethyl acetate, dimethylformamide, hexamethylphosphoramide, acetonitrile, acetone, dimethoxy-ethane or pyridine.", "It is likewise possible to use mixtures of the solvents mentioned.", "1,2-Dichloroethane is preferred.", "The reaction temperature can be varied within a relatively wide range.", "In general, the reaction is carried out in a range from −20° C. to 200° C., preferably from −20° C. to 90° C. The process steps according to the invention are in general carried out at normal pressure.", "However, it is also possible to work at elevated pressure or at reduced pressure (e.g.", "in a range from 0.5 to 5 bar).", "The reduction according to reaction step [B] can be carried out according to customary methods.", "The reductions are in general carried out using hydrides or using boranes, diboranes or their complex compounds in inert solvents.", "The reductions can also be carried out by means of hydrogen in water or in inert solvents such as alcohols, ethers or halogenohydrocarbons, or their mixtures, using catalysts such as Raney nickel, palladium, palladium on active carbon or platinum.", "Preferably, the reductions are carried out using hydrides, such as complex borohydrides or aluminium hydrides.", "Particularly preferably, sodium borohydride, lithium borohydride, sodium cyanoborohydride, lithium aluminium hydride, sodium bis(2-methoxyethoxy)aluminium hydride or borane/tetrahydrofuran are employed here.", "Suitable solvents here for the reduction are all solvents which do not change under the reaction conditions.", "These preferably include alcohols such as methanol, ethanol, n-propanol or isopropanol, or ethers such as diethyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether, diethylene glycol dimethyl ether or amides such as hexamethylphosphoramide or dimethylformamide, or acetic acid.", "It is likewise possible to use mixtures of the solvents mentioned.", "The reduction is in general carried out in a temperature range from −50° C. up to the respective boiling point of the solvent, preferably from −20° C. to +90° C., particularly preferably from −5° C. to 30° C. If necessary, the compounds of the general formula (I) can be separated into the pure diastereomers and/or pure enantiomers.", "For example, chromatographic separation under normal-, medium- or high-pressure conditions on stationary phases such as, for example, silica gel or reversed phase-modified silica gels or chirally modified silica gels is suitable for this purpose.", "This is preferably carried out by the high-performance liquid chromatography (=HPLC) method using chiral stationary silica gel phases.", "Chiral polyamide/silica gel phases based on the monomers N-methacryloyl-L-leucine-d-menthylamide or N-methacryloyl-L-leucine-1-menthyl-amide are particularly suitable for the separation of the racemates (cf.", "EP-A-0 379 917).", "It can also prove favourable to employ diastereomerically and/or enantiomerically pure compounds of the general formula (IIa) in reaction step [A] and/or to separate the compounds of the general formula (Ia) into the pure diastereomers and/or enantiomers, if appropriate, before carrying out reaction step [B].", "It is likewise possible to carry out the reduction [B] diastereoselectively.", "For this purpose, the reduction is expediently carried out using hydrides, such as complex borohydrides or aluminium hydrides and also boranes in the presence of metal salts.", "Particularly preferred metal salts are those whose cations are capable of bidentate coordination, such as, for example, metals of the main groups IIa and IIIa or metals of the subgroups including the lanthanoids.", "Salts of Zn, Mn, Mg or Ca are particularly preferred.", "Anions which can be used are, for example, halides or acetates.", "The reaction is expediently carried out in an alcohol or a mixture of an alcohol and a further inert solvent.", "Mixtures of methanol or ethanol and dichloromethane are preferred.", "The reduction is in general carried out in a temperature range from −50° C. up to the respective boiling point of the solvent, preferably from −20° C. to +90° C., particularly preferably from −5° C. to 30° C. The reduction is carried out, inter alia, using 1 to 20 equivalents of the reducing agent in the presence of 0.1 to 10 equivalents of metal salt.", "In a preferred embodiment, 0.2 to 3 equivalents of metal salt are used.", "Preferred reducing agents are, for example, sodium borohydride, lithium borohydride, sodium cyanoborohydride or zinc borohydride.", "The intermediates of the general formula (II) are new.", "A further aspect of the present invention therefore relates to the new compounds of the general formula (II), in which R1, R2, R3, R4, R5, R6, R7 and L have the meaning indicated above, and their salts.", "The compounds of the general formula (IIa) can be prepared, for example, according to known methods by the oxidation of corresponding compounds of the general formula (IIb), in which R1, R2, R3, R4, R5, R6 and R7 have the meaning indicated in claim 1, for example by Swern oxidation or Collins oxidation (for further oxidation methods also see March, J. M., “Advanced Organic Chemistry”, 3rd Edition, John Wiley, New York, 1985, pp.", "1057-1060 and literature cited therein).", "The preparation of the compounds of the general formula (II) can be illustrated by way of example by the following synthesis scheme: The compounds of the general formulae (III), (IV), (V), (VI), (VII), (VIII), (IX), (X) and (XI) are known or can be prepared by known processes.", "According to this reaction scheme, the aminomethyltriazinones (Mb) are condensed with the carboxylic acids (IV) under the conditions customary for the formation of amide bonds using a dehydrating reagent in an inert solvent, if appropriate in the presence of a base.", "Suitable dehydrating reagents are carbodiimides, such as, for example, diiso-propylcarbodiimide, dicyclohexylcarbodiimide (DCC) or N-(3-dimethylamino-propyl)-N′-ethylcarbodiimide hydrochloride (EDC) or carbonyl compounds such as carbonyldiimidazole or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium-3-sulphonate or propanephosphonic anhydride or isobutyl chloroformate or benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate or diphenyl phosphoramidate or methanesulphonyl chloride, if appropriate in the presence of bases such as triethylamine, N-ethylmorpholine, N-methylmorpholine or N-methylpiperidine, and if appropriate in the presence of a catalyst such as N-hydroxysuccinimide or N-hydroxybenzotriazole (HOBT).", "The condensation with EDC is preferably carried out in the presence of NMM and HOBT.", "Suitable solvents are the customary inert solvents described above.", "Dichloromethane is preferred.", "The aminomethyltriazinones (IIIb) are obtainable by deprotection of the corresponding N-protected aminomethyltriazinones (IIIa), which in turn are accessible via cyclo-condensation of the corresponding amidrazones (Vb) and α-keto esters (VI).", "Suitable amino protective groups for the intermediates (IIIa), (VI) and (VIII) are, for example, acyl radicals, in particular the acetyl group.", "These groups can be cleaved into the N-protected aminomethyltriazinones (IIIa) under acidic conditions, for example by heating in hydrochloric acid.", "The cyclocondensation to give the N-protected aminomethyltriazinones (IIIa) can be brought about by heating the individual components, the amidrazones (Vb) and α-keto esters (VI), in an alcoholic solvent, preferably by heating to reflux in ethanol.", "The amidrazones (Vb) can be prepared by reaction of the corresponding amidines (Va) with, for example, hydrazine hydrate and are either isolated or employed in situ in the following reaction.", "The amidines (Va) are accessible from the corresponding nitriles (VII) according to customary methods, for example by reaction of the nitrites (VII) with ammonium chloride and a solution of trimethylaluminium in hexane firstly in a temperature range from −20° C. to room temperature, preferably at 0° C. and then at 60 to 100° C., preferably 70 to 90° C., and preferably at normal pressure.", "The nitrites (VII) are known or can be prepared according to customary methods.", "For example, aryl-difluoro-acetonitriles can be prepared from arylacetonitriles or aryloxo-acetonitriles (cf.", "J. Org.", "Chem.", "1998, 63, 8052-8057 or J. Fluorine Chem.", "1996, 76, 15-20).", "The α-keto esters (VI) can be prepared from the corresponding N-protected α-amino acids (VIII), for example by reaction with ethyl oxalyl chloride.", "The carboxylic acids (IV) are accessible by alkylation of the corresponding β-keto esters (IX) with the electrophiles (X) and if appropriate (XI), followed by ester hydrolysis and, if appropriate, reduction of the β-carbonyl function.", "For alkylation, the β-ketoester (IX) is deprotonated for example using a base, preferably a hydride such as sodium hydride, in an inert solvent such as, for example, tetrahydrofuran in a temperature range from preferably 0° C. to room temperature and, after isolation or in situ, treated with a solution of the electrophile (X) or (XI) in, preferably, 1,3-dimethyltetrahydro-2-(1H)-pyrimidone with addition of a catalytic amount of potassium iodide.", "If R6 is not hydrogen, the alkylation can be repeated using a second electrophile after the monoalkylation product has optionally been isolated.", "The leaving group Y in the electrophile (X) or (XI) is preferably a halogen, particularly preferably bromine.", "The β-carbonyl function can be reduced according to the conditions described above for reaction step [B].", "The hydrolysis of the ester to the carboxylic acid (IV) is carried out according to customary conditions, in the case of the methyl or ethyl ester preferably using sodium or potassium hydroxide solution.", "Substituents, for example in R1, can be introduced via the starting materials, such as, for example, via the nitrile (VII), but can also be introduced or modified in a later process stage.", "Thus the substituent —SO2NR10R11, for example, can be introduced into R1 by chlorosulphonating an appropriate N-protected aminomethyltriazinone (IIIa) with chlorosulphonic acid and then further reacting it with an appropriate amine HNR10R11 to give the corresponding sulphonamide.", "This can be illustrated by the following reaction scheme: The compounds according to the invention show an unforeseeable, valuable spectrum of pharmacological action: they preferably inhibit PDE 2, and/or exhibit a favourable pharmacokinetic profile.", "The inhibition of PDE 2 leads to a differentiated increase in cGMP.", "The differentiating action is additionally determined by the distribution of the isoenzymes in the tissue.", "The compounds according to the invention moreover intensify the action of substances, such as, for example, EDRF (endothelium-derived relaxing factor) and ANP (atrial natriuretic peptide), which increase the cGMP level.", "Because of their selective PDE 2 inhibition, the compounds according to the invention are particularly suitable for improving perception, concentration power, learning power or memory power, in particular after cognitive disorders, such as occur, for example, in situations/illnesses/syndromes such as mild cognitive impairment, age-associated learning and memory disorders, age-associated memory losses, vascular dementia, craniocerebral trauma, stroke, dementia which occurs after strokes (post-stroke dementia), post-traumatic craniocerebral trauma, general concentration disorders, concentration disorders in children with learning and memory problems, Alzheimer's disease, vascular dementia, dementia with Lewy bodies, dementia with degeneration of the frontal lobes including Pick's disease, Parkinson's disease, progressive nuclear palsy, dementia with corticobasal degeneration, amyolateral sclerosis (ALS), Huntington's disease, multiple sclerosis, thalamic degeneration, Creutzfeld-Jacob dementia, HIV dementia, schizophrenia with dementia or Korsakoff psychosis.", "The compounds according to the invention are generally suitable for the treatment and/or prophylaxis of dementia.", "The active compound can act systemically and/or locally.", "For this purpose, it can be administered in a suitable manner, such as, for example, orally, parenterally, pulmonarily, nasally, sublingually, lingually, buccally, rectally, transdermally, conjunctivally, otically or as an implant.", "For these administration routes, the active compound can be administered in suitable administration forms.", "For oral administration, known administration forms releasing the active compound rapidly and/or in modified form are suitable, such as, for example, tablets (non-coated and coated tablets, e.g.", "enteric coatings), capsules, sugar-coated tablets, granules, pellets, powders, emulsions, suspensions and solutions.", "The parenteral administration can take place with circumvention of an absorption step (intravenous, intraarterial, intracardiac, intraspinal or intralumbar) or with involvement of an absorption (intramuscular, subcutaneous, intracutaneous, percutaneous, or intraperitoneal).", "For parenteral administration, suitable administration forms are, inter alia, injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilizates and sterile powders.", "For the other administration routes, for example, inhalation pharmaceutical forms (inter alia powder inhalers, nebulizers), nasal drops/solutions, sprays; tablets or capsules to be applied lingually, sublingually or buccally, suppositories, ear and eye preparations, vaginal capsules, aqueous suspensions (lotions, shake lotions), lipphilic suspensions, ointments, creams, milk, pastes, dusting powder or implants are suitable.", "The active compounds can be converted into the administration forms mentioned in a known manner.", "This takes place using inert non-toxic, pharmaceutically suitable excipients.", "These include, inter alia, vehicles (e.g.", "microcrystalline cellulose), solvents (e.g.", "liquid polyethylene glycols), emulsifiers (e.g.", "sodium dodecylsulphate), dispersants (e.g.", "polyvinylpyrrolidone), synthetic and natural biopolymers (e.g.", "albumin), stabilizers (e.g.", "antioxidants such as ascorbic acid), colourants (e.g.", "inorganic pigments such as iron oxides) or taste and/or odour corrigents.", "In general, it has proved advantageous in the case of parenteral administration to administer amounts of approximately 0.001 to 30 mg/kg, preferably approximately 0.01 to 10 mg/kg, of body weight to achieve effective results.", "In the case of oral administration, the amount is approximately 0.01 to 100 mg/kg, preferably approximately 0.1 to 30 mg/kg, of body weight.", "In spite of this, it may be necessary, if appropriate, to deviate from the amounts mentioned, namely depending on the body weight, route of application, individual behaviour towards the active compound, manner of preparation and time or interval at which administration takes place.", "Measurement of the PDE Inhibition The cGMP-stimulable PDE (PDE 2), the cGMP-inhibitable PDE (PDE 3) and the cAMP-specific PDE (PDE 4) were isolated either from porcine or bovine heart myocardium.", "The Ca2+ calmodulin-stimulable PDE 1 was isolated from porcine aorta, porcine brain or preferably from bovine aorta.", "The cGMP-specific PDE (PDE 5) was preferably obtained from porcine small intestine, porcine aorta, human blood platelets and preferably from bovine aorta.", "Purification was carried out by anion exchange chromatography on mono QR Pharmacia essentially according to the method of Hoey, M; Houslay, M. D., Biochem.", "Pharmacol.", "1990, 40, 193-202 and Lugman et al.", "Biochem.", "Pharmacol.", "1986, 35, 1743-1751.The enzyme activity was determined in a test batch of 100 μl in 20 mM tris/HCl buffer pH 7.5 which contains 5 mM MgCl2, 0.1 mg/ml of bovine serum albumin and either 800 Bq of [3H]-cAMP or [3H]-cGMP.", "The final concentration of the corresponding nucleotides is 10−6 mol/l.", "The reaction is started by addition of the enzyme and the amount of enzyme is proportioned such that about 50% of the substrate are reacted during the incubation time of 30 min.", "In order to test the cGMP-stimulable PDE 2, [3H]-cAMP is used as a substrate and 10−6 mol/l of non-labelled cGMP is added to the batch.", "In order to test the Ca calmodulin-dependent PDE 1, additionally CaCl2 1 μM and calmodulin 0.1 μM and are added to the reaction batch.", "The reaction is stopped by addition of 100 μl of acetonitrile which contains 1 mM cAMP and 1 mM AMP.", "100 μl of the reaction batch are separated on the HPLC and the cleavage products are determined quantitatively online using a flow-through scintillation counter.", "The substance concentration at which the reaction rate is decreased by 50% is measured.", "In addition, the phosphodiesterase [3H] cAMP SPA enzyme assay and the phosphodiesterase [3H] cGMP SPA enzyme assay from Amersham Life Sciences were used for testing.", "The test was carried out according to the experimental protocol indicated by the manufacturer.", "The activity of the test substances on PDE 2 was determined using the [3H] cAMP Scintillation Proximity Assay (SPA) kit (TRKQ7090) from Amersham International (Little Chalfont, England) or on PDE1 and PDE5 using the [3H] cGMP Scintillation Proximity Assay (SPA) Kit (TRKQ7100) from Amersham International (Little Chalfont, England).", "Test substances were dissolved in 100% DMSO (10 mM), and this solution was further diluted with H2O (highest final concentration in the test: 10 μM).", "For the prestimulation of the PDE 2, cGMP is additionally added (final concentration in the test: 10−6 M).", "The enzyme is diluted in PDE buffer (20 mM TRIS/HCl, 5 mM MgCl2, 0.1 mg/ml of albumin, pH 7.5).", "The following volumes per hole are pipetted into a 96-hole plate (Wallac, 1450-401): 10 μl of substance solution (at the 100% value 10 μl of H2O), 10 μl of cGMP (10−5 M), 70 μl of [3H]-cAMP test mixture (see kit), 10 μl of enzyme (at the 0 value no enzyme, instead of this +10 μl of H2O) at the start of the reaction.", "After incubation at 30° C. for 15 min, the reaction was stopped using 50 μl of SPA bead solution (see kit), and the plate was sealed with a film and shaken for 30 seconds.", "After the beads had settled (about 15 min), the plate was measured in a beta counter.", "For the measurement of PDE 1, calmodulin 10−7 M and CaCl2 1 μM were added to the reaction batch.", "The PDE 5 was measured using the [3H] cGMP SPA Assay.", "Under the conditions indicated above the working examples inhibit the PDE 2 with IC50 values of less than 1 μm.", "Measurement of the Increase in the Intracellular Neuronal cGMP Concentration in Cell Cultures PDE 2 inhibitors increase the intracellular neuronal cGMP concentration after prestimulation of the guanylate cyclase using 10−4 M sodium nitroprusside (SNP) in primary rat brain cell cultures.", "Rat embryos were decapitated and the heads were transferred to preparation dishes.", "The scalp and cranium were removed, and the exposed brains were transferred to a further Petri dish.", "With the aid of a binocular microscope and two pairs of forceps, hippocampi were isolated from the cortex and cooled to 4° C. using ice.", "This preparation and the isolation of the hippocampal neurons were then carried out according to a standard protocol using the papain dissociation system (Worthington Biochemical Corporation, Lakewood, N.J. 08701, USA) (Huettner et al.", "J. Neurosci.", "1986, 6, 3044-3060).", "The mechanically isolated neurons were cultured under standard conditions (37° C., 5% CO2) to 150,000 cells/hole in 200 μl of neurobasal medium/hole (neurobasal; Gibco/BRL; 2 mM L-glutamine; in the presence of penicillin/streptomycin) for 7 days in 96-hole plates (pretreated with poly-D-lysine 100 μg/ml for 20 min).", "After 7 days, the medium was removed and the cells were washed with HBS buffer (Gibco/BRL).", "Subsequently, 100 μl each of SNP solution and 100 μl of the racemate of Example 1 (dissolved in 100% DMSO beforehand: 10 mM) were added in HBS to the cells such that the final concentration of SNP was 100 mM and that of the racemate of Example 1 was as indicated in FIG.", "1 and the mixture was incubated at 37° C. for 20 min.", "The cells were then lysed in 200 μl of lysis buffer (cGMP kit code RPN 226; from Amersham Pharmacia Biotech.)", "and the cGMP concentration was measured according to the instructions of the manufacturer.", "All measurements were carried out in triplicate.", "Statistical analysis was carried out using Prism Software Version 2.0 (GraphPad Software Inc., San Diego, Calif. USA; *** p<0.001).", "Object Recognition Test The object recognition test is a memory test.", "It measures the ability of rats (and mice) to differentiate between known and unknown objects and is therefore suitable for the determination of the memory-improving action of the compounds according to the invention.", "The test is carried out as described (Blokland et al.", "NeuroReport 1998, 9, 4205-4208; Ennaceur, A., Delacour, J., Behav.", "Brain Res.", "1988, 31, 47-59; Ennaceur, A., Meliani, K., Psychopharmacology 1992, 109, 321-330; Prickaerts, et al.", "Eur.", "J. Pharmacol.", "1997, 337, 125-136).", "In a first passage, a rat in an otherwise empty relatively large observation arena is confronted with two identical objects.", "The rat will extensively examine, i.e.", "sniff and touch, both objects.", "In a second passage, after an interval of 24 hours, the rat is again tested in the observation arena.", "One of the known objects is now replaced by a new, unknown object.", "When a rat recognizes the known object, it will especially examine the unknown object.", "After 24 hours, a rat, however, has normally forgotten which object it has already examined in the first passage, and will therefore inspect both objects equally intensively.", "The administration of a substance having learning- and memory-improving action will lead to a rat recognizing the object already seen 24 hours beforehand, in the first passage, as known.", "It will examine the new, unknown object in greater detail than the already known one.", "This memory power is expressed in a discrimination index.", "A discrimination index of zero means that the rat examines both objects, the old and the new one, for the same length of time; i.e.", "it has not recognized the old object and reacts to both objects as if they were both unknown and new.", "A discrimination index of greater than zero means that the rat has inspected the new object for longer than the old one; i.e.", "the rat has recognized the old object.", "Definitions of Terms Chromatography, if not mentioned otherwise, was carried out on silica gel Si 60.In the case of flash chromatography, the described conditions were normally followed (cf.", "Still J. Org.", "Chem.).", "If not described otherwise, the reactions were carried out under argon and, where necessary, under anhydrous conditions.", "HPLC=high-pressure liquid chromatography MS=mass spectrometry NMR=nuclear magnetic resonance spectroscopy LC-MS=liquid chromatography combined with mass spectrometry MeOH=methanol DMSO=dimethyl sulphoxide THF=tetrahydrofuran of th.=of theory Starting Compounds EXAMPLE 1A N-Acetylalanine 134 g (1.50 mol) of DL-alanine are introduced into acetic acid and treated dropwise with 230 g (2.25 mol) of acetic anhydride.", "The mixture is additionally stirred at 100° C. for 2 h to complete the reaction and the solvent is then stripped off in vacuo.", "The solid obtained is suspended in ethyl acetate and filtered off with suction.", "For purification, the solid is washed several times with diethyl ether.", "Yield: 162 g (82.6% of th.)", "1H-NMR (methanol-d4): δ=1.38 (d, 3H), 1.97 (s, 3H), 4.37 (q, 1H).", "EXAMPLE 2A 2-(Acetylamino)butanoic Acid 163 g (1.58 mol) of 2-aminobutyric acid are reacted analogously to Example 1A with 242 g (2.37 mol) of acetic anhydride to give 2-(acetylamino)butanoic acid.", "Yield: 220 g (95.9% of th.)", "1H-NMR (CD3OD): δ=0.97 (t, 3H), 1.65-1.93 (m, 2H), 1.99 (s, 3H), 4.29 (q, 1H).", "EXAMPLE 3A 2-(4-Methylphenyl)ethanamidine hydrochloride 10.8 g (201 mmol) of ammonium chloride are suspended in 200 ml of dry toluene and the suspension is cooled to 0° C. 100 ml of a 2M solution of trimethylaluminium in hexane are added dropwise and the mixture is stirred at room temperature until the evolution of gas is complete.", "After addition of 13.2 g (100 mmol) of 4-methylbenzyl cyanide, the reaction mixture is stirred overnight at 80° C. (bath).", "The cooled reaction mixture is treated with ice-cooling with 35 ml of methanol and then stirred at room temperature for a further 1 h. The solid is then first filtered off with suction, and the filter cake is washed several times with methanol.", "The filtrate is concentrated, the residue is resuspended in dichloromethane/methanol 10/1 and the insoluble solid is separated off.", "The filtrate is then again evacuated from the solvent in vacuo.", "Yield: 16.4 g (88.1% of th.)", "1H-NMR (methanol-d4): δ=2.35 (s, 3H), 3.77 (s, 2H), 7.21-7.29 (m, 4H).", "3,4-dimethoxybenzyl cyanide, 112 g (71.7% of th.)", "of 2-(3,4-dimethoxy-phenyl)ethanamidine hydrochloride are obtained.", "1H-NMR (DMSO-d6): δ=3.62 (s, 2H), 3.74 (s, 3H), 3.76 (s, 3H), 6.92-7.14 (m, 3H).", "EXAMPLE 6A Ethyl 3-(acetylamino)-2-oxobutanoate 10.65 g (81.2 mmol) of acetylalanine (Example 1A) are taken up in 150 ml of tetrahydrofuran and heated under reflux with 19.3 g (244 mmol) of pyridine and a spatula tipful of N,N-dimethylaminopyridine.", "At boiling heat, 22.2 g (162 mmol) of ethyl oxalyl chloride are added dropwise.", "The mixture is then heated at reflux until the evolution of gas can no longer be observed.", "After cooling, the batch is added to ice water and the organic phase is extracted in ethyl acetate.", "The dried organic phase is concentrated and, dissolved directly in ethanol, reacted further.", "EXAMPLE 4A 2-(4-Methoxyphenyl)ethanamidine hydrochloride Analogously to Example 3A, starting from 21.4 g (400 mmol) of ammonium chloride, 200 ml of a 2M solution of trimethylaluminium in hexane and 29.4 g (200 mmol) of 4-methoxybenzyl cyanide, 28.5 g (71.3% of th.)", "of 2-(4-methoxy-phenyl)ethanamidine hydrochloride are obtained.", "Melting point: 126° C. EXAMPLE 5A 2-(3,4-Dimethoxyphenyl)ethanamidine hydrochloride Analogously to Example 3A, starting from 72.5 g (1.35 mol) of ammonium chloride, 672 ml of a 2M solution of trimethylaluminium in hexane and 120 g (677 mmol) of EXAMPLE 7A N-{1-[3-(4-Methylbenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}acetamide 10 g (54.2 mmol) of 2-(4-methylphenyl)ethanamidine hydrochloride (Example 3A) are taken up in 100 ml of ethanol and treated with 3.25 g (65.0 mmol) of hydrazine hydrate.", "The mixture is stirred for 45 min, then the compound of Example 6A is added.", "It is then stirred for 4 h at 80° C. (bath) and overnight at room temperature.", "The substance is purified by flash chromatography, preliminary fractions first being separated off using ethyl acetate.", "The product is eluted with dichloromethane/methanol 30/1.Yield: 5.63 g (36.3% of th.)", "1H-NMR (methanol-d4): δ=1.40 (d, 3H), 1.93 (s, 3H), 2.29 (s, 3H), 3.85 (s, 2H), 5.12 (q, 1H), 7.12-7.23 (m, 4H).", "EXAMPLE 8A 6-(1-Aminoethyl)-3-(4-methylbenzyl)-1,2,4-triazin-5(4H)-one 20 g (69.9 mmol) of N-{1-[3-(4-methylbenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]-ethyl}acetamide (Example 7A) are stirred under reflux in 200 ml of 2 N hydrochloric acid for 18 h. The cooled mixture is then neutralized using 6 N NaOH and evaporated to dryness in vacuo.", "The residue is suspended in methanol and the salt is separated off.", "The concentrated filtrate is flash-chromatographed using dichloromethane/methanol 20/1 and 5/1.Yield: 8 g (46.9% of th.)", "1H-NMR (methanol-d4): δ=1.50 (d, 3H), 2.20 (s, 3H), 3.84 (s, 2H), 4.52 (q, 1H), 7.03 (d, 2H), 7.13 (d, 2H).", "EXAMPLE 9A N-{1-[3-(4-Methoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}acetamide Analogously to Example 7A, 5.1 g (25.4 mmol) of 2-(4-methoxyphenyl)-ethanamidine hydrochloride (Example 4A) are reacted with 1.53 g (30.5 mmol) of hydrazine hydrate and 7.14 g (38.1 mol) of ethyl 3-(acetylamino)-2-oxobutanoate (Example 6A) to give N-{1-[3-(4-methoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}acetamide.", "Yield: 2.97 g (38.7% of th.)", "1H-NMR (methanol-d4): δ=1.44 (d, 3H), 1.99 (s, 3H), 3.78 (s, 3H), 3.91 (s, 2H), 5.23 (q, 1H), 6.90 (d, 2H), 7.28 (d, 2H).", "EXAMPLE 10A 6-(1-Aminoethyl)-3-(4-methoxybenzyl)-1,2,4-triazin-5(4H)-one Analogously to Example 8A, 17 g (56.2 mmol) of N-{1-[3-(4-methoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}acetamide (Example 9A) are reacted to give 6-(1-aminoethyl)-3-(4-methoxybenzyl)-1,2,4-triazin-5(4H)-one.", "Yield: 5 g (34.2% of th.)", "1H-NMR (methanol-d4): δ=1.55 (d, 3H), 3.74 (s, 3H), 3.84 (s, 2H), 4.51 (q, 1H), 6.83 (d, 2H), 7.24 (d, 2H).", "EXAMPLE 11A N-{1[-3-(3,4-Dimethoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}-acetamide Analogously to Example 7A, 20.0 g (86.7 mmol) of 2-(3,4-dimethoxyphenyl)-ethanamidine hydrochloride (Example 5A) are reacted with 5.21 g (104 mmol) of hydrazine hydrate and 24.3 g (130 mmol) of ethyl 3-(acetylamino)-2-oxobutanoate (Example 6A) to give N-{1-[3-(3,4-dimethoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}acetamide.", "Yield: 15.5 g (77.5% of th.)", "1H-NMR (methanol-d4): δ=1.40 (d, 3H), 1.95 (s, 3H), 3.78 (s, 3H), 3.81 (s, 3H), 3.82 (s, 2H), 5.16 (q, 1H), 6.86-6.97 (m, 3H).", "EXAMPLE 12A 6-(1-Aminoethyl)-3-(3,4-dimethoxybenzyl)-1,2,4-triazin-5(4H)-one Analogously to Example 8A, 23 g of N-{1-[3-(3,4-dimethoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}acetamide (Example 11A) are reacted to give 6-(1-aminoethyl)-3-(3,4-dimethoxybenzyl)-1,2,4-triazin-5(4H)-one.", "Yield: 10.1 g (50.4% of th.)", "1H-NMR (methanol-d4): δ=1.55 (d, 3H), 3.78 (s, 3H), 3.80 (s, 3H), 3.83 (s, 2H), 4.52 (q, 1H), 6.83-6.98 (m, 3H).", "EXAMPLE 13A Sodium (2E)-4-methoxy-4-oxo-2-buten-2-olate 60 g of a 30% strength sodium hydride suspension in mineral oil (744 mmol of NaH) are suspended in 250 ml of dry THF in an inert gas atmosphere.", "86.4 g (744 mmol) of methyl acetoacetate in 200 ml of THF are slowly added dropwise, the resulting hydrogen being led directly into the waste air.", "After dropwise addition has taken place, the mixture is stirred at reflux for half an hour and then cooled.", "The solvent is stripped off in vacuo and the residual solid is washed with diethyl ether.", "Yield: 81.9 g (79.7% of th.)", "Melting point: the substance decomposes at 200° C. EXAMPLE 14A Methyl 2-acetylhexanoate 30 g (217 mmol) of sodium (2E)-4-methoxy-4-oxo-2-buten-2-olate (Example 13A) suspended in 1,3-dimethyltetrahydro-2(1H)-pyrimidone and 1.24 g (7.5 mmol) of potassium iodide are treated dropwise with 29.8 g (217 mmol) of butyl bromide and the mixture is stirred at 80° C. for 1 h under reflux.", "The cooled mixture is then added to ice water and extracted with diethyl ether.", "The ether phase is washed with sodium thiosulphate solution, dried, concentrated and chromatographed.", "The eluent used is cyclohexane having an increasing ethyl acetate content.", "Yield: 11.6 g (30.9% of th.)", "1H-NMR (CDCl3): δ=0.90 (t, 3H), 1.21-1.41 (m, 4H), 1.80-1.90 (m, 2H), 2.22 (s, 3H), 3.41 (t, 1H), 3.74 (s, 3H).", "EXAMPLE 15A 2-Acetylhexanoic Acid 3.08 g (17.9 mmol) of methyl 2-acetylhexanoate (Example 14A) is dissolved in 10 ml of dioxane and cooled to 0° C. 7.00 ml of a 3.5 M potassium hydroxide solution are added with cooling.", "After a reaction time of 5 h, the batch is concentrated, treated with 20 ml of ethyl acetate and 20 ml of water and extracted with shaking.", "The water phase is recovered, cooled to 0° C. and slowly treated with 1 N hydrochloric acid until a pH of 1 is reached.", "The mixture is then extracted with dichloromethane.", "The dichloromethane phase is dried and directly reacted further without concentrating.", "EXAMPLE 16A Methyl 2-acetylheptanoate Analogously to Example 14A, 30 g (217 mmol) of sodium (2E)-4-methoxy-4-oxo-2-buten-2-olate (Example 13A) and 1.24 g (7.5 mmol) of potassium iodide are reacted with 32.8 g (217 mmol) of n-pentyl bromide to give methyl 2-acetylheptanoate.", "Yield: 10.5 g (26.0% of th.)", "1H-NMR (CDCl3): δ=0.89 (t, 3H), 1.20-1.38 (m, 6H), 1.84 (m, 2H), 2.22 (s, 3H), 3.42 (t, 1H), 3.73 (s, 3H).", "EXAMPLE 17A 2-Acetylheptanoic Acid Analogously to Example 15A, 900 mg (5.23 mmol) of methyl 2-acetylheptanoate (Example 16A) are reacted with 2.5 ml of a 3.5 M potassium hydroxide solution to give 2-acetylheptanoic acid in dichloromethane.", "EXAMPLE 18A Methyl 2-acetyloctanoate Analogously to Example 14A, 30 g (217 mmol) of sodium (2E)-4-methoxy-4-oxo-2-buten-2-olate (Example 13A) and 1.24 g (7.5 mmol) of potassium iodide are reacted with 37.7 g (228 mmol) of hexyl bromide to give methyl 2-acetyloctanoate.", "Yield: 16.03 g (36.8% of th.)", "1H-NMR (CDCl3: δ=0.89 (t, 3H), 1.19-1.39 (m, 8H), 1.84 (m, 2H), 2.22 (s, 3H), 3.42 (t, 1H), 3.73 (s, 3H).", "EXAMPLE 19A 2-Acetyloctanoic Acid Analogously to Example 15A, 3.16 g (15.8 mmol) of methyl 2-acetyloctanoate (Example 18A) are reacted with 7 ml of a 3.5 M potassium hydroxide solution to give 2-acetyloctanoic acid in dichloromethane.", "EXAMPLE 20A Methyl 2-acetyl-6-heptenoate Analogously to Example 14A, 10 g (72.4 mmol) of sodium (2E)-4-methoxy-4-oxo-2-buten-2-olate (Example 13A) and 0.4 g (2.41 mmol) of potassium iodide are reacted with 10.8 g (72.4 mmol) of 1-bromopentene to give methyl 2-acetyl-6-heptenoate.", "Yield: 5.0 g (37.5% of th.)", "1H-NMR (CDCl3: δ=1.33-1.47 (m, 2H), 1.79-1.94 (m, 2H), 1.99-2.15 (m, 2H), 2.23 (s, 3H), 3.43 (t, 1H), 3.74 (s, 3H), 4.93-5.08 (m, 2H), 5.67-5.88 (m, 1H).", "EXAMPLE 21A Methyl 2-(1-hydroxyethyl)-6-heptenoate 5.00 g (27.1 mmol) of methyl 2-acetyl-6-heptenoate (Example 20A) are introduced into 50 ml of methanol and ice-cooled.", "0.56 g (14.9 mmol) of sodium borohydride are added in portions and the mixture is stirred for a further 1 h. The batch is then evacuated from the solvent, taken up in diethyl ether and washed with 1 N hydrochloric acid.", "The organic phase is dried, concentrated and flash-chromatographed using the eluent petroleum ether/ethyl acetate 10/1.Yield: 4.9 g (96.9% of th.)", "1H-NMR (CDCl3, diastereomer mixture): δ=1.17-1.25 (d, 3H), 1.33-1.48 (m, 2H), 1.55-1.71 (m, 2H), 2.00-2.14 (m, 2H), 2.29-2.49 (m, 1H), 3.73 (s, 3H), 3.85-3.99 (m, 1H), 4.91-5.07 (m, 2H), 5.67-5.90 (m, 1H).", "EXAMPLE 22A 2-(1-Hydroxyethyl)-6-heptenoic Acid Analogously to Example 15A, 4.80 g (25.8 mmol) of methyl 2-(1-hydroxyethyl)-6-heptenoate (Example 21A) are reacted with 39.0 ml of a 1 M sodium hydroxide solution to give 2-(1-hydroxyethyl)-6-heptenoic acid.", "EXAMPLE 23A 2-Acetyl-N-{1-[3-(4-methylbenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}-hexanamide The amount of 2-acetylhexanoic acid in dichloromethane from Example 15A is treated with 2.3 g (17.0 mmol) of 1-hydroxy-1H-benzotriazole and 3.44 g (34 mmol) of 4-methylmorpholine and cooled to −20° C. After addition of 3.26 g (17.0 mmol) of N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, the mixture is stirred for 30 min.", "The cooling bath is removed in the course of this.", "1.60 g (6.55 mmol) of 6-(1-aminoethyl)-3-(4-methylbenzyl)-1,2,4-triazin-5(4H)-one (Example 8A) are then added after fresh cooling to −20° C. and the mixture is stirred overnight while warming to room temperature.", "For work-up, the dichloromethane phase is washed with 1 N potassium hydrogensulphate solution and then with saturated sodium hydrogencarbonate solution.", "The dried organic phase is concentrated and chromatographed using the eluent dichloromethane/methanol 100/1 to 30/1.Yield: 1.69 g (67.1% of th.)", "1H-NMR (methanol-d4, diastereomer mixture): δ=0.83-0.93 (m, 3H), 1.16-1.40 (m, 4H), 1.45 (d, 3H), 1.74 (m, 2H), 2.17 (s, 3H), 2.30 (s, 3H), 3.70 (m, 1H), 3.85 (s, 2H), 5.12 (m, 1H), 7.10-7.24 (m, 4H).", "EXAMPLE 24A 2-Acetyl-N-{1-[3-(4-methylbenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}-heptanamide The amount of 2-acetylheptanoic acid in dichloromethane from Example 17A is reacted analogously to Example 23A with 680 mg (5.0 mmol) of 1-hydroxy-1H-benzotriazole, 1.52 g (15.0 mmol) of 4-methylmorpholine, 960 mg (5.0 mmol) of N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 1.22 g (5.00 mmol) of 6-(1-aminoethyl)-3-(4-methylbenzyl)-1,2,4-triazin-5(4H)-one (Example 8A) to give 2-acetyl-N-{1-[3-(4-methylbenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]-ethyl}heptanamide.", "Yield: 533 mg (26.8% of th.)", "1H-NMR (methanol-d4, diastereomer mixture): δ=0.82-0.93 (m, 3H), 1.19-1.34 (m, 6H), 1.44 (d, 3H), 1.74 (m, 2H), 2.17 (s, 3H), 2.30 (s, 3H), 3.43 (m, 1H), 3.85 (s, 2H), 5.13 (m, 1H), 7.11-7.24 (m, 4H).", "EXAMPLE 25A 2-Acetyl-N-{1-[3-(4-methylbenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}-octanamide The amount of 2-acetyloctanoic acid in dichloromethane from Example 19A is reacted analogously to Example 23A with 2.14 g (15.8 mmol) of 1-hydroxy-1H-benzotriazole, 1.90 g (18.8 mmol) of 4-methylmorpholine, 3.03 g (15.8 mmol) of N′-(3-dimethylaminopropyl-N-ethylcarbodiimide hydrochloride and 3.80 mg (15.6 mmol) of 6-(1-aminoethyl)-3-(4-methylbenzyl)-1,2,4-triazin-5(4H)-one (Example 8A) to give 2-acetyl-N-{1-[3-(4-methylbenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}octanamide.", "Yield: 909 mg (14.2% of th.)", "LC-MS: retention time 3.89 and 3.94 min., m/z 413.3 [M+H]+ LC parameters: soln.", "A acetonitrile+0.1% formic acid Soln.", "B water+0.1% formic acid Column oven 40° C.; Column symmetry C18 50 mm×2.1 mm Gradient: Time % A % B Flow 0 10 90 0.5 4 90 10 0.5 6 90 10 0.5 6.1 10 90 1.0 7.5 10 90 0.5 9 90 10 0.8 EXAMPLE 26A 2-Acetyl-N-{1-[3-(4-methoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}-heptanamide 1.29 g (6.91 mmol) of methyl 2-acetylheptanoate (Example 16A) are hydrolysed to 2-acetylheptanoic acid according to Example 17A.", "The acid in 20 ml of dichloro-methane is reacted analogously to Example 23A with 903 mg (6.90 mmol) of 1-hydroxy-1H-benzotriazole, 2.02 g (20 mmol) of 4-methylmorpholine, 1.32 g (6.90 mmol) of N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 890 mg (3.4 mmol) of 6-(1-aminoethyl)-3-(4-methoxybenzyl)-1,2,4-triazin-5(4H)-one (Example 10A) to give 2-acetyl-N-{1-[3-(4-methoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}heptanamide.", "Yield: 357 mg (25.3% of th.)", "1H-NMR (CDCl3, diastereomer mixture): δ=0.78-0.94 (m, 3H), 1.19-1.34 (m, 6H), 1.46 (d, 3H), 1.83 (m, 2H), 2.20 and 2.24 (each s, 3H), 3.31 (m, 1H), 3.81 (s, 3H), 3.94 (s, 2H), 5.11 (m, 1H), 6.83-7.31 (m, 4H, under CHCl3 signal).", "EXAMPLE 27A 2-Acetyl-N-{1-[3-(4-methoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}-octanamide 1.18 g (5.91 mmol) of methyl 2-acetyloctanoate (Example 18A) are hydrolysed to 2-acetyloctanoic acid according to Example 19A.", "The acid in 20 ml of dichloro-methane is reacted analogously to Example 23A with 800 mg (5.90 mmol) of 1-hydroxy-1H-benzotriazole, 1.66 g (16.4 mmol) of 4-methylmorpholine, 1.13 g (5.9 mmol) of N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 900 mg (3.5 mmol) of 6-(1-aminoethyl)-3-(4-methoxybenzyl)-1,2,4-triazin-5(4H)-one (Example 10A) to give 2-acetyl-N-{1-[3-(4-methoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}octanamide.", "Yield: 722 mg (48.7% of th.)", "1H-NMR (CDCl3, diastereomer mixture): δ=0.80-0.90 (m, 3H), 1.16-1.33 (m, 8H), 1.46 (d, 3H), 1.84 (m, 2H), 2.20 and 2.23 (each s, 3H), 3.32 (m, 1H), 3.79 (s, 3H), 3.94 (s, 2H), 5.13 (m, 1H), 6.85-7.30 (m, 4H, under CHCl3 signal).", "EXAMPLE 28A 2-Acetyl-N-{1-[3-(3,4-dimethoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}octanamide 601 g (3.0 mmol) of methyl 2-acetyloctanoate (Example 18A) are hydrolysed to 2-acetyloctanoic acid according to Example 19A.", "The acid in 20 ml of dichloro-methane is reacted analogously to Example 23A with 1.08 g (8.0 mmol) of 1-hydroxy-1H-benzotriazole, 1.62 g (16.0 mmol) of 4-methylmorpholine, 1.53 g (8.0 mmol) of N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 870 mg (3.0 mmol) of 6-(1-aminoethyl)-3-(3,4-methoxybenzyl)-1,2,4-triazin-5(4H)-one (Example 12A) to give 2-acetyl-N-{1-[3-(3,4-dimethoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}octanamide.", "Yield: 149 mg (10.8% of th.)", "1H-NMR (DMSO-d6, diastereomer mixture): δ=0.79-0.90 (m, 3H), 1.08-1.34 (m, 11H), 1.59 (m, 2H), 2.08 and 2.09 (each s, 3H), 3.41 (m, 1H), 3.72 (s, 3H), 3.74 (s, 3H), 3.76 (s, 2H), 4.98 (m, 1H), 6.79-6.99 (m, 3H).", "EXAMPLE 29A N-{1-[3-(3,4-Dimethoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}-2-(1-hydroxyethyl)-6-heptenamide 742 mg (4.31 mmol) of 2-(1-hydroxyethyl)-6-heptenoic acod (Example 22A) are reacted analogously to Example 23A with 580 mg (4.31 mmol) of 1-hydroxy-1H-benzotriazole, 870 mg (8.61 mmol) of 4-methylmorpholine, 830 mg (4.31 mmol) of N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 500 mg (1.72 mmol) of 6-(1-aminoethyl)-3-(3,4-dimethoxybenzyl)-1,2,4-triazin-5(4H)-one (Example 12A) to give N-{1-[3-(3,4-dimethoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}-2-(1-hydroxyethyl)-6-heptenamide.", "Yield: 285 mg (37.2% of th.)", "1H-NMR (methanol-d4, diastereomer mixture): δ=1.10-1.19 (m, 3H), 1.25-1.79 (m, 7H), 1.96-2.10 (m, 2H), 2.15-2.26 (m, 1H), 3.66-3.85 (m, 9H), 4.90-5.02 (m, 2H), 5.10-5.21 (m, 1H), 5.67-5.85 (m, 1H), 6.85-6.99 (m, 3H).", "EXAMPLE 30A 2-Acetyl-N-{1-[3-(3,4-dimethoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]-ethyl}-6-heptenamide 100 mg (0.76 mmol) of oxalyl chloride in 5 ml of dichloromethane are treated dropwise at −70° C. with 130 mg (1.64 mmol) of dimethyl sulphoxide.", "The mixture is stirred at −70° C. for 30 min, then 280 mg (0.63 mmol) of N-{1-[3-(3,4-dimethoxy-benzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}-2-(1-hydroxyethyl)-6-hepten-amide (Example 29A) are added.", "After a further 30 min, during which the temperature in the batch rises to about −60° C., 320 mg (3.15 mmol) of triethylamine are added and the cooling bath is then removed.", "If the batch temperature is warmed almost to room temperature, 10 ml of water are added and the phases are separated after stirring briefly.", "The dried organic phase is chromatographed in dichloromethane/methanol 50/1.Yield: 175 mg (55.9% of th.)", "1H-NMR (methanol-d4, diastereomer mixture): δ=1.27-1.38 (m, 2H), 1.46 (d, 3H), 1.71-1.80 (m, 2H), 1.99-2.21 (m, 2H), 2.17 (s, 3H), 3.44 (m, 1H), 3.78-3.86 (m, 8H), 4.87-5.03 (m, 2H), 5.09-5.17 (m, 1H), 5.68-5.84 (m, 1H), 6.85-6.99 (m, 3H).", "Working Examples EXAMPLE 1 7-(1-Acetylpentyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one 1.66 g (4.31 mmol) of 2-acetyl-N-{1-[3-(4-methylbenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}hexanamide (Example 23A) in 20 ml of dichloroethane are treated with 3.31 g (21.6 mmol) of phosphorus oxychloride and the mixture is stirred at 100° C. under reflux for 1 h. The cooled mixture is neutralized with saturated sodium hydrogencarbonate solution and the solvent is stripped off.", "The product is chromatographed using dichloromethane/methanol 70/1.Yield: 1.58 g (quant.)", "Rf value (dichloromethane/methanol 10/1): 0.58 1H-NMR (400 MHz, methanol-d4): δ=0.90 (t, 3H), 1.26-1.40 (m, 4H), 1.92-2.27 (m, 2H), 2.29 (s, 3H), 2.32 (s, 3H), 2.71 (s, 3H), 3.88 (s, 2H), 4.74 (m, 1H), 7.17 (d, 2H), 7.24 (d, 2H).", "EXAMPLE 2 7-(1-Acetylhexyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one Analogously to Example 1, 520 mg (1.30 mmol) of 2-acetyl-N-{1-[3-(4-methyl-benzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}heptanamide (Example 24A) and 1.99 g (13.0 mmol) of phosphorus oxychloride are reacted to give 7-(1-acetylhexyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one.", "Yield: 495 mg (quant.)", "Rf value (dichloromethane/methanol 10/1): 0.56 1H-NMR (300 MHz, methanol-d4): δ=0.88 (t, 3H), 1.18-1.38 (m, 6H), 1.93-2.24 (m, 2H), 2.28 (s, 3H), 2.32 (s, 3H), 2.71 (s, 3H), 3.89 (s, 2H), 4.75 (m, 1H), 7.17 (d, 2H), 7.25 (d, 2H).", "EXAMPLE 3 7-(1-Acetylheptyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one Analogously to Example 1, 910 mg (2.20 mmol) of 2-acetyl-N-{1-[3-(4-methyl-benzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}octanamide (Example 25A) and 1.65 g (10.7 mmol) of phosphorus oxychloride are reacted to give 7-(1-acetylheptyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one.", "Yield: 870 mg (quant.)", "Rf value (dichloromethane/methanol 10/1): 0.64 1H-NMR (400 MHz, methanol-d4): δ=0.88 (t, 3H), 1.15-1.38 (m, 8H), 1.93-2.24 (m, 5H, s at 2.17), 2.31 (s, 3H), 2.64 (s, 3H), 3.86 (s, 2H), 4.55 (m, 1H), 7.16 (d, 2H), 7.24 (d, 2H).", "EXAMPLE 4 7-(1-Acetylhexyl)-2-(4-methoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one Analogously to Example 1, 340 mg (0.82 mmol) of 2-acetyl-N-{1-[3-(4-methoxy-benzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}heptanamide (Example 26A) and 540 g (3.50 mmol) of phosphorus oxychloride are reacted to give 7-(1-acetylhexyl)-2-(4-methoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one.", "Yield: 218 mg (67.0% of th.)", "Rf value (dichloromethane/methanol 10/1): 0.56 1H-NMR (400 MHz, methanol-d4): δ=0.85 (t, 3H), 1.18-1.35 (m, 6H), 1.95-2.12 (m, 5H, s at 2.03), 2.53 (s, 3H), 3.75 (s, 2H), 3.77 (s, 3H), 4.27 (m, 1H), 6.88 (d, 2H), 7.25 (d, 2H).", "EXAMPLE 5 7-(1-Acetylheptyl)-2-(4-methoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one Analogously to Example 1, 710 mg (1.65 mmol) of 2-acetyl-N-{1-[3-(4-methoxy-benzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}octanamide (Example 27A) and 1.23 g (8.00 mmol) of phosphorus oxychloride are reacted to give 7-(1-acetylheptyl)-2-(4-methoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one.", "Yield: 507 mg (75.1% of th.)", "Rf value (dichloromethane/methanol 10/1): 0.47 1H-NMR (400 MHz, methanol-d4): δ=0.86 (t, 3H), 1.15-1.35 (m, 8H), 1.95-2.13 (m, 5H, s at 2.03), 2.53 (s, 3H), 3.77 (s, 3H), 3.79 (s, 2H), 4.29 (m, 1H), 6.88 (d, 2H), 7.25 (d, 2H).", "EXAMPLE 6 7-(1-Acetylheptyl)-2-(3,4-dimethoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one Analogously to Example 1, 150 mg (0.32 mmol) of 2-acetyl-N-{1-[3-(3,4-dimethoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}octanamide (Example 28A) and 250 g (1.61 mmol) of phosphorus oxychloride are reacted to give 7-(1-acetylheptyl)-2-(3,4-dimethoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one.", "Yield: 80 mg (55.9% of th.)", "Rf value (dichloromethane/methanol 10/1): 0.62 1H-NMR (400 MHz, methanol-d4): δ=0.85 (t, 3H), 1.15-1.32 (m, 8H), 1.99-2.14 (m, 5H, s at 2.03), 2.54 (s, 3H), 3.78 (s, 2H), 3.80 (s, 3H), 3.82 (s, 3H) 4.27 (m, 1H), 6.87-6.97 (m, 3H).", "EXAMPLE 7 7-(1-Acetyl-5-hexenyl)-2-(3,4-dimethoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]-triazin-4(3H)-one Analogously to Example 1, 160 mg (0.36 mmol) of 2-acetyl-N-{1-[3-(3,4-di-methoxybenzyl)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl]ethyl}-6-heptenamide (Example 30A) and 0.06 g (0.36 mmol) of phosphorus oxychloride are reacted to give 7-(1-acetyl-5-hexenyl)-2-(3,4-dimethoxybenzyl)-5-methylimidazo[5,1-f]-[1,2,4]triazin-4(3H)-one.", "Yield: 115 mg (74.9% of th.)", "Rf value (dichloromethane/methanol 10/1): 0.58 1H-NMR (400 MHz, methanol-d4): δ=1.22-1.37 (m, 2H), 1.97-2.15 (m, 7H, s at 2.02), 2.54 (s, 3H), 3.78 (s, 2H), 3.82 (s, 3H), 3.83 (s, 3H) 4.28 (m, 1H), 4.87-4.98 (m, 2H), 5.68-5.80 (m, 1H), 6.89-6.95 (m, 3H).", "EXAMPLE 8 7-[1-(1-Hydroxyethyl)pentyl]-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]-triazin-4(3H)-one 200 mg (0.55 mmol) of 7-(1-acetylpentyl)-5-methyl-2-(4-methyl-benzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one (Example 1) are dissolved in 5 ml of ethanol and treated in portions with 38 mg (1.00 mmol) of sodium borohydride.", "The batch is stirred at room temperature for 1 h, then it is neutralized with a few drops of 2 N hydrochloric acid.", "The solvent is stripped off in vacuo, then the residue is chromatographed using the eluent dichloromethane/methanol 40/1.Yield: 46 mg (22.9% of th.)", "Rf value (dichloromethane/methanol 10/1): 0.44 1H-NMR (400 MHz, methanol-d4, diastereomer mixture): δ=0.82 (t, 3H), 0.91-1.37 (m, 7H), 1.68-2.09 (m, 2H), 2.31 (s, 3H), 2.53 and 2.54 (each s, 3H), 3.37 (m, 1H), 3.79 (s, 2H), 3.97-4.13 (m, 1H), 7.10-7.26 (m, 4H).", "EXAMPLE 9 7-[1-(1-Hydroxyethyl)hexyl]-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]-triazin-4(3H)-one 100 mg (0.26 mmol) of 7-(1-acetylhexyl)-5-methyl-2-(4-methylbenzyl)-imidazo[5,1-f][1,2,4]triazin-4(3H)-one (Example 2) are reacted analogously to Example 8 with 15 mg (0.39 mmol) of sodium borohydride to give 7-[1-(1-hydroxy-ethyl)hexyl]-5-methyl-2-(4-methylbenzyl)-imidazo[5,1-f][1,2,4]triazin-4(3H)-one.", "Yield: 43 mg (42.8% of th.)", "Rf value (dichloromethane/methanol 10/1): 0.44 1H-NMR (400 MHz, methanol-d4, diastereomer mixture): δ=0.81-0.91 (m, 3H), 1.05 and 1.12 (each d, 3H), 1.18-1.36 (m, 6H), 1.80-2.08 (m, 2H), 2.31 (s, 3H), 2.62 and 2.67 (each s, 3H), 3.44-3.57 (m, 1H) 3.84 and 3.86 (each s, 2H), 3.97-4.16 (m, 1H), 7.16 (d, 2H), 7.25 (d, 2H).", "EXAMPLE 10 7-[1-(1-Hydroxyethyl)heptyl]-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f]-[1,2,4]triazin-4(3H)-one 100 mg (0.25 mmol) of 7-(1-acetylheptyl)-5-methyl-2-(4-methylbenzyl)-imidazo[5,1-f][1,2,4]triazin-4(3H)-one (Example 3) are reacted analogously to Example 8 with 10 mg (0.25 mmol) of sodium borohydride to give 7-[1-(1-hydroxy-ethyl)heptyl]-5-methyl-2-(4-methylbenzyl)-imidazo[5,1-f][1,2,4]triazin-4(3H)-one.", "Yield: 33 mg (32.8% of th.)", "Rf value (dichloromethane/methanol 10/1): 0.54 1H-NMR (400 MHz, methanol-d4, diastereomer mixture): δ=0.80-0.94 (m, 3H), 1.05 and 1.12 (each d, 3H), 1.18-1.38 (m, 8H), 1.78-2.09 (m, 2H), 2.31 (s, 3H), 2.62 and 2.67 (each s, 3H), 3.44-3.57 (m, 1H) 3.84 and 3.88 (each s, 2H), 3.97-4.15 (m, 1H), 7.16 (d, 2H), 7.25 (d, 2H).", "EXAMPLE 11 7-[1-(1-Hydroxyethyl)heptyl]-2-(4-methoxybenzyl)-5-methylimidazo[5,1-f]-[1,2,4]triazin-4(3H)-one 100 mg (0.24 mmol) of 7-(1-acetylheptyl)-2-(4-methoxybenzyl)-5-methyl-imidazo[5,1-f][1,2,4]triazin-4(3H)-one (Example 5) are reacted analogously to Example 8 with 17 mg (0.45 mmol) of sodium borohydride to give 7-[1-(1-hydroxy-ethyl)heptyl]-2-(4-methoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one.", "Yield: 14 mg (13.9% of th.)", "1H-NMR (400 MHz, methanol-d4, diastereomer mixture): δ=0.84 (t, 3H), 0.84-1.37 (m, 11H), 1.67-2.09 (m, 2H), 2.53 and 2.54 (each s, 3H), 3.32-3.41 (m, 1H) 3.76-3.78 (m, 5H), 3.98-4.13 (m, 1H), 6.88 (d, 2H), 7.26 (d, 2H).", "EXAMPLE 12 2-(3,4-Dimethoxybenzyl)-7-[1-(1-hydroxyethyl)-5-hexenyl]-5-methylimidazo[5,1-f]-[1,2,4]triazin-4(3H)-one 90 mg (0.20 mmol) of 7-(1-acetyl-5-hexenyl)-2-(3,4-dimethoxylbenzyl)-5-methyl-imidazo[5,1-f][1,2,4]triazin-4(3H)-one (Example 7) are reacted analogously to Example 8 with 8 mg (0.20 mmol) of sodium borohydride to give 2-(3,4-dimethoxy-benzyl)-7-[1-(1-hydroxyethyl)-5-hexenyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one.", "Yield: 43 mg (50.4% of th.)", "Rf value (dichloromethane/methanol 10/1): 0.40 1H-NMR (400 MHz, methanol-d4, diastereomer mixture): δ=0.92-1.25 (m, 5H, each d at 0.94 and 1.19), 1.70-2.12 (m, 4H), 2.54 (s, 3H), 3.34-3.42 (m, 1H), 3.76-3.84 (m, 8H), 3.98-4.13 (m, 1H) 4.85-4.94 (m, 2H), 5.64-5.76 (m, 1H), 6.87-6.98 (m, 3H)." ] ]	Patent_10468511
[ [ "Water level indicating device for a dishwasher", "A water level indicating device for a dishwasher provided with a cabinet, inside which is placed a washer tub (1), and presenting a control unit, which determines the operational conditions of providing or interrupting the water supply to the inside of the washer tub (1), said device comprising: a first chamber (10) in fluid communication with the washer tub (1); a second chamber (20) having a lower portion (21) maintained in fluid communication with a lower portion (11) of the first chamber (10); and a level sensor (2), which is operatively associated with both the second chamber (20) and the control unit, in order to inform the latter about a water level detected inside said second chamber (20) and corresponding to a desired filling level of the washer tub (1)." ], [ "1.A water level indicating device for a dishwasher provided with a cabinet, inside which is placed a washer tub (1), and presenting a control unit, which determines the operational conditions of providing or interrupting the water supply to the inside of the washer tub (1), characterized in that it comprises: a first chamber (10) in fluid communication with the washer tub (1); a second chamber (20) having a lower portion (21) maintained in fluid communication with a lower portion (11) of the first chamber (10); and a level sensor (2), which is operatively associated with both the second chamber (20) and the control unit, in order to inform said control unit about a water level detected inside said second chamber (20) and corresponding to a desired filling level of the washer tub (1).", "2.Device, according to claim 1, characterized in that it comprises a hollow body (40) defining, internally, first and second chambers (10, 20), said hollow body (40) presenting a first end (41a) opened to the washer tub (1) and providing fluid communication between the latter and the first chamber (10).", "3.Device, according to claim 2, characterized in that the hollow body (40) presents a vented second end (41b), whereto is mounted the level sensor (2), which is defined in the second chamber (20) spaced from the lower portion of the latter that is in fluid communication with the first chamber (10).", "4.Device, according to claim 1, characterized in that the level sensor (2) is displaceable inside the second chamber (20) between a first position and a second position, which are axially spaced from each other and respectively correspond to the conditions of presence of water in the second chamber (20), below and on a certain predetermined filling level of said second chamber (20).", "5.Device, according to claim 4, characterized in that the level sensor (2) is mounted to a first float (50), provided inside the second chamber (20).", "6.Device, according to claim 1, characterized in that the hollow body (40) is coupled to the washer tub (1) through its first end (41a).", "7.Device, according to claim 6, characterized in that the first and the second chambers (10, 20) are separated from each other by a divider (80) carried by the hollow body (40).", "8.Device, according to claim 7, characterized in that the divider (80) is a piece mounted inside the hollow body (40) defining inside the latter the first and the second chambers (10, 20).", "9.Device, according to claim 6, characterized in that it includes a safety sensor (70) provided in a third chamber (30) having a respective lower portion in fluid communication with the first chamber (10) through a lower opening in the latter, said safety sensor (70) being operatively associated with both the control unit and said third chamber (30), in order to detect the water level existing therein, informing this detected level to the control unit.", "10.Device, according to claim 9, characterized in that the safety sensor (70) is mounted to a second float (60) provided in said third chamber (30), said safety sensor (70) being actuated when the washing liquid inside said third chamber (30) reaches a level in the latter that is higher than that level actuating the level sensor (20).", "11.Device, according to claim 9, characterized in that the third chamber (30) is defined in the hollow body (40) by the divider (80) and that said third chamber (30) is positioned in said hollow body (40) in order to have a common wall with the first chamber (10).", "12.Device, according to claim 9, characterized in that the hollow body (40) is superiorly closed by a cover (43), through which is mounted at least one of the level sensor (2) and safety sensor (70).", "13.Device, according to claim 12, characterized in that the cover (43) carries a first and a second tubular projection (46, 47) for respectively mounting the first and the second floats (50, 60)." ], [ "<SOH> FIELD OF THE INVENTION <EOH>The present invention refers to a water level indicating device for a dishwasher, particularly to be applied in small and medium size dishwashers." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Thus, it is an object of the present invention to provide a water level indicating device for a dishwasher, which minimizes the contact of the level sensor with the impurities existing in the washing liquid, particularly grease, thus preventing the inadequate operation of the float that carries said level sensor and consequently of said level sensor and said dishwasher.", "A more specific object of the present invention is to provide a water level indicating device, such as that mentioned above which, in case of failure of its level sensor, prevents the dishwasher from working when the latter does not present effective operational conditions, for example, due to insufficient water therein.", "These and other objectives are achieved by a water level indicating device for a dishwasher having a cabinet, inside which is provided a washer tub, and presenting a control unit, which determines the operational conditions of providing or interrupting the water supply to the inside of the washer tub, said device comprising: a first chamber, in fluid communication with the washer tub; a second chamber, having a lower portion maintained in fluid communication with a lower portion of the first chamber; and a level sensor, which is operatively associated with both the second chamber and the control unit, in order to inform said control unit about a water level detected inside said second chamber and corresponding to a desired filling level of the washer tub." ], [ "FIELD OF THE INVENTION The present invention refers to a water level indicating device for a dishwasher, particularly to be applied in small and medium size dishwashers.", "PRIOR ART Dishwashers have a cabinet, which is usually parallelepipedic and closed by a vertical door, and inside which is mounted a washer tub designed to receive a determined quantity and type of dishes to be cleaned.", "For the washing operation, after closing the door and starting the washing operation, the washer tub is filled with water, up to a certain predetermined filling level, which, when achieved, is informed to a control unit that commands the interruption of water supply to the dishwasher.", "The small and medium size dishwashers present, mounted inside the cabinet and adjacent and external to the washer tub, a level sensor in fluid communication with said washer tub through a duct arranged in a vessel communicating system.", "In the known constructions, the level sensor is mounted to a float provided inside a level duct in fluid communication with the washer tub and which is disposed substantially vertical, parallel and adjacent to a rear wall of said washer tub, said level duct having a lower end opened to a lower portion of the washer tub, in order to receive the filling water for the latter, and a vented upper end, through which is mounted the float with the level sensor.", "In these constructions, the level sensor informs the control unit when a predetermined water level has been achieved inside the washer tub, allowing said control unit to start the washing operation.", "In such constructions, the level sensor may also, for example, inform the control unit about a minimum water level, indicating that either the washer tub has been drained, or, in a filling condition, that the minimum water level has not been achieved to allow the washing operation to start.", "In these dishwashers, the grease of the dishes contained in the washing water inside the washer tub also reaches the level duct where the level sensor is located, impregnating in these elements and gradually interfering with the adequate operation of the level sensor, or even causing operational defects in the float that carries said level sensor.", "SUMMARY OF THE INVENTION Thus, it is an object of the present invention to provide a water level indicating device for a dishwasher, which minimizes the contact of the level sensor with the impurities existing in the washing liquid, particularly grease, thus preventing the inadequate operation of the float that carries said level sensor and consequently of said level sensor and said dishwasher.", "A more specific object of the present invention is to provide a water level indicating device, such as that mentioned above which, in case of failure of its level sensor, prevents the dishwasher from working when the latter does not present effective operational conditions, for example, due to insufficient water therein.", "These and other objectives are achieved by a water level indicating device for a dishwasher having a cabinet, inside which is provided a washer tub, and presenting a control unit, which determines the operational conditions of providing or interrupting the water supply to the inside of the washer tub, said device comprising: a first chamber, in fluid communication with the washer tub; a second chamber, having a lower portion maintained in fluid communication with a lower portion of the first chamber; and a level sensor, which is operatively associated with both the second chamber and the control unit, in order to inform said control unit about a water level detected inside said second chamber and corresponding to a desired filling level of the washer tub.", "BRIEF DESCRIPTION OF THE DRAWINGS The following invention will be described below, with reference to the attached drawings, in which: FIG.", "1 illustrates a longitudinal sectional view of an embodiment of the water level indicating device for a dishwasher of the present invention, to be mounted inside the cabinet of the dishwasher, externally to the washer tub thereof and carrying two floats therewithin; FIG.", "2 is an upper view of the embodiment for the water level indicating device illustrated in FIG.", "1, without the cover upper portion of said device; FIG.", "3 is a perspective view of the device of FIG.", "2; FIG.", "4 is a perspective view of a chamber divider of the device of the present invention; FIG.", "5 is a perspective view of the cover of the present device; and FIGS.", "6 and 7 illustrate a perspective view of each float with the respective level sensor of the device of the present invention.", "DESCRIPTION OF THE ILLUSTRATED EMBODIMENT The present invention will be described in relation to a small or medium size dishwasher of the type comprising, inside a non-illustrated cabinet, a washer tub 1, which receives the dishes to be cleaned, and external and adjacent to said washer tub, and inside the cabinet, the water level device of the present invention.", "The dishwasher further comprises a control unit, not illustrated, which determines the operational conditions for providing and interrupting the water supply to the inside of the washer tub 1, based on the information received from a level sensor 2, provided inside the present device, as described below and which indicates when the filling water of the dishwasher achieves a determined level in said water level indicating device corresponding to a certain water level within the washer tub 1.Upon receiving this information from the level sensor 2, the control unit commands the interruption of water supply to the washer tub 1 and the start of the washing operation.", "In a possible option, the level sensor 2 further informs the control unit when the water inside the device of the present invention achieves a minimum water level, indicating the washer tub 1 has been drained and that it may thus be refilled.", "According to the present invention, the water level indicating device of the present invention comprises a first chamber 10 in fluid communication with the washer tub 1, and a second chamber 20, having a lower portion 21 maintained in fluid communication with a lower portion 11 of the first chamber 10 and to which are operatively associated the level sensor 2 and the control unit, so that the level sensor 2 detects and informs said control unit about at least one water level, which is previously defined and achieved by the water being supplied to the inside of said second chamber 20 corresponding to a respective and desired filling level of the washer tub 1.The level sensor 2 is displaceable inside the second chamber 20 between a first position and a second position, which are axially spaced form each other and respectively correspond to the conditions of presence of water inside the second chamber 20, below and on a certain predetermined filling level of said second chamber 20.In the illustrated constructive option, the water level indicating device comprises a hollow body 40, inside which are defined at least two, for example three chambers 10, 20, 30, in fluid communication with each other and with the washer tub 1, as described below, said hollow body 40 presenting a first end 41a, opened to the washer tub 1 and providing fluid communication with the latter and with the first chamber 10, and a second end 41b, vented to the atmosphere and whereto is mounted the level sensor 2.In the illustrated constructive option, the hollow body 40 is coupled to the washer tub 1 through its first end 41a and receives, through its second end 41b, a first float 50, which is displaceable inside the second chamber 20 and carries the level sensor 2, and, through a third end 41c, a second float 60 actuating inside the third chamber 30, and a safety sensor 70 associated with said second float 60.The safety sensor 70 is provided inside the third chamber 30 in a vertical higher position than that of the level sensor 2 in its position corresponding to a maximum filling condition of the washer tub 1, so as to inform the control unit that a maximum filling limit of the washer tub 1 has been achieved.", "Each float is made of plastic material, for example.", "The first float 50 carries a magnetic element, such as a magnet, which allows its movement in the respective chamber.", "In the illustrated construction, the hollow body 40 is closed by a cover 43 carrying both the level sensor 2 and the safety sensor 70 and which is affixed to the hollow body 40, closing the latter by fitting each of a plurality of ears 44 provided in a lower skirt portion of said cover 43, into a corresponding tooth 45 provided in an upper portion of the hollow body 40.In this construction, the cover 43 presents, from an external face thereof, first and second vented tubular projections 46, 47, said first tubular projection 46 lodging the first float 50 and the level sensor 2 and said second tubular projection 47 lodging the safety sensor 70 and the second float 60.The first float 50 presents a buoy portion 51 and a rod portion 52 projecting from said buoy portion 51 and provided with grooves 53, for example orthogonal to each other, a free end of said rod portion 52 presenting a magnet 54 and said second float 60 comprising a respective buoy 61 and an arm 62 projecting from an upper end edge of the respective buoy 61 and presenting, for example, a curved shape, particularly spacing away from said buoy 61, said arm 62 being articulated by an end 63 to a portion of the second tubular projection 47.The first tubular projection 46 presents, internally along its length, at least one pair of longitudinal guides 48, which are for example, continuous and opposite to each other, and which will receive respective grooves 53 of the rod portion of the first float 50, in order to avoid that, when water is supplied to and drained from said chamber 20, said first float 50 presents other movements than the vertical displacement to and from said first tubular projection 46.The safety sensor 70 is mounted to the vented portion of the second tubular projection 47 of the cover 43 and is actuated by a switch, whose operation is defined as a function of the movement of the second float 60 inside the third chamber 30.According to the illustrated constructive option, the hollow body 40 of the water level indicating device 10 of the present invention carries a divider 80, for example in a single piece mounted inside said hollow body 40, in order to define therein the first, the second and the third chambers 10, 20 and 30, said first chamber 10 being positioned between the other chambers and having its lower portion opened to the first end 41a of the hollow body 40.As a function of the construction of the divider 80, the latter defines, for each chamber of the hollow body 40, a respective lower opening on the same level inside the hollow body 40.The hollow body 40 presents, internally, in a lower portion of each lateral wall, internal projections 40a, which define guides for the introduction and fixation of the divider 80.In the illustrated construction, each lateral wall includes three internal projections 40a, one of each being vertically longer and determining the support for the portion of the divider 80 that defines the third chamber 30.The hollow body 40 presents a bottom wall with a “V” profile, in order to allow water to be drained when said hollow body 40 is emptied.", "In the vertex region of said “V” profile is provided a lateral nozzle 49 located in the hollow body 40 and which is opened to the first end 41a, for example being tooted and receiving a sealing ring, not illustrated, for mounting the present device to the washer tub 1.The divider 80 presents a flat central structure, from which upper contour project, to each side of said structure, flanges 81 provided with holes 82, each hole for receiving a fitting end of an internal projection 40a provided in an internal lateral wall of the hollow body 40.In a way of carrying out the present invention, the level sensor 2 is a magnetic sensor and the safety sensor 70 is an electrical switch, said sensors being provided in order to, respectively, detect a minimum water level and a maximum water level in the dishwasher, and each sensor being mounted on a respective printed circuit board provided in the cover 43 of the hollow body 40 of the present device.", "The safety sensor 70 operates independently of the level sensor 2, preventing the water from flowing out of the dishwasher in case the level sensor 2 fails to detect the water level.", "After receiving the information from said safety sensor 70 that the water level has reached a predetermined maximum value and, therefore, indicating the operation of the level sensor 2 has failed, the control unit commands the deenergization of a water inlet valve and the energization of a drainage pump, for promoting the partial or total emptying of the washer tub 1.In the illustrated construction, the water supply and water discharge in the hollow body 40 is made through the same opening, represented by the first end 41a of said hollow body 40.The device of the present invention minimizes reading errors of the water levels and reduces risks of water overflowing from said washer tub 1." ] ]	Patent_10468521
[ [ "Pyrazole derivative, intermediate therefor, processes for producing these, and herbicide containing these as active ingredient", "The present invention provides a pyrazole derivative of the general formula (1), which has an excellent efficacy as an active component for a herbicide, an intermediate for the production thereof, processes for the production thereof, and a herbicide containing the derivative as an active ingredient." ], [ "1.A pyrazole derivative of the general formula (1), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R2 is a hydrogen atom, a halogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, R3 is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms, an optionally substituted phenyl group, an optionally substituted alkyloxy group having 1 to 6 carbon atoms, an optionally substituted cycloalkyloxy group having 3 to 8 carbon atoms, an optionally substituted aralkyloxy group having 7 to 11 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms or an optionally substituted phenyloxy group, R4 is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms or an optionally substituted phenyl group, or, R3 and R4 may form a heterocyclic ring together with a nitrogen atom to which they bond, R5 is an optionally substituted phenyl group or an optionally substituted pyridyl group, and Y is an oxygen atom or a sulfur atom.", "2.A pyrazole derivative of the general formula (2), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R2 is a hydrogen atom, a halogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, and R5 is an optionally substituted phenyl group or an optionally substituted pyridyl group.", "3.A process for the preparation of a pyrazole derivative of claim 2 which comprises reacting a 3-hydroxypyrazole derivative of the general formula (3), and a compound of the general formula (4), R5-Z (4) wherein Z is a leaving group, in the presence of a base.", "4.The process of claim 3, wherein R5 in the general formula (4) is a phenyl group or a pyridyl group substituted with an electron-withdrawing group such as a halogen atom, a haloalkyl group, a cyano group or a nitro group.", "5.A process for the preparation of a pyrazole derivative of the general formula (2b), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, and R5a is a hydrogen atom, an optionally substituted phenyl group or an optionally substituted pyridyl group and R2a is a halogen atom, which comprises halogenating a pyrazole derivative of the general formula (2a), 6.A process for the preparation of a pyrazole derivative of the general formula (1a) in the present invention, wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R2 is a hydrogen atom, a halogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, and R5a is a hydrogen atom, an optionally substituted phenyl group or an optionally substituted pyridyl group and R2a is a halogen atom, Y is an oxygen atom or a sulfur atom, and R3a is an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms, an optionally substituted phenyl group, an optionally substituted alkyloxy group having 1 to 6 carbon atoms, an optionally substituted cycloalkyloxy group having 3 to 8 carbon atoms, an optionally substituted aralkyloxy group having 7 to 11 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms or an optionally substituted phenyloxy group, which comprises reacting a pyrazole derivative of the general formula (2c), and isocyanate or isothiocyanates of the general formula (5), R3a—NCY (5) optionally in the presence of a base.", "7.A process for the preparation of a pyrazole derivative of the general formula (1b), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R2 is a hydrogen atom, a halogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, and R3a is an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms, an optionally substituted phenyl group, an optionally substituted alkyloxy group having 1 to 6 carbon atoms, an optionally substituted cycloalkyloxy group having 3 to 8 carbon atoms, an optionally substituted aralkyloxy group having 7 to 11 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms or an optionally substituted phenyloxy group, and R5 is an optionally substituted phenyl group or an optionally substituted pyridyl group, and which comprises reacting a 3-hydroxypyrazole derivative of the general formula (3a), and a compound of the general formula (4), R5-Z (4) wherein Z is a leaving group, in the presence of a base.", "8.A process for the preparation of a pyrazole derivative of the general formula (1c), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R2 is a hydrogen atom, a halogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, and R3a is an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms, an optionally substituted phenyl group, an optionally substituted alkyloxy group having 1 to 6 carbon atoms, an optionally substituted cycloalkyloxy group having 3 to 8 carbon atoms, an optionally substituted aralkyloxy group having 7 to 11 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms or an optionally substituted phenyloxy group, and R5 is an optionally substituted phenyl group or an optionally substituted pyridyl group, and Y is an oxygen atom or a sulfur atom, R4a is an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms or an optionally substituted alkynyl group having 3 to 6 carbon atoms, which comprises reacting the thus-obtained pyrazole derivative of the general formula (1b), and a compound of the general formula (6), R4a-Z (6) wherein Z is a leaving group, in the presence of a base.", "9.A process for the preparation of a pyrazole-1-carboxamide derivative of the general formula (1d), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R2 is a hydrogen atom, a halogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, and R5 is an optionally substituted phenyl group or an optionally substituted pyridyl group, and each of R3b and R4b is an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms or an optionally substituted phenyl group, or R3b and R4b may form a heterocyclic ring together with a nitrogen atom to which they bond, which comprises reacting a pyrazole derivative of the general formula (2), and carbamic acid chlorides of the general formula (7), in the presence of a base.", "10.A pyrazole derivative of the general formula (2d), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R2 is a hydrogen atom, a halogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, and R5 is an optionally substituted phenyl group or an optionally substituted pyridyl group.", "11.A process for the preparation of a pyrazole derivative of claim 10, which comprises reacting a pyrazole derivative of the general formula (2), with phosgene or a material equivalent to phosgene.", "12.A process for the preparation of a pyrazole-1-carboxamide derivative of the general formula (1e), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R2 is a hydrogen atom, a halogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, R4 is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms or an optionally substituted phenyl group, or, R3 and R4 may form a heterocyclic ring together with a nitrogen atom to which they bond, R5 is an optionally substituted phenyl group or an optionally substituted pyridyl group, R3c is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms, an optionally substituted phenyl group, an optionally substituted alkyloxy group having 1 to 6 carbon atoms, an optionally substituted cycloalkyloxy group having 3 to 8 carbon atoms, an optionally substituted aralkyloxy group having 7 to 11 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms or an optionally substituted phenyloxy group, or R3c and R4 may form a heterocyclic ring together with a nitrogen atom to which they bond, which comprises reacting a pyrazole derivative of the general formula (2d) as an intermediate in production, with amines of the general formula (8) optionally in the presence of a base.", "13.A process for the preparation of a pyrazole derivative of the general formula (1g), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R3 is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms, an optionally substituted phenyl group, an optionally substituted alkyloxy group having 1 to 6 carbon atoms, an optionally substituted cycloalkyloxy group having 3 to 8 carbon atoms, an optionally substituted aralkyloxy group having 7 to 11 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms or an optionally substituted phenyloxy group, R4 is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms or an optionally substituted phenyl group, or, R3 and R4 may form a heterocyclic ring together with a nitrogen atom to which they bond, R5 is an optionally substituted phenyl group or an optionally substituted pyridyl group, Y is an oxygen atom or a sulfur atom, and and R2b is a halogen atom, which comprises halogenating a pyrazole derivative of the general formula (1f), 14.A process for the preparation of a pyrazole-1-carboxamide derivative of the general formula (1i), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R2 is a hydrogen atom, a halogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, R3 is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms, an optionally substituted phenyl group, an optionally substituted alkyloxy group having 1 to 6 carbon atoms, an optionally substituted cycloalkyloxy group having 3 to 8 carbon atoms, an optionally substituted aralkyloxy group having 7 to 11 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms or an optionally substituted phenyloxy group, R4 is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms or an optionally substituted phenyl group, or, R3 and R4 may form a heterocyclic ring together with a nitrogen atom to which they bond, and R5 is an optionally substituted phenyl group or an optionally substituted pyridyl group, which comprises oxidizing a pyrazole-1-carbothioamide derivative of the general formula (1h), 15.A herbicide containing, as an active ingredient, a pyrazole derivative of claim 1" ], [ "<SOH> TECHNICAL BACKGROUND <EOH>Conventionally, there are known a number of pyrazole derivatives having pesticidal activity such as herbicidal activity or insecticidal activity.", "However, nothing has been reported on any pyrazole derivative having a substituted oxy group at the 3-position of a pyrazole ring and a substituted carbamoyl group or a substituted thiocarbamoyl group on a nitrogen atom at the 1-position as shown in the following general formula (1) in the present invention, nor is there any report on biological activities thereof.", "As pyrazole derivatives structurally similar to the pyrazole derivative (1) of the present invention, there are known pyrazole derivatives described in WO97/24332 (EP876351, JP2000/502686, U.S. Pat.", "No.", "6,075,149) and EP256667 (JP63/044571, U.S. Pat.", "No.", "5,374,644).", "However, these pyrazole derivatives are completely different from the pyrazole derivative (1) of the present invention in that they have an alkyl group on a nitrogen atom of their pyrazole ring.", "Further, it is described that the pyrazole derivatives described in the above WO97/24332 and EP256667 have pesticidal activity against fungi and harmful insects, but these Publications describe nothing concerning any other biological activity." ], [ "TECHNICAL FIELD The present invention relates to a novel pyrazole derivative, an intermediate thereof, processes for the preparation thereof and a herbicide containing any one of them as an active ingredient.", "TECHNICAL BACKGROUND Conventionally, there are known a number of pyrazole derivatives having pesticidal activity such as herbicidal activity or insecticidal activity.", "However, nothing has been reported on any pyrazole derivative having a substituted oxy group at the 3-position of a pyrazole ring and a substituted carbamoyl group or a substituted thiocarbamoyl group on a nitrogen atom at the 1-position as shown in the following general formula (1) in the present invention, nor is there any report on biological activities thereof.", "As pyrazole derivatives structurally similar to the pyrazole derivative (1) of the present invention, there are known pyrazole derivatives described in WO97/24332 (EP876351, JP2000/502686, U.S. Pat.", "No.", "6,075,149) and EP256667 (JP63/044571, U.S. Pat.", "No.", "5,374,644).", "However, these pyrazole derivatives are completely different from the pyrazole derivative (1) of the present invention in that they have an alkyl group on a nitrogen atom of their pyrazole ring.", "Further, it is described that the pyrazole derivatives described in the above WO97/24332 and EP256667 have pesticidal activity against fungi and harmful insects, but these Publications describe nothing concerning any other biological activity.", "DISCLOSURE OF THE INVENTION The present invention provides a novel pyrazole derivative having excellent herbicidal activity and high selectivity to crops, an intermediate thereof and processes for the preparation thereof, and further provides a herbicide containing the derivative as an active ingredient.", "The present inventors have made diligent studies for herbicides having excellent herbicidal activity and crop selectivity.", "As a result, it has been found that the pyrazole derivative of the following general formula (1) in the present invention exhibits excellent herbicidal activity at a low dosage without causing phytotoxicity, and a simple process for the preparation thereof has been also found.", "The present invention has been accordingly completed.", "That is, the present invention is directed to a pyrazole derivative of the general formula (1), wherein R1 is a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, R2 is a hydrogen atom, a halogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, R3 is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms, an optionally substituted phenyl group, an optionally substituted alkyloxy group having 1 to 6 carbon atoms, an optionally substituted cycloalkyloxy group having 3 to 8 carbon atoms, an optionally substituted aralkyloxy group having 7 to 11 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms or an optionally substituted phenyloxy group, R4 is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms or an optionally substituted phenyl group, or, R3 and R4 may form a heterocyclic ring together with a nitrogen atom to which they bond, R5 is an optionally substituted phenyl group or an optionally substituted pyridyl group, and Y is an oxygen atom or a sulfur atom.", "Further, the present invention is also directed to a pyrazole derivative of the general formula (2), wherein R1, R2 and R5 are as defined above.", "Further, the present invention is directed to a process for the preparation of a pyrazole derivative of the general formula (2), wherein R1, R2 and R5 are as defined above, which comprises reacting a 3-hydroxypyrazole derivative of the general formula (3), wherein R1 and R2 are as defined above, and a compound of the general formula (4), R5-Z (4) wherein R5 is as defined above and Z is a leaving group, in the presence of a base.", "Further, the present invention is directed to a process for the preparation of a pyrazole derivative of the general formula (2b), wherein R1 is as defined above, R5a is a hydrogen atom, an optionally substituted phenyl group or an optionally substituted pyridyl group and R2a is a halogen atom, which comprises halogenating a pyrazole derivative of the general formula (2a), wherein R1 and R5a are as defined above.", "Further, the present invention is directed to a process for the preparation of a pyrazole derivative of the general formula (1a) in the present invention, wherein R1, R2, R5a and Y are as defined above, and R3a is an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms, an optionally substituted phenyl group, an optionally substituted alkyloxy group having 1 to 6 carbon atoms, an optionally substituted cycloalkyloxy group having 3 to 8 carbon atoms, an optionally substituted aralkyloxy group having 7 to 11 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms or an optionally substituted phenyloxy group, which comprises reacting a pyrazole derivative of the general formula (2c), wherein R1, R2 and R5a are as defined above, and isocyanates or isothiocyanates of the general formula (5), R3a—NCY (5) wherein R3a and Y are as defined above, optionally in the presence of a base.", "Further, the present invention is directed to a process for the preparation of a pyrazole derivative of the general formula (1b), wherein R1, R2, R3a and R5 are as defined above, which comprises reacting a 3-hydroxypyrazole derivative of the general formula (3a), wherein R1, R2 and R3a are as defined above, and a compound of the general formula (4), R5-Z (4) wherein R5 is as defined above and Z is a leaving group, in the presence of a base.", "Further, the present invention is directed to a process for the preparation of a pyrazole derivative of the general formula (1c), wherein R1, R2, R3a, R5 and Y are as defined above, and R4a is an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms or an optionally substituted alkynyl group having 3 to 6 carbon atoms, which comprises reacting the thus-obtained pyrazole derivative of the general formula (1b), wherein R1, R2, R3a, R5 and Y are as defined above, and a compound of the general formula (6), R4a-Z (6) wherein R4a is as defined above and Z is a leaving group.", "Further, the present invention is directed to a process for the preparation of a pyrazole-1-carboxamide derivative of the general formula (1d) in the present invention, wherein R1, R2 and R5 are as defined above and each of R3b and R4b is an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms or an optionally substituted phenyl group, or R3b and R4b may form a heterocyclic ring together with a nitrogen atom to which they bond, which comprises reacting a pyrazole derivative of the general formula (2), wherein R1, R2 and R5 are as defined above, and carbamic acid chlorides of the general formula (7), wherein R3b and R4b are as defined above, in the presence of a base.", "Further, the present invention is directed to a pyrazole derivative of the general formula (2d), wherein R1, R2 and R5 are as defined above, which is an intermediate in the production of a pyrazole-1-carboxamide derivative in the present invention, and, said pyrazole derivative can be produced by reacting a pyrazole derivative of the general formula (2), wherein R1, R2 and R5 are as defined above, with phosgene or a material equivalent to phosgene.", "Further, the present invention is directed to a process for the preparation of a pyrazole-1-carboxamide derivative of the general formula (1e) in the present invention, wherein R1, R2, R4 and R5 are as defined above and R3c is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 3 to 8 carbon atoms, an optionally substituted aralkyl group having 7 to 11 carbon atoms, an optionally substituted alkenyl group having 3 to 6 carbon atoms, an optionally substituted alkynyl group having 3 to 6 carbon atoms, an optionally substituted phenyl group, an optionally substituted alkyloxy group having 1 to 6 carbon atoms, an optionally substituted cycloalkyloxy group having 3 to 8 carbon atoms, an optionally substituted aralkyloxy group having 7 to 11 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms or an optionally substituted phenyloxy group, or R3c and R4 may form a heterocyclic ring together with a nitrogen atom to which they bond, which comprises reacting a pyrazole derivative of the general formula (2d) as an intermediate in production, wherein R1, R2 and R5 are as defined above, with amines of the general formula (8).", "wherein R3c and R4 are as defined above, optionally in the presence of a base.", "Further, the present invention is directed to a process for the preparation of a pyrazole derivative of the general formula (1g), wherein R1, R3, R4, R5 and Y are as defined above and R2b is a halogen atom, which comprises halogenating a pyrazole derivative of the general formula (1f), wherein R1, R3, R4, R5 and Y are as defined above.", "Further, the present invention is directed to a process for the preparation of a pyrazole-1-carboxamide derivative of the general formula (1i), wherein R1, R2, R3, R4 and R5 are as defined above, which comprises oxidizing a pyrazole-1-carbothioamide derivative of the general formula (1h), wherein R1, R2, R3, R4 and R5 are as defined above.", "Further, the present invention is directed to a herbicide containing, as an active ingredient, a pyrazole derivative of the general formula (1), wherein R1, R2, R3, R4, R5 and Y are as defined above.", "Preferred Embodiments of the Invention In compounds of the present invention, definitions of substituents represented by R1 to R5, Y and Z are as follows.", "The alkyl group having 1 to 6 carbon atoms, represented by R1, R2 and R2a, may be linear or branched, and examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, tert-butyl, pentyl, isoamyl, neopentyl, 1-ethylpropyl, 1-methylbutyl, 2-methylbutyl, hexyl, isohexyl, 2-ethylbutyl and 4-methylpentyl.", "The above alkyl group may have at least one substituent such as a halogen atom, an alkyloxy group having 1 to 6 carbon atoms, a formyl group, an alkyloxycarbonyl group having 1 to 6 carbon atoms or a heterocyclic ring.", "More specifically, examples of such alkyl groups include trichloromethyl, trifluoromethyl, methoxymethyl, ethoxymethyl, 2-methoxyethyl, formylmethyl, methoxycarbonylmethyl, ethoxycarbonylmethyl, furfuryl, tetrahydrofurfuryl, 2-picolyl, 3-picolyl, 4-picolyl, 2-thienylmethyl and 2-morpholinoethyl.", "The alkyl group having 1 to 12 carbon atoms, represented by R3, R3a, R3b, R3c, R4, R4a and R4b, may be linear or branched, and examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, tert-butyl, pentyl, isoamyl, neopentyl, 1-ethylpropyl, 1-methylbutyl, 2-methylbutyl, hexyl, isohexyl, 2-ethylbutyl, 4-methylpentyl, heptyl, 1-methylhexyl, octyl, decyl, undecyl and dodecyl.", "The above alkyl group may have at least one substituent such as a halogen atom, a cycloalkyl group having 3 to 8 carbon atoms, a cyano group, a nitro group, an alkylthio group having 1 to 6 carbon atoms, an alkyloxy group having 1 to 6 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms, a carboxy group or an acyl group.", "More specifically, examples of such alkyl groups include 2-chloroethyl, 2-bromoethyl, 3-chloropropyl, 3-fluoropropyl, cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyanomethyl, 2-cyanoethyl, 3-cyanopropyl, nitromethyl, 2-methylthioethyl, methoxymethyl, ethoxymethyl, 2-methoxyethyl, 2-chloroethoxymethyl, methoxycarbonylmethyl, ethoxycarbonylmethyl, 1-methoxycarbonylethyl, 1-ethoxycarbonylethyl, 2-ethoxycarbonylethyl, carboxymethyl, acetonyl, 1-acetylethyl, 3-acetylpropyl, phenacyl, 4-chlorophenacyl, 2,4-difluorophenacyl, 4-methylphenacyl, 4-isopropylphenacyl, 4-isobutylphenacyl, 4-cyclohexylphenacyl, 4-cyanophenacyl and 4-nitrophenacyl.", "Examples of the cycloalkyl group having 3 to 8 carbon atoms, represented by R1, R3, R3a, R3b, R3c, R4, R4a and R4b, include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cyclooctyl.", "The above cycloalkyl group may have a substituent such as a halogen atom, an alkyl group having 1 to 4 carbon atoms, an alkyloxycarboyl group having 1 to 4 carbon atoms or a cyano group.", "More specifically, examples of such cycloalkyl groups include 1-methylcyclopropyl, 2,2-dimethylcyclopropyl, 2-chlorocyclopropyl, 2,2-dichlorocyclopropyl, 2-methoxycarbonylcyclopropyl, 2-cyanocyclopropyl, 2-methylcyclopentyl and 3-methylcyclopentyl.", "Examples of the aralkyl group having 7 to 11 carbon atoms, represented by R3, R3a, R3b, R3c, R4, R4a and R4b, include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, 1-naphthylmethyl and 2-naphthylmethyl.", "The above aralkyl group may have, on its aromatic ring, at least one substituent such as a halogen atom, an alkyl group having 1 to 12 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, an alkyloxy group having 1 to 6 carbon atoms, a haloalkyloxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, an alkylsulfonyl group having 1 to 6 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms, an carboxy group, an optionally substituted carbamoyl group, a cyano group or a nitro group.", "More specifically, examples of such aralkyl groups include benzyl, 2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 2-bromobenzyl, 3-bromobenzyl, 4-bromobenzyl, 3,5-difluorobenzyl, 3,5-dichlorobenzyl, 3,5-dibromobenzyl, 2-methylbenzyl, 3-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,5-dimethylbenzyl, 2-trifluoromethylbenzyl, 3-trifluoromethylbenzyl, 4-trifluoromethylbenzyl, 3,5-bis(trifluoromethyl)benzyl, 2,4-bis(trifluoromethyl)benzyl, 2-methoxycarbonylbenzyl, 3-methoxycarbonylbenzyl, 4-methoxycarbonylbenzyl, 3-carboxybenzyl, 4-carboxybenzyl, 3-(N,N-dimethylcarbamoyl)benzyl, 4-(N,N-dimethylcarbamoyl)benzyl, 3-(N,N-diethylcarbamoyl)benzyl, 3-(N-ethyl-N-propylcarbamoyl)benzyl, 3-cyanobenzyl, 4-cyanobenzyl, 2-methoxybenzyl, 3-methoxybenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 4-trifluoromethoxybenzyl, 4-phenoxybenzyl, 4-methylthiobenzyl, 4-methylsulfonylbenzyl, 2-nitrobenzyl, 3-nitrobenzyl, 4-nitrobenzyl, 1-(2-fluorophenyl)ethyl, 1-(2-chlorophenyl)ethyl, 1-(2-bromophenyl)ethyl, 1-(3-fluorophenyl)ethyl, 1-(3-chlorophenyl)ethyl, 1-(3-bromophenyl)ethyl, 1-(4-fluorophenyl)ethyl, 1-(4-chlorophenyl)ethyl, 1-(4-bromophenyl)ethyl, 1-(2-trifluoromethylphenyl)ethyl, 1-(3-trifluoromethylphenyl)ethyl, 1-(4-trifluoromethylphenyl)ethyl, 2-(3-bromophenyl)ethyl, 2-(3-trifluoromethylphenyl)ethyl, 3-phenylpropyl and 4-phenylbutyl.", "Examples of the alkenyl group having 3 to 6 carbon atoms, represented by R3, include allyl, 2-methyl-2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 2-hexenyl and 3-hexenyl.", "The alkenyl group may be substituted with a halogen atom or the like.", "Examples of the substituted alkenyl group include 2-chloro-2-propenyl, 3-chloropropenyl and 4-chloro-2-butenyl.", "Examples of the alkenyl group having 3 to 6 carbon atoms, represented by R3a, R3b, R3c, R4, R4a and R4b, include allyl, 2-methyl-2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 2-hexenyl and 3-hexenyl.", "The alkenyl group may be substituted with a halogen atom or the like.", "Examples of the substituted alkenyl group include 2-chloro-2-propenyl, 3-chloropropenyl and 4-chloro-2-butenyl.", "The alkynyl group having 3 to 6 carbon atoms, represented by R3, R3a, R3b, R3c, R4, R4a and R4b, may be linear or branched, and examples thereof include propargyl, 1-butyn-3-yl, 3-methyl-1-butyn-3-yl, 2-butynyl, 2-pentynyl and 3-pentynyl.", "The alkynyl group may be substituted with a halogen atom, or the like.", "Examples of the substituted alkynyl group include 3-fluoro-2-propynyl, 3-chloro-2-propynyl, 3-bromo-2-propynyl, 4-bromo-2-butynyl and 4-bromo-3-butynyl.", "The optionally substituted phenyl group represented by R1, R3, R3a, R3b, R3c, R4 and R4b is, for example, a phenyl group having, as a substituent on a benzene ring, a halogen atom, an alkyl group having 1 to 12 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 12 carbon atoms and being substituted with an alkyloxyimino group having 1 to 4 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms, a carboxy group, a cyano group, an alkyloxy group having 1 to 6 carbon atoms, an aryloxy group, a haloalkyloxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, an alkylsulfinyl group having 1 to 6 carbon atoms, an alkylsulfonyl group having 1 to 6 carbon atoms, a haloalkylthio group having 1 to 6 carbon atoms, a haloalkylsulfinyl group having 1 to 6 carbon atoms, a haloalkylsulfonyl group having 1 to 6 carbon atoms or a nitro group.", "More specifically, examples of such optionally substituted phenyl group include 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-fluorophenyl, 3-chlorophenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 2,4-difluorophenyl, 2,4-dichlorophenyl, 3,5-difluorophenyl, 3,5-dichlorophenyl, 3-chloro-2,4-difluorophenyl, 2,4,5-trichlorophenyl, 2,4-dichloro-3-methylphenyl, 2,4-dichloro-5-methoxyphenyl, 2,4-dichloro-5-isopropyloxyphenyl, 2-fluoro-4-chloro-5-methoxyphenyl, 2-fluoro-4-chloro-5-isopropyloxyphenyl, 2-fluoro-4-chloro-5-cyclopentyloxyphenyl, 2-fluoro-4-chloro-5-propargyloxyphenyl, 2-fluoro-4-chloro-5-(1-butyn-3-yloxy)phenyl, 2-fluoro-4-trifluoromethylphenyl, 2-chloro-4-trifluoromethylphenyl, 2-chloro-5-trifluoromethylphenyl, 4-fluoro-3-phenoxyphenyl, 2-fluoro-5-nitrophenyl, 2,4-difluoro-5-nitrophenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2,4-dimethylphenyl, 4-ethylphenyl, 4-isopropylphenyl, 4-tert-butylphenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2,4-bis(trifluoromethyl)phenyl, 3,5-bis(trifluoromethyl)phenyl, 2-acetylphenyl, 4-acetylphenyl, 4-isovalerylphenyl, 2-methoxycarbonylphenyl, 2-ethoxycarbonylphenyl, 3-methoxycarbonylphenyl, 4-methoxycarbonylphenyl, 2-carboxyphenyl, 4-carboxyphenyl, 2-cyanophenyl, 4-cyanophenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3,4-dimethoxyphenyl, 4-isopropyloxyphenyl, 4-tert-butyloxyphenyl, 3-trifluoromethyloxyphenyl, 4-trifluoromethyloxyphenyl, 3-phenoxyphenyl, 4-phenoxyphenyl, 2-methylthiophenyl, 4-methylthiophenyl, 2-methylsulfinylphenyl, 4-methylsulfinylphenyl, 2-methylsulfonylphenyl, 4-methylsulfonylphenyl, 4-trifluoromethylthiophenyl, 4-trifluoromethylsulfinylphenyl, 4-trifluoromethylsulfonylphenyl, 2-nitrophenyl and 4-nitrophenyl.", "The optionally substituted phenyl group represented by R5 and R5a is, for example, a phenyl group having, as a substituent on a benzene ring, a halogen atom, an alkyl group having 1 to 12 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 12 carbon atoms and being substituted with an alkyloxyimino group having 1 to 4 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms, a carboxy group, a cyano group, a substituted amino group, an alkyloxy group having 1 to 6 carbon atoms, an aryloxy group, a haloalkyloxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, an alkylsulfinyl group having 1 to 6 carbon atoms, an alkylsulfonyl group having 1 to 6 carbon atoms, a haloalkylthio group having 1 to 6 carbon atoms, a haloalkylsulfinyl group having 1 to 6 carbon atoms, a haloalkylsulfonyl group having 1 to 6 carbon atoms or a nitro group.", "As a substituent on a benzene ring, electron-withdrawing groups such as a trifluoromethyl group, a nitro group, a cyano group, a chlorine atom, a fluorine atom and an alkyloxycarbonyl group are preferred, since a good reaction yield is attained and since raw materials can be easily obtained.", "Further, such electron-withdrawing groups are preferably substituted at an o-position and/or p-position.", "More specifically, examples of the above substituted phenyl include 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 2,4-difluorophenyl, 2,4-dichlorophenyl, 2,4,5-trichlorophenyl, 2-chloro-5-trifluoromethylphenyl, 2-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2,4-bis(trifluoromethyl)phenyl, 2,6-dichloro-4-trifluoromethylphenyl, 4-cyanophenyl, 4-cyano-2-trifluoromethylphenyl, 2-methylthiophenyl, 4-methylthiophenyl, 2-methylsulfinylphenyl, 4-methylsulfinylphenyl, 2-methylsulfonylphenyl, 4-methylsulfonylphenyl, 4-trifluoromethylthiophenyl, 4-trifluoromethylsulfinylphenyl, 4-trifluoromethylsulfonylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2-nitro-4-trifluoromethylphenyl, 4-nitro-2-trifluoromethylphenyl, 4-nitro-3-trifluoromethylphenyl, 2,6-dichloro-4-trifluoromethylphenyl, 2-chloro-6-fluoro-4-trifluoromethylphenyl, 2-chloro-6-nitro-4-trifluoromethylphenyl and 2,4-dinitro-6-trifluoromethylphenyl.", "Concerning these substituents on a benzene ring, for example, a nitro group is convertible to an amino group by reduction, and the amino group is further convertible to a halogen atom or a substituted alkyl group through a diazonium salt, and these can be included in the substituents on a benzene ring.", "The optionally substituted pyridyl group represented by R5 and R5a is, for example, a pyridyl group having, as a substituent on a pyridine ring, a halogen atom, an alkyl group having 1 to 12 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 12 carbon atoms and being substituted with an alkyloxyimino group having 1 to 4 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms, a carboxy group, a cyano group, an alkyloxy group having 1 to 6 carbon atoms, an aryloxy group, a haloalkyloxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, an alkylsulfinyl group having 1 to 6 carbon atoms, an alkylsulfonyl group having 1 to 6 carbon atoms, a haloalkylthio group having 1 to 6 carbon atoms, a haloalkylsulfinyl group having 1 to 6 carbon atoms, a haloalkylsulfonyl group having 1 to 6 carbon atoms or a nitro group.", "As a substituent on a pyridine ring, electron-withdrawing groups such as a trifluoromethyl group, a nitro group, a cyano group, a chlorine atom, a fluorine atom and an alkyloxycarbonyl group are preferred, since a good reaction yield is attained and since raw materials can be easily obtained.", "Further, such electron-withdrawing groups are preferably substituted at the 3-position and/or the 5-position of a pyridine ring.", "More specifically, examples of the substituted pyridyl group include 3-chloropyridin-2-yl, 5-chloropyridin-2-yl, 3,5-dichloropyridin-2-yl, 4-amino-3,5-dichloropyridin-2-yl, 3-cyano-6-methylpyridin-2-yl, 5-trifluoromethylpyridin-2-yl, 3-chloro-5-trifluoromethylpyridin-2-yl, 3-nitropyridin-2-yl, 5-nitropyridin-2-yl, 3-nitro-4-methylpyridin-2-yl, 3-nitro-6-methoxypyridin-2-yl, 2-chloro-3-nitropyridin-6-yl, 6-chloro-3-nitropyridin-2-yl and 3,5-dinitropyridin-2-yl.", "Concerning these substituents on a pyridine ring, for example, a nitro group is convertible to an amino group by reduction, and the amino group is further convertible to a halogen atom or a substituted alkyl group through a diazonium salt, and these can be included in the substituents on a pyridine ring.", "Examples of the alkyloxy group having 1 to 6 carbon atoms, represented by R3, R3a and R3c, include methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy, sec-butyloxy, tert-butyloxy, pentyloxy and hexyloxy.", "The alkyl group constituting such an alkyloxy group may be substituted with at least one of a halogen atom, a cycloalkyl group having 3 to 8 carbon atoms, an alkyloxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms, a carboxy group, a cyano group, a nitro group, an optionally substituted amino group and an optionally substituted phenyl group.", "Examples of the cycloalkyloxy group having 3 to 8 carbon atoms, represented by R3, R3a and R3c, include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy and cyclooctyloxy.", "These cycloalkyl groups may be substituted with a halogen atom, an alkyl group having 1 to 4 carbon atoms, an alkyloxycarbonyl group having 1 to 4 carbon atoms or a cyano group.", "More specifically, examples of such substituted cycloalkyloxy groups include 1-methylcyclopropyloxy, 2,2-dimethylcyclopropyloxy, 2-chlorocyclopropyloxy, 2,2-dichlorocyclopropyloxy, 2-methoxycarbonylcyclopropyloxy, 2-cyanocyclopropyloxy, 2-methylcyclopentyloxy and 3-methylcyclopentyloxy.", "Examples of the aralkyloxy group having 7 to 11 carbon atoms, represented by R3, R3a and R3c, include benzyloxy, 1-phenylethyloxy, 2-phenylethyloxy, 1-phenylpropyloxy, 1-naphthylmethyloxy and 2-naphthylmethyloxy.", "The aromatic ring of the aralkyloxy group may be substituted with at least one of a halogen atom, an alkyl group having 1 to 12 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, an alkyloxy group having 1 to 6 carbon atoms, a haloalkyloxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, an alkylsulfonyl group having 1 to 6 carbon atoms, an alkyloxycarbonyl group having 1 to 6 carbon atoms, a carboxy group, an optionally substituted carbamoyl group, a cyano group and a nitro group.", "The alkenyloxy group having 3 to 12 carbon atoms, represented by R3, R3a and R3c, may be linear or branched, and examples thereof include 1-propenyloxy, allyloxy, 2-methyl-2-propenyloxy, 2-butenyloxy, 3-butenyloxy, 2-pentenyloxy, 3-pentenyloxy, 1-cyclopentenyloxy, 2-hexenyloxy, 3-hexenyloxy, 1-cyclohexenyloxy, 2-heptenyloxy and 1-cyclooctenyloxy.", "The alkenyloxy group may be substituted with a halogen atom, or the like, and examples of the substituted alkenyloxy group include 2-chloro-2-propenyloxy, 3-chloropropenyloxy and 4-chloro-2-butenyloxy.", "The alkynyloxy group having 3 to 6 carbon atoms, represented by R3, R3a and R3c, may be linear or branched, and examples thereof include propargyloxy, 1-butyn-3-yloxy, 3-methyl-1-butyn-3-yloxy, 2-butynyloxy, 2-pentynyloxy and 3-pentynyloxy.", "The above alkynyloxy group may be substituted with a halogen atom, or the like, and examples of the substituted alkynyloxy group include 3-fluoro-2-propynyloxy, 3-chloro-2-propynyloxy, 3-bromo-2-propynyloxy, 4-bromo-2-butynyloxy and 4-bromo-3-butynyloxy.", "Examples of the heterocyclic ring that a combination of R3 and R4, a combination of R3c and R4 or a combination of R3b and R4b forms together with a nitrogen atom to which they bond include pyrrole, pyrroline, pyrrolidine, imidazole, imidazoline, imidazolioine, pyrazole, pyrazoline, pyrazolidine, piperidine, piperazine, indole, indoline, isoindole, 1H-indazole, purine, oxazoline, isoxazoline, isoxazolidine, thiazoline, morpholine, thiomorpholine, aziridine, azocane and tetrahydrooxazine.", "The above heterocyclic ring may be substituted with at least one of an alkyl group having 1 to 6 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, a halogen atom and a cyano group.", "Examples of the halogen atom represented by R2 and R2b include a chlorine atom, a bromine atom and a fluorine atom.", "Examples of the leaving group represented by Z include halogen atoms such as a chlorine atom, a bromine atom and iodine atom and alkyl- or arylsulfonyloxy groups such as methylsulfonyloxy, trifluoromethylsulfonyloxy, phenylsulfonyloxy and p-tolylsulfonyloxy.", "The method of producing the pyrazole derivative of the present invention and the intermediate thereof will be explained in detail below.", "Production method-1 (Step-1) shows a method in which a reaction of a pyrazole derivative (3) and a compound (4) is carried out in the presence of a base, to produce a pyrazole derivative (2) of the present invention.", "wherein R1, R2, R5 and Z are as defined above.", "The above reaction is essentially carried out in the presence of a base.", "The base can be selected from alkali metal bases such as sodium hydride, sodium amide, sodium carbonate, potassium carbonate, sodium methoxide, sodium ethoxide, potassium-t-butoxide, sodium hydroxide and potassium hydroxide, and organic amines such as triethylamine, tributylamine, N-methylmorpholine, pyridine and dimethylaniline.", "The base is preferably used in an amount of one to excess equivalents to the reaction substrate, since a good yield is attained.", "The reaction is preferably carried out in a solvent, and any solvent can be used so long as it has no adversary effect on the reaction.", "For example, the solvent can be selected from ether solvents such as diethyl ether, tetrahydrofuran (THF), dioxane and 1,2-dimethoxyethane (DME), nitriles such as acetonitrile and propionitrile, esters such as ethyl acetate and ethyl propionate, aromatic hydrocarbon solvents such as benzene, toluene, xylene and chlorobenzene, amides such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone, dimethylsulfoxide (DMSO), water or mixtures of these.", "While the reaction temperature is not specially limited, the desired product can be obtained in good yield by carrying out the reaction at a temperature that is determined in the range of 0° C. to 150° C. as required.", "After completion of the reaction, the desired product can be obtained by ordinary post-treatment procedures.", "The product can be purified by column chromatography or recrystallization as required.", "The pyrazole derivative represented by the general formula (3), as a raw material in the above step, can be easily prepared by a cyclization reaction of hydrazine and a β-ketoester derivative as described, for example, in Organic Synthesis Vol.", "6, 791 (1988).", "While the thus-obtained 3-hydroxypyrazole derivative (3) is present in the form of an equilibrium mixture with a tautomer, the general formula shows a structure of an alcohol form (3) for the convenience.", "Production method-2 (step-2) is a method in which a pyrazole derivative (2a) is halogenated to produce a pyrazole derivative (2b) of the present invention.", "wherein R1, R2a and R5a are as defined above.", "The halogenation can be carried out with a halogenating reagent.", "The halogenating reagent can be selected from sulfuryl chloride, N-chlorosuccinimide or N-bromosuccinimide.", "The reaction is preferably carried out in a solvent, and any solvent can be used so long as it has no adversary effect on the reaction.", "Examples of the solvent include aliphatic hydrocarbon solvents such as pentane, hexane and octane, ether solvents such as diethyl ether, tetrahydrofuran, dioxane and DME, halogen-containing solvents such as dichloromethane, chloroform and carbon tetrachloride, aromatic hydrocarbon solvents such as chlorobenzene and dichlorobenzene, organic acid solvents such as acetic acid and propionic acid, water and mixtures of these.", "While the reaction temperature differs depending upon the halogenating reagent used, it is determined at a temperature in the range of −10° C. to 150° C. The reaction is preferably carried out at a temperature determined in the range of 0° C. to the reflux temperature of a solvent as required, since it gives a good yield.", "After completion of the reaction, the desired product can be obtained by ordinary post-treatment procedures.", "The product can be purified by column chromatography or recrystallization as required.", "Production method-3 (step-3) is a step in which a pyrazole derivative (2c) reacts with isocyanates or isothiocyanates (5), to produce a pyrazole derivative (1a) of the present invention.", "wherein R1, R2, R3a, R5a and Y are as defined above.", "The above reaction can be carried out in the presence of a base, and the base can be selected from alkali metal bases such as sodium hydride, sodium amide, sodium carbonate, potassium carbonate, sodium methoxide, sodium ethoxide, potassium-t-butoxide, sodium hydroxide and potassium hydroxide, and organic amines such as triethylamine, tributylamine, N-methylmorpholine, pyridine and dimethylaniline.", "The amount of the base is not specially limited.", "The reaction can be carried out in a solvent, and any solvent can be used so long as it has no adversary effect on the reaction.", "Examples of the solvent include ether solvents such as diethyl ether, THF, dioxane and DME, nitriles such as acetonitrile and propionitrile, esters such as ethyl acetate and ethyl propionate, aromatic hydrocarbon solvents such as benzene, toluene, xylene and chlorobenzene, halogen-containing solvents such as dichloromethane, chloroform and carbon tetrachloride, amides such as DMF and N-methylpyrrolidone, DMSO or mixtures of these.", "While the reaction temperature is not specially limited, the desired product can be obtained in good yield by carrying out the reaction at a temperature that is determined in the range of 0° C. to 150° C. as required.", "After completion of the reaction, the desired product can be obtained by ordinary post-treatment procedures.", "The product can be purified by column chromatography or recrystallization as required.", "Production-4 (step-4) shows a method in which a reaction of a pyrazole derivative (3a) and a compound (4) is carried out in the presence of a base, to produce a pyrazole derivative (1b) of the present invention.", "wherein R1, R2, R3a, R5 and Z are as defined above.", "It is essential to carry out the above reaction in the presence of a base.", "The base can be selected from alkali metal bases such as sodium hydride, sodium amide, sodium carbonate, potassium carbonate, sodium methoxide, sodium ethoxide, potassium-t-butoxide, sodium hydroxide and potassium hydroxide, and organic amines such as triethylamine, tributylamine, N-methylmorpholine, pyridine and dimethylaniline.", "The base is preferably used in an amount of one to excess equivalents to the reaction substrate, since a good yield is attained.", "The reaction is preferably carried out in a solvent, and any solvent can be used so long as it has no adversary effect on the reaction.", "For example, the solvent can be selected from ether solvents such as diethyl ether, tetrahydrofuran (THF), dioxane and 1,2-dimethoxyethane (DME), nitriles such as acetonitrile and propionitrile, esters such as ethyl acetate and ethyl propionate, aromatic hydrocarbon solvents such as benzene, toluene, xylene and chlorobenzene, amides such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone, dimethylsulfoxide (DMSO), water or mixtures of these.", "While the reaction temperature is not specially limited, the desired product can be obtained in good yield by carrying out the reaction at a temperature that is determined in the range of 0° C. to 150° C. as required.", "After completion of the reaction, the desired product can be obtained by ordinary post-treatment procedures.", "The product can be purified by column chromatography or recrystallization as required.", "Production method-5 (step-5) shows a method in which a reaction of a pyrazole derivative (1b) and a compound (6) is carried out in the presence of a base, to produce a pyrazole derivative (1c) of the present invention.", "wherein R1, R2, R3a, R4a, R5, Y and Z are as defined above.", "It is essential to carry out the above reaction in the presence of a base.", "The base can be selected from alkali metal bases such as sodium hydride, sodium amide, sodium carbonate, potassium carbonate, sodium methoxide, sodium ethoxide, potassium-t-butoxide, sodium hydroxide and potassium hydroxide, and organic amines such as triethylamine, tributylamine, N-methylmorpholine, pyridine and dimethylaniline.", "When the base is used in an amount of one to excess equivalents to the reaction substrate, the desired product can be obtained in good yield.", "The reaction is preferably carried out in a solvent, and any solvent can be used so long as it has no adversary effect on the reaction.", "Examples of the solvent include ether solvents such as diethyl ether, THF, DME and dioxane, nitriles such as acetonitrile and propionitrile, aromatic hydrocarbon solvents such as benzene, toluene, xylene and chlorobenzene, amides such as DMF and N-methylpyrrolidone, DMSO, water or mixtures of these.", "While the reaction temperature is not specially limited, the desired product can be obtained in good yield by carrying out the reaction at a temperature that is determined in the range of 0° C. to 100° C. as required.", "After completion of the reaction, the desired product can be obtained by ordinary post-treatment procedures.", "The product can be purified by column chromatography or recrystallization as required.", "Production method-6 (step-6) shows a method in which a reaction of a pyrazole derivative (2) and a carbamic acid chlorides (7) is carried out in the presence of a base, to produce a pyrazole derivative (1d) of the present invention.", "wherein R1, R2, R3b, R4b and R5 are as defined above.", "It is essential to carry out the above reaction in the presence of a base.", "The base can be selected from alkali metal bases such as sodium hydride, sodium amide, sodium carbonate, potassium carbonate, sodium methoxide, sodium ethoxide, potassium-t-butoxide, sodium hydroxide and potassium hydroxide, and organic amines such as triethylamine, tributylamine, N-methylmorpholine, pyridine and dimethylaniline.", "When the base is used in an amount of one to excess equivalents to the reaction substrate, the desired product can be obtained in good yield.", "The reaction is preferably carried out in a solvent, and any solvent can be used so long as it has no adversary effect on the reaction.", "Examples of the solvent include ether solvents such as diethyl ether, THF, DME and dioxane, nitriles such as acetonitrile and propionitrile, aromatic hydrocarbon solvents such as benzene, toluene, xylene and chlorobenzene, amides such as DMF and N-methylpyrrolidone, DMSO, water or mixtures of these.", "While the reaction temperature is not specially limited, the desired product can be obtained in good yield by carrying out the reaction at a temperature that is determined in the range of 0° C. to 100° C. as required.", "After completion of the reaction, the desired product can be obtained by ordinary post-treatment procedures.", "The product can be purified by column chromatography or recrystallization as required.", "Production method-7 shows a method in which a pyrazole derivative (2) reacts with phosgene or a phosgene equivalent material such as a phosgene dimer or a phosgene trimer to prepare a pyrazole derivative (2d) of the present invention which is a production intermediate (step-7) and then the pyrazole derivative (2d) reacts with amines (8) in the presence of a base, to produce a pyrazole derivative (1e) of the present invention (step-8).", "wherein R1, R2, R3c, R4 and R5 are as defined above.", "In the reaction in the step-7, the pyrazole derivative (2) reacts with phosgene or a phosgene equivalent material in a halogen-containing solvent such as dichloromethane, chloroform or carbon tetrachloride, an aromatic hydrocarbon solvent such as benzene, toluene, xylene or chlorobenzene or an ester solvent such as ethyl acetate or propyl acetate, whereby the desired product can be synthesized.", "While the reaction temperature is not specially limited, the desired product can be obtained in good yield by carrying out the reaction at a temperature that is determined in the range of −30° C. to 150° C. as required.", "After completion of the reaction, the desired product can be obtained by ordinary post-treatment procedures.", "The product can be used for the subsequent reaction without its isolation.", "It is essential to carry out the react-on in the step-8 in the presence of a base.", "The base can be selected from alkali metal bases such as sodium hydride, sodium amide, sodium carbonate, potassium carbonate, potassium-t-butoxide, sodium hydroxide and potassium hydroxide, and organic amines such as triethylamine, tributylamine, N-methylmorpholine, pyridine and dimethylaniline.", "The base is used in an amount of one to excess equivalents to the reaction substrate, whereby the desired product can be obtained in good yield.", "The reaction is preferably carried out in an organic solvent, and the solvent can be selected from benzene, toluene, xylene, THF, diethyl ether, chloroform, dichloromethane, methanol, ethanol, propyl alcohol, isopropyl alcohol, tert-butyl alcohol, ethyl acetate, DMF or DMSO.", "The reaction can be carried out at a temperature that is determined in the range of room temperature to the reflux temperature of a solvent as required.", "After completion of the reaction, the desired product can be obtained by ordinary post-treatment procedures.", "The product can be purified by column chromatography or recrystallization as required.", "Production method-8 (step 9) shows a method in which a pyrazole derivative (1f) is halogenated to produce a pyrazole derivative (1g) of the present invention.", "[Production Method-8] wherein R1, R2b, R3, R4, R5 and Y are as defined above.", "The halogenation can be carried out with a halogenating reagent.", "The halogenating reagent can be selected from sulfuryl chloride, N-chlorosuccinimide or N-bromosuccinimide.", "The reaction is preferably carried out in a solvent, and any solvent can be used so long as it has no adversary effect on the reaction.", "Examples of the solvent include aliphatic hydrocarbon solvents such as pentane, hexane and octane, ether solvents such as diethyl ether, tetrahydrofuran, dioxane and DME, halogen-containing solvents such as dichloromethane, chloroform and carbon tetrachloride, aromatic hydrocarbon solvents such as chlorobenzene and dichlorobenzene, organic acid solvents such as acetic acid and propionic acid, water and mixtures of these.", "While the reaction temperature differs depending upon the halogenating reagent used, it is determined at a temperature in the range of −10° C. to 150° C. The reaction is preferably carried out at a temperature determined in the range of 0° C. to the reflux temperature of a solvent as required, since a good yield is attained.", "After completion of the reaction, the desired product can be obtained by ordinary post-treatment procedures.", "The product can be purified by column chromatography or recrystallization as required.", "Production method-9 (step-10) shows a method in which a thiocarbonyl group of a pyrazole derivative (1 h) is oxidized to produce a pyrazole derivative (1i) of the present invention.", "wherein R1, R2, R3, R4 and R5 are as defined above.", "The thiocarbonyl group can be oxidized with an oxidizing reagent.", "As an oxidizing reagent, for example, hydrogen peroxide can be used.", "The reaction is preferably carried out in an aqueous alcohol with a suitable water-concentration.", "The alcohol can be selected from methanol, ethanol or propanol.", "The above reaction is preferably carried out in the presence of a base, and the base can be selected from sodium hydroxide or potassium hydroxide.", "The reaction can be carried out at a temperature that is determined in the range of −30° C. to 60° C. as required.", "After completion of the reaction, the desired product can be obtained by ordinary post-treatment procedures.", "The product can be purified by column chromatography or recrystallization as required.", "The present invention will be explained further in detail with reference to Referential Examples and Examples hereinafter, while the present invention shall not be limited thereto.", "EXAMPLES Hydrazine monohydrate (10.0 g, 20.0 mmol) was added to a solution of ethyl 3-oxobutanoate (26.0 g, 20.0 mmol) in ethanol (50 ml) at 0° C., and the mixture was stirred at room temperature for 1 hour.", "After completion of the reaction, a precipitated solid was filtered and washed with diethyl ether, to give a white solid of 3-hydroxy-5-methylpyrazole (16.9 g, yield: 86.0%).", "mp: 215-216° C.; 1H-NMR(DMSO-d6, DMSO, ppm): δ 2.10(s, 3H), 5.22(s, 1H), 8.50-11.90(br s, 2H).", "Referential Examples 2-9 Reactions of hydrazine monohydrate with β-ketoester derivative (Referential Example 2: ethyl 3-oxopentanoate, Referential Example 3: ethyl isobutylylacetate, Referential Example 4: methyl 4,4-dimethyl-3-oxopentanate, Referential Example 5: methyl 4-methoxyacetate, Referential Example 6: diethyl 3-oxoglutarate, Referential Example 7: ethyl 4,4,4-trifluoro-3-oxobutanate, Referential Example 8: ethyl 2-methyl-3-oxobutanate, Referential Example 9: ethyl 2-ethyl-3-oxobutanate) were carried out in the same manner as in Referential Example 1, to give corresponding 3-hydroxypyrazole derivatives.", "Products/forms/yields/melting points/NMR spectra are described below.", "Referential Example 2 5-ethyl-3-hydroxypyrazole/white solid/yield: 74.8%/mp: 191-193° C./1H-NMR(DMSO-d6, DMSO, ppm): δ 1.13(t, J=7.6 Hz, 3H), 2.46(q, J=7.6 Hz, 2H), 5.24(s, 1H), 9.10-11.60(br s, 1H).", "(Amino proton was not assigned.).", "Referential Example 3 3-hydroxy-5-isopropylpyrazole/white solid/yield: 57.5%/mp: 189-193° C./1H-NMR(DMSO-d6, DMSO, ppm): δ 1.15(d, J=6.9 Hz, 6H), 2.79(sep, J=6.9 Hz, 1H), 5.23(s, 1H), 8.80-12.40(br s, 1H).", "Referential Example 4 5-tert-butyl-3-hydroxypyrazole/white solid/yield: 74.2%/mp: 205-206° C./1H-NMR(DMSO-d6, DMSO, ppm): δ 1.26(s, 9H), 5.22(s, 1H), 8.60-12.20(br s, 2H).", "Referential Example 5 3-hydroxy-5-(methoxymethyl)pyrazole/white solid/yield: 80.1%/mp: 123-125° C./1H-NMR(DMSO-d6, DMSO, ppm): δ 3.23(s, 3H), 4.25(s, 2H), 5.42(s, 1H), 9.00-13.00(m, 2H).", "Referential Example 6 ethyl(3-hydroxypyrazol-5-yl)acetate/white solid/yield: 35.4%/mp: 114-117° C./1H-NMR(DMSO-d6, DMSO, ppm): δ 1.20(t, J=7.1 Hz, 3H), 3.34(s, 2H), 4.10(q, J=7.1 Hz, 2H), 5.35(s, 1H), 8.60-12.20(m, 2H).", "Referential Example 7 3-hydroxy-5-trifluoromethylpyrazole/white solid/yield: 81.5%/mp: 211-215° C./1H-NMR(DMSO-d6, DMSO, ppm): δ 5.69(s, 1H), 10.70-11.60(brs, 1H), 12.20-13.40(br s, 1H).", "Referential Example 8 4,5-dimethyl-3-hydroxypyrazole/white solid/yield: 93.7%/mp: 268-270° C./1H-NMR(DMSO-d6, DMSO, TMS, ppm): δ 1.73(s, 3H), 2.03(s, 3H), 9.20-12.50(br s, 2H).", "Referential Example 9 4-ethyl-3-hydroxy-5-methylpyrazole/white solid/yield: 86.7%/mp: 232-234° C./1H-NMR(DMSO-d6, DMSO, ppm): δ 1.00(t, J=7.5 Hz, 3H), 2.05(s, 3H), 2.21(q, J=7.5 Hz, 2H), 8.90-11.50(br s, 2H).", "Referential Example 10 Hydrazine monohydrate (1.80 g, 45.0 mmol) was added to a solution of tert-butyl 4-cyclopropyl-3-oxopropionate (5.53 g, 30.0 mmol) in ethanol (50 ml) at 0° C., and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, a precipitated solid was filtered and washed with diethyl ether, to give a white solid of 5-cyclopropyl-3-hydroxypyrazole (2.08 g, yield: 55.9%).", "mp: 213-215° C.; 1H-NMR(DMSO-d6, DMSO, ppm): δ 0.55-0.65(m, 2H), 0.75-0.95(m, 2H), 1.65-1.85(m, 1H), 5.12(s, 1H).", "(Amino proton and hydroxy proton were not assigned.)", "Referential Example 11 Hydrazine monohydrate (0.80 g, 20 mmol) was added to a solution of methyl 4-chlorobenzoylacetate (3.19 g, 15.0 mmol) in ethanol (40 ml) at 0° C., and the mixture was stirred at room temperature for 2 days.", "After completion of the reaction, a precipitated solid was filtered and washed with diethyl ether, to give a white solid of 5-(4-chlorophenyl)-3-hydroxypyrazole (2.02 g, yield: 55.1%).", "mp: 240-241° C.; 1H-NMR(DMSO-d6, DMSO, ppm): δ 5.92(s, 1H), 7.47(d, J=8.5 Hz, 2H), 7.70(d, J=8.5 Hz, 2H), 9.30-10.60(br s, 1H), 11.60-12.70(br s, 1H).", "Referential Example 12 Hydrazine monohydrate (6.7 g, 210 mmol) was added to a solution of dimethyl acetylenedicarboxylate (24.7 g, 175 mmol) in toluene (90 ml) at 0° C., and the thus-obtained mixture was gradually temperature-increased up to room temperature, stirred for 2 hours and further stirred under heating at 130° C. for 4 hours.", "After completion of the reaction, a precipitated solid was filtered and washed with diethyl ether, to give a white solid of methyl 3-hydroxypyrazole-5-carboxylate (18.2 g, yield: 73.8%).", "mp: 229-231° C.; 1H-NMR(DMSO-d6, DMSO, ppm): δ 3.79(s, 3H), 5.91(s, 1H), 10.10-10.45(br s, 1H).", "(Hydroxy proton was not assigned.)", "Referential Example 13 Sulfuryl chloride (4.86 g, 36 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (2.94 g, 30.0 mmol) in acetic acid (25 ml), and the mixture was stirred at room temperature for 6 hours.", "After completion of the reaction, the reaction mixture was poured into ice water, and a precipitated solid was filtered and washed with water, to give a yellowish solid of 4-chloro-3-hydroxy-5-methylpyrazole (1.48 g, yield: 37.2%).", "mp: 235-238° C. (dec.); 1H-NMR(CDCl3, TMS, ppm): δ 2.10(s, 3H), 8.75-9.15(m, 2H).", "Referential Example 14 Potassium carbonate (3.52 g, 25.5 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (5.00 g, 51.0 mmol) in DMF (80 ml).", "Then, allylisocyanate (4.51 ml, 51.0 mmol) was added, and the mixture was stirred at room temperature for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid and extracted with ethyl acetate (50 ml×2).", "An organic layer was washed with water (50 ml×3), dried over anhydrous magnesium sulfate and then filtered to remove a desiccant, and the solvent was distilled off from a filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a white solid of N-allyl-3-hydroxy-5-methylpyrazole-1-carboxamide (4.72 g, yield: 51.1%).", "mp: 80-83° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.55(d, J=0.8 Hz, 3H), 3.97(dddt, J=1.6, 1.6, 5.8 and 5.8 Hz, 2H), 5.19(ddt, J=1.6, 2.7 and 10.3 Hz, 1H), 5.28(ddt, J=1.6, 2.7 and 17.2 Hz, 1H), 5.63(d, J=0.8 Hz, 1H), 5.90(ddt, J=5.8, 10.3 and 17.2 Hz, 1H), 6.70(m, 1H).", "(Hydroxy proton was not assigned.)", "Referential Example 15 Reaction of 3-hydroxy-5-methylpyrazole with phenyl isocyanate was carried out in the same manner as in Referential Example 14, to give a white solid of N-phenyl-3-hydroxy-5-methylpyrazol-1-carboxamide (yield: 18.9%).", "mp: 232-234° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.62(d, J=0.8 Hz, 3H), 5.72(d, J=0.8 Hz, 1H), 7.11-7.17(m, 1H), 7.33-7.39(m, 2H), 7.53-7.57(m, 2H), 8.64(br s, 1H).", "(Hydroxy proton was not assigned.)", "Example 1 Potassium carbonate (0.5 g, 3.6 mmol) and 4-fluoronitrobenzene (1.6 g, 1.0 mmol) were added to a solution of 3-hydroxy-5-methylpyrazole (0.33 g, 3.4 mmol) in DMF (50 ml) at room temperature, and further, the mixture was stirred at room temperature for 8 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (100 ml) and extracted with ethyl acetate (30 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7→1/3), to give a white solid of 5-methyl-3-(4-nitrophenyloxy)pyrazole (0.4 g, yield: 63.3%) mp: 124-125° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.33(s, 3H), 5.75(s, 1H), 7.20(dd, J=2.2 and 7.1 Hz, 2H), 8.22(dd, J=2.2 and 7.1 Hz, 2H), 9.30-9.90(br s, 1H).", "Example 2 Potassium carbonate (3.19 g, 23.1 mmol) and 2,5-difluoronitrobenzene (7.34 g, 46.1 mmol) were added to a solution of 3-hydroxy-5-methylpyrazole (4.52 g, 46.1 mmol) in DMF (80 ml) at room temperature, and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (100 ml) and extracted with ethyl acetate (100 ml×2).", "An organic layer was washed with water (100 ml×2), dried over anhydrous magnesium sulfate and filtered to remove magnesium sulfate, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellow solid of 3-(4-fluoro-2-nitrophenyloxy)-5-methylpyrazole (5.33 g, yield: 48.8%).", "mp: 81-84° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.28(d, J=0.5 Hz, 3H), 5.71(d, J=0.5 Hz, 1H), 7.26-7.38(m, 2H), 7.69(dd, J=2.8 and 7.9 Hz, 1H), 9.33(br s, 1H).", "Example 3 Potassium carbonate (2.23 g, 16.1 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (3.16 g, 32.2 mmol) and 5-fluoro-2-nitrotoluene (5.00 g, 32.2 mmol) in DMF (50 ml), and the mixture was stirred at 70° C. for 3 hours.", "After completion of the reaction, a reaction mixture was poured into 2N hydrochloric acid (50 ml) and extracted with ethyl acetate (30 ml×2).", "An organic layer was washed with water (50 ml×3), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellowish solid of 5-methyl-3-(4-nitro-3-methylphenyloxy)pyrazole (2.51 g, yield: 27.8%).", "mp: 114-117° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.30(d, J=0.5 Hz, 3H), 2.61(s, 3H), 5.72(d, J=0.5 Hz, 1H), 6.98-7.04(m, 2H), 8.05(d, J=9.8 Hz, 1H), 10.12(br s, 1H).", "Example 4 Reaction of 3-hydroxy-5-methylpyrazole with 5-chloro-2-nitroanisole was carried out in the same manner as Example 3, to give a yellow viscous substance of 5-methyl-3-(3-methoxy-4-nitrophenyloxy)pyrazole (yield: 13.2%).", "1H-NMR(CDCl3, TMS, ppm): δ 2.34(s, 3H), 3.93(s, 3H), 5.74(s, 1H), 6.70(dd, J=2.4 and 9.1 Hz, 1H), 6.85(d, J=2.4 Hz, 1H), 7.95(d, J=9.1 Hz, 1H), 9.45(br s, 1H).", "Example 5 Potassium carbonate (9.12 g, 66.0 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (5.88 g, 60.0 mmol) and 4-fluoro-3-nitrobenzotrifluoride (12.6 g, 60.0 mmol) in DMF (120 ml), and the mixture was stirred at 50° C. for 4 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (200 ml) and extracted with ethyl acetate (70 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellow solid of 5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole (12.1 g, yield: 69.0%).", "mp: 89-91° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.28(s, 3H), 5.77(s, 1H), 7.40(d, J=8.8 Hz, 1H), 7.76(dd, J=2.0 and 8.8 Hz, 1H), 8.21(d, J=2.0 Hz, 1H), 10.10-11.05(br s, 1H).", "Examples 6-9 Reactions of 4-fluoro-3-nitrobenzotrifluoride with 3-hydroxypyrazole derivative (Example 6: 3-hydroxy-5-trifluoromethylpyrazole, Example 7: 5-ethyl-3-hydroxypyrazole, Example 8: 4,5-dimethyl-3-hydroxypyrazole, Example 9: 4-ethyl-3-hydroxy-5-methylpyrazole) were carried out in the same manner as in Example 5, to give corresponding 3-aryloxypyrazole derivatives.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 6 3-(2-nitro-4-trifluoromethylphenyloxy)-5-trifluoromethylpyrazole/yellow viscous substance/yield: 28.7%/1H-NMR(CDCl3, TMS, ppm): δ 6.35(s, 1H), 7.48(d, J=8.5 Hz, 1H), 7.85(dd, J=2.0 and 8.8 Hz, 1H), 8.27(m, 1H), 10.10-10.90(br s, 1H).", "Example 7 5-ethyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole/yellow viscous substance/yield: 77.0%/1H-NMR(CDCl3, TMS, ppm): δ 1.27(t, J=7.6 Hz, 3H), 2.67(dq, J=0.4 and 7.6 Hz, 2H), 5.81(s, 1H), 7.44(d, J=8.8 Hz, 1H), 7.77(dd, J=1.9 and 8.8 Hz, 1H), 8.22(d, J=1.9 Hz, 1H).", "(Amino proton was not assigned.)", "Example 8 4,5-dimethyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole/yellowish solid/yield: 50.8%/mp: 92-95° C./1H-NMR(CDCl3, TMS, ppm): δ 1.89(s, 3H), 2.23(s, 3H), 7.38(d, J=8.8 Hz, 1H), 7.74(dd, J=1.9 and 8.8 Hz, 1H), 8.22(d, J=1.9 Hz, 1H), 8.90-10.35(br s, 1H).", "Example 9 4-ethyl-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole/yellow solid/yield: 49.8%/mp: 92-93° C./1H-NMR(CDCl3, TMS, ppm): δ 1.10(t, J=7.6 Hz, 3H), 2.24(s, 3H), 2.36(q, J=7.6 Hz, 2H), 7.42(d, J=8.8 Hz, 1H), 7.74(dd, J=2.0 and 8.8 Hz, 1H), 8.22(d, J=2.0 Hz, 1H), 9.10-9.75(brs, 1H).", "Example 10 Potassium carbonate (7.60 g, 55.0 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (4.90 g, 50.0 mmol) and 2-fluoro-5-nitrobenzotrifluoride (10.5 g, 50.0 mmol) in DMF (50 ml), and the mixture was stirred under heating at 60° C. for 4 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (100 ml) and extracted with ethyl acetate (50 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellowish viscous substance of 5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole (8.70 g, yield: 60.6%).", "1H-NMR(CDCl3, TMS, ppm): δ 2.30(d, J=0.5 Hz, 3H), 5.78(d, J=0.5 Hz, 1H), 7.31(d, J=9.2 Hz, 1H), 8.31(dd, J=2.7 and 9.2 Hz, 1H), 8.57(d, J=2.7 Hz, 1H), 10.05-10.50(br s, 1H).", "Examples 11-13 Reactions of 2-fluoro-5-nitrobenzotrifluoride with 3-hydroxypyrazole derivative (Example 11: 5-ethyl-3-hydroxypyrazole, Example 12: 4,5-dimethyl-3-hydroxypyrazole, Example 13: 4-ethyl-3-hydroxy-5-methylpyrazole) were carried out in the same manner as in Example 10, to give corresponding 3-aryloxypyrazole derivatives.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 11 5-ethyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole/yellow viscous substance/yield: 79.3%/1H-NMR(CDCl3, TMS, ppm): δ 1.29(t, J=7.6 Hz, 3H), 2.69(q, J=7.6 Hz, 2H), 5.82(s, 1H), 7.38(d, J=9.2 Hz, 1H), 8.35(dd, J=2.7 and 9.2 Hz, 1H), 8.57(d, J=2.7 Hz, 1H), 9.45-10.15(br s, 1H).", "Example 12 4,5-dimethyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole/yellow solid/yield: 51.1%/mp: 97-99° C./1H-NMR(CDCl3, TMS, ppm): δ 1.84(s, 3H), 2.25(s, 3H), 7.33(d, J=9.2 Hz, 1H), 8.33(dd, J=2.7 and 9.2 Hz, 1H), 8.57(d, J=2.7 Hz, 1H), 9.05˜10.20(br s, 1H).", "Example 13 4-ethyl-5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole/orange viscous substance/yield: 50.8%/1H-NMR(CDCl3, TMS, ppm): δ 1.05(t, J=7.6 Hz, 3H), 2.26(s, 3H), 2.31(q, J=7.6 Hz, 2H), 7.36(d, J=9.2 Hz, 1H), 8.33(dd, J=2.7 and 9.2 Hz, 1H), 8.57(d, J=2.7 Hz, 1H).", "(Amino proton was not assigned.)", "Example 14 Potassium carbonate (3.52 g, 25.5 mmol) and 2,4-dichloronitrobenzene (9.79 g, 51.0 mmol) were added to a solution of 3-hydroxy-5-methylpyrazole (5.00 g, 51.0 mmol) in DMF (130 ml) at room temperature, and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (200 ml) and extracted with ethyl acetate (100 ml×3).", "An organic layer was washed with water (100 ml×3), dried over anhydrous magnesium sulfate and filtered to remove magnesium sulfate, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellow solid of 3-(3-chloro-4-nitrophenyloxy)-5-methylpyrazole (6.33 g, yield: 48.9%) [mp: 91-93° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.30(s, 3H), 5.74(d, J=0.6 Hz, 1H), 7.10(dd, J=2.8 and 9.1 Hz, 1H), 7.25(d, J=2.5 Hz, 1H), 7.97(d, J=9.1 Hz, 1H), 10.73(br d, J=8.8 Hz, 1H)] and a yellow solid of 3-(5-chloro-2-nitrophenyloxy)-5-methylpyrazole (2.31 g, yield: 17.9%) [mp: 87-90° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.27(s, 3H), 5.73(d, J=0.6 Hz, 1H), 7.17(dd, J=2.2 and 8.8 Hz, 1H), 7.26(d, J=2.2 Hz, 1H), 7.91(d, J=8.8 Hz, 1H), 10.77(br s, 1H)].", "Example 15 Potassium carbonate (3.46 g, 25.0 mmol) and 2,4-difluoronitrobenzene (7.95 g, 50.0 mmol) were added to a solution of 3-hydroxy-5-methylpyrazole (4.91 g, 50.0 mmol) in DMSO (80 ml) at room temperature, and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (100 ml) and extracted with ethyl acetate (100 ml×2).", "An organic layer was washed with water (100 ml×2), dried over anhydrous magnesium sulfate and filtered to remove magnesium sulfate, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellow solid of 3-(3-fluoro-4-nitrophenyloxy)-5-methylpyrazole (1.52 g, yield: 12.8%) [mp: 80-83° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.33(d, J=0.5 Hz, 3H), 5.77(d, J=0.5 Hz, 1H), 6.96-7.03(m, 2H), 8.06-8.13(m, 1H), 10.03(br s, 1H)] and a yellow solid of 3-(5-fluoro-2-nitrophenyloxy)-5-methylpyrazole (0.97 g, yield: 8.2%) [mp: 95-97° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.31(s, 3H), 5.77(s, 1H), 6.90(ddd, J=2.7, 5.8 and 9.1 Hz, 1H), 7.01(dd, J=2.7 and 9.6 Hz, 1H), 8.03(dd, J=5.8 and 9.1 Hz, 1H), 9.86(br s, 1H)].", "Example 16 Potassium carbonate (2.49 g, 18.0 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (1.47 g, 15.0 mmol) and 5-fluoro-2-nitrobenzotrifluoride (3.14 g, 15.0 mmol) in DMF (20 ml), and the mixture was stirred under heating at 70° C. for 3 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (40 ml) and extracted with ethyl acetate (30 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellowish solid of 5-methyl-3-(4-nitro-3-trifluoromethylphenyloxy)pyrazole (2.74 g, yield: 63.6%).", "mp: 96-98° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.34(s, 3H), 5.77(s, 1H), 7.40(dd, J=2.6 and 8.9 Hz, 1H), 7.56(d, J=2.6 Hz, 1H), 7.98(d, J=8.9 Hz, 1H), 9.20-10.15(br s, 1H).", "Example 17 Potassium carbonate (2.8 g, 20.0 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (1.47 g, 15.0 mmol) and 4-fluoro-3-trifluoromethylbenzonitrile (2.8 g, 15.0 mmol) in DMF (30 ml), and the mixture was stirred under heating at 80° C. for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (70 ml) and extracted with ethyl acetate (30 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a yellowish solid of 3-(4-cyano-2-trifluoromethylphenyloxy)-5-methylpyrazole (1.7 g, yield: 42.4%).", "mp: 123-125° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.28(d, J=0.5 Hz, 3H), 5.75(d, J=0.5 Hz, 1H), 7.30(d, J=8.8 Hz, 1H), 7.75(dd, J=2.0 and 8.8 Hz, 1H), 7.96(d, J=2.0 Hz, 1H), 9.60-11.60(br s, 1H).", "Example 18 Potassium carbonate (2.8 g, 20.0 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (1.47 g, 15.0 mmol) and 3,5-dichloro-4-fluorobenzotrifluoride (3.5 g, 15.0 mmol) in DMF (30 ml), and the mixture was stirred under heating at 60° C. for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (70 ml) and extracted with ethyl acetate (30 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10→1/7), to give a white solid of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole (2.5 g, yield: 54.0%).", "mp: 153-155° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.27(s, 3H), 5.68(s, 1H), 7.64(s, 2H), 8.75-9.70(br s, 1H).", "Examples 19-29 Reactions of 3,5-dichloro-4-fluorobenzotrifluoride with 3-hydroxypyrazole derivative (Example 19: 5-ethyl-3-hydroxypyrazole, Example 20: 3-hydroxy-5-isopropylpyrazole, Example 21: 5-tert-butyl-3-hydroxypyrazole, Example 22: 5-cyclopropyl-3-hydroxypyrazole, Example 23: 4-ethyl-3-hydroxy-5-methylpyrazole, Example 24: 3-hydroxy-5-(methoxymethyl)pyrazole, Example 25: ethyl(3-hydroxypyrazol-5-yl)acetate, Example 26: methyl 3-hydroxypyrazole-5-carboxylate, Example 27: 4,5-dimethyl-3-hydroxypyrazole, Example 28: 4-ethyl-3-hydroxy-5-methylpyrazole, Example 29: 5-(4-chlorophenyl)-3-hydroxypyrazole) were carried out in the same manner as in Example 18, to give corresponding 3-aryloxypyrazole derivatives.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 19 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-ethylpyrazole/white solid/yield: 35.7%/mp: 118-121° C./1H-NMR(CDCl3, TMS, ppm): δ 1.26(t, J=7.5 Hz, 3H), 2.62(q, J=7.5 Hz, 2H), 5.69(s, 1H), 7.65(s, 2H), 8.90-9.30(br s, 1H).", "Example 20 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-isopropylpyrazole/white solid/yield: 59.7%/mp: 98-101° C./1H-NMR(CDCl3, TMS, ppm): δ 1.26(d, J=6.9 Hz, 6H), 2.90(sep, J=6.9 Hz, 1H), 5.68(d, J=0.2 Hz, 1H), 7.65(d, J=0.2 Hz, 2H), 9.06(br s, 1H).", "Example 21 5-tert-butyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazole/white solid/yield: 68.3%/mp: 170-172° C./1H-NMR(CDCl3, TMS, ppm): δ 1.30(s, 9H), 5.69(s, 1H), 7.64(s, 2H), 8.85-9.15(m, 1H).", "Example 22 5-cyclopropyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazole/white solid/yield: 16.8%/mp: 119-121° C./1H-NMR(CDCl3, TMS, ppm): δ 0.65-0.77(m, 2H), 0.90-1.05(m, 2H), 1.70-1.85(m, 1H), 5.54(s, 1H), 7.65(s, 2H), 8.80-9.75(br s, 1H).", "Example 23 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-(methoxymethyl)pyrazole/white solid/yield: 64.5%/mp: 141-143° C./1H-NMR(CDCl3, TMS, ppm): δ 3.41(s, 3H), 4.46(s, 2H), 5.82(s, 1H), 7.64(s, 2H), 9.20˜9.65(br s, 1H).", "Example 24 methyl 3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazole-5-carboxylate/white solid/yield: 43.0%/mp: 114-116° C./1H-NMR(CDCl3, TMS, ppm): δ 3.93(s, 3H), 6.41(s, 1H), 7.65(d, J=0.4 Hz, 2H), 10.10(br s, 1H).", "Example 25 ethyl {3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazol-5-yl}acetate/colorless viscous substance/yield: 51.1%/1H-NMR(CDCl3, TMS, ppm): δ 1.30(t, J=7.2 Hz, 3H), 3.71(s, 2H), 4.23(q, J=7.2 Hz, 2H), 5.79(s, 1H), 7.65(s, 2H).", "(Amino proton was not assigned.)", "Example 26 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-4,5-dimethylpyrazole/white solid/yield: 34.0%/mp: 220-223° C./1H-NMR(CDCl3, TMS, ppm): δ 1.90(s, 3H), 1.93(s, 3H), 7.71(s, 2H).", "(Amino proton was not assigned.)", "Example 27 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-4-ethyl-5-methylpyrazole/white solid/yield: 38.3%/mp: 215-218° C./1H-NMR(CDCl3, TMS, ppm): δ 1.10(t, J=7.5 Hz, 3H), 1.93(s, 3H), 2.34(q, J=7.5 Hz, 2H), 7.69(s, 2H), 9.50-12.50(br s, 1H).", "Example 28 5-(4-chlorophenyl)-3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazole/white solid/yield: 55.1%/mp: 181-183° C./1H-NMR(CDCl3, TMS, ppm): δ 5.99(s, 1H), 7.20-7.40(m, 4H), 7.51(s, 2H), 10.35-11.50(br s, 1H).", "Example 29 Potassium carbonate (1.66 g, 12 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (2.45 g, 25.0 mmol) and 3-chloro-4,5-difluorobenzotrifluoride (2.5 g, 25.0 mmol) in DMF (40 ml), and the mixture was stirred under heating at 70° C. for 4 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (100 ml) and extracted with ethyl acetate (50 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a white solid of 3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole (2.29 g, yield: 33.8%).", "mp: 171-174° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.27(s, 3H), 5.71(s, 1H), 7.37(dd, JHF=1.8 and 9.4 Hz, 1H), 7.54(s, 1H), 8.85-9.55(br s, 1H).", "Example 30 Sodium hydride (60% in oil, 0.2 g, 5.5 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (0.49 g, 5.0 mmol) and 3,5-dinitro-2-fluorobenzotrifluoride (1.35 g, 5.0 mmol) in DMF (10 ml), and the mixture was stirred under heating at 70° C. for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (30 ml) and extracted with ethyl acetate (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7), to give a yellowish solid of 3-(2,4-dinitro-6-trifluoromethylphenyloxy)-5-methylpyrazole (0.58 g, yield: 34.9%).", "mp: 145-147° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.29(s, 3H), 5.84(s, 1H), 8.82(m, 2H), 9.01(d, J=2.7 Hz, 1H).", "Example 31 Sodium hydride (60% in oil, 0.44 g, 11.0 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (0.98 g, 10.0 mmol) and 3-chloro-4-fluoro-5-nitrobenzotrifluoride (1.5 g, 10.0 mmol) in DMF (30 ml), and the mixture was stirred under heating at 60° C. for 7 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (80 ml) and extracted with ethyl acetate (30 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7→1/3), to give a yellowish solid of 3-(2-chloro-6-nitro-4-trifluoromethylphenyloxy)-5-methylpyrazole (1.8 g, yield: 56.6%).", "mp: 120-123° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.29(s, 3H), 5.79(s, 1H), 7.98(d, J=1.8 Hz, 1H), 8.17(d, J=1.8 Hz, 1H), 8.65-9.30(br s, 1H).", "Example 32 Sodium hydride (60% in oil, 0.2 g, 5.5 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (0.49 g, 5.0 mmol) in DMF (10 ml) at 0° C., and the mixture was stirred for 30 minutes while it was allowed to have room temperature gradually.", "Then, 2,3,5-trichloropyridine (0.9 g, 5.0 mmol) was added, and the mixture was stirred under heating at 70° C. for 2 days.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (20 ml) and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7→1/2), to give a white solid of 3-(3,5-dichloropyridin-2-yloxy)-5-methylpyrazole (0.7 g, yield: 57.4%).", "mp: 109-111° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.32(s, 3H), 5.86(s, 1H), 7.77(d, J=2.3 Hz, 1H), 8.03(d, J=2.3 Hz, 1H), 9.40-11.50(br s, 1H).", "Examples 34-35 Reactions of 3-hydroxy-5-methylpyrazole with pyridine derivative (Example 34: 2-chloro-6-methoxy-3-nitropyridine, Example 35: 2-chloro-4-methyl-5-nitropyridine) were carried out in the same manner as in Example 33, to give corresponding 3-aryloxypyrazole derivatives.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 33 5-methyl-3-(6-methoxy-3-nitropyridin-2-yloxy)pyrazole/yellow solid/yield: 63.6%/mp: 133-136° C./1H-NMR(CDCl3, TMS, ppm): δ 2.32(d, J=0.6 Hz, 3H), 3.80(s, 3H), 5.90(d, J=0.6 Hz, 1H), 6.51(dd, J=1.7 and 8.8 Hz, 1H), 8.39(d, J=8.8 Hz, 1H), 10.90(br s, 1H).", "Example 34 5-methyl-3-(4-methyl-5-nitropyridin-2-yloxy)pyrazole/white solid/yield: 39.8%/mp: 136-139° C./1H-NMR(CDCl3, TMS, ppm): δ 2.35(d, J=0.5 Hz, 3H), 2.67(s, 3H), 5.91(d, J=0.5 Hz, 1H), 6.94(s, 1H), 8.93(s, 1H), 9.59(br s, 1H).", "Example 35 Potassium carbonate (7.60 g, 55.0 mmol) was added to a solution of 3-hydroxy-5-methylpyrazole (4.90 g, 50.0 mmol) and 2,3-dichloro-5-trifluoromethylpyridine (10.8 g, 50.0 mmol) in DMF (50 ml) at room temperature, and the mixture was stirred under heating at 60° C. for 4 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (100 ml) and extracted with ethyl acetate (30 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/2), to give a white solid of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole (10.5 g, yield: 76.0%).", "mp: 93-95° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.32(s, 3H), 5.90(s, 1H), 7.98(d, J=2.0 Hz, 1H), 8.33(m, 1H), 10.80-11.60(br s, 1H).", "Examples 36-39 Reactions of 2,3-dichloro-5-trifluoromethylpyridine with 3-hydroxypyrazole derivative (Example 36: 3-hydroxy-5-trifluoromethylpyrazole, Example 37: 5-ethyl-3-hydroxypyrazole, Example 38: 4,5-dimethylpyrazole-3-hydroxy, Example 39: 4-ethyl-3-hydroxy-5-methylpyrazole) were carried out in the same manner as in Example 35, to give corresponding 3-aryloxypyrazole derivatives.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 36 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-trifluoromethylpyrazole/colorless viscous substance/yield: 41.5%/1H-NMR(CDCl3, TMS, ppm): δ 6.53(s, 1H), 8.05(d, J=2.5 Hz, 1H), 8.38(m, 1H), 10.90-11.65(br s, 1H).", "Example 37 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-ethylpyrazole/white solid/yield: 85.2%/mp: 55-57° C./1H-NMR(CDCl3, TMS, ppm): δ 1.27(t, J=7.5 Hz, 3H), 2.70(q, J=7.5 Hz, 2H), 5.92(s, 1H), 7.98(d, J=2.5 Hz, 1H), 8.34(m, 1H), 10.15-10.70(br s, 1H).", "Example 38 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4,5-dimethylpyrazole/white solid/yield: 86.9%/mp: 102-103° C./1H-NMR(DMSO-d6, DMSO, ppm): δ 1.68(s, 3H), 2.17(s, 3H), 8.53(m, 1H), 8.57(m, 1H), 12.05-12.25(br s, 1H).", "Example 39 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4-ethyl-5-methylpyrazole/white solid/yield: 77.6%/mp: 74-75° C./1H-NMR(DMSO-d6, DMSO, ppm): δ 0.93(t, J=7.6 Hz, 3H), 2.14(q, J=7.6 Hz, 2H), 2.19(s, 3H), 8.45-8.65(m, 2H), 12.00-12.30(br s, 1H).", "Example 40 Potassium hydrogencarbonate (0.15 g, 1.5 mmol) was added to a solution of 3-(3-fluoro-4-nitrophenyloxy)-5-methylpyrazole (0.36 g, 1.5 mmol) in ethanol (5 ml), and the mixture was refluxed under heating for 10 hours.", "After completion of the reaction, the reaction mixture was poured into water (10 ml) and extracted with ethyl acetate (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove magnesium sulfate, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a yellow solid of 3-(3-ethoxy-4-nitrophenyloxy)-5-methylpyrazole (0.17 g, yield: 42.3%).", "mp: 123-124° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.47(t, J=7.0 Hz, 3H), 2.33(d, J=0.5 Hz, 3H), 4.13(q, J=7.0 Hz, 2H), 5.73(d, J=0.5 Hz, 1H), 6.68(dd, J=2.5 and 9.1 Hz, 1H), 6.81(d, J=2.5 Hz, 1H), 7.91(d, J=9.1 Hz, 1H), 9.53(br s, 1H).", "Example 41 Reaction of 3-(5-fluoro-2-nitrophenyloxy)-5-methylpyrazole with ethanol was carried out in the presence of potassium hydrogencarbonate in the same manner as in Example 40, to give a yellow viscous substance of 3-(5-ethoxy-2-nitrophenyloxy)-5-methylpyrazole (yield: 59.8%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.39(t, J=7.0 Hz, 3H), 2.28(s, 3H), 4.04(q, J=7.0 Hz, 2H), 5.72(s, 1H), 6.68(dd, J=2.6 and 9.1 Hz, 1H), 6.73(d, J=2.6 Hz, 1H), 8.04(d, J=9.1 Hz, 1H), 9.62(br s, 1H).", "Example 42 A solution of 5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole (5.3 g, 18.5 mmol) in ethanol (200 ml) was placed in an autoclave, and 10% palladium carbon (2.0 g) was added.", "An atmosphere in the autoclave was fully replaced with hydrogen gas, and hydrogen gas was filled up to 5 kg/cm2.Then, the reaction solution was stirred at room temperature for 4 hours.", "After completion of the reaction, the catalyst was separated by filtration using Celite, and the solvent was distilled off from the filtrate under reduced pressure, to give a white solid of 3-(4-amino-2-trifluoromethylphenyloxy)-5-methylpyrazole (3.6 g, yield: 76.1%).", "mp: 143-145° C.; 1H-NMR(DMSO-d6, DMSO, ppm): δ 2.17(s, 3H), 5.33(br s, 2H), 5.44(s, 1H), 6.78(dd, J=2.6 and 8.8 Hz, 1H), 6.88(d, J=2.6 Hz, 1H), 6.95(d, J=8.8 Hz, 1H), 11.80-11.95(br s, 1H).", "Examples 43-45 3-(Substituted phenyloxy)pyrazole derivative having a nitro group (Example 43: 5-methyl-3-(4-nitrophenyloxy)pyrazole, Example 44: 3-(3-chloro-4-nitrophenyloxy)-5-methylpyrazole, Example 45: 5-methyl-(4-nitro-3-trifluoromethylphenyloxy)pyrazole) was reduced in a hydrogen gas atmosphere in the same manner as in Example 42, to give corresponding 3-(substituted phenyloxy)pyrazole derivative having an amino group.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 43 3-(4-aminophenyloxy)-5-methylpyrazole/brown solid/yield: 49.7%/mp: 170-173° C./1H-NMR(CDCl3, TMS, ppm): δ 1.48-2.13(br s, 2H), 2.23(d, J=0.7 Hz, 3H), 5.47(d, J=0.7 Hz, 1H), 6.64(dd, J=2.2 and 7.1 Hz, 2H), 6.95(dd, J=2.2 and 7.1 Hz, 2H).", "(Amino proton was not assigned.)", "Example 44 3-(4-amino-3-chlorophenyloxy)-5-methylpyrazole/brown viscous substance/yield: 27.4%/1H-NMR(CDCl3, TMS, ppm): δ 2.26(d, J=0.4 Hz, 3H), 3.63(br s, 2H), 5.60(d, J=0.4 Hz, 1H), 6.70(d, J=8.5 Hz, 1H), 6.91(dd, J=2.3 Hz and 8.5 Hz, 1H), 7.00(d, J=2.3 Hz, 1H).", "(Amino proton was not assigned.)", "Example 45 3-(4-amino-3-trifluoromethylphenyloxy)-5-methylpyrazole/brown viscous substance/yield: 97.8%/1H-NMR(CDCl3, TMS, ppm): δ 2.24(s, 3H), 3.85-4.25(br s, 2H), 5.53(s, 1H), 6.71(d, J=8.8 Hz, 1H), 7.14(dd, J=2.8 and 8.8 Hz, 1H), 7.25(d, J=2.8 Hz, 1H).", "(Amino proton was not assigned.)", "Example 46 Concentrated hydrochloric acid (3 ml) and water (6 ml) were added to 3-(4-amino-3-trifluoromethylphenyloxy)-5-methylpyrazole, and the mixture was cooled to 0° C. Then, a solution of sodium nitrite (0.54 g, 7.8 mmol) in concentrated hydrochloric acid (2 ml) and water (2 ml) was dropwise added, and the mixture was stirred at an ambient temperature for 1 hour.", "Then, hypophosphorous acid (2.57 g, 38.9 mmol) was added, and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 1N sodium hydroxide (50 ml) and extracted with ethyl acetate (50 ml×2).", "An organic layer was washed with water (50 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/4), to give a yellow viscous substance of 5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole (1.47 g, yield: 78.0%).", "1H-NMR(CDCl3, TMS, ppm): δ 2.2.3(s, 3H), 5.63(s, 1H), 7.27˜7.46(m, 4H), 10.91(br s, 1H).", "Examples 47-49 3-(Substituted phenyloxy)pyrazole derivative having an amino group (Example 47: 3-(4-aminophenyloxy)-5-methylpyrazole, Example 48: 3-(4-amino-3-chlorophenyloxy)-5-methylpyrazole, Example 49: 3-(4-amino-2-trifluoromethylphenyloxy)-5-methylpyrazole) was de-aminated via a diazonium salt in the same manner as in Example 46, to give 3-(substituted phenyloxy)pyrazole derivative.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 47 5-methyl-3-phenyloxypyrazole/brown viscous substance/yield: 72.2%/1H-NMR(CDCl3, TMS, ppm): δ 2.22(s, 3H), 5.58(s, 1H), 7.05-7.12(m, 3H), 7.27-7.35(m, 2H).", "(Amino proton was not assigned.)", "Example 48 3-(3-chlorophenyloxy)-5-methylpyrazole/yellow viscous substance/yield: 22.4%/1H-NMR(CDCl3, TMS, ppm): δ 2.23(s, 3H), 5.62(s, 1H), 6.95-7.13(m, 3H), 7.21(d, J=8.1 Hz, 1H), 10.95(br s, 1H).", "Example 49 5-methyl-3-(2-trifluoromethylphenyloxy)pyrazole/yellowish viscous substance/yield: 75.8%/1H-NMR(CDCl3, TMS, ppm): δ 2.20(d, J=0.4 Hz, 3H), 5.59(d, J=0.4 Hz, 1H), 7.12-7.20(m, 2H), 7.42-7.49(m, 1H), 7.64(dd, J=0.9 and 7.7 Hz, 1H), 10.70-12.19(br s, 1H).", "Example 50 3-(4-Amino-3-trifluoromethylphenyloxy)-5-methylpyrazole (2.00 g, 7.8 mmol) was added to a mixed solution of concentrated hydrochloric acid (2.1 ml) and acetone (20 ml) at room temperature, and the mixture was stirred at room temperature for 20 minutes.", "After the mixture was cooled below 0° C., a solution of sodium nitrite (0.54 g, 7.8 mmol) in water (3 ml) was dropwise added, and the mixture was stirred at an ambient temperature for 30 minutes.", "Then, cuprous chloride (0.85 g, 8.6 mmol) was little by little added at 0° C., and the mixture was stirred at room temperature for 1 hour.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (50 ml) and extracted with ethyl acetate (50 ml×2).", "An organic layer was washed with water (50 ml×3), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellowish viscous substance of 3-(4-chloro-3-trifluoromethylphenyloxy)-5-methylpyrazole (2.12 g, yield: 98.7%).", "1H-NMR(CDCl3, TMS, ppm): δ 2.25(s, 3H), 5.64(s, 1H), 7.23(dd, J=3.0 and 8.8 Hz, 1H), 7.31-7.46(m, 2H), 10.56(br s, 1H).", "Example 51 A diazonium salt was prepared from 3-(4-amino-2-trifluoromethylphenyloxy)-5-methylpyrazole and reacted with cuprous chloride in the same manner as in Example 50, to give a yellowish viscous substance of 3-(4-chloro-2-trifluoromethylphenyloxy)-5-methylpyrazole (yield: 56.0%).", "1H-NMR(CDCl3, TMS, ppm): δ 2.24(s, 3H), 5.63(s, 1H), 7.16(d, J=8.9 Hz, 1H), 7.41(dd, J=2.5 and 8.9 Hz, 1H), 7.61(d, J=2.5 Hz, 1H), 10.93(br s, 1H).", "Example 52 Sulfuryl chloride (0.48 g, 3.6 mmol) was added to a solution of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.93 g, 3.0 mmol) in acetic acid (10 ml), and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of 4-chloro-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.57 g, yield: 55.0%).", "mp: 156-158° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.28(s, 3H), 7.65(s, 2H), 8.75-9.15(br s, 1H).", "Example 53 Triethylamine (0.13 g, 1.3 mmol) was added to a solution of 5-methyl-3-phenyloxypyrazole (0.20 g, 1.2 mmol) in ethyl acetate (5 ml), and the mixture cooled to 0° C. Then, ethyl isocyanate (0.09 g, 1.3 mmol) was added, and the mixture was stirred for 30 minutes at an ambient temperature.", "And, the mixture was stirred for 4 hours while it was allowed to have room temperature gradually.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml) and extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/15), to give a yellow viscous substance of N-ethyl-5-methyl-3-phenyloxypyrazole-1-carboxamide (0.16 g, yield: 56.4%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.22 (t, J=7.3 Hz, 3H), 2.58 (d, J=0.8 Hz, 3H), 3.38(dq, J=1.3 and 7.3 Hz, 2H), 5.67(q, J=0.8 Hz, 1H), 6.99-7.07(br s, 1H), 7.12-7.19(m, 3H), 7.33-7.39(m, 2H).", "Example 54 Potassium carbonate (0.19 g, 1.5 mmol) and ethyl isocyanate (0.09 g, 1.3 mmol) were added to a solution of 5-methyl-3-(4-nitrophenyloxy)pyrazole (0.29 g, 1.3 mmol) in ethyl acetate (15 ml), and the mixture was stirred at room temperature for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with diethyl ether (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/20), to give a white solid of N-ethyl-5-methyl-3-(4-nitrophenyloxy)pyrazole-1-carboxamide (0.38 g, yield: 99.2%).", "mp: 105-107° C., 1H-NMR(CDCl3, TMS, ppm): δ 1.24(t, J=7.3 Hz, 3H), 2.64(d, J=0.7 Hz, 3H), 3.40(dq, J=5.9 and 7.2 Hz, 2H), 5.83(q, J=0.7 Hz, 1H), 6.75(m, 1H), 7.20-7.30(m, 2H), 8.26(dd, J=2.2 and 7.0 Hz, 2H).", "Example 55 Triethylamine (0.19 g, 1.4 mmol) and phenyl isocyanate (0.17 g, 1.4 mmol) were added to a solution of 5-methyl-3-(2-trifluoromethylphenyloxy)pyrazole (0.30 g, 1.2 mmol) in ethyl acetate (5 ml) at 0° C., and the mixture was allowed to have room temperature gradually and stirred for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml) and extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/15), to give an orange solid of N-phenyl-5-methyl-3-(2-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.39 g, yield: 87.5%).", "mp: 87-89° C., 1H-NMR(CDCl3, TMS, ppm): δ 2.65(d, J=0.7 Hz, 3H), 5.78(d, J=0.7 Hz, 1H), 7.14(ddd, J=1.2, 2.3 and 7.3 Hz, 1H), 7.28-7.38(m, 4H), 7.51-7.60(m, 3H), 7.69-7.73(m, 1H), 8.90(br s, 1H).", "Example 56 Triethylamine (0.17 g, 1.7 mmol) and isopropyl isocyanate (0.14 g, 1.7 mmol) were added to a solution of 5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole (0.36 g, 1.5 mmol) in ethyl acetate (5 ml), and the mixture was stirred at room temperature for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml) and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/15), to give a colorless viscous substance of N-isopropyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.15 g, yield: 30.6%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.25(d, J=6.6 Hz, 6H), 2.60(d, J=0.6 Hz, 3H), 4.05(sep, J=6.6 and 6.6 Hz, 1H), 5.71(q, J=0.6 Hz, 1H), 6.81(br d, J=6.6 Hz, 1H), 7.30-7.35(m, 1H), 7.40-7.52(m, 3H).", "Examples 57-82 Reactions of 5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole and isocyanates (Example 57: ethyl isocyanate, Example 58: tert-butyl isocyanate, Example 59: hexyl isocyanate, Example 60: cyclohexyl isocyanate, Example 61: allyl isocyanate, Example 62: 2-chloroethyl isocyanate, Example 63: ethyl isocyanatoacetate, Example 64: phenyl isocyanate, Example 65: 2-chlorophenyl isocyanate, Example 66: 3-chlorophenyl isocyanate, Example 67: 3-methylphenyl isocyanate, Example 68: 3-nitrophenyl isocyanate, Example 69: 4-chlorophenyl isocyanate, Example 70: 4-fluorophenyl isocyanate, Example 71: 4-trifluoromethylphenyl isocyanate, Example 72: 2,4-dichlorophenyl isocyanate, Example 73: 2,4-difluorophenyl isocyanate, Example 74: 3,4-dichlorophenyl isocyanate, Example 75: 2,6-dichlorophenyl isocyanate, Example 76: 4-chloro-2-methylphenyl isocyanate, Example 77: 2-methyl-4-nitrophenyl isocyanate, Example 78: 2-chloro-6-methylphenyl isocyanate, Example 79: 2,3,4-trifluorophenyl isocyanate, Example 80: 4-chloro-5-cyclopentyloxy-2-fluorophenyl isocyanate, Example 81: benzyl isocyanate, Example 82: α-phenethyl isocyanate) were carried out in ethyl acetate in the presence of potassium carbonate in the same manner as in Example 56, to give corresponding N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 57 N-ethyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellow viscous substance/yield: 63.8%/1H-NMR(CDCl3, TMS, ppm): δ 1.23(t, J=7.3 Hz, 3H), 2.60(d, J=0.8 Hz, 3H), 3.39(dq, J=6.0 and 7.3 Hz, 2H), 5.73(q, J=0.8 Hz, 1H), 6.80-7.05(m, 1H), 7.25-7.55(m, 4H).", "Example 58 N-tert-butyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/colorless viscous substance/yield: 11.7%/1H-NMR(CDCl3, TMS, ppm): δ 1.43(s, 9H), 2.59(d, J=0.6 Hz, 3H), 5.69(q, J=0.6 Hz, 1H), 6.95(br s, 1H), 7.29-7.34(m, 1H), 7.45-7.51(m, 3H).", "Example 59 N-hexyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellow viscous substance/yield: 53.1%/1H-NMR(CDCl3, TMS, ppm): δ 0.86-0.91(m, 3H), 1.26-1.41(m, 6H), 1.52-1.64(m, 2H), 2.60(d, J=0.7 Hz, 3H), 3.29-3.37(m, 2H), 5.73(q, J=0.7 Hz, 1H), 6.95(br s, 1H), 7.32-7.51(m, 4H).", "Example 60 N-cyclohexyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellow viscous substance/yield: 26.7%/1H-NMR(CDCl3, TMS, ppm): δ 1.11-1.47(m, 5H), 1.59-1.80(m, 3H), 1.94-2.04(m, 2H), 2.60(d, J=0.7 Hz, 3H), 3.65-3.80(m, 1H), 5.71(q, J=0.7 Hz, 1H), 6.88(br d, J=7.5 Hz, 1H), 7.30-7.35(m, 1H), 7.40-7.51(m, 3H).", "Example 61 N-allyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/colorless viscous substance/yield: 83.9%/1H-NMR(CDCl3, TMS, ppm): δ 2.61(d, J=0.8 Hz, 3H), 3.97(tt, J=1.5 and 5.8 Hz, 2H), 5.15-5.30(m, 2H), 5.75(q, J=0.8 Hz, 1H), 5.80-5.97(m, 1H), 7.06(br s, 1H), 7.32-7.36(m, 1H), 7.40-7.52(m, 3H).", "Example 62 N-(2-chloroethyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/colorless viscous substance/yield: 71.0%/1H-NMR(CDCl3, TMS, ppm): δ 2.60(d, J=0.7 Hz, 3H), 3.67-3.70(m, 4H), 5.76(q, J=0.7 Hz, 1H), 7.30-7.38(m, 2H), 7.41-7.49(m, 3H).", "Example 63 ethyl [{3-(3-trifluoromethylphenyloxy)-5-methylpyrazol-1-yl}carbonylamino]acetate/white solid/yield: 73.7%/mp: 66-68° C./1H-NMR(CDCl3, TMS, ppm): δ 1.29(t, J=7.2 Hz, 3H), 2.60(d, J=0.8 Hz, 3H), 4.10(d, J=5.8 Hz, 2H), 4.24(q, J=7.2 Hz, 2H), 5.76(q, J=0.8 Hz, 1H), 7.31-7.49(m, 5H).", "Example 64 N-phenyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellowish solid/yield: 94.0%/mp: 53-54° C./1H-NMR(CDCl3, TMS, ppm): δ 2.67(d, J=0.8 Hz, 3H), 5.80(q, J=0.8 Hz, 1H), 7.15(ddt, J=1.1, 7.4 and 7.4 Hz, 1H), 7.30-7.60(m, 8H), 8.89(br s, 1H).", "Example 65 N-2-chlorophenyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellowish solid/yield: 70.7%/mp: 84-86° C./1H-NMR(CDCl3, TMS, ppm): δ 2.67(d, J=0.7 Hz, 3H), 5.85(q, J=0.7 Hz, 1H), 7.06(ddt, J=1.5, 7.8 and 7.8 Hz, 1H), 7.20-7.60(m, 6H), 8.30(dd, J=1.5 and 8.3 Hz, 1H), 9.56(br s, 1H).", "Example 66 N-3-chlorophenyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellow viscous substance/yield: 94.0%/1H-NMR(CDCl3, TMS, ppm): δ 2.66(d, J=0.9 Hz, 3H), 5.81(q, J=0.9 Hz, 1H), 7.11(ddd, J=1.3, 1.9 and 7.5 Hz, 1H), 7.23-7.39(m, 3H), 7.45-7.55(m, 3H), 7.70(t, J=1.9 Hz, 1H), 8.91(br s, 1H).", "Example 67 N-(3-methylphenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellow solid/yield: 92.2%/mp: 47-49° C./1H-NMR(CDCl3, TMS, ppm): δ 2.36(s, 3H), 2.66(d, J=0.7 Hz, 3H), 5.80(q, J=0.7 Hz, 1H), 6.96(dd, J=0.4 and 7.3 Hz, 1H), 7.20-7.29(m, 2H), 7.29-7.41(m, 2H), 7.43-7.54(m, 3H), 8.84(br s, 1H).", "Example 68 N-(3-nitrophenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellow solid/yield: 80.5%/mp: 83-86° C./1H-NMR(CDCl3, TMS, ppm): δ 2.68(d, J=0.7 Hz, 3H), 5.84(q, J=0.7 Hz, 1H), 7.36-7.40(m, 1H), 7.46-7.57(m, 4H), 7.83(ddd, J=0.9, 2.2 and 8.2 Hz, 1H), 7.99(ddd, J=0.9, 2.2 and 8.2 Hz, 1H), 8.52(dd, J=2.2 and 2.2 Hz, 1H), 9.11(br s, 1H).", "Example 69 N-(4-chlorophenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/white solid/yield: 88.9%/mp: 73-74° C./1H-NMR(CDCl3, TMS, ppm): δ 2.66(d, J=0.8 Hz, 3H), 5.81(q, J=0.8 Hz, 1H), 7.29-7.39(m, 3H), 7.44-7.55(m, 5H), 8.88(br s, 1H).", "Example 70 N-(4-fluorophenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellow solid/yield: 77.0%/mp: 70-72° C./1H-NMR(CDCl3, TMS, ppm): δ 2.66(d, J=0.8 Hz, 3H), 5.80(q, J=0.8 Hz, 1H), 7.00˜7.09(m, 2H), 7.35-7.39(m, 1H), 7.43-7.54(m, 5H), 8.84(br s, 1H).", "Example 71 N-(4-trifluoromethylphenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellowish solid/yield: 90.8%/mp: 82-84° C./1H-NMR(CDCl3, TMS, ppm): δ 2.67(d, J=0.9 Hz, 3H), 5.83(q, J=0.9 Hz, 1H), 7.32-7.40(m, 1H), 7.45-7.56(m, 3H), 7.64(dd, J=8.9 and 15.6 Hz, 4H), 9.06(br s, 1H).", "Example 72 N-(2,4-dichlorophenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/colorless viscous substance/yield: 63.7%/mp: 113-114° C./1H-NMR(CDCl3, TMS, ppm): δ 2.66(d, J=0.6 Hz, 3H), 5.85(1H), 7.25-7.30(m, 1H), 7.39-7.56(m, 5H), 8.27(d, J=8.8 Hz, 1H), 9.52(br s, 1H).", "Example 73 N-(2,4-difluorophenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellowish solid/yield: 45.3%/mp: 84-85° C./1H-NMR(CDCl3, TMS, ppm): δ 2.66(d, J=0.8 Hz, 3H), 5.83(q, J=0.8 Hz, 1H), 6.80-6.95(m, 2H), 7.35-7.60(m, 4H), 8.05-8.20(m, 1H), 9.00(br s, 1H).", "Example 74 N-(3,4-dichlorophenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/white solid/yield: 85.2%/mp: 97-99° C./1H-NMR(CDCl3, TMS, ppm): δ 2.65(d, J=0.7 Hz, 3H), 5.82(q, J=0.7 Hz, 1H), 7.32(dd, J=2.5 and 8.8 Hz, 1H), 7.35-7.56(m, 5H), 7.81(d, J=2.4 Hz, 1H), 8.90(br s, 1H).", "Example 75 N-(2,6-dichlorophenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellowish viscous substance/yield: 50.2%/1H-NMR(CDCl3, TMS, ppm): δ 2.64(d, J=0.8 Hz, 3H), 5.84(q, J=0.8 Hz, 1H), 7.21(dd, J=7.5 and 8.8 Hz, 1H), 7.39-7.53(m, 6H), 8.57(br s, 1H).", "Example 76 N-(4-chloro-2-methylphenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/white solid/yield: 83.7%/mp: 95-96° C./1H-NMR(CDCl3, TMS, ppm): δ 2.21(s, 3H), 2.65(d, J=0.8 Hz, 3H), 5.83(q, J=0.8 Hz, 1H), 7.17-7.23(m, 2H), 7.37-7.54(m, 4H), 7.91(d, J=8.4 Hz, 1H), 8.82(br s, 1H).", "Example 77 N-(2-methyl-4-nitrophenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellowish solid/yield: 16.7%/mp: 165-166° C./1H-NMR(CDCl3, TMS, ppm): δ 2.29(s, 3H), 2.68(d, J=0.8 Hz, 3H), 5.88(q, J=0.8 Hz, 1H), 7.39-7.43(m, 1H), 7.47-7.57(m, 3H), 8.08-8.16(m, 2H), 8.37(d, J=9.0 Hz, 1H), 9.24(br s, 1H).", "Example 78 N-(2-chloro-6-methylphenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/white solid/yield: 66.1%/mp: 55-58° C./1H-NMR(CDCl3, TMS, ppm): δ 2.36(s, 3H), 2.64(d, J=0.8 Hz, 3H), 5.83(q, J=0.8 Hz, 1H), 7.12-7.20(m, 2H), 7.30(dd, J=2.9 and 6.5 Hz, 1H), 7.39-7.54(m, 4H), 8.52(br s, 1H).", "Example 79 N-(2,3,4-trifluorophenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/white solid/yield: 73.4%/mp: 102-104° C./1H-NMR(CDCl3, TMS, ppm): δ 2.65(d, J=0.8 Hz, 3H), 5.84(q, J=0.8 Hz, 1H), 6.93-7.05(m, 1H), 7.38-7.42(m, 1H), 7.45-7.56(m, 3H), 7.85˜7.92(m, 1H), 9.30(br s, 1H).", "Example 80 N-(2-fluoro-4-chloro-5-cyclopentyloxyphenyl)-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/white solid/yield: 52.0%/mp: 81-83° C./1H-NMR(CDCl3, TMS, ppm): δ 1.55-1.70(m, 2H), 1.75-2.00(m, 6H), 2.66(d, J=0.8 Hz, 3H), 4.75-4.90(m, 1H), 5.82(q, J=0.8 Hz, 1H), 7.14(d, J=10.2 Hz, 1H), 7.35-7.55(m, 4H), 7.97(d, J=7.3 Hz, 1H), 9.00-9.20(m, 1H).", "Example 81 N-benzyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/colorless viscous substance/yield: 56.8%/1H-NMR(CDCl3, TMS, ppm): δ 2.62(d, J=0.8 Hz, 3H), 4.53(d, J=6.0 Hz, 2H), 5.74(q, J=0.8 Hz, 1H), 7.20˜7.50(m, 10H).", "Example 82 N-α-phenethyl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/colorless viscous substance/yield: 63.7%/1H-NMR(CDCl3, TMS, ppm): δ 1.57(d, J=6.9 Hz, 3H), 2.58(s, 3H), 5.06(dq, J=7.1 and 7.1 Hz, 1H), 5.71(s, 1H), 7.26-7.36(m, 7H), 7.43-7.51(m, 3H).", "Example 83 Triethylamine (0.13 g, 1.3 mmol) and phenyl isocyanate (0.15 g, 1.3 mmol) were added to a solution of 3-(3-chlorophenyloxy)-5-methylpyrazole (0.25 g, 1.2 mmol) in ethyl acetate (5 ml) at 0° C., and while the mixture was allowed to have room temperature gradually, it was stirred for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml) and extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/15), to give a yellow viscous substance of N-phenyl-3-(3-chlorophenyloxy)-5-methylpyrazole-1-carboxamide (0.34 g, yield: 84.5%).", "1H-NMR(CDCl3, TMS, ppm): δ 2.66(d, J=0.8 Hz, 3H), 5.78(q, J=0.8 Hz, 1H), 7.05-7.11(m, 1H), 7.14-7.21(m, 3H), 7.28-7.39(m, 3H), 7.52-7.56(m, 2H), 8.91(br s, 1H).", "Example 84 Triethylamine (0.17 g, 1.65 mmol) and ethyl isocyanate (0.31 g, 2.2 mmol) were added to a solution of 3-(4-chloro-2-trifluoromethylphenyloxy)-5-methylpyrazole (0.55 g, 2.0 mmol) in ethyl acetate (5 ml), and the mixture was stirred at room temperature for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml) and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/15), to give a colorless viscous substance of N-ethyl-3-(4-chloro-2-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.53 g, yield: 75.9%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.22(t, J=7.3 Hz, 3H), 2.59(d, J=0.7 Hz, 3H), 3.37(q, J=7.3 Hz, 2H), 5.73(q, J=0.7 Hz, 1H), 6.91(br s, 1H), 7.22(d, J=8.8 Hz, 1H), 7.49(dd, J=2.5 and 8.8 Hz, 1H), 7.65(d, J=2.5 Hz, 1H).", "Example 85 Triethylamine (0.32 g, 3.2 mmol) and ethyl isocyanate (0.23 g, 3.2 mmol) were added to a solution of 3-(4-fluoro-2-nitrophenyloxy)-5-methylpyrazole (0.69 g, 2.9 mmol) in ethyl acetate (10 ml) at 0° C., and the mixture was allowed to have room temperature gradually and stirred for 5 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml) and extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/15), to give a yellow solid of N-ethyl-3-(4-fluoro-2-nitrophenyloxy)-5-methylpyrazole-1-carboxamide (0.72 g, yield: 79.7%).", "mp: 81-84° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.20(t, J=7.2 Hz, 3H), 2.59(d, J=0.8 Hz, 3H), 3.34(dq, J=1.4 and 7.2 Hz, 2H), 5.78(q, J=0.8 Hz, 1H), 6.71(br s, 1H), 7.34-7.37(m, 2H), 7.73(ddd, J=1.2, 2.2 and 7.7 Hz, 1H).", "Example 86 Potassium carbonate (0.83 g, 6.0 mmol) and tert-butyl isocyanate (0.59 g, 6.0 mmol) were added to a solution of 5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole (1.15 g, 4.0 mmol) in DMF (10 ml), and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a white solid of N-tert-butyl-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)-pyrazole-1-carboxamide (0.80 g, yield: 51.8%).", "mp: 104-106° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.41(s, 9H), 2.61(s, 3H), 5.82(s, 1H), 6.65-6.90(br s, 1H), 7.43(d, J=8.7 Hz, 1H), 7.82(dd, J=2.1 and 8.7 Hz, 1H), 8.23(d, J=2.1 Hz, 1H).", "Examples 87-90 Reactions of 5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole and isocyanates (Example 87: methyl isocyanate, Example 88: ethyl isocyanate, Example 89: propyl isocyanate, Example 90: isopropyl isocyanate) were carried out in the same manner as in Example 86, to give corresponding N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 87 N-methyl-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide/white solid/yield: 89.0%/mp: 70-72° C./1H-NMR(CDCl3, TMS, ppm): δ 2.62(d, J=0.8 Hz, 3H), 2.91(d, J=5.0 Hz, 3H), 5.88(q, J=0.8 Hz, 1H), 6.55-6.85(m, 1H), 7.49(d, J=8.7 Hz, 1H), 7.83(dd, J=2.0 and 8.7 Hz, 1H), 8.25(d, J=2.0 Hz, 1H).", "Example 88 N-ethyl-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellow viscous substance/yield: 25.2%/1H-NMR(CDCl3, TMS, ppm): δ 1.21(t, J=7.3 Hz, 3H), 2.62(d, J=0.7 Hz, 3H), 3.36(dq, J=6.0 and 7.3 Hz, 2H), 5.87(q, J=0.7 Hz, 1H), 6.60-6.95(m, 1H), 7.48(d, J=8.5 Hz, 1H), 7.85(d, J=2.0 and 8.5 Hz, 1H), 8.25(d, J=2.0 Hz, 1H).", "Example 89 N-propyl-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellowish solid/yield: 13.6%/mp: 50-51° C./1H-NMR(CDCl3, TMS, ppm): δ 0.95(t, J=7.4 Hz, 3H), 1.60(tq, J=7.1 and 7.4 Hz, 2H), 2.62(d, J=0.7 Hz, 3H), 3.28(dt, J=6.3 and 7.1 Hz, 2H), 5.87(q, J=0.7 Hz, 1H), 6.65˜6.95(m, 1H), 7.48(d, J=8.8 Hz, 1H), 7.85(dd, J=2.0 and 8.8 Hz, 1H), 8.25(d, J=2.0 Hz, 1H).", "Example 90 N-isopropyl-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellow viscous substance/yield: 70.6%/1H-NMR(CDCl3, TMS, ppm): δ 1.24(d, J=6.6 Hz, 6H), 2.62(d, J=0.7 Hz, 3H), 3.90-4.10(m, 1H), 5.86(q, J=0.7 Hz, 1H), 6.50-6.80(m, 1H), 7.46(d, J=8.6 Hz, 1H), 7.84(dd, J=2.0 and 8.6 Hz, 1H), 8.25(d, J=2.0 Hz, 1H).", "Example 91 Potassium carbonate (0.61 g, 4.4 mmol) and propyl isocyanate (0.34 g, 4.0 mmol) were added to a solution of 5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole (1.15 g, 4.0 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature for 3 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered co remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a yellow viscous substance of N-propyl-5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole-1-carboxamide (1.08 g, yield: 72.5%).", "1H-NMR(CDCl3, TMS, ppm): δ 0.97(t, J=7.4 Hz, 3H), 1.62(tq, J=7.4 and 7.4 Hz, 2H), 2.64(d, J=0.7 Hz, 3H), 3.25-3.40(m, 2H), 5.88(q, J=0.7 Hz, 1H), 6.80-7.05(m, 1H), 7.42(d, J=9.2 Hz, 1H), 8.40(dd, J=2.7 and 9.2 Hz, 1H), 8.59(d, J=2.7 Hz, 1H).", "Examples 93-95 Reactions of 5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole and isocyanates (Example 93: methyl isocyanate, Example 94: ethyl isocyanate, Example 95: isopropyl isocyanate) were carried out in the same manner as in Example 92, to give corresponding N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 92 N-methyl-5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole-1-carboxamide/white solid/yield: 79.7%/mp: 142-144° C./1H-NMR(CDCl3, TMS, ppm): δ 2.64(d, J=0.8 Hz, 3H), 2.95(d, J=5.0 Hz, 3H), 5.89(q, J=0.8 Hz, 1H), 6.75-6.95(m, 1H), 7.43(d, J=9.2 Hz, 1H), 8.40(dd, J=2.7 and 9.2 Hz, 1H), 8.59(d, J=2.7 Hz, 1H).", "Example 93 N-ethyl-5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellowish solid/yield: 86.5%/mp: 95-97° C./1H-NMR(CDCl3, TMS, ppm): δ 1.24(t, J=7.3 Hz, 3H), 2.64(d, J=0.6 Hz, 3H), 3.40(dq, J=5.9 and 7.3 Hz, 2H), 5.88(q, J=0.6 Hz, 1H), 6.75-7.00(m, 1H), 7.42(d, J=9.2 Hz, 1H), 8.38(dd, J=2.8 and 9.2 Hz, 1H), 8.59(d, J=2.8 Hz, 1H).", "Example 94 N-isopropyl-5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole-1-carboxamide/white solid/yield: 77.9%/mp: 90-92° C./1H-NMR(CDCl3; TMS, ppm): δ 1.26(d, J=6.6 Hz, 6H), 2.64(d, J=0.8 Hz, 3H), 4.05(dq, J=1.4 and 6.6 Hz, 1H), 5.86(q, J=0.8 Hz, 1H), 6.60-6.90(m, 1H), 7.40(d, J=9.2 Hz, 1H), 8.40(dd, J=2.7 and 9.2 Hz, 1H), 8.59(d, J=2.7 Hz, 1H).", "Example 95 Potassium carbonate (0.23 g, 1.7 mmol) and ethyl isocyanate (0.11 g, 1.5 mmol) were added to a solution of 3-(4-cyano-2-trifluoromethylphenyloxy)-5-methylpyrazole (0.40 g, 1.5 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a yellowish solid of N-ethyl-3-(4-cyano-2-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.35 g, yield: 69.0%).", "mp: 103-105° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.23(t, J=7.3 Hz, 3H), 2.63(d, J=0.7 Hz, 3H), 3.39(dq, J=5.9 and 7.2 Hz, 2H), 5.85(q, J=0.7 Hz, 1H), 6.80-7.00(m, 1H), 7.38(d, J=8.7 Hz, 1H), 7.81(dd, J=1.8 and 8.7 Hz, 1H), 7.99(d, J=1.8 Hz, 1H).", "Example 96 Triethylamine (0.18 g, 1.8 mmol) and ethyl isocyanate (0.13 g, 1.8 mmol) were added to a solution of 5-methyl-3-(3-methyl-4-nitrophenyloxy)pyrazole (0.35 g, 1.5 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature for 8 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7), to give a white solid of N-ethyl-5-methyl-3-(3-methyl-4-nitrophenyloxy)pyrazole-1-carboxamide (0.27 g, yield: 59.2%).", "mp: 67-69° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.24(t, J=7.3 Hz, 3H), 2.63(s, 6H), 3.40(dq, J=5.9 and 7.3 Hz, 2H), 5.79(q, J=0.8 Hz, 1H), 6.85-7.00(m, 1H), 7.00-7.15(m, 2H), 8.07(d, J=8.6 Hz, 1H).", "Example 97 Reaction of 5-methyl-3-(3-methoxy-4-nitrophenyloxy)pyrazole with ethyl isocyanate was carried out in the same manner as in Example 96, to give a yellow viscous substance of N-ethyl-5-methyl-3-(3-methoxy-4-nitrophenyloxy)pyrazole-1-carboxamide (yield: 62.9%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.24(t, J=7.2 Hz, 3H), 2.63(d, J=0.6 Hz, 3H), 3.40(dq, J=5.9 and 7.2 Hz, 2H), 3.95(s, 3H), 5.81(q, J=0.6 Hz, 1H), 6.75(dd, J=2.4 and 9.0 Hz, 1H), 6.84(d, J=2.4 Hz, 1H), 6.93(br s, 1H), 7.96(d, J=9.0 Hz, 1H).", "Example 98 Triethylamine (0.07 g, 0.7 mmol) and ethyl isocyanate (0.12 g, 0.7 mmol) were added to a solution of 3-(3-ethoxy-4-nitrophenyloxy)-5-methylpyrazole (0.16 g, 0.6 mmol) in ethyl acetate (5 ml) at 0° C., and the mixture was allowed to have room temperature gradually and stirred for 5 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml) and extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/15), to give a yellow solid of N-ethyl-3-(3-ethoxy-4-nitrophenyloxy)-5-methylpyrazole-1-carboxamide (0.13 g, yield: 61.8%).", "mp: 110-112° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.24(t, J=7.3 Hz, 3H), 1.49(t, J=7.0 Hz, 3H), 2.63(d, J=0.5 Hz, 3H), 3.40(dq, J=6.0 and 7.3 Hz, 2H), 4.14(q, J=7.0 Hz, 2H), 5.80(q, J=0.5 Hz, 1H), 6.73(dd, J=2.4 and 9.0 Hz, 1H), 6.82(d, J=2.4 Hz, 1H), 6.92(br s, 1H), 7.92(d, J=9.0 Hz, 1H).", "Example 99 Reaction of 3-(5-ethoxy-2-nitrophenyloxy)-5-methylpyrazole with ethyl isocyanate was carried out in ethyl acetate in the presence of potassium carbonate in the same manner as in Example 99, to give a yellow solid of N-ethyl-3-(5-ethoxy-2-nitrophenyloxy)-5-methylpyrazole-1-carboxamide (yield: 75.9%).", "mp: 73-75° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.20(t, J=7.2 Hz, 3H), 1.44(t, J=7.0 Hz, 3H), 2.59(d, J=0.7 Hz, 3H), 3.35(dq, J=5.9 and 7.2 Hz, 2H), 4.09(q, J=7.0 Hz, 2H), 5.78(q, J=0.7 Hz, 1H), 6.73(d, J=2.4 Hz, 1H), 6.77(dd, J=2.4 and 8.9 Hz, 1H), 6.81(br s, 1H), 8.06(d, J=8.9 Hz, 1H).", "Example 100 Triethylamine (0.13 g, 1.3 mmol) and ethyl isocyanate (0.09 g, 1.3 mmol) were added to a solution of 3-(4-amino-3-trifluoromethylphenyloxy)-5-methylpyrazole (0.30 g, 1.2 mmol) in ethyl acetate (5 ml) at 0° C., and the mixture was allowed to have room temperature gradually and stirred for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml) and extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/15), to give a yellowish solid of N-ethyl-3-(4-amino-3-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.19 g, yield: 50.5%).", "mp: 102-104° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.22(t, J=7.3 Hz, 3H), 2.56(d, J=0.8 Hz, 3H), 3.37(dq, J=5.9 and 7.3 Hz, 2H), 4.11(br s, 2H), 5.61(q, J=0.8 Hz, 1H), 6.74(d, J=8.8 Hz, 1H), 6.93(br s, 1H), 7.14(dd, J=2.7 and 8.8 Hz, 1H), 7.27(d, J=2.7 Hz, 1H).", "Example 101 Triethylamine (0.17 g, 1.7 mmol) and ethyl isocyanate (0.12 g, 1.7 mmol) were added to a solution of 3-(4-chloro-3-trifluoromethylphenyloxy)-5-methylpyrazole (0.42 g, 1.5 mmol) in ethyl acetate (5 ml) at 0° C., and the mixture was allowed to have room temperature gradually and stirred for 5 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml) and extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/15), to give a yellow viscous substance of N-ethyl-3-(4-chloro-3-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.42 g, yield: 80.1%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.22(t, J=7.2 Hz, 3H), 2.59(d, J=0.8 Hz, 3H), 3.38(dq, J=5.9 and 7.2 Hz, 2H), 5.73(q, J=0.8 Hz, 1H), 6.91(br s, 1H), 7.22(d, J=8.8 Hz, 1H), 7.49(dd, J=2.4 and 8.8 Hz, 1H), 7.65(d, J=2.4 Hz, 1H).", "Example 102 Potassium carbonate (0.30 g, 2.2 mmol) and propyl isocyanate (0.17 g, 2.0 mmol) were added to a solution of 5-methyl-3-(4-nitro-3-trifluoromethylphenyloxy)pyrazole (0.57 g, 2.0 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a yellow viscous substance of N-propyl-5-methyl-3-(4-nitro-3-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.64 g, yield: 69.0%).", "1H-NMR(CDCl3, TMS, ppm): δ 0.97(t, J=7.3 Hz, 3H), 1.62(q, J=7.3 Hz, 2H), 2.64(s, 3H), 3.32(dt, J=7.3 and 7.3 Hz, 2H), 5.85(m, 1H), 6.80˜7.05(m, 1H), 7.45(dd, J=2.6 and 9.0 Hz, 1H), 7.64(d, J=2.6 Hz, 1H), 8.00(d, J=9.0 Hz, 1H).", "Examples 103-104 Reactions of 5-methyl-3-(4-nitro-3-trifluoromethylphenyloxy)pyrazole with isocyanates (Example 103: methyl isocyanate, Example 104: ethyl isocyanate) were carried out in ethyl acetate in the presence of potassium carbonate in the same manner as in Example 102, to give corresponding N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 103 N-methyl-5-methyl-3-(4-nitro-3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellow solid/yield: 42.1%/mp: 139-141° C./1H-NMR(CDCl3, TMS, ppm): δ 2.65(d, J=0.4 Hz, 3H), 2.95(d, J=5.0 Hz, 3H), 5.86(m, 1H), 6.65-7.00(m, 1H), 7.47(dd, J=2.6 and 8.9 Hz, 1H), 7.61(d, J=2.6 Hz, 1H), 8.00(d, J=8.9 Hz, 1H).", "Example 104 N-ethyl-5-methyl-3-(4-nitro-3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/yellowish solid/yield: 34.9%/mp: 95-97° C./1H-NMR(CDCl3, TMS, ppm): δ 1.24(t, J=7.3 Hz, 3H), 2.65(s, 3H), 3.41(dq, J=5.9 and 7.2 Hz, 2H), 5.85(m, 1H), 6.75-7.00(m, 1H), 7.45(dd, J=2.6 and 8.9 Hz, 1H), 7.63(d, J=2.6 Hz, 1H), 8.00(d, J=8.9 Hz, 1H).", "Example 105 Triethylamine (0.40 g, 4.0 mmol) and methyl isocyanate (0.25 g, 4.4 mmol) were added to a solution of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole (1.24 g, 4.0 mmol) in ethyl acetate (20 ml), and the mixture was stirred at room temperature for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-methyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.87 g, yield: 59.0%).", "mp: 113-114° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.60(d, J=0.7 Hz, 3H), 2.88(d, J=4.9 Hz, 3H), 5.77(d, J=0.7 Hz, 1H), 6.50-6.75(m, 1H), 7.66(s, 2H).", "Example 106 Potassium carbonate (0.30 g, 2.2 mmol) and ethyl isocyanate (0.14 g, 2.0 mmol) were added to a solution of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.62 g, 2.0 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.48 g, yield: 62.8%).", "mp: 80-82° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.19(t, J=7.3 Hz, 3H), 2.59(s, 3H), 3.34(dq, J=6.1 and 7.3 Hz, 2H), 5.72(s, 1H), 6.60-6.85(m, 1H), 7.67(s, 2H).", "Examples 107-117 Reactions of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole with isocyanates (Example 107: propyl isocyanate, Example 108: isopropyl isocyanate, Example 109: tert-butyl isocyanate, Example 110: hexyl isocyanate, Example 111: octyl isocyanate, Example 112: allyl isocyanate, Example 113: 2-chloroethyl isocyanate, Example 114: 2-bromoethyl isocyanate, Example 115: benzyl isocyanate, Example 116: ethyl isocyanatoacetate, Example 117: ethyl 3-isocyanatopropionate) were carried out in ethyl acetate in the presence of a base in the same manner as in Example 105 or 106, to give corresponding N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 107 N-propyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 68.4%/1H-NMR(CDCl3, TMS, ppm): δ 0.92(t, J=7.5 Hz, 3H), 1.59(tq, J=7.5 and 7.8 Hz, 2H), 2.58(d, J=0.8 Hz, 3H), 3.25(m, 2H), 5.71(q, J=0.8 Hz, 1H), 6.70-6.90(m, 1H), 7.66(s, 2H).", "Example 108 N-isopropyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 68.4%/mp: 103-105° C./1H-NMR(CDCl3, TMS, ppm): δ 1.22(d, J=6.5 Hz, 6H), 2.57(d, J=0.8 Hz, 3H), 3.90-4.10(m, 1H), 5.62(q, J=0.8 Hz, 1H), 6.50-6.75(m, 1H), 7.67(s, 2H).", "Example 109 N-tert-butyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 54.8%/mp: 116-117° C./1H-NMR(CDCl3, TMS, ppm): δ 1.40(s, 9H), 2.56(s, 3H), 5.57(s, 1H), 6.70-6.85(m, 1H), 7.67(s, 2H).", "Example 110 N-hexyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 69.5%/1H-NMR(CDCl3, TMS, ppm): δ 0.88(t, J=6.8 Hz, 3H), 1.15-1.40(m, 6H), 1.45-1.65(m, 2H), 2.58(s, 3H), 3.15-3.40(m, 2H), 5.71(s, 1H), 6.65-6.85(m, 1H), 7.66(s, 2H).", "Example 111 N-octyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 64.3%/mp: 43-45° C./1H-NMR(CDCl3, TMS, ppm): δ 0.88(t, J=6.8 Hz, 3H), 1.10-1.45(m, 10H), 1.45-1.65(m, 2H), 2.58(s, 3H), 3.15-3.35(m, 2H), 5.70(s, 1H), 6.65-6.85(m, 1H), 7.66(s, 2H).", "Example 112 N-allyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 83.1%/1H-NMR(CDCl3, TMS, ppm): δ 2.59(d, J=0.8 Hz, 3H), 3.80-4.00(m, 2H), 5.05-5.30(m, 2H), 5.75(q, J=0.8 Hz, 1H), 5.75-5.95(m, 1H), 6.70-7.00(m, 1H), 7.66(s, 2H).", "Example 113 N-(2-chloroethyl)-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 62.4%/mp: 116-118° C./1H-NMR(CDCl3, TMS, ppm): δ 2.58(s, 3H), 3.50-3.75(m, 4H), 5.76(s, 1H), 6.95-7.20(m, 1H), 7.67(s, 2H).", "Example 114 N-(2-bromoethyl)-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 58.6%/mp: 106-107° C./1H-NMR(CDCl3, TMS, ppm): δ 2.58(s, 3H), 3.48(t, J=6.1 Hz, 2H), 3.69(dt, J=6.1 and 6.1 Hz, 2H), 5.77(s, 1H), 6.95-7.20(m, 1H), 7.67(s, 2H).", "Example 115 N-benzyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 76.5%/mp: 50-53° C./1H-NMR(CDCl3, TMS, ppm): δ 2.61(d, J=0.7 Hz, 3H), 4.40(d, J=6.2 Hz, 2H), 5.74(q, J=0.7 Hz, 1H), 7.13(br t, J=6.2 Hz, 1H), 7.25-7.40(m, 5H), 7.65(s, 2H).", "Example 116 ethyl [{3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazol-1-yl}carbonylamino]acetate/white solid/yield: 76.5%/mp: 50-53° C./1H-NMR(CDCl3, TMS, ppm): δ 1.27(t, J=7.1 Hz, 3H), 2.58(d, J=0.7 Hz, 3H), 4.04(d, J=5.9 Hz, 2H), 4.21(q, J=7.2 Hz, 2H), 5.80(q, J=0.7 Hz, 1H), 7.10(br t, J=5.9 Hz, 1H), 7.66(s, 2H).", "Example 117 ethyl 3-[{3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazol-1-yl}carbonylamino]propionate/white solid/yield: 77.2%/mp: 73-74° C./1H-NMR(CDCl3, TMS, ppm): δ 1.20(t, J=7.5 Hz, 3H), 2.50-2.65(m, 4H), 3.56(q, J=7.5 Hz, 2H), 4.00-4.20(m, 2H), 5.76(q, J=0.8 Hz, 1H), 7.00-7.20(m, 2H), 7.66(s, 2H).", "Example 118 Triethylamine (0.13 g, 1.3 mmol) and methyl isocyanate (0.07 g, 1.2 mmol) were added to a solution of 3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.32 g, 1.1 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-methyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.22 g, yield: 56.9%).", "mp: 87-88° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.59(s, 3H), 2.88(d, J=5.0 Hz, 3H), 5.80(s, 1H), 6.55-6.75(m, 1H), 7.40(dd, JHF=2.0 and 9.3 Hz, 1H), 7.56(s, 1H).", "Examples 119-123 Reactions of 3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole with isocyanates (Example 119: ethyl isocyanate, Example 120: isopropyl isocyanate, Example 121: propyl isocyanate, Example 122: allyl isocyanate, Example 123: 2-bromoethyl isocyanate) were carried out in the same manner as in Example 118, to give corresponding N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 119 N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 42.8%/mp: 58-60° C./1H-NMR(CDCl3, TMS, ppm): δ 1.19(t, J=7.3 Hz, 3H), 2.59(d, J=0.7 Hz, 3H), 3.34(dq, J=6.0 and 7.2 Hz, 2H), 5.76(q, J=0.7 Hz, 1H), 6.60-6.85(m, 1H), 7.40(dd, JHF=1.8 and 9.2 Hz, 1H), 7.57(s, 1H).", "Example 120 N-isopropyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 31.1%/1H-NMR(CDCl3, TMS, ppm): δ 1.22(d, J=6.6 Hz, 6H), 2.58(d, J=0.8 Hz, 3H), 4.01(dsep, J=6.6 and 6.6 Hz, 1H), 5.69(q, J=0.8 Hz, 1H), 6.60(br d, J=6.6 Hz, 1H), 7.41(dd, JHF=1.8 and 9.2 Hz, 1H), 7.58(s, 1H).", "Example 121 N-propyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substrate/yield: 49.5%/1H-NMR(CDCl3, TMS, ppm): δ 0.92(t, J=7.4 Hz, 3H), 1.58(tq, J=7.4 and 13.3 Hz, 3H), 2.58(d, J=0.8 Hz, 3H), 3.25(dt, J=6.4 and 13.3 Hz, 1H), 5.76(q, J=0.8 Hz, 1H), 6.65-6.90(m, 1H), 7.40(dd, J=2.0 and 9.4 Hz, 1H), 7.57(s, 1H).", "Example 122 N-allyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substrate/yield: 50.7%/1H-NMR(CDCl3, TMS, ppm): δ 2.59(d, J=0.8 Hz, 3H), 3.92(ddt, J=1.6, 5.8 and 5.8 Hz, 2H), 5.10-5.25(m, 2H), 5.79(q, J=0.8 Hz, 1H), 5.75-5.95(m, 1H), 6.70-7.00(m, 1H), 9.40(dd, JHF=2.1 and 9.5 Hz, 1H), 7.57(s, 1H).", "Example 123 N-(2-bromoethyl)-3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 63.7%/mp: 85-86° C./1H-NMR(CDCl3, TMS, ppm): δ 2.58(d, J=0.8 Hz, 3H), 3.48(t, J=6.3 Hz, 2H), 3.70(dt, J=6.3 and 6.3 Hz, 2H), 5.81(d, J=0.8 Hz, 1H), 7.09(br t, J=6.3 Hz, 1H), 7.40(dd, JHF=1.8 and 9.3 Hz, 1H), 7.57(s, 1H).", "Example 124 Triethylamine (0.20 g, 2.0 mmol) and ethyl isocyanate (0.14 g, 2.0 mmol) were added to a solution of 3-(2,6-difluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.42 g, 1.5 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature for 12 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (chloroform), to give a white solid of N-ethyl-3-(2,6-difluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.11 g, yield: 21.0%).", "mp: 102-104° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.21(t, J=7.3 Hz, 3H), 2.19 (s, 3H), 3.32(dq, J=6.0 and 7.3 Hz, 2H), 5.00-5.25(m, 1H), 6.25(s, 1H), 7.36(d, JHF=7.0 Hz, 2H).", "Example 125 Potassium carbonate (0.15 g, 1.1 mmol) and ethyl isocyanate (0.07 g, 1.0 mmol) were added to a solution of 3-(2,4-dinitro-6-trifluoromethylphenyloxy)-5-methylpyrazole (0.33 g, 1.0 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature for 12 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a yellowish solid of N-ethyl-3-(2,4-dinitro-6-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.20 g, yield: 49.6%).", "mp: 169-171° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.18(t, J=7.2 Hz, 3H), 2.59(d, J=0.4 Hz, 3H), 3.31(dq, J=6.0 and 7.2 Hz, 2H), 5.95(q, J=0.4 Hz, 1H), 6.25-6.50(m, 1H), 8.85(d, J=2.7 Hz, 1H), 9.02(d, J=2.7 Hz, 1H).", "Example 126 Potassium carbonate (0.30 g, 2.2 mmol) and ethyl isocyanate (0.14 g, 2.0 mmol) were added to a solution of 3-(2-chloro-6-nitro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.64 g, 2.0 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a yellowish solid of N-ethyl-3-(2-chloro-6-nitro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.13 g, yield: 16.6%).", "mp: 98-99° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.18(t, J=7.2 Hz, 3H), 2.60(d, J=0.5 Hz, 3H), 3.32(dq, J=6.0 and 7.2 Hz, 2H), 5.87(q, J=0.5 Hz, 1H), 6.40-6.65(m, 1H), 8.01(d, J=2.0 Hz, 1H), 8.17(m, 1H).", "Examples 127-128 Reactions of 3-(2-chloro-6-nitro-4-trifluoromethylphenyloxy)-5-methylpyrazole and isocyanates (Example 127: methyl isocyanate, Example 128: propyl isocyanate) were carried out in ethyl acetate in the presence of potassium carbonate in the same manner as in Example 126, to give corresponding N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 127 N-methyl-3-(2-chloro-6-nitro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 60.9%/mp: 140-142° C./1H-NMR(CDCl3, TMS, ppm): δ 2.60(d, J=0.6 Hz, 3H), 2.87(d, J=5.0 Hz, 3H), 5.89(q, J=0.6 Hz, 1H), 6.40-6.55(m, 1H), 8.01(d, J=2.0 Hz, 1H), 8.19(m, 1H).", "Example 128 N-propyl-3-(2-chloro-6-nitro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 34.4%/mp: 111-113° C./1H-NMR(CDCl3, TMS, ppm): δ 0.92(t, J=7.4 Hz, 3H), 1.57(m, 2H), 2.19(s, 3H), 3.23(dt, J=7.4 and 7.4 Hz, 2H), 5.88(s, 1H), 6.45-6.70(m, 1H), 8.01(d, J=2.1 Hz, 1H), 8.17(m, 1H).", "Example 129 Potassium carbonate (0.30 g, 2.2 mmol) and ethyl isocyanate (0.14 g, 2.0 mmol) were added to a solution of 3-(3,5-dichloropyridin-2-yloxy)-5-methylpyrazole (0.49 g, 2.0 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-ethyl-3-(3,5-dichloropyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (0.44 g, yield: 69.8%).", "mp: 74-76° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.23(t, J=7.2 Hz, 3H), 2.64(s, 3H), 3.39(dq, J=5.8 and 7.2 Hz, 2H), 6.00(s, 1H), 6.90-7.15(m, 1H), 7.80(d, J=2.3 Hz, 1H), 8.05(d, 2.3 Hz, 1H).", "Example 130 Triethylamine (0.22 g, 2.2 mmol) was added to a solution of 5-methyl-3-(6-methoxy-3-nitropyridin-2-yloxy)pyrazole (0.5 g, 2.0 mmol) in ethyl acetate (10 ml), and the mixture was cooled to 0° C. Ethyl isocyanate (0.16 g, 2.2 mmol) was added, and the mixture was stirred at an ambient temperature for 30 minutes.", "The mixture was allowed to have room temperature.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml) and extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×2), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/6), to give a yellow viscous substance of N-ethyl-5-methyl-3-(6-methoxy-3-nitropyridin-2-yloxy)pyrazole-1-carboxamide (0.38 g, yield: 59.8%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.24(t, J=7.3 Hz, 3H), 2.65(d, J=0.8 Hz, 3H), 3.40(dq, J=1.4 and 7.3 Hz, 2H), 3.84(s, 3H), 6.02(q, J=0.8 Hz, 1H), 6.57(dd, J=3.5 and 8.8 Hz, 1H), 7.05(br s, 1H), 8.41(dd, J=3.5 and 8.8 Hz, 1H).", "Example 131 Reaction of 5-methyl-3-(4-methyl-5-nitropyridin-2-yloxy)pyrazole with ethyl isocyanate was carried out in the presence of triethylamine in the same manner as in Example 130, to give a yellow viscous substance of N-ethyl-5-methyl-3-(4-methyl-5-nitropyridin-2-yloxy)pyrazole-1-carboxamide (yield: 19.3%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.23(t, J=7.3 Hz, 3H), 2.65(d, J=0.7 Hz, 3H), 2.70(s, 3H), 3.40(dq, J=5.9 and 7.3 Hz, 2H), 6.03(q, J=0.7 Hz, 1H), 6.95(s, 1H), 6.99(br s, 1H), 8.92(s, 1H).", "Example 132 Potassium carbonate (0.75 g, 5.4 mmol) and isopropyl isocyanate (0.46 g, 5.4 mmol) were added to a solution of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole (1.00 g, 3.6 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature for 3 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a colorless viscous substance of N-isopropyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (0.91 g, yield: 69.7%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.25(d, J=6.5 Hz, 6H), 2.65(d, J=0.8 Hz, 3H), 3.95-4.20(m, 1H), 6.04(q, J=0.8 Hz, 1H), 6.80˜7.00(m, 1H), 8.02(d, J=2.3 Hz, 1H), 8.35(m, 1H).", "Examples 133-150 Reactions of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole and isocyanates (Example 133: methyl isocyanate, Example 134: ethyl isocyanate, Example 135: propyl isocyanate, Example 136: tert-butyl isocyanate, Example 137: pentyl isocyanate, Example 138: hexyl isocyanate, Example 139: heptyl isocyanate, Example 140: octyl isocyanate, Example 141: dodecyl isocyanate, Example 142: cyclohexyl isocyanate, Example 143: allyl isocyanate, Example 144: 2-chloroethyl isocyanate, Example 145: phenyl isocyanate, Example 146: 3-chlorophenyl isocyanate, Example 147: 3,4-dichlorophenyl isocyanate, Example 148: 3-trifluoromethylphenyl isocyanate, Example 149: 4-trifluoromethylphenyl isocyanate, Example 150: 3-nitrophenyl isocyanate) were carried out in the same manner as in Example 132, to give corresponding N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 133 N-methyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 81.7%/mp: 85-86° C./1H-NMR(CDCl3, TMS, ppm): δ 2.66(d, J=0.8 Hz, 3H), 2.95(d, J=5.0 Hz, 3H), 6.05(q, J=0.8 Hz, 1H), 6.85-7.15(m, 1H), 8.01(d, J=2.0 Hz, 1H), 8.35(m, 1H).", "Example 134 N-ethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 55.8%/mp: 85-86° C./1H-NMR(CDCl3, TMS, ppm): δ 1.23(t, J=7.3 Hz, 3H), 2.66(s, 3H), 3.40(dq, J=6.0 and 7.3 Hz, 2H), 6.04(s, 1H), 6.90-7.15(m, 1H), 8.02(d, J=2.0 Hz, 1H), 8.35(m, 1H).", "Example 135 N-propyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 86.3%/1H-NMR(CDCl3, TMS, ppm): δ 0.97(t, J=7.3 Hz, 3H), 1.60(tq, J=7.3 and 7.3 Hz, 2H), 2.66(d, J=0.8 Hz, 3H), 3.30(dt, J=6.5 and 7.3 Hz, 2H), 6.05(q, J=0.8 Hz, 1H), 6.95-7.20(m, 1H), 8.02(d, J=2.0 Hz, 1H), 8.35(m, 1H).", "Example 136 N-tert-butyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 74.5%/1H-NMR(CDCl3, TMS, ppm): δ 1.44(s, 9H), 2.64(d, J=0.7 Hz, 3H), 6.02(q, J=0.7 Hz, 1H), 6.90-7.15(br s, 1H), 8.01(d, J=2.1 Hz, 1H), 8.34(m, 1H).", "Example 137 N-pentyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/yellowish solid/yield: 73.2%/mp: 142-144° C./1H-NMR(CDCl3, TMS, ppm): δ 0.90(t, J=7.0 Hz, 3H), 1.20-1.45(m, 4H), 1.50-1.70(m, 2H), 2.66(d, J=0.8 Hz, 3H), 3.25-3.40(m, 2H), 6.04(q, J=0.8 Hz, 1H), 6.95-7.15(m, 1H), 8.02(d, J=2.3 Hz, 1H), 8.25-8.40(m, 1H).", "Example 138 N-hexyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 65.2%/1H-NMR(CDCl3, TMS, ppm): δ 0.89(t, J=6.8 Hz, 3H), 1.15-1.45(m, 6H), 1.45-1.70(m, 2H), 2.65(d, J=0.8 Hz, 3H), 3.25-3.45(m, 2H), 6.04(q, J=0.8H, 1H), 6.95-7.15(m, 1H), 8.02(d, J=2.0 Hz, 1H), 8.35(m, 1H).", "Example 139 N-heptyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 84.9%/1H-NMR(CDCl3, TMS, ppm): δ 0.75-1.00(m, 3H), 1.10-1.50(m, 8H), 1.50-1.70(m, 2H), 2.65(s, 3H), 3.20-3.45(m, 2H), 6.04(s, 1H), 6.95-7.15(m, 1H), 8.02(d, J=2.0 Hz, 1H), 8.36(m, 1H).", "Example 140 N-octyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 81.5%/1H-NMR(CDCl3, TMS, ppm): δ 0.88(t, J=7.0 Hz, 3H), 1.15-1.45(m, 10H), 1.45-1.75(m, 2H), 2.66(d, J=0.5 Hz, 3H), 3.20-3.45(m, 2H), 6.04(s, 1H), 6.95-7.15(m, 1H), 8.02(d, J=2.0 Hz, 1H), 8.35(m, 1H).", "Example 141 N-dodecyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 80.7%/mp: 56-57° C./1H-NMR(CDCl3, TMS, ppm): δ 0.88(t, J=6.9 Hz, 3H), 1.20-1.40(m, 18H), 1.50-1.65(m, 2H), 2.65(d, J=0.8 Hz, 3H), 3.25-3.40(m, 2H), 6.04(q, J=0.8 Hz, 1H), 6.95-7.15 (m, 1H), 8.03 (d, J=2.1 Hz, 1H), 8.38 (m, 1H).", "Example 142 N-cyclohexyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 65.5%/mp: 69-71° C./1H-NMR(CDCl3, TMS, ppm): δ 1.05-1.50(m, 6H), 1.55-1.85(m, 2H), 1.90-2.10(m, 2H), 2.65(d, J=0.8 Hz, 3H), 3.60-3.85(m, 1H), 6.03(q, J=0.8 Hz, 1H), 6.80-7.05(m, 1H), 8.01(d, J=2.0 Hz, 1H), 8.35(m, 1H).", "Example 143 N-allyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 62.2%/1H-NMR(CDCl3, TMS, ppm): δ 2.66(d, J=0.8 Hz, 3H), 3.85-4.05(m, 2H), 5.05-5.35(m, 2H), 5.75-6.00(m, 1H), 6.06(q, J=0.8 Hz, 1H), 7.05-7.25(m, 1H), 8.02(d, J=2.3 Hz, 1H), 8.35(m, 1H).", "Example 144 N-(2-chloroethyl)-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 65.2%/mp: 98-100° C./1H-NMR(CDCl3, TMS, ppm): δ 2.65(d, J=0.5 Hz, 3H), 3.65-3.75(m, 4H), 6.08(q, J=0.5 Hz, 1H), 7.30-7.50(m, 1H), 8.03(d, J=1.8 Hz, 1H), 8.36(m, 1H).", "Example 145 N-phenyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 4.9%/mp: 116-118° C./1H-NMR(CDCl3, TMS, ppm): δ 2.72(d, J=0.8 Hz, 3H), 6.12(q, J=0.8 Hz, 1H), 7.10-7.20(m, 1H), 7.30-7.45(m, 2H), 7.50-7.60(m, 2H), 8.05(d, J=2.2 Hz, 1H), 8.40(m, 1H), 8.85-9.10(m, 1H).", "Example 146 N-(3-chlorophenyl)-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 36.0%/mp: 129-130° C./1H-NMR(CDCl3, TMS, ppm): δ 2.72(d, J=0.7 Hz, 3H), 6.15(q, J=0.7 Hz, 1H), 7.05-7.15(m, 1H), 7.20-7.30(m, 1H), 7.30-7.40(m, 1H), 7.73(t, J=2.0 Hz, 1H), 8.05(d, J=1.8 Hz, 1H), 8.30-8.40(m, 1H), 8.90-9.10(br s, 1H).", "Example 147 N-(3,4-dichlorophenyl)-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 38.1%/mp: 150-152° C./1H-NMR(CDCl3, TMS, ppm): δ 2.71(s, 3H), 6.15(s, 1H), 7.30-7.45(m, 2H), 7.83(d, J=2.3 Hz, 1H), 8.00˜8.10(m, 1H), 8.25-8.45(m, 1H), 9.01(s, 1H).", "Example 148 N-(3-trifluoromethylphenyl)-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 18.0%/mp: 123-124° C./1H-NMR(CDCl3, TMS, ppm): δ 2.73(d, J=0.8 Hz, 3H), 6.16(q, J=0.8 Hz, 1H), 7.45(dt, J=8.0 and 8.0 Hz, 2H), 7.72(d, J=8.0 Hz, 1H), 7.93(s, 1H), 8.05(d, J=8.0 Hz, 1H), 8.37(m, 1H), 9.00-9.25(br s, 1H).", "Example 149 N-(4-trifluoromethylphenyl)-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 24.4%/mp: 164-167° C./1H-NMR(CDCl3, TMS, ppm): δ 2.73(d, J=0.7 Hz, 3H), 6.16(q, J=0.7 Hz, 1H), 7.62(d, J=8.8 Hz, 2H), 7.70(d, J=8.8 Hz, 2H), 8.05(d, J=2.1 Hz, 1H), 8.30-8.45(m, 1H), 9.10-9.25(br s, 1H).", "Example 150 N-(3-nitrophenyl)-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 13.6%/mp: 172-174° C./1H-NMR(CDCl3, TMS, ppm): δ 2.73(s, 3H), 6.18(s, 1H), 7.54(t, J=8.2 Hz, 1H), 7.88(dd, J=0.9 and 2.2 Hz, 1H), 8.03(dd, J=0.9 and 2.2 Hz, 1H), 8.06(d, J=2.1 Hz, 1H), 8.37(m, 1H), 8.54(t, J=2.1 Hz, 1H), 9.15-9.30(m, 1H).", "Example 151 Potassium carbonate (0.50 g, 3.6 mmol) and methyl isocyanate (0.17 g, 3.0 mmol) were added to a solution of 4,5-dimethyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole (1.08 g, 3.6 mmol) in ethyl acetate (20 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with diethyl ether (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellow solid of N-methyl-4,5-dimethyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.93 g, yield: 86.5%).", "mp: 117-119° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.95(s, 3H), 2.55(s, 3H), 2.90(d, J=5.0 Hz, 3H), 6.50-6.80(m, 1H), 7.49(d, J=8.7 Hz, 1H), 7.84(dd, J=2.0 and 8.7 Hz, 1H), 8.25(d, J=2.0 Hz, 1H).", "Example 152 Potassium carbonate (0.50 g, 3.6 mmol) and methyl isocyanate (0.17 g, 3.0 mmol) were added to a solution of 4,5-dimethyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole (1.08 g, 3.6 mmol) in ethyl acetate (20 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with diethyl ether (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a white solid of N-methyl-4,5-dimethyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.88 g, yield: 81.9%).", "mp: 113-115° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.88(s, 3H), 2.57(s, 3H), 2.94(d, J=5.0 Hz, 3H), 6.70-6.95(m, 1H), 7.44(d, J=9.2 Hz, 1H), 8.39(dd, J=2.7 and 9.2 Hz, 1H), 8.60(d, J=2.7 Hz, 1H).", "Example 153 Potassium carbonate (0.62 g, 4.5 mmol) and isopropyl isocyanate (0.26 g, 3.0 mmol) were added to a solution of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4,5-dimethylpyrazole (0.88 g, 3.0 mmol) in DMF (10 ml), and the mixture was stirred at room temperature for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a white solid of N-isopropyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4,5-dimethylpyrazole-1-carboxamide (0.55 g, yield: 48.7%) mp: 78-80° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.24 (d, J=6.6 Hz, 6H), 1.78(s, 3H), 2.58(s, 3H), 3.95-4.15(m, 1H), 6.70-6.95(m, 1H), 8.02(d, J=2.1 Hz, 1H), 8.32(m, 1H).", "Examples 154-155 Reactions of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4,5-dimethylpyrazole and isocyanates (Example 154: methyl isocyanate, Example 155: ethyl isocyanate) were carried out in the same manner as in Example 153, to give corresponding N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 154 N-methyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4,5-dimethylpyrazole-1-carboxamide/white solid/yield: 92.3%/mp: 117-118° C./1H-NMR(CDCl3, TMS, ppm): δ 1.80(s, 3H), 2.58(s, 3H), 2.93(d, J=4.9 Hz, 3H), 6.80-7.10(m, 1H), 8.02(d, J=2.0 Hz, 1H), 8.32(m, 1H).", "Example 155 N-ethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4,5-dimethylpyrazole-1-carboxamide/white solid/yield: 38.6%/mp: 124-125° C./1H-NMR(CDCl3, TMS, ppm): δ 1.21(t, J=7.3 Hz, 3H), 1.79(s, 3H), 2.58(s, 3H), 3.38(dq, J=6.0 and 7.2 Hz, 2H), 6.85-7.10(m, 1H), 8.02(d, J=2.1 Hz, 1H), 8.32(m, 1H).", "Example 156 Potassium carbonate (0.50 g, 3.6 mmol) and methyl isocyanate (0.17 g, 3.0 mmol) were added to a solution of 4-ethyl-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole (1.13 g, 3.6 mmol) in ethyl acetate (20 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with diethyl ether (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellow solid of N-methyl-4-ethyl-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.62 g, yield: 55.5%).", "mp: 81-82° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.14(t, J=7.6 Hz, 3H), 2.41(q, J=7.6 Hz, 2H), 2.56(s, 3H), 2.90(d, J=5.0 Hz, 3H), 6.50-6.75(m, 1H), 7.51(d, J=8.7 Hz, 1H), 7.84(dd, J=2.1 and 8.7 Hz, 1H), 8.26(d, J=2.1 Hz, 1H).", "Example 157 Potassium carbonate (0.50 g, 3.6 mmol) and methyl isocyanate (0.17 g, 3.0 mmol) were added to a solution of 4-ethyl-5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole (1.13 g, 3.6 mmol) in ethyl acetate (15 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with diethyl ether (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/3), to give a yellow solid of N-methyl-4-ethyl-5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.71 g, yield: 63.6%).", "mp: 96-97° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.08(t, J=7.6 Hz, 3H), 2.35(q, J=7.6 Hz, 2H), 2.59(s, 3H), 2.94(d, J=5.0 Hz, 3H), 6.70-6.90(m, 1H), 7.48(d, J=9.2 Hz, 1H), 8.40(dd, J=2.7 and 9.2 Hz, 1H), 8.60(d, J=2.7 Hz, 1H).", "Example 158 Potassium carbonate (0.83 g, 6.0 mmol) and methyl isocyanate (0.29 g, 5.0 mmol) were added to a solution of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4-ethyl-5-methylpyrazole (1.83 g, 6.0 mmol) in ethyl acetate (25 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with diethyl ether (30 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a white solid of N-methyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4-ethyl-5-methylpyrazole-1-carboxamide (1.14 g, yield: 62.9%).", "mp: 84-86° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.04(t, J=7.6 Hz, 3H), 2.26(q, J=7.6 Hz, 2H), 2.59(s, 3H), 2.93(d, J=5.0 Hz, 3H), 6.80-7.05(m, 1H), 8.02(d, J=2.0 Hz, 1H), 8.32(m, 1H).", "Example 159 Reaction of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4-ethyl-5-methylpyrazole with ethyl isocyanate was carried out in the same manner as in Example 158, to give a white solid of N-ethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-4-ethyl-5-methylpyrazole-1-carboxamide (0.36 g, yield: 31.9%).", "mp: 101-102° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.03(t, J=7.6 Hz, 3H), 1.21(t, J=7.3 Hz, 3H), 2.25(q, J=7.6 Hz, 2H), 2.59(s, 3H), 3.38(dq, J=5.9 and 7.3 Hz, 2H), 6.85-7.05(m, 1H), 8.02(d, J=2.0 Hz, 1H), 8.32(m, 1H).", "Example 160 Triethylamine (0.10 g, 1.0 mmol) and 2-chloroethyl isocyanate (0.12 g, 1.1 mmol) were added to a solution of 4-chloro-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.35 g, 1.0 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature for 8 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-(2-chloroethyl)-4-chloro-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.44 g, yield: 97.6%).", "mp: 141-143° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.62(s, 3H), 3.55-3.65(m, 4H), 6.80-7.05(m, 1H), 7.67(s, 2H).", "Example 161 Potassium carbonate (0.72 g, 5.2 mmol) and methyl isocyanate (0.29 g, 5.0 mmol) were added to a solution of 5-ethyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole (1.58 g, 5.2 mmol) in ethyl acetate (20 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with diethyl ether (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a yellow solid of N-methyl-5-ethyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.68 g, yield: 38.0%).", "mp: 89-91° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.29(t, J=7.4 Hz, 3H), 2.91(d, J=5.0 Hz, 3H), 3.09(q, J=7.4 Hz, 2H), 5.92(s, 1H), 6.55-6.85(m, 1H), 7.50(d, J=8.7 Hz, 1H), 7.85(dd, J=2.1 and 8.7 Hz, 1H), 8.26(d, J=2.1 Hz, 1H).", "Example 162 Potassium carbonate (0.83 g, 6.0 mmol) and methyl isocyanate (0.29 g, 5.0 mmol) were added to a solution of 5-ethyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole (1.81 g, 6.0 mmol) in ethyl acetate (15 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with diethyl ether (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7), to give a yellow solid of N-methyl-5-ethyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole-1-carboxamide (1.42 g, yield: 79.3%).", "mp: 97-100° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.30(t, J=7.5 Hz, 3H), 2.95(d, J=5.0 Hz, 3H), 3.12(q, J=7.5H, 2H), 5.93(s, 1H), 6.75-7.00(m, 1H), 7.45(d, J=9.2 Hz, 1H), 8.41(dd, J=2.7 and 9.2 Hz, 1H), 8.60(d, J=2.7 Hz, 1H).", "Example 163 Potassium carbonate (0.46 g, 3.3 mmol) and ethyl isocyanate (0.21 g, 3.0 mmol) were added to a solution of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-ethylpyrazole (0.98 g, 3.0 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-ethylpyrazole-1-carboxamide (0.79 g, yield: 66.5%).", "mp: 86-89° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.19(t, J=7.3 Hz, 3H), 1.27(t, J=7.5 Hz, 3H), 3.06(q, J=7.5 Hz, 2H), 3.34(dq, J=6.1 and 7.3 Hz, 2H), 5.75(s, 1H), 6.60-6.85(m, 1H), 7.68(s, 2H).", "Example 164 Potassium carbonate (1.16 g, 8.4 mmol) and methyl isocyanate (0.40 g, 7.0 mmol) were added to a solution of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-ethylpyrazole (2.45 g, 8.4 mmol) in ethyl acetate (30 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with diethyl ether (30 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a white solid of N-methyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-ethylpyrazole-1-carboxamide (1.97 g, yield: 80.7%).", "mp: 85-87° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.31(t, J=7.5 Hz, 3H), 2.95(d, J=5.0 Hz, 3H), 3.13(dq, J=6.0 and 7.5 Hz, 2H), 6.08(s, 1H), 6.85-7.15(m, 1H), 8.02(d, J=2.1 Hz, 1H), 8.36(m, 1H).", "Example 165 Reaction of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-ethylpyrazole with ethyl isocyanate was carried out in the same manner as in Example 164, to give a white solid of N-ethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-ethylpyrazole-1-carboxamide (0.25 g, yield: 23.0%).", "mp: 85-86° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.23(t, J=7.3 Hz, 3H), 1.30(t, J=7.5 Hz, 3H), 3.13(q, J=7.5 Hz, 2H), 3.40(dq, J=5.9 and 7.3 Hz, 2H), 6.08(s, 1H), 6.90-7.15(m, 1H), 8.02(d, J=2.1 Hz, 1H), 8.37(m, 1H).", "Example 166 Potassium carbonate (0.23 g, 1.7 mmol) and ethyl isocyanate (0.11 g, 1.5 mmol) were added to a solution of 5-cyclopropyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazole (0.51 g, 1.5 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a colorless viscous substance of N-ethyl-5-cyclopropyl-3-(2,6-dichloro-4-trifluoromethylpheyloxy)pyrazole-1-carboxamide (0.54 g, yield: 88.2%).", "1H-NMR(CDCl3, TMS, ppm): δ 0.70(m, 2H), 1.07(m, 2H), 1.20(t, J=7.3 Hz, 3H), 2.81(m, 1H), 3.36(dq, J=6.0 and 7.3 Hz, 2H), 5.45(s, 1H), 6.60-6.85(m, 1H), 7.66(s, 2H).", "Examples 167-171 Reactions of 3-(substituted phenyloxy)pyrazole derivatives (Example 167: 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-isopropylpyrazole, Example 168: 5-tert-butyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazole, Example 169: 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-4-ethyl-5-methylpyrazole, Example 170: 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methoxymethylpyrazole, Example 171: {3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazol-5-yl}acetate) with ethyl isocyanate were carried out in the presence of a base in the same manner as in Example 166, to give corresponding N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 167 N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-isopropylpyrazole-1-carboxamide/white solid/yield: 21.9%/mp: 74-76° C./1H-NMR(CDCl3, TMS, ppm): δ 1.19(t, J=7.3 Hz, 3H), 1.28(d, J=6.8 Hz, 6H), 3.34(dq, J=6.0 and 7.3 Hz, 2H), 3.85(sep, J=6.8 Hz, 1H), 5.77(m, 1H), 6.77(br t, J=6.0 Hz, 1H), 7.67(d, 0.4 Hz, 2H).", "Example 168 N-ethyl-5-tert-butyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide/colorless viscous substance/yield: 31.4%/1H-NMR(CDCl3, TMS, ppm): δ 1.18(t, J=7.2 Hz, 3H), 1.46(s, 9H), 3.33(dq, J=5.9 and 7.2 Hz, 2H), 5.79(s, 1H), 6.80-6.95(m, 1H), 7.67(d, J=0.4 Hz, 2H).", "Example 169 N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-4-ethyl-5-methylpyrazole-1-carboxamide/white solid/yield: 27.6%/mp: 120-123° C./1H-NMR(CDCl3, TMS, ppm): δ 1.15(t, J=7.5 Hz, 3H), 1.19(t, J=6.3 Hz, 3H), 1.93(s, 3H), 2.39(q, J=7.5 Hz, 2H), 3.29(dq, J=6.0 and 6.3 Hz, 2H), 7.63(s, 2H), 8.85(br t, J=6.0 Hz, 1H).", "Example 170 N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methoxymethylpyrazole-1-carboxamide/white solid/yield: 76.0%/mp: 106-108° C./1H-NMR(CDCl3, TMS, ppm): δ 1.20(t, J=7.2 Hz, 3H), 3.35(dq, J=6.0 and 7.2 Hz, 2H), 3.49(s, 3H), 4.83(d, J=1.0 Hz, 2H), 5.99(t, J=1.0 Hz, 1H), 6.60-6.80(m, 1H), 7.67(d, J=0.4 Hz, 2H).", "Example 171 N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-ethoxycarbonylmethylpyrazole-1-carboxamide/white solid/yield: 26.9%/mp: 100-102° C./1H-NMR(CDCl3, TMS, ppm): δ 1.18(t, J=7.3 Hz, 3H), 1.27(t, J=7.2 Hz, 3H), 3.33(dq, J=6.0 and 7.3 Hz, 2H), 4.04(s, 2H), 4.20(q, J=7.2 Hz, 2H), 5.94(s, 1H), 6.60-6.80(m, 1H), 7.67(d, J=0.5 Hz, 2H).", "Example 172 Potassium carbonate (0.58 g, 4.2 mmol) and methyl isocyanate (0.20 g, 3.5 mmol) were added to a solution of 3-(2-nitro-4-trifluoromethylphenyloxy)-5-trifluoromethylpyrazole (1.43 g, 4.2 mmol) in ethyl acetate (15 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5→1/1), to give a yellowish solid of N-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)-5-trifluoromethylpyrazole-1-carboxamide (0.70 g, yield: 50.4%).", "mp: 102-104° C./1H-NMR(CDCl3, TMS, ppm): δ 3.53(s, 3H), 5.63(s, 1H), 7.56(d, J=8.3 Hz, 1H), 7.95(d, J=8.3 Hz, 1H), 8.39(s, 1H), 10.05-10.75(m, 1H).", "Example 173 Potassium carbonate (0.69 g, 5.0 mmol) and ethyl isocyanate (0.28 g, 5.0 mmol) were added to a solution of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-trifluoromethylpyrazole (1.66 g, 5.0 mmol) in ethyl acetate (20 ml), and the mixture was stirred at room temperature for 3 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5→1/1), to give a white solid of N-ethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-trifluoromethylpyrazole-1-carboxamide (0.83 g, yield: 51.6%).", "mp: 52-54° C./1H-NMR(CDCl3, TMS, ppm): δ 1.34(t, J=7.1 Hz, 3H), 3.75-4.30(m, 2H), 5.66(s, 1H), 7.98(d, J=1.5 Hz, 1H), 8.64(m, 1H), 10.20-10.40(m, 1H).", "Example 174 Reaction of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-trifluoromethylpyrazole with methyl isocyanate was carried out in the same manner as in Example 173, to give a white solid of N-methyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-trifluoromethylpyrazole-1-carboxamide (0.39 g, yield: 20.1%).", "mp: 61-63° C.; 1H-NMR(CDCl3, TMS, ppm): δ 3.52(s, 3H), 5.66(s, 1H), 8.00(d, J=11.8 Hz, 1H), 8.61(d, J=1.8 Hz, 1H), 10.10-10.40(m, 1H).", "Example 175 Triethylamine (0.20 g, 2.0 mmol) and ethyl isocyanate (0.14 g, 2.0 mmol) were added to a solution of methyl 3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazole-5-carboxylate (0.53 g, 1.5 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature for 6 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (chloroform), to give a white solid of methyl 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-1(N-ethylcarbamoyl)pyrazole-5-carboxylate (0.15 g, yield: 23.5%).", "mp: 88-90° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.20(t, J=7.3 Hz, 3H), 3.37(dq, J=6.0 and 7.3 Hz, 2H), 3.95(s, 3H), 6.21(s, 1H), 6.40-6.60(m, 1H), 7.68(d, J=0.5 Hz, 2H).", "Example 176 Potassium carbonate (0.25 g, 1.8 mmol) and ethyl isocyanate (0.11 g, 1.5 mmol) were added to a solution of 5-(4-chlorophenyl)-3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazole (0.61 g, 1.5 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7), to give a white solid of N-ethyl-5-(4-chlorophenyl)-3-(2,6-dichloro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.39 g, yield: 54.3%).", "mp: 88-89° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.17(t, J=7.3 Hz, 3H), 3.30(dq, J=6.1 and 7.3 Hz, 2H), 5.96(s, 1H), 6.65-6.85(m, 1H), 7.30-7.50(m, 4H), 7.70(s, 2H).", "Example 177 Potassium carbonate (0.23 g, 1.7 mmol) and methoxymethyl isothiocyanate (0.15 g, 1.5 mmol) were added to a solution of 3-(4-cyano-2-trifluoromethylphenyloxy)-5-methylpyrazole (0.40 g, 1.5 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-methoxymethyl-3-(4-cyano-2-trifluoromethylphenyloxy)-5-methylpyrazole-1-carbothioamide (0.18 g, yield: 32.4%) mp: 89-90° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.84(s, 3H), 3.44(s, 3H), 5.13(d, J=6.2 Hz, 2H), 5.98(m, 1H), 7.44(d, J=8.7 Hz, 1H), 7.84(dd, J=2.0 and 8.7 Hz, 1H), 8.01(d, J=2.0 Hz, 1H), 8.95-9.30(m, 1H).", "Example 178 Potassium carbonate (0.23 g, 1.7 mmol) and methoxymethyl isothiocyanate (0.16 g, 1.5 mmol) were added to a solution of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.47 g, 1.5 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-methoxymethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carbothioamide (0.29 g, yield: 46.7%).", "mp: 95-96° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.82(d, J=0.6 Hz, 3H), 3.41(s, 3H), 5.10(d, J=6.3 Hz, 2H), 5.91(q, J=0.6 Hz, 1H), 7.68(s, 2H), 8.80-9.20(m, 1H).", "Examples 179-185 Reactions of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole with isothiocyanates (Examples 179: methyl isothiocyanate, Example 180: ethyl isothiocyanate, Example 181: propyl isothiocyanate, Example 182: isopropyl isothiocyanate, Example 183: butyl isothiocyanate, Example 184: 2-methoxyethyl isothiocyanate, Example 185: tetrahydrofurfuryl isothiocyanate) were carried out in the same manner as in Example 178, to give corresponding N-substituted carbothioamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 179 N-methyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 39.9%/mp: 121-123° C./1H-NMR(CDCl3, TMS, ppm): δ 2.82(d, J=0.8 Hz, 3H), 3.15(d, J=5.0 Hz, 3H), 5.86(q, J=0.8 Hz, 1H), 7.67(s, 2H), 8.40-8.70(m, 1H).", "Example 180 N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 29.8%/mp: 94-96° C./1H-NMR(CDCl3, TMS, ppm): δ 1.25(t, J=7.5 Hz, 3H), 2.81(s, 3H), 3.67(dq, J=6.3 and 7.5 Hz, 2H), 5.78(s, 1H), 7.67(s, 2H), 8.40-8.70(m, 1H).", "Example 181 N-propyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 22.7%/mp: 71-73° C./1H-NMR(CDCl3, TMS, ppm): δ 0.93(t, J=7.3 Hz, 3H), 1.66(tq, J=7.3 and 7.3 Hz, 2H), 2.81(s, 3H), 3.57(dt, J=6.0 and 7.3 Hz, 2H), 5.79(s, 1H), 7.67(s, 2H), 8.45-8.75(m, 1H).", "Example 182 N-isopropyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 9.7%/mp: 92-94° C./1H-NMR(CDCl3, TMS, ppm): δ 1.27(d, J=6.5 Hz, 6H), 2.80(s, 3H), 4.52(m, 1H), 5.69(s, 1H), 7.68(s, 2H), 8.20-8.70(m, 1H).", "Example 183 N-butyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carbothioamide/colorless viscous substance/yield: 20.3%/1H-NMR(CDCl3, TMS, ppm): δ 0.92(t, J=7.3 Hz, 3H), 1.25-1.80(m, 4H), 2.81(d, J=0.6 Hz, 3H), 3.61(dt, J=5.7 and 7.2 Hz, 2H), 5.80(q, J=0.6 Hz, 1H), 7.67(s, 2H), 8.45-8.75(m, 1H).", "Example 184 N-(2-methoxyethyl)-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 46.7%/mp: 83-86° C./1H-NMR(CDCl3, TMS, ppm): δ 2.81(d, J=0.5 Hz, 3H), 3.29(s, 3H), 3.56(t, J=5.3 Hz, 2H), 3.81(dt, J=5.3 and 5.3 Hz, 2H), 5.84(q, J=0.5 Hz, 1H), 7.67(d, J=0.3 Hz, 2H), 8.65-9.00(m, 1H).", "Example 185 N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 16.5%/mp: 69-71° C./1H-NMR(CDCl3, TMS, ppm): δ 1.50-1.65(m, 1H), 1.65-2.05(m, 3H), (s, 3H), 3.55-3.70(m, 2H), 3.70-3.80(m, 2H), 4.05-4.20(m, 1H), 5.86(q, J=0.8 Hz, 1H), 7.66(d, J=0.5 Hz, 2H), 8.77(br s, 1H).", "Example 186 Potassium carbonate (0.22 g, 1.6 mmol) and ethyl isothiocyanate (0.13 g, 1.8 mmol) were added to a solution of 3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.44 g, 1.5 mmol) in ethyl acetate (10 ml), and the mixture was stirred at room temperature overnight.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carbothioamide (0.05 g, yield: 9.4%).", "mp: 114-115° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.25(t, J=7.3 Hz, 3H), 2.81(d, J=0.6 Hz, 3H), 3.67(dq, J=5.7 and 7.3 Hz, 2H), 5.84(q, J=0.6 Hz, 1H), 7.41(dd, JHF=2.1 and 9.4 Hz, 1H), 7.62(s, 1H), 8.54(br s, 1H).", "Example 187 Potassium carbonate (0.75 g, 5.4 mmol) and methyl isothiocyanate (0.39 g, 5.4 mmol) were added to a solution of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole (1.00 g, 3.6 mmol) in DMF (10 ml), and the mixture was stirred at room temperature for 8 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-methyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide (0.35 g, yield: 37.7%) mp: 63-65° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.87(d, J=0.6 Hz, 3H), 3.21(d, J=4.9 Hz, 3H), 6.14(q, J=0.6 Hz, 1H), 8.02(d, J=2.1 Hz, 1H), 8.36(m, 1H), 8.80-9.05(m, 1H).", "Examples 188-196 Reactions of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole with isothiocyanates (Example 188: ethyl isothiocyanate, Example 189: propyl isothiocyanate, Example 190: isopropyl isothiocyanate, Example 191: butyl isothiocyanate, Example 192: allyl isothiocyanate, Example 193: 2-methoxymethyl isothiocyanate, Example 194: 2-methoxyethyl isothiocyanate, Example 195: methyl 2-isothiocyanato-3-methylbutanoate, Example 196: 4-chlorobenzyl isothiocyanate) were carried out in the same manner as in Example 187, to give corresponding N-substituted carbothioamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 188 N-ethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 11.2%/mp: 74-75° C./1H-NMR(CDCl3, TMS, ppm): δ 1.31 (t, J=7.3 Hz, 3H), 2.87(s, 3H), 3.72(dq, J=5.5 and 7.3 Hz, 2H), 6.13(s, 1H), 8.03(d, J=2.0 Hz, 1H), 8.36(m, 1H), 8.70-9.05(m, 1H).", "Example 189 N-propyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 6.4%/mp: 51-53° C./1H-NMR(CDCl3, TMS, ppm): δ 1.00(t, J=7.3 Hz, 3H), 1.71(tq, J=7.3 and 7.3 Hz, 2H), 2.87(d, J=0.8 Hz, 3H), 3.64(dt, J=5.8 and 7.3 Hz, 2H), 6.13(q, J=0.8 Hz, 1H), 8.02(d, J=2.0 Hz, 1H), 8.36(m, 1H), 8.80-9.05(m, 1H).", "Example 190 N-isopropyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide/colorless viscous substance/yield: 5.4%/1H-NMR(CDCl3, TMS, ppm): δ 1.32(d, J=6.6 Hz, 6H), 2.87(d, J=0.5 Hz, 3H), 4.50-4.70(m, 1H), 6.12(m, 1H), 8.03(d, J=2.0 Hz, 1H), 8.36(m, 1H), 8.60-8.85(m, 1H).", "Example 191 N-butyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide/colorless viscous substance/yield: 29.7%/1H-NMR(CDCl3, TMS, ppm): δ 0.96(t, J=7.3 Hz, 3H), 1.30-1.55(m, 2H), 1.68-1.80(m, 2H), 2.87(s, 3H), 3.60-3.85(m, 2H), 6.12(s, 1H), 8.02(d, J=1.8 Hz, 1H), 8.36(m, 1H), 8.75-9.05(m, 1H).", "Example 192 N-allyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 5.9%/mp: 46-48° C./1H-NMR(CDCl3, TMS, ppm): δ 2.87(s, 3H), 4.25-4.40(m, 2H), 5.25-5.35(m, 2H), 5.80-6.10(m, 1H), 6.15(q, J=0.5 Hz, 1H), 8.02(d, J=2.0 Hz, 1H), 8.36(m, 1H), 8.85-9.10(m, 1H).", "Example 193 N-(2-methoxymethyl)-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 31.3%/mp: 102-104° C./1H-NMR(CDCl3, TMS, ppm): δ 2.87(s, 3H), 3.44(s, 3H), 5.16(d, J=6.2 Hz, 2H), 6.18(d, J=0.4 Hz, 1H), 8.04(d, J=2.1 Hz, 1H), 8.37(m, 1H), 9.15-9.55(m, 1H).", "Example 194 N-(2-methoxyethyl)-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 16.1%/mp: 108-109° C./1H-NMR(CDCl3, TMS, ppm): δ 2.86(d, J=0.6 Hz, 3H), 3.38(s, 3H), 3.63(t, J=5.4 Hz, 2H), 3.90(dt, J=5.2 and 5.4 Hz, 2H), 6.12(q, J=0.6 Hz, 1H), 8.03(d, J=1.8 Hz, 1H), 8.36(m, 1H), 9.00-9.25(m, 1H).", "Example 195 methyl 2-[{3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazol-1-yl}thiocarbonylamino]-3-methylbutanoate/white solid/yield: 7.1%/mp: 109-110° C./1H-NMR(CDCl3, TMS, ppm): δ 0.95-1.15(m, 6H), 2.25-2.55(m, 1H), 2.84(m, 3H), 3.78(s, 3H), 5.01(dd, J=5.2 and 8.3 Hz, 1H), 6.14(q, J=0.5 Hz, 1H), 8.03(d, J=2.1 Hz, 1H), 8.36(m, 1H), 9.10-9.40(m, 1H).", "Example 196 N-(4-chlorobenzyl)-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide/white solid/yield: 12.2%/mp: 119-121° C./1H-NMR(CDCl3, TMS, ppm): δ 2.88(d, J=0.5 Hz, 3H), 4.86(d, J=5.3 Hz, 2H), 6.16(s, 1H), 7.25-7.35(m, 4H), 8.01(d, J=2.0 Hz, 1H), 8.34(m, 1H), 9.05-9.30(m, 1H).", "Example 197 Potassium carbonate (0.76 g, 5.5 mmol) and 2-bromoethyl isothiocyanate (0.83 g, 5 mmol) were added to a solution of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole (1.39 g, 5.0 mmol) in DMF (15 ml), and the mixture was stirred at room temperature for 3 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid (30 ml) and extracted with ethyl acetate (30 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/20→1/10), to give a white solid of N-vinyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide (0.11 g, yield: 5.0%).", "mp: 90-92° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.87(d, J=0.8 Hz, 3H), 4.79(d, J=8.6 Hz, 1H), 5.00(dd, J=0.9 and 15.7 Hz, 1H), 6.19(q, J=0.8 Hz, 1H), 7.45(ddd, J=0.9, 8.6 and 15.7 Hz, 1H), 8.04(d, J=2.1 Hz, 1H), 8.36(m, 1H), 10.15-10.55(m, 1H).", "Example 198 Trichloromethyl chloroformate (0.59 g, 3.0 mmol) was added to a solution of 5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole (0.48 g, 2.0 mmol) in ethyl acetate (10 ml) at 0° C., and while the mixture was allowed to have room temperature gradually, the mixture was further stirred at room temperature for 3 hours.", "To the reaction mixture were added O-ethylhydroxylamine hydrochloride (0.98 g, 10.0 mmol) and triethylamine (0.61 g, 6.0 mmol), and the resulting mixture was refluxed under heating.", "After completion of the reaction, the reaction mixture was poured into ice water, and the mixture was extracted with ethyl acetate (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a yellowish viscous substance of N-ethoxy-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.30 g, yield: 45.4%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.31(t, J=7.0 Hz, 3H), 2.61(d, J=0.8 Hz, 3H), 4.05(q, J=7.0 Hz, 2H), 5.78(q, J=0.8 Hz, 1H), 7.33-7.37(m, 1H), 7.42-7.50(m, 3H), 9.25(br s, 1H).", "Example 199 5-Methyl-3-(3-trifluoromethylphenyloxy)pyrazole reacted with trichloromethyl chloroformate in the same manner as in Example 198 and then reacted with tetrahydrofurfurylamine to give a white solid of N-tetrahydrofurfuryl-5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide (yield: 70.3%).", "mp: 101-102° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.56-1.67(m, 1H), 1.84-2.05(m, 3H), 2.60(s, 3H), 3.29-3.39(m, 1H), 3.55(ddd, J=3.8, 6.1 and 13.8 Hz, 1H), 3.70-3.90(m, 2H), 4.00-4.10(m, 1H), 5.74(s, 1H), 7.21(br s, 1H), 7.33-7.37(m, 1H), 7.40-7.51(m, 3H).", "Example 200 Trichloromethyl chloroformate (0.58 g, 3.0 mmol) was added to a solution of 3-(4-chloro-2-trifluoromethylphenyloxy)-5-methylpyrazole (0.55 g, 2.0 mmol) in ethyl acetate (10 ml) at 0° C., and the mixture was further stirred at an ambient temperature for 30 minutes.", "Then, the mixture was refluxed under heating for 1 hour.", "The solvent was distilled off from the reaction mixture and then the reaction mixture was cooled to 0° C. and dissolved in toluene (10 ml).", "Triethylamine (0.61 g, 6.0 mmol) and tetrahydrofurfurylamine (1.01 g, 10.0 mmol) were added, and while the mixture was allowed to have room temperature gradually, it was stirred for 1 hour.", "Then, the mixture was refluxed under heating for 2 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml), and the mixture was extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×3), dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a colorless viscous substance of N-tetrahydrofurfuryl-3-(4-chloro-2-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.40 g, yield: 48.9%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.55-1.66(m, 1H), 1.84-2.02(m, 3H), 2.59(d, J=0.8 Hz, 3H), 3.26-3.37(m, 1H), 3.54(ddd, J=3.7, 6.2 and 13.8 Hz, 1H), 3.71-3.90(m, 2H), 4.00-4.16(m, 1H), 5.74(q, J=0.8 Hz, 1H), 7.18(br s, 1H), 7.25(d, J=8.7 Hz, 1H), 7.48(dd, J=2.5 and 8.7 Hz, 1H), 7.64(d, J=2.5 Hz, 1H).", "Example 201 Trichloromethyl chloroformate (0.59 g, 3.0 mmol) was added to a solution of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.83 g, 3.0 mmol) in chloroform (10 ml) at 0° C., and while the mixture was allowed to have room temperature gradually, it was further stirred at room temperature for 3 hours.", "Cyclopropylamine (0.17 g, 3.0 mmol) and potassium carbonate (0.50 g, 3.6 mmol) were added to the reaction mixture, and the reaction mixture was refluxed under heating for 5 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with chloroform (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7), to give a colorless viscous substance of N-cyclopropyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.27 g, yield: 22.8%).", "1H-NMR(CDCl3, TMS, ppm): δ 0.55-0.65(m, 2H), 0.75-0.85(m, 2H), 2.59(d, J=0.4 Hz, 3H), 2.60-2.75(m, 1H), 5.71(m, 1H), 6.75-6.90(m, 1H), 7.67(s, 2H).", "Examples 202-216 3-(2,6-Dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole reacted with trichloromethyl chloroformate, and then reacted with amines (Example 202: s-butylamine, Example 203: 3-aminopentane, Example 204: cyclopentylamine, Example 205: propargylamine, Example 206: O-ethylhydroxylamine hydrochloride, Example 207: O-tert-butylhydroxylamine, Example 208: O-allylhydroxylamine, Example 209: O-benzylhydroxylamine, Example 210: ethanolamine, Example 211: 2,2,2-trifluoroethylamine, Example 212: tetrahydrofurfurylamine, Example 213: furfurylamine, Example 214: 2-morpholinoethylamine, Example 215: 2-picolylamine, Example 216: 2-thienylmethylamine) in the same manner as in Example 201, to give N-substituted carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 202 N-s-butyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 48.8%/mp: 73-75° C./1H-NMR(CDCl3, TMS, ppm): δ 0.92(t, J=7.5 Hz, 3H), 1.19(d, J=6.7 Hz, 3H), 1.45-1.60(m, 2H), 2.57(d, J=0.7 Hz, 3H), 3.82(dq, J=6.7 and 8.4 Hz, 1H), 5.62(d, J=0.7 Hz, 1H), 6.62(br d, J=8.4 Hz, 1H), 7.57(s, 2H).", "Example 203 N-(3-pentyl)-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 43.6%/mp: 65-67° C./1H-NMR(CDCl3, TMS, ppm): δ 0.91(t, J=7.4 Hz, 6H), 1.53(m, 4H), 2.58(s, 3H), 3.67(m, 1H), 5.63(s, 1H), 6.58(br d, J=9.0 Hz, 1H), 7.67(s, 2H).", "Example 204 N-cyclopentyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 47.4%/mp: 90-92° C./1H-NMR(CDCl3, TMS, ppm): δ 1.35-1.80(m, 6H), 1.90-2.10(m, 2H), 2.57(d, J=0.4 Hz, 3H), 4.13(dt, J=7.0 and 7.0 Hz, 1H), 5.61(d, J=0.6 Hz, 1H), 6.72(br d, J=7.0 Hz, 1H), 7.67(s, 2H).", "Example 205 N-propargyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 21.7%/1H-NMR(CDCl3, TMS, ppm): δ 2.25(t, J=2.5 Hz, 1H), 2.59(s, 3H), 4.09(dd, J=2.5 and 6.0 Hz, 2H), 5.78(s, 1H), 6.85-7.00(m, 1H), 7.67(s, 2H).", "Example 206 N-ethoxy-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 5.0%/mp: 105-107° C./1H-NMR(CDCl3, TMS, ppm): δ 1.28(t, J=7.1 Hz, 3H), 2.59(s, 3H), 4.03(q, J=7.1 Hz, 2H), 5.82(s, 1H), 7.67(s, 2H), 9.05(s, 1H).", "Example 207 N-t-butoxy-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 40.7%/mp: 92-94° C./1H-NMR(CDCl3, TMS, ppm): δ 1.29(s, 9H), 2.58(d, J=0.8 Hz, 3H), 5.76(q, J=0.8 Hz, 1H), 7.67(s, 2H), 8.73(s, 1H).", "Example 208 N-allyloxy-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 37.8%/mp: 68-70° C./1H-NMR(CDCl3, TMS, ppm): δ 2.59(d, J=0.8 Hz, 3H), 4.35-4.50(m, 2H), 5.20-5.45(m, 2H), 5.82(d, J=0.8 Hz, 1H), 5.85-6.10(m, 1H), 7.67(d, J=0.4 Hz, 2H), 9.06(br s, 1H).", "Example 209 N-benzyloxy-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 26.1%/mp: 98-100° C./1H-NMR(CDCl3, TMS, ppm): δ 2.61(d, J=0.7 Hz, 3H), 4.95(s, 2H), 5.83(q, J=0.9 Hz, 1H), 7.30-7.45(m, 5H), 7.63(d, J=0.5 Hz, 2H), 9.00(br s, 1H).", "Example 210 N-(2-hydroxyethyl)-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 20.1%/mp: 124-126° C./1H-NMR(CDCl3, TMS, ppm): δ 2.25(m, 1H), 2.58(s, 3H), 3.40-3.55(m, 2H), 3.70-3.85(m, 2H), 5.76(q, J=0.5 Hz, 1H), 6.90-7.15(m, 1H), 7.66(s, 2H).", "Example 211 N-2,2,2-trifluoroethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 51.6%/mp: 104-106° C./1H-NMR(CDCl3, TMS, ppm): δ 2.60(d, J=0.5 Hz, 3H), 3.93(dq, J=6.8 and 8.8 Hz, 2H), 5.80(q, J=0.7 Hz, 1H), 7.05(br t, J=6.8 Hz, 1H), 7.68(s, 2H).", "Example 212 N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 13.7%/mp: 74-76° C./1H-NMR(CDCl3, TMS, ppm): δ 1.45-1.65(m, 1H), 1.75-2.05(m, 3H), 2.58(d, J=0.8 Hz, 3H), 3.32(dt, J=6.2 and 13.9 Hz, 1H), 3.47(ddd, J=3.7, 5.6 and 13.9 Hz, 1H), 3.60-3.85(m, 2H), 3.95-4.15(m, 1H), 5.75(q, J=0.8 Hz, 1H), 6.85-7.15(m, 1H), 7.66(s, 2H).", "Example 213 N-furfuryl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/orange viscous substance/yield: 24.0%/1H-NMR(CDCl3, TMS, ppm): δ 2.59(d, J=0.6 Hz, 3H), 4.47(d, J=6.0 Hz, 2H), 5.73(q, J=0.8 Hz, 1H), 6.25(m, 1H), 6.32(dd, J=1.8 and 3.2 Hz, 1H), 7.07(br t, J=6.0 Hz, 1H), 7.35(dd, J=0.8 and 1.8 Hz, 1H), 7.66(s, 2H).", "Example 214 N-(2-morpholinoethyl)-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 25.7%/mp: 99-101° C./1H-NMR(CDCl3, TMS, ppm): δ 2.40(t, J=4.7 Hz, 4H), 2.50(t, J=6.2 Hz, 2H), 2.59(d, J=0.8 Hz, 3H), 3.37(dt, J=5.4 and 6.1 Hz, 2H), 3.57(t, J=4.7 Hz, 4H), 5.80(q, J=0.8 Hz, 1H), 7.10-7.25(m, 1H), 7.67(s, 2H).", "Example 215 N-2-picolyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 27.0%/mp: 121-123° C./1H-NMR(CDCl3, TMS, ppm): δ 2.60(d, J=0.8 Hz, 3H), 4.59(d, J=5.8 Hz, 2H), 5.77(q, J=0.8 Hz, 1H), 7.19(dd, J=5.8 and 7.8 Hz, 1H), 7.31(d, J=7.8 Hz, 1H), 7.55-7.70(m, 4H), 8.50(d, J=4.2 Hz, 1H).", "Example 216 N-(2-thienylmethyl)-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/yellow viscous substance/yield: 34.1%/1H-NMR(CDCl3, TMS, ppm): δ 2.61(d, J=0.8 Hz, 3H), 4.65(d, J=5.9 Hz, 2H), 5.74(q, J=0.8 Hz, 1H), 6.90-7.05(m, 2H), 7.05-7.20(m, 1H), 7.20-7.30(m, 1H), 7.66(s, 2H).", "Example 217 Trichloromethyl chloroformate (0.39 g, 2.0 mmol) was added to a solution of 3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.59 g, 2.0 mmol) in chloroform (10 ml) at 0° C., and while the mixture was allowed to have room temperature gradually, it was further stirred at room temperature for 3 hours.", "Tetrahydrofurfurylamine (0.61 g, 6.0 mmol) and triethylamine (0.4 g, 4.0 mmol) were added to the reaction mixture, the reaction mixture was refluxed under heating for 5 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with chloroform (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10→1/5), to give a colorless viscous substance of N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.25 g, yield: 29.6%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.50-1.65(m, 1H), 1.75-2.05(m, 3H), 2.58(s, 3H), 3.32(dt, J=6.3 and 13.9 Hz, 1H), 3.48(ddd, J=3.7, 5.7 and 13.9 Hz, 1H), 3.74(dq, J=7.0 and 8.6 Hz, 2H), 4.03(dq, J=3.7 and 7.0 Hz, 1H), 5.79(s, 1H), 6.85-7.10(m, 1H), 7.39(dd, JHF=1.9 and 9.3 Hz, 1H), 7.56(s, 1H).", "Example 218 Trichloromethyl chloroformate (0.49 g, 2.5 mmol) was added to a solution of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole (0.69 g, 2.5 mmol) in chloroform (10 ml) at 0° C., and while the mixture was allowed to have room temperature gradually, it was further stirred at room temperature for 3 hours.", "Sec-butylamine (0.22 g, 3.0 mmol) and potassium carbonate (0.42 g, 3.0 mmol) were added to the reaction mixture, and the mixture was stirred at 50° C. for 8 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with chloroform (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7), to give a colorless viscous substance of N-s-butyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (0.24 g, yield: 25.5%).", "1H-NMR(CDCl3, TMS, ppm): δ 0.96(t, J=7.5 Hz, 3H), 1.22(d, J=6.6 Hz, 3H), 1.45-1.65(m, 2H), 2.65(s, 3H), 3.75-4.00(m, 1H), 6.04(s, 1H), 6.75-7.05(m, 1H), 8.02(d, J=1.8 Hz, 1H), 8.30-8.40(m, 1H).", "Examples 219-222 3-(3-Chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole reacted with trichloromethyl chloroformate, and then reacted with amines (Example 219: cyclopropylamine, Example 220: isobutylamine, Example 221: O-methylhydroxylamine hydrochloride, Example 223: O-ethylhydroxylamine hydrochloride) in the same manner as in Example 218, to give corresponding carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 219 N-cyclopropyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 37.9%/mp: 98-99° C./1H-NMR(CDCl3, TMS, ppm): δ 0.55-0.70(m, 2H), 0.75-0.90(m, 2H), 2.66(s, 3H), 2.70-2.85(m, 1H), 6.05(s, 1H), 7.05-7.20(m, 1H), 8.02(d, J=2.1 Hz, 1H), 8.35(d, J=2.1 Hz, 1H).", "Example 220 N-isobutyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 29.7%/mp: 45-47° C./1H-NMR(CDCl3, TMS, ppm): δ 0.96(d, J=6.7 Hz, 6H), 1.87(sep, J=6.7 Hz, 1H), 2.65(d, J=0.6 Hz, 3H), 3.18(t, J=6.5 Hz, 2H), 6.04(q, J=0.6 Hz, 1H), 7.00-7.20(m, 1H), 8.02(d, J=2.1 Hz, 1H), 8.35(m, 1H).", "Example 221 N-methoxy-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/colorless viscous substance/yield: 19.4%/1H-NMR(CDCl3, TMS, ppm): δ 2.66(d, J=0.7 Hz, 3H), 3.86(s, 3H), 6.10(q, J=0.7 Hz, 1H), 8.03(d, J=2.1 Hz, 1H), 8.35(m, 1H), 9.35-9.55(m, 1H).", "Example 222 N-ethoxy-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/yellow viscous substance/yield: 38.5%/1H-NMR(CDCl3, TMS, ppm): δ 1.32(t, J=7.0 Hz, 3H), 2.66(s, 3H), 4.07(q, J=7.0 Hz, 2H), 6.10(s, 1H), 8.03(d, J=2.1 Hz, 1H), 8.35(m, 1H), 9.38(br s, 1H).", "Example 223 Trichloromethyl chloroformate (3.0 g, 15.0 mmol) was added to a solution of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole (4.67 g, 15.0 mmol) in chloroform (50 ml) at 0° C., and while the mixture was allowed to have room temperature gradually, it was further stirred at room temperature for 4 hours.", "30% ammonia solution (10 ml) was added to the reaction mixture, and the mixture was further stirred at room temperature for 1 hour.", "After completion of the reaction, water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7), to give a white solid of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (2.82 g, yield: 53.1%).", "mp: 152-154° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.58(d, J=0.4 Hz, 3H), 4.80-5.15(br s, 1H), 5.80(q, J=0.4 Hz, 1H), 6.45-6.80(br s, 1H), 7.66(s, 2H).", "Examples 224-226 3-(Substituted phenyloxy)-5-methylpyrazoles (Example 224: 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole, Example 225: 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole, Example 226: 5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole) reacted with trichloromethyl chloroformate, and then reacted with ammonia solution in the same manner as in Example 223, to give corresponding carboxamide derivatives.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 224 3-(2-chloro-6-fluoro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 35.5%/mp: 132-135° C./1H-NMR(CDCl3, TMS, ppm): δ 2.59(d, J=0.8 Hz, 3H), 4.75-5.20(br s, 1H), 5.84(q, J=0.8 Hz, 1H), 6.45-6.80(br s, 1H), 7.39(dd, JHF=2.0 and 9.3 Hz: 1H), 7.56(s, 1H).", "Example 225 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 76.7%/mp: 101-104° C./1H-NMR(CDCl3, TMS, ppm): δ 2.65(d, J=0.6 Hz, 3H), 5.00-5.40(br s, 1H), 6.08(m, 1H), 6.75-7.15(br s, 1H), 8.03(d, J=2.1 Hz, 1H), 8.36(m, 1H).", "Example 226 5-methyl-3-(3-trifluoromethylphenyloxy)pyrazole-1-carboxamide/white solid/yield: 35.8%/mp: 76-77° C./1H-NMR(CDCl3, TMS, ppm): δ 2.60(d, J=0.8 Hz, 3H), 5.09(br s, 2H), 5.77(q, J=0.8 Hz, 1H), 7.33-7.38(m, 1H), 7.41-7.52(m, 3H).", "Example 227 Trichloromethyl chloroformate (0.46 g, 2.3 mmol) was added to a solution of 3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole (0.47 g, 1.5 mmol) in ethyl acetate (10 ml) at 0° C., and while the mixture was allowed to have room temperature gradually, it was further stirred under reflux for 4 hours.", "The reaction mixture was concentrated, and further, toluene (10 ml), triethylamine (0.46 g, 4.5 mmol) and pyrrolidine (0.43 g, 6.0 mmol) were added.", "Further, the mixture was refluxed under heating for 3 hours.", "After completion of the reaction, water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (50 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant, and the solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a white solid of N,N-tetramethylene-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.11 g, yield: 18.0%).", "mp: 102-104° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.70-1.95(m, 4H), 2.50(d, J=0.6 Hz, 3H), 3.40-3.60(m, 4H), 5.78(q, J=0.6 Hz, 1H), 7.64(s, 2H).", "Example 228 3-(2,6-Dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole reacted with trichloromethyl chloroformate to obtain an intermediate, and further, the intermediate reacted with 2,6-dimethylmorpholine, in the same manner as in Example 228, to give a white solid of 3-(2,6-dichloro-4-trifluoromethylphenyloxy-1-(2,6-dimethylmorpholino)carbonyl-5-methylpyrazole (0.14 g, yield: 20.6%).", "mp: 87˜89° C.; 1H-NMR(CDCl3, TMS, ppm): δ 0.90-1.30(m, 6H), 2.46(d, J=0.7 Hz, 3H), 2.50-2.65(m, 2H), 3.20-3.75(m, 2H), 3.80-4.25(m, 2H), 5.85(q, J=0.7 Hz, 1H), 7.64(s, 2H).", "Example 229 N-allyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (4.3 g, 10.9 mmol) was dissolved in a solution mixture of ether (50 ml) and water (50 ml), an aqueous solution (60 ml) of osmium tetraoxide (254 mg, 1.0 mmol) and sodium periodate (4.7 g, 21.8 mmol) were added, and the mixture was stirred at room temperature overnight.", "After completion of the reaction, a 10% sodium thiosulfate aqueous solution (100 ml) and ethyl acetate (100 ml) were added to the reaction solution, and an organic layer was separated and an aqueous layer was extracted with ethyl acetate (50 ml×2).", "These organic layers combined were washed with a saturated sodium chloride aqueous solution, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7→1/5), to give a white solid of N-formylmethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (2.12 g, yield: 49.1%).", "mp: 111-113° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.59(d, J=0.8 Hz, 3H), 4.17(d, J=5.5 Hz, 2H), 5.81(q, J=0.8 Hz, 1H), 7.21(br t, J=5.5 Hz, 1H), 7.67(s, 2H), 9.66(s, 1H).", "Example 230 p-Toluenesulfonic acid monohydrate (20 mg, 0.1 mmol) and ethylene glycol (0.31 g, 5 mmol) were added to a solution of N-formylmethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.40 g, 1.0 mmol) in benzene (10 ml), and the mixture was refluxed under heating for 5 hours.", "After completion of the reaction, the solvent was distilled off from the reaction mixture, water (30 ml) was added to the residue, and the mixture was extracted with ethyl acetate (30 ml×3).", "An organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7→chloroform), to give a white solid of N,N-(1,3-dioxa-2-cyclopentyl)methyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.33 g, yield: 75.0%).", "mp: 80-81° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.59(d, J=0.8 Hz, 3H), 3.53(dd, J=3.3 and 6.1 Hz, 2H), 3.80-3.95(m, 4H), 5.03(t, J=3.3 Hz, 1H), 5.76(q, J=0.8 Hz, 1H), 6.92(br t, J=6.1 Hz, 1H), 7.66(s, 2H).", "Examples 231-232 N-Formylmethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide reacted with alcohols (Example 231: 1,3-propanediol, Example 232: methanol) in the same manner as in Example 230, to give corresponding carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 231 N,N-(1,3-dioxa-2-cyclohexyl)methyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 74.9%/mp: 87-89° C./1H-NMR(CDCl3, TMS, ppm): δ 1.25-1.45(m, 1H), 1.95-2.15(m, 1H), 2.58(d, J=0.8 Hz, 3H), 3.41(dd, J=4.7 and 6.1 Hz, 2H), 3.65-3.85(m, 1H), 4.00-4.15(m, 1H), 4.67(t, 4.7 Hz, 1H), 5.73(q, J=0.8 Hz, 1H), 6.90-7.10(br t, J=6.1 Hz, 1H), 7.67(s, 2H).", "Example 232 N,N-2,2-dimethoxyethyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/mp: 57-59° C./1H-NMR(CDCl3, TMS, ppm): δ 2.58(d, J=0.7 Hz, 3H), 3.38(s, 6H), 3.35-3.45(m, 2H), 4.41(t, J=5.4 Hz, 1H), 5.75(q, J=0.8 Hz, 1H), 6.80-7.00(m, 1H), 7.66(d, J=0.4 Hz, 2H).", "Example 233 Potassium hydroxide (0.42 g, 7.5 mmol) and 30% hydrogen peroxide (2.7 ml) were added to a solution of N-butyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carbothioamide (0.98 g, 2.5 mmol) in methanol (10 ml), and the mixture was stirred at room temperature for 3 hours.", "Concentrated hydrochloric acid was added to the reaction mixture up to a pH of 1, and the mixture was further stirred at room temperature for 2 hours.", "After completion of the reaction, the reaction mixture was extracted with ethyl acetate (15 ml×3), and the organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10→1/1), to give a colorless viscous substance of N-butyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (0.17 g, yield: 18.1%).", "1H-NMR(CDCl3, TMS, ppm): δ 0.94(t, J=7.3 Hz, 3H), 1.30-1.50(m, 2H), 1.50-1.70(m, 2H), 2.65(s, 3H), 3.35(dt, J=6.3 and 6.3 Hz, 2H), 6.04(s, 1H), 6.90-7.20(m, 1H), 8.02(d, J=2.0 Hz, 1H), 8.35(m, 1H).", "Example 234 Potassium carbonate (0.07 g, 0.50 mmol) was added to a solution of N-allyl-3-hydroxy-5-methylpyrazole-1-carboxamide (0.18 g, 1.0 mmol) in DMF (5 ml), then, 3,5-dichloro-4-fluorotrifluoromethylbenzene (0.23 g, 1.0 mmol) was added, and the mixture was stirred at room temperature for 5 hours.", "After completion of the reaction, the reaction mixture was poured into 1N hydrochloric acid (10 ml), and the mixture was extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×3) and dried over anhydrous magnesium sulfate, and a desiccant was removed from a filtrate.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a yellow viscous substance of N-allyl-3-(2,6-dichloro-4-trifluoromethyloxy)-5-methylpyrazole-1-carboxamide (0.18 g, yield: 45.7%).", "The product had NMR spectrum as shown in Example 112.Example 235 Diethylcarbamoyl chloride (1.52 g, 7.8 mmol) was added to a solution of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole (1.39 g, 5.0 mmol) and potassium carbonate (1.98 g, 14.3 mmol) in DMF (15 ml), the mixture was stirred at 70° C. for 9 hours and further stirred at room temperature for 2 days.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid, and the mixture was extracted with ethyl acetate (20 ml×3).", "An organic-layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a colorless viscous substance of N,N-diethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (0.21 g, yield: 11.2%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.21(t, J=7.0 Hz, 6H), 2.48(d, J=0.6 Hz, 3H), 3.47(q, J=7.0 Hz, 4H), 6.00(q, J=0.6 Hz, 1H), 7.99(d, J=2.1 Hz, 1H), 8.33(m, 1H).", "Example 236 A solution of diisopropylcarbamoyl chloride (1.47 g, 9.0 mmol) in pyridine (5 ml) was added to a solution of 3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole (1.67 g, 6.0 mmol) in pyridine (10 ml), and the mixture was stirred under heating at 80° C. for 20 hours.", "After completion of the reaction, the reaction mixture was poured into 2N hydrochloric acid, and the mixture was extracted with ethyl acetate (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a white solid of N,N-diisopropyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (2.1 g, yield: 86.7%).", "mp: 58-60° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.28(d, J=7.5 Hz, 12H), 2.29(s, 3H), 3.50-4.10(m, 2H), 6.00(s, 1H), 7.98(d, J=2.5 Hz, 1H), 8.31(m, 1H).", "Example 237 N,N-Dimethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide was obtained in the same manner as in Example 235.White solid/yield: 30.6%/mp: 102-104° C./1H-NMR(CDCl3, TMS, ppm): δ 2.32(s, 3H), 2.85-3.30(br s, 6H), 6.01(s, 1H), 7.99(d, J=2.0 Hz, 1H), 8.30(m, 1H).", "Examples 238-240 3-(3-Chloro-5-trifluoromethylpyridin-2-yloxy)-5-trifluoromethylpyrazole reacted with carbamoyl chloride derivatives (Example 238: dimethylcarbamoyl chloride, Example 240 diethylcarbamoyl chloride, Example 241: diisopropylcarbamoyl chloride) in the same manner as in Example 236, to give corresponding carboxamides.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 238 N,N-dimethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-trifluoromethylpyrazole-1-carboxamide/orange viscous substance/yield: 25.0%/1H-NMR(CDCl3, TMS, ppm): δ 3.13(s, 6H), 6.51(s, 1H), 8.00-8.10(m, 1H), 8.25-8.35(m, 1H).", "Example 239 N,N-diethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-trifluoromethylpyrazole-1-carboxamide/white solid/yield: 58.1%/mp: 58-60° C./1H-NMR(CDCl3, TMS, ppm): δ 1.27(m, 6H), 3.40(q, J=6.9 Hz, 4H), 6.51(s, 1H), 8.03(d, J=2.1 Hz, 1H), 8.30(m, 1H).", "Example 240 N,N-diisopropyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-trifluoromethylpyrazole-1-carboxamide/colorless viscous substance/yield: 50.9%/1H-NMR(CDCl3, TMS, ppm): δ 1.10-1.55(m, 12H), 3.35-4.05(m, 2H), 6.53(s, 1H), 8.04(d, J=2.2 Hz, 1H), 8.34(m, 1H).", "Example 241 N,N-Diisopropyl-3-(2-nitro-4-trifluoromethylphenyloxy)-5-trifluoromethylpyrazole-1-carboxamide was obtained in the same manner as in Example 236.White solid/yield: 22.9%/mp: 118-120° C./1H-NMR(CDCl3, TMS, ppm): δ 1.33(d, J=5.9 Hz, 12H), 2.27(s, 3H), 3.30-4.25(m, 2H), 5.70(s, 1H), 7.43(d, J=8.8 Hz, 1H), 7.78(dd, J=2.0 and 8.8 Hz, 1H), 8.24(d, J=2.0 Hz, 1H).", "Example 242 A solution of N-methyl-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide (2.1 g, 6.0 mmol) in ethanol (50 ml) was placed in an autoclave, and 10% palladium carbon (0.7 g) was added.", "An atmosphere in the autoclave was fully replaced with hydrogen gas, and hydrogen gas was charged up to 5 kg/cm2.Then, the reaction solution was stirred at room temperature for 5 hours.", "After completion of the reaction, the catalyst was removed by filtration using Celite, and the solvent was distilled off from the filtrate under reduced pressure, to give a yellowish solid of N-methyl-3-(2-amino-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (1.6 g, yield: 82.2%).", "mp: 93-94; 1H-NMR(CDCl3, TMS, ppm): δ 2.59(d, J=0.7 Hz, 3H), 2.93(d, J=5.0 Hz, 1H), 3.90-4.15(br s, 2H), 5.71(q, J=0.7 Hz, 1H), 6.80-6.95(m, 1H), 6.97(dd, J=1.7 and 8.4 Hz, 1H), 7.11(d, J=8.4 Hz, 1H).", "Example 243 Sulfuryl chloride (0.24 g, 1.8 mmol) was added to a solution of N-isopropyl-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.56 g, 1.5 mmol) in acetic acid (5 ml), and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with ethyl acetate (20 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a yellowish solid of N-isopropyl-4-chloro-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.60 g, yield: 98.4%).", "mp: 89-91° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.24(d, J=6.6 Hz, 6H), 2.63(s, 3H), 3.90-4.10(m, 1H), 6.40-6.65(m, 1H), 7.38(d, J=8.7 Hz, 1H), 7.85(dd, J=2.0 and 8.7 Hz, 1H), 8.29(d, J=2.0 Hz, 1H).", "Example 244 A white solid of N-methyl-4-chloro-5-methyl-3-(2-nitro-4-trifluoromethylphenyloxy)pyrazole-1-carboxamide was obtained in the same manner as in Example 243 (0.36 g, yield: 79.3%).", "mp: 107-108° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.63(s, 3H), 2.91(d, J=5.0 Hz, 3H), 6.50-6.75(m, 1H), 7.46(d, J=8.7 Hz, 1H), 7.88(dd, J=2.0 and 8.7 Hz, 1H), 8.30(d, J=2.0 Hz, 1H).", "Example 245 Sulfuryl chloride (0.16 g, 1.2 mmol) was added to a solution of N-methyl-5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.34 g, 1.0 mmol) in acetic acid (5 ml), and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-methyl-4-chloro-5-methyl-3-(4-nitro-2-trifluoromethylphenyloxy)pyrazole-1-carboxamide (0.27 g, yield: 71.2%).", "mp: 157-158° C.; 1H-NMR(CDCl3, TMS, ppm): δ 2.66(s, 3H), 2.96(d, J=5.0 Hz, 3H), 6.70-6.95(m, 1H), 7.31(d, J=9.2 Hz, 1H), 8.41(dd, J=2.7 and 9.2 Hz, 1H), 8.62(d, J=2.7 Hz, 1H).", "Example 246 Sulfuryl chloride (0.16 g, 1.2 mmol) was added to a solution of N-hexyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.44 g, 1.0 mmol) in acetic acid (5 ml), and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane 1/10), to give a white solid of N-hexyl-4-chloro-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.40 g, yield: 84.8%).", "mp: 86-88° C.; 1H-NMR(CDCl3, TMS, ppm): δ 0.88(t, J=6.8 Hz, 3H), 1.26(m, 6H), 1.40-1.60(m, 2H), 2.62(s, 3H), 3.15-3.35(m, 2H), 6.50-6.70(m, 1H), 7.67(s, 2H).", "Examples 247-248 N-Substituted-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide derivatives (Example 247: N-methyl compound, Example 248: N-ethyl compound) reacted with sulfuryl chloride in the same manner as in Example 246, to give corresponding 4-chloro derivatives.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 247 N-methyl-4-chloro-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 66.5%/mp: 125-127° C./1H-NMR(CDCl3, TMS, ppm): δ 2.62(s, 3H), 2.87(d, J=5.0 Hz, 3H), 6.35-6.70(m, 1H), 7.67(s, 2H).", "Example 248 N-ethyl-4-chloro-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 48.0%/mp: 85-86° C./1H-NMR(CDCl3, TMS, ppm): δ 1.17(t, J=7.2 Hz, 3H), 2.62(s, 3H), 3.32(dq, J=6.1 and 7.2 Hz, 2H), 6.40-6.70(m, 1H), 7.67(m, 2H).", "Example 249 Sulfuryl chloride (0.16 g, 1.2 mmol) was added to a solution of N-isopropyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.40 g, 1.0 mmol) in acetic acid (5 ml), and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/20), to give a white solid of N-isopropyl-4-chloro-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.19 g, yield: 44.1%) [mp: 109-111° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.20(d, J=7.5 Hz, 6H), 2.61(s, 3H), 3.85-4.10(m, 1H), 6.25-6.50(m, 1H), 7.67(s, 2H).]", "and a white solid of N-isopropyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-trichloromethylpyrazole-1-carboxamide (0.04 g, yield: 8.0%) [mp: 105-106° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.22(d, J=6.5 Hz, 6H), 3.85-4.15(m, 1H), 6.25-6.55(m, 1H), 7.69(s, 2H), 8.24(s, 1H)].", "Example 250 Sulfuryl chloride (0.09 g, 0.7 mmol) was added to a solution of N-propyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazole-1-carboxamide (0.24 g, 0.6 mmol) in acetic acid (5 ml), and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/20), to give a white solid of N-propyl-3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-trichloromethylpyrazole-1-carboxamide (0.11 g, yield: 36.7%).", "mp: 89-91° C.; 1H-NMR(CDCl3, TMS, ppm): δ 0.90(t, J=7.3 Hz, 3H), 1.56(tq, J=7.3 and 8.0 Hz, 2H), 3.25(dt, J=6.3 and 8.0 Hz, 2H), 6.50-6.80(m, 1H), 7.69(s, 2H), 8.23(s, 1H).", "Example 251 Sulfuryl chloride (0.16 g, 1.2 mmol) was added to a solution of N-ethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (0.35 g, 1.0 mmol) in acetic acid (5 ml), and the mixture was stirred at room temperature for 4 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-ethyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (0.35 g, yield: 92.1%).", "mp: 111-112° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.23(t, J=7.3 Hz, 3H), 2.66(s, 3H), 3.41(dq, J=5.8 and 7.3 Hz, 2H), 6.85-7.10(m, 1H), 8.03(d, J=2.0 Hz, 1H), 8.31(m, 1H).", "Examples 252-260 N-Substituted-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide derivatives reacted with sulfuryl chloride in the same manner as in Example 251, to give corresponding 4-chloro derivatives.", "Products/forms/yields/melting points/NMR spectra are described below.", "Example 252 N-methyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 97.1%/mp: 81-83° C./1H-NMR(CDCl3, TMS, ppm): δ 2.66(s, 3H), 2.96(d, J=5.0 Hz, 3H), 6.80-7.15(m, 1H), 8.04(d, J=2.1 Hz, 1H), 8.32(m, 1H).", "Example 253 N-propyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 90.4%/mp: 54-56° C./1H-NMR(CDCl3, TMS, ppm): δ 0.97(t, J=7.3 Hz, 3H), 1.62(tq, J=7.0 and 7.3 Hz, 2H), 2.65(s, 3H), 3.32(dt, J=7.0 and 7.3 Hz, 2H), 6.90-7.15(m, 1H), 8.03(d, J=2.1 Hz, 1H), 8.31(m, 1H).", "Example 254 N-isopropyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 89.2%/mp: 57-59° C./1H-NMR(CDCl3, TMS, ppm): δ 1.25(d, J=6.5 Hz, 6H), 2.65(s, 3H), 3.95-4.20(m, 1H), 6.75-6.95(m, 1H), 8.04(d, J=2.0 Hz, 1H), 8.32(m, 1H).", "Example 255 N-pentyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 80.0%/mp: 76-78° C./1H-NMR(CDCl3, TMS, ppm): δ 0.90(t, J=6.9 Hz, 3H), 1.20-1.45(m, 4H), 1.50-1.70(m, 2H), 2.65(s, 3H), 3.25-3.45(m, 2H), 6.85-7.10(m, 1H), 8.03(d, J=2.0 Hz, 1H), 8.32(m, 1H).", "Example 256 N-hexyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 70.6%/mp: 57-59° C./1H-NMR(CDCl3, TMS, ppm): δ 0.88(t, J=6.9 Hz, 3H), 1.15-1.45(m, 6H), 1.45-1.70(m, 2H), 2.65(s, 3H), 3.20-3.45(m, 2H), 6.90-7.05(m, 1H), 8.04(d, J=2.5 Hz, 1H), 8.32(m, 1H).", "Example 257 N-heptyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 80.0%/mp: 67-68° C./1H-NMR(CDCl3, TMS, ppm): δ 0.88(t, J=7.0 Hz, 3H), 1.10-1.45(m, 8H), 1.45-1.70(m, 2H), 2.65(s, 3H), 3.20-3.50(m, 2H), 6.80-7.10(m, 1H), 8.03(d, J=2.0 Hz, 1H), 8.32(m, 1H).", "Example 258 N-octyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 88.6%/mp: 61-62° C./1H-NMR(CDCl3, TMS, ppm): δ 0.87(t, J=6.3 Hz, 3H), 1.15-1.45(m, 10H), 1.45-1.70(m, 2H), 2.65(s, 3H), 3.25-3.45(m, 2H), 6.90-7.10(m, 1H), 8.03(d, J=1.8 Hz, 1H), 8.32(m, 1H).", "Example 259 N-dodecyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 89.9%/mp: 68-70° C./1H-NMR(CDCl3, TMS, ppm): δ 0.88(t, J=6.9 Hz, 3H), 1.15-1.40(m, 18H), 1.50-1.65(m, 2H), 2.65(s, 3H), 3.25(m, 2H), 6.90-7.10(m, 1H), 8.04(d, J=1.9 Hz, 1H), 8.25-8.35(m, 1H).", "Example 260 N-cyclohexyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 85.7%/mp: 94-96° C./1H-NMR(CDCl3, TMS, ppm): δ 1.05-1.50(m, 6H), 1.55-1.90(m, 2H), 1.90-2.10(m, 2H), 2.65(s, 3H), 3.60-3.85(m, 1H), 6.75-7.00(m, 1H), 8.03(d, J=1.8 Hz, 1H), 8.32(m, 1H).", "Example 261 N-(2-chloroethyl)-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide/white solid/yield: 86.2%/mp: 112-114° C./1H-NMR(CDCl3, TMS, ppm): δ 2.65(s, 3H), 3.60-3.80(m, 4H), 7.30-7.45(m, 1H), 8.04(d, J=2.1 Hz, 1H), 8.32(m, 1H).", "Example 262 Sulfuryl chloride (0.17 g, 1.2 mmol) was added to a solution of N-methyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-ethylpyrazole-1-carboxamide (0.35 g, 1.0 mmol) in acetic acid (5 ml), and the mixture was stirred at room temperature for 5 hours.", "After completion of the reaction, the reaction mixture was poured into ice water and extracted with ethyl acetate (10 ml×3).", "The organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/10), to give a white solid of N-methyl-4-chloro-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-ethylpyrazole-1-carboxamide (0.31 g, yield: 80.9%).", "mp: 88-89° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.30(t, J=7.4 Hz, 3H), 2.96(d, J=5.0 Hz, 3H), 3.14(q, J=7.4 Hz, 2H), 6.90-7.10(m, 1H), 8.04(d, J=2.0 Hz, 1H), 8.32(m, 1H).", "Example 263 N-Bromosuccinimide (0.64 g, 3.6 mmol) was added to a solution of N-ethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (1.05 g, 3.0 mmol) in dichloromethane (20 ml), and the mixture was stirred at room temperature for 6 hours.", "After completion of the reaction, water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/7), to give a white solid of N-ethyl-4-bromo-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (0.86 g, yield: 67.0%).", "mp: 123-125° C.; 1H-NMR(CDCl3, TMS, ppm): δ 1.23(t, J=7.3 Hz, 3H), 2.67(s, 3H), 3.40(dq, J=5.9 and 7.3 Hz, 2H), 6.90-7.10(m, 1H), 8.04(d, J=2.0 Hz, 1H), 8.32 (m, 1H).", "Example 264 Sodium hydride (60% in oil, 0.03 g, 0.7 mmol) was added to a solution of ethyl 3-[{3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazol-1-yl}carbonylamino]propionate (0.30 g, 0.7 mmol) in DMF (5 ml) at 0° C., and the mixture was stirred at an ambient temperature for 30 minutes.", "Then, methyl iodide (0.10 g, 0.7 mmol) was added, and the mixture was allowed to have room temperature gradually and stirred overnight.", "After completion of the reaction, the reaction mixture was poured into water (10 ml) and extracted with ethyl acetate (10 ml×2).", "An organic layer was washed with water (10 ml×3), dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/15), to give a colorless viscous substance of ethyl 3-[{3-(2,6-dichloro-4-trifluoromethylphenyloxy)-5-methylpyrazol-1-yl}carbonyl]methylamino]propionate (0.23 g, yield: 74.4%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.28(t, J=7.1 Hz, 3H), 2.22-2.40(m, 2H), 2.45(s, 3H), 2.96(s, 3H), 3.57(m, 2H), 4.12(q, J=7.1 Hz, 2H), 5.83(s, 1H), 7.63(s, 2H).", "Example 265 Chloromethyl methyl ether (0.16 g, 2.0 mmol) was added to a solution of sodium hydride (60% in oil, 0.09 g, 2.2 mmol) and N-methyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (0.67 g, 2.0 mmol) in DMF (10 ml) at 0° C., and while the mixture was allowed to have room temperature gradually, the mixture was stirred overnight.", "After completion of the reaction, water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (10 ml×3).", "An organic layer was washed with water, dried over anhydrous magnesium sulfate and filtered to remove a desiccant.", "The solvent was distilled off from the filtrate under reduced pressure.", "The resultant crude product was purified with a silica gel column (ethyl acetate/hexane=1/5), to give a colorless viscous substance of N-methyl-N-methoxymethyl-3-(3-chloro-5-trifluoromethylpyridin-2-yloxy)-5-methylpyrazole-1-carboxamide (0.18 g, yield: 23.8%).", "1H-NMR(CDCl3, TMS, ppm): δ 1.87(s, 3H), 3.52(s, 3H), 3.53(s, 3H), 5.18(s, 2H), 5.46(s, 1H), 7.80-7.95(m, 1H), 8.50-8.65(m, 1H).", "The following Tables 1 to 4 show compounds of the present invention that can be synthesized by those processes shown in the above Production Examples, Referential Examples and Examples.", "The present invention shall not be limited to these compounds.", "Abbreviations in Tables have the following meanings.", "Me: methyl; Et: ethyl, Pr: propyl; i-Pr: isopropyl; c-Pr: cyclopropyl, Bu: butyl; i-Bu: isobutyl; s-Bu: secondary butyl; t-Bu: tertiary butyl; Pent: pentyl; c-Pent: cyclopentyl; Hex: hexyl; c-Hex: cyclohexyl; Hep: heptyl; Oct: octyl; Dod: dodecyl; 1): tetrahydrofurfuryl; 2): (1,3-dioxa-2-cyclopentyl)methyl; 3): (1,3-dioxa-2-cyclohexyl)methyl; 4): furfuryl; 5): 2-morpholinoethyl; 6): 2-picolyl; 7): 2-thienylmethyl; 8): (4-chloro-5-cyclopentyloxy-2-fluoro)phenyl; TABLE 1 7/26 3-(Substituted phenyloxy)pyrazole derivatives Compound No.", "Example No.", "R1 R2 Xn 1-1 H H 2,6-Cl2-4-CF3 1-2 H H 2-Cl-6-F-4-CF3 1-3 47 Me H H 1-4 3 Me H 3-Me-4-NO2 1-5 4 Me H 3-MeO-4-NO2 1-6 40 Me H 3-EtO-4-NO2 1-7 41 Me H 5-EtO-2-NO2 1-8 48 Me H 3-Cl 1-9 Me Cl 3-Cl 1-10 Me H 2-Cl-4-CF3 1-11 Me Cl 2-Cl-4-CF3 1-12 51 Me H 4-Cl-2-CF3 1-13 Me Cl 4-Cl-2-CF3 1-14 50 Me H 4-Cl-3-CF3 1-15 Me Cl 4-Cl-3-CF3 1-16 Me H 4-F-3-CF3 1-17 18 Me H 2,6-Cl2-4-CF3 1-18 52 Me Cl 2, 6-Cl2-4-CF3 1-19 29 Me H 2-Cl-6-F-4-CF3 1-20 Me Cl 2-Cl-6-F-4-CF3 1-21 31 Me H 2-Cl-6-NO2-4-CF3 1-22 44 Me H 3-Cl-4-NH2 1-23 14 Me H 3-Cl-4-NO2 1-24 Me Cl 3-Cl-4-NO2 1-25 14 Me H 5-Cl-2-NO2 1-26 15 Me H 3-F-4-NO2 1-27 Me Cl 3-F-4-NO2 1-28 15 Me H 5-F-2-NO2 1-29 2 Me H 4-F-2-NO2 1-30 17 Me H 4-CN-2-CF3 1-31 49 Me H 2-CF3 1-32 46 Me H 3-CF3 1-33 Me Cl 3-CF3 1-34 Me H 2,4-(CF3)2 1-35 Me Cl 2,4-(CF3)2 1-36 43 Me H 4-NH2 1-37 42 Me H 4-NH2-2-CF3 1-38 45 Me H 4-NH2-3-CF3 1-39 Me H 2-NH2-4-CF3 1-40 10 Me H 4-NO2-2-CF3 1-41 Me Cl 4-NO2-2-CF3 1-42 16 Me H 4-NO2-3-CF3 1-43 Me Cl 4-NO2-3-CF3 1-44 5 Me H 2-NO2-4-CF3 1-45 Me Cl 2-NO2-4-CF3 1-46 30 Me H 2,4-(NO2)2-6-CF3 1-47 1 Me H 4-NO2 1-48 26 Me Me 2,6-Cl2-4-CF3 1-49 Me Me 2-Cl-6-F-4-CF3 1-50 12 Me Me 4-NO2-2-CF3 1-51 8 Me Me 2-NO2-4-CF3 1-52 27 Me Et 2,6-Cl2-4-CF3 1-53 Me Et 2-Cl-6-F-4-CF3 1-54 13 Me Et 4-NO2-2-CF3 1-55 9 Me Et 2-NO2-4-CF3 1-56 19 Et H 2,6-Cl2-4-CF3 1-57 Et Cl 2,6-Cl2-4-CF3 1-58 Et H 2-Cl-6-F-4-CF3 1-59 Et Cl 2-Cl-6-F-4-CF3 1-60 11 Et H 4-NO2-2-CF3 1-61 Et Cl 4-NO2-2-CF3 1-62 7 Et H 2-NO2-4-CF3 1-63 Et Cl 2-NO2-4-CF3 1-64 Pr H 2,6-Cl2-4-CF3 1-65 Pr H 2-Cl-6-F-4-CF3 1-66 20 i-Pr H 2,6-Cl2-4-CF3 1-67 i-Pr H 2-Cl-6-F-4-CF3 1-68 22 c-Pr H 2,6-Cl2-4-CF3 1-69 c-Pr H 2-Cl-6-F-4-CF3 1-70 21 t-Bu H 2,6-Cl2-4-CF3 1-71 t-Bu H 2-Cl-6-F-4-CF3 1-72 23 MeOCH2 H 2,6-Cl2-4-CF3 1-73 MeOCH2 H 2-Cl-6-F-4-CF3 1-74 25 MeO2CCH2 H 2,6-Cl2-4-CF3 1-75 MeO2CCH2 H 2-Cl-6-F-4-CF3 1-76 CF3 H 2,6-Cl2-4-CF3 1-77 6 CF3 H 2-NO2-4-CF3 1-78 CF3 H 2-Cl-6-F-4-CF3 1-79 24 MeO2C H 2,6-Cl2-4-CF3 1-80 MeO2C H 2-Cl-6-F-4-CF3 1-81 EtO2C H 2,6-Cl2-4-CF3 1-82 EtO2C H 2-Cl-6-F-4-CF3 1-83 28 4-Cl-C6H4 H 2,6-Cl2-4-CF3 1-84 4-Cl-C6H4 H 2-Cl-6-F-4-CF3 TABLE 2 7/26 3-(Substituted pyridyloxy)pyrazole derivatives Compound No.", "Example No.", "R1 R2 Xn 2-1 H H 3-Cl-5-CF3 2-2 34 Me H 4-Me-5-NO2 2-3 33 Me H 6-MeO-3-NO2 2-4 32 Me H 3,5-Cl2 2-5 35 Me H 3-Cl-5-CF3 2-6 Me Cl 3-Cl-5-CF3 2-7 Me H 5-CF3 2-8 Me H 3,5-(NO2)2 2-9 38 Me Me 3-Cl-5-CF3 2-10 39 Me Et 3-Cl-5-CF3 2-11 37 Et H 3-Cl-5-CF3 2-12 Et Cl 3-Cl-5-CF3 2-13 Pr H 3-Cl-5-CF3 2-14 i-Pr H 3-Cl-5-CF3 2-15 c-Pr H 3-Cl-5-CF3 2-16 t-Bu H 3-Cl-5-CF3 2-17 MeOCH2 H 3-Cl-5-CF3 2-18 MeO2CCH2 H 3-Cl-5-CF3 2-19 36 CF3 H 3-Cl-5-CF3 2-20 MeO2C H 3-Cl-5-CF3 2-21 4-Cl-C6H4 H 3-Cl-5-CF3 TABLE 3 Synthesis of 3-(substituted Phenyloxy)pyrazole derivatives Compound Example No.", "No.", "R1 R2 R3 R4 Xn Y 3-1 H H H H 2,6-Cl2-4-CF3 O 3-2 H H H H 2-Cl-6-F-4-CF3 O 3-3 223 Me H H H 2,6-Cl2-4-CF3 O 3-4 Me H H H 2,6-Cl2-4-CF3 S 3-5 224 Me H H H 2-Cl-6-F-4-CF3 O 3-6 Me H H H 2-Cl-6-F-4-CF3 S 3-7 Me H H H 2-Cl-4-NO2-6-CF3 O 3-8 226 Me H H H 3-CF3 O 3-9 Me H H H 4-NO2-2-CF3 O 3-10 Me H H H 4-NO2-3-CF3 O 3-11 Me H H H 2-NO2-4-CF3 O 3-12 118 Me H Me H 2-Cl-6-F-4-CF3 O 3-13 Me H Me H 2-Cl-6-F-4-CF3 S 3-14 105 Me H Me H 2,6-Cl2-4-CF3 O 3-15 179 Me H Me H 2,6-Cl2-4-CF3 S 3-16 Me H Me Me 2,6-Cl2-4-CF3 O 3-17 Me H Me Me 2,6-Cl2-4-CF3 S 3-18 Me H Me Et 2,6-Cl2-4-CF3 O 3-19 Me H Me Et 2,6-Cl2-4-CF3 S 3-20 Me H Me MeOCH2 2,6-Cl2-4-CF3 O 3-21 Me H Me MeOCH2 2,6-Cl2-4-CF3 S 3-22 Me H Me EtOCH2 2,6-Cl2-4-CF3 O 3-23 Me H Me EtOCH2 2,6-Cl2-4-CF3 S 3-24 127 Me H Me H 2-Cl-4-NO2-6-CF3 O 3-25 Me H Me H 2-Cl-4-NO2-6-CF3 S 3-26 Me H Me H 4-NH2-2-CF3 O 3-27 Me H Me H 4-NH2-3-CF3 O 3-28 242 Me H Me H 2-NH2-4-CF3 O 3-29 92 Me H Me H 4-NO2-2-CF3 O 3-30 Me H Me H 4-NO2-2-CF3 S 3-31 103 Me H Me H 4-NO2-3-CF3 O 3-32 Me H Me H 4-NO2-3-CF3 S 3-33 87 Me H Me H 2-NO2-4-CF3 O 3-34 Me H Me Me 2-NO2-4-CF3 O 3-35 Me H Me H 2-NO2-4-CF3 S 3-36 Me H Me H 2,4-(NO2)2-6-CF3 O 3-37 Me H Me H 2,4-(NO2)2-6-CF3 S 3-38 Me H Me H 4-NO2 O 3-39 Me H Me H 4-NO2 S 3-40 53 Me H Et H H O 3-41 97 Me H Et H 3-Me-4-NO2 O 3-42 98 Me H Et H 3-EtO-4-NO2 O 3-43 99 Me H Et H 5-EtO-2-NO2 O 3-44 106 Me H Et H 2,6-Cl2-4-CF3 O 3-45 180 Me H Et H 2,6-Cl2-4-CF3 S 3-46 119 Me H Et H 2-Cl-6-F-4-CF3 O 3-47 186 Me H Et H 2-Cl-6-F-4-CF3 S 3-48 Me H Et H 2-Cl-4-CF3 O 3-49 Me H Et H 2-Cl-4-CF3 S 3-50 84 Me H Et H 4-Cl-2-CF3 O 3-51 101 Me H Et H 4-Cl-3-CF3 O 3-52 124 Me H Et H 2,6-F2-4-CF3 O 3-53 Me H Et Me 2,6-Cl2-4-CF3 O 3-54 Me H Et Et 2,6-Cl2-4-CF3 O 3-55 Me H Et MeOCH2 2,6-Cl2-4-CF3 O 3-56 Me H Et EtOCH2 2,6-C12-4-CF3 O 3-57 126 Me H Et H 2-Cl-4-NO2-6-CF3 O 3-58 Me H Et H 2-Cl-4-NO2-6-CF3 S 3-59 85 Me H Et H 4-F-2-NO2 O 3-60 57 Me H Et H 3-CF3 O 3-61 95 Me H Et H 4-CN-2-CF3 O 3-62 Me H Et H 4-NH2-2-CF3 O 3-63 100 Me H Et H 4-NH2-3-CF3 O 3-64 Me H Et H 2-NH2-4-CF3 O 3-65 93 Me H Et H 4-NO2-2-CF3 O 3-66 104 Me H Et H 4-NO2-3-CF3 O 3-67 88 Me H Et H 2-NO2-4-CF3 O 3-68 125 Me H Et H 2,4-(NO2)2-6-CF3 O 3-69 54 Me H Et H 4-NO2 O 3-70 Me H Pr H 2-Cl-4-CF3 O 3-71 Me H Pr H 4-Cl-3-CF3 O 3-72 121 Me H Pr H 2-Cl-6-F-4-CF3 O 3-73 Me H Pr H 2-Cl-6-F-4-CF3 S 3-74 107 Me H Pr H 2,6-Cl2-4-CF3 O 3-75 181 Me H Pr H 2,6-Cl2-4-CF3 S 3-76 128 Me H Pr H 2,Cl-4-NO2-6-CF3 O 3-77 Me H Pr H 2-Cl-4-NO2-6-CF3 S 3-78 91 Me H Pr H 4-NO2-2-CF3 O 3-79 Me H Pr H 4-NO2-2-CF3 S 3-80 102 Me H Pr H 4-NO2-3-CF3 O 3-81 Me H Pr H 4-NO2-3-CF3 S 3-82 89 Me H Pr H 2-NO2-4-CF3 O 3-83 Me H Pr H 2-NO2-4-CF3 S 3-84 56 Me H i-Pr H 3-CF3 O 3-85 Me H i-Pr H 2-Cl-4-CF3 O 3-86 Me H i-Pr H 4-Cl-3-CF3 O 3-87 120 Me H i-Pr H 2-Cl-6-F-4-CF3 O 3-88 Me H i-Pr H 2-Cl-6-F-4-CF3 S 3-89 108 Me H i-Pr H 2,6-Cl2-4-CF3 0 3-90 182 Me H i-Pr H 2,6-Cl2-4-CF3 S 3-91 94 Me H i-Pr H 2-NO2-2-CF3 O 3-92 Me H i-Pr H 4-NO2-2-CF3 S 3-93 90 Me H i-Pr H 2-NO2-4-CF3 O 3-94 Me H i-Pr H 2-NO2-4-CF3 S 3-95 132 Me H i-Pr i-Pr 2-NO2-4-CF3 O 3-96 Me H Bu H 2-Cl-6-F-4-CF3 O 3-97 Me H Bu H 2-Cl-6-F-4-CF3 S 3-98 Me H Eu H 2,6-Cl2-4-CF3 O 3-99 183 Me H Bu H 2,6-Cl24-CF3 S 3-100 Me H i-Bu H 2-Cl-6-F-4-CF3 O 3-101 Me H i-Bu H 2-Cl-6-F-4-CF3 S 3-102 Me H i-Eu H 2,6-Cl2-4-CF3 O 3-103 Me H s-Bu H 2-Cl-6-F-4-CF3 0 3-104 Me H s-Bu H 2-Cl-6-F-4-CF3 S 3-105 202 Me H s-Bu H 2,6-Cl2-4-CF3 O 3-106 58 Me H t-Bu H 3-CF3 O 3-107 109 Me H t-Bu H 2,6-Cl2-4-CF3 O 3-108 Me H t-Bu H 2-Cl-6-F-4-CF3 O 3-109 86 Me H t-Bu H 2-NO2-4-CF3 O 3-110 Me H Pent H 2,6-Cl2-4-CF3 O 3-111 Me H 3-Pent H 2-Cl-6-F-4-CF3 O 3-112 203 Me H 3-Pent H 2,6-Cl2-4-CF3 O 3-113 59 Me H Hex H 3-CF3 O 3-114 110 Me H Hex H 2,6-Cl2-4-CF3 O 3-115 Me H Hex H 2-Cl-6-F-4-CF3 O 3-116 Me H Hep H 2,6-Cl2-4-CF3 O 3-117 Me H Hep H 2-Cl-6-F-4-CF3 O 3-118 111 Me H Oct H 2,6-Cl2-4-CF3 O 3-119 Me H Oct H 2-Cl-6-F-4-CF3 O 3-120 Me H Dod H 2,6-Cl2-4-CF3 O 3-121 Me H Dod H 2-Cl-6-F-4-CF3 O 3-122 201 Me H c-Pr H 2,6-Cl2-4-CF3 O 3-123 Me H c-Pr H 2,6-Cl2-4-CF3 S 3-124 Me H c-Pr H 2-Cl-6-F-4-CF3 O 3-125 204 Me H c-Pent H 2,6-Cl2-4-CF3 O 3-126 Me H c-Pent H 2-Cl-6-F-4-CF3 O 3-127 60 Me H c-Hex H 3-CF3 O 3-128 Me H c-Hex H 2,6-Cl2-4-CF3 O 3-129 Me H c-Hex H 2-Cl-6-F-4-CF3 O 3-130 112, 234 Me H H2C = CHCH2 H 2,6-Cl2-4-CF3 O 3-131 Me H H2C = CHCH2 H 2,6-Cl2-4-CF3 S 3-132 122 Me H H2C = CHCH2 H 2-Cl-6-F-4-CF3 O 3-133 Me H H2C = CHCH2 H 2-Cl-6-F-4-CF3 S 3-134 61 Me H H2C = CHCH2 H 3-CF3 O 3-135 Me H H2C = CHCH2 H 3-CF3 S 3-136 205 Me H HC = CCH2 H 2,6-Cl2-4-CF3 O 3-137 Me H HC = CCH2 H 2-Cl-6-F-4-CF3 O 3-138 229 Me H HCOCH2 H 2,6-Cl2-4-CF3 O 3-139 Me H HCOCH2 H 2-Cl-6-F-4-CF3 O 3-140 211 Me H CF3CH2 H 2,6-Cl2-4-CF3 O 3-141 Me H CF3CH2 H 2-Cl-6-F-4-CF3 O 3-142 62 Me H 2-ClC2H4 H 3-CF3 O 3-143 113 Me H 2-ClC2H4 H 2,6-Cl2-4-CF3 O 3-144 Me H 2-ClC2H4 H 2,6-Cl2-4-CF3 S 3-145 Me H 2-ClC2H4 H 2-Cl-6-F-4-CF3 O 3-146 114 Me H 2-BrC2H4 H 2,6-Cl2-4-CF3 O 3-147 Me H 2-BrC2H4 H 2,6-Cl2-4-CF3 S 3-148 123 Me H 2-BrC2H4 H 2-Cl-6-F-4-CF3 O 3-149 Me H 2-FC2H4 H 2,6-Cl2-4-CF3 O 3-150 Me H 2-FC2H4 H 2-Cl-6-F-4-CF3 O 3-151 210 Me H 2-HOC2H4 H 2,6-Cl2-4-CF3 O 3-152 Me H 2-HOC2H4 H 2-Cl-6-F-4-CF3 O 3-153 Me H MeO H 2,6-Cl2-4-CF3 O 3-154 Me H MeO H 2,6-Cl2-4-CF3 S 3-155 Me H MeO H 2-Cl-6-F-4-CF3 O 3-156 Me H MeG H 2-Cl-6-F-4-CF3 S 3-157 198 Me H EtO H 3-CF3 O 3-158 206 Me H EtO H 2,6-Cl2-4-CF3 O 3-159 Me H EtO H 2,6-Cl2-4-CF3 S 3-160 Me H EtO H 2-Cl-6-F-4-CF3 O 3-161 Me H EtO H 2-Cl-6-F-4-CF3 S 3-162 207 Me H t-BuO H 2,6-Cl2-4-CF3 O 3-163 Me H t-BuO H 2-Cl-6-F-4-CF3 O 3-164 208 Me H H2C = CHCH2O H 2,6-Cl2-4-CF3 O 3-165 Me H H2C = CHCH2O H 2-Cl-6-F-4-CF3 O 3-166 209 Me H C6H5CH2O H 2,6-Cl2-4-CF3 O 3-167 Me H H2C = CHCH2O H 2-Cl-6-F-4-CF3 O 3-168 Me H MeOCH2 H 2,6-Cl2-4-CF3 O 3-169 178 Me H MeOCH2 H 2,6-Cl2-4-CF3 S 3-170 Me H MeOCH2 H 2-Cl-6-F-4-CF3 O 3-171 Me H MeOCH2 H 2-Cl-6-F-4-CF3 S 3-172 177 Me H MeOCH2 H 4-CN-2-CF3 S 3-173 Me H MeOCH2 H 4-NO2-2-CF3 O 3-174 Me H MeOCH2 H 2-NO2-4-CF3 O 3-175 Me H MeOCH2CH2 H 2,6-Cl2-4-CF3 O 3-176 184 Me H MeOCH2CH2 H 2,6-Cl2-4-CF3 S 3-177 Me H MeOCH2CH2 H 2-Cl-6-F-4-CF3 O 3-178 Me H MeOCH2CH2 H 2-Cl-6-F-4-CF3 S 3-179 232 Me H (MeO)2CHCH2 H 2,6-Cl2-4-CF3 O 3-180 Me H (MeO)2CHCH2 H 2-Cl-6-F-4-CF3 O 3-181 199 Me H 1) H 3-CF3 O 3-182 200 Me H 1) H 4-Cl-2-CF3 O 3-183 212 Me H 1) H 2,6-Cl2-4-CF3 O 3-184 185 Me H 1) H 2,6-Cl2-4-CF3 S 3-185 217 Me H 1) H 2-Cl-6-F-4-CF3 O 3-186 230 Me H 2) H 2,6-Cl2-4-CF3 O 3-187 Me H 2) H 2,Cl-6-F-4-CF3 O 3-188 231 Me H 3) H 2,6-Cl2-4-CF3 O 3-189 Me H 3) H 2,Cl-6-F-4-CF3 O 3-190 213 Me H 4) H 2,6-Cl2-4-CF3 O 3-191 Me H 4) H 2-Cl-6-F-4-CF3 O 3-192 214 Me H 5) H 2,6-Cl2-4-CF3 O 3-193 Me H 5) H 2-Cl-6-F-4-CF3 O 3-194 215 Me H 6) H 2,6-Cl2-4-CF3 O 3-195 Me H 6) H 2-Cl-6-F-4-CF3 O 3-196 216 Me H 7) H 2,6-Cl2-4-CF3 O 3-197 Me H 7) H 2-Cl-6-F-4-CF3 O 3-198 116 Me H EtO2CCH2 H 2,6-Cl2-4-CF3 O 3-199 Me H EtO2CCH2 H 2-Cl-6-F-4-CF3 O 3-200 63 Me H EtO2CCH2 H 3-CF3 O 3-201 117 Me H EtO2C(CH2)2 H 2,6-Cl2-4-CF3 O 3-202 Me H EtO2C(CH2)2 H 2-Cl-6-F-4-CF3 O 3-203 264 Me H EtO2C(CH2)2 Me 2,6-Cl2-4-CF3 O 3-204 83 Me H C6H5 H 3-Cl O 3-205 55 Me H C6H5 H 2-CF3 O 3-206 64 Me H C6H5 H 3-CF3 O 3-207 67 Me H 3-Me-C6H4 H 3-CF3 O 3-208 76 Me H 2-Cl-4-Me-C6H3 H 3-CF3 O 3-209 78 Me H 2-Cl-6-Me-C6H3 H 3-CF3 O 3-210 77 Me H 2-Me-4-NO2-C6H3 H 3-CF3 O 3-211 65 Me H 2-Cl-C6H4 H 3-CF3 O 3-212 66 Me H 3-Cl-C6H4 H 3-CF3 O 3-213 Me H 3-Cl-C6H4 H 2,6-Cl2-4-CF3 O 3-214 69 Me H 4-Cl-C6H4 H 3-CF3 O 3-215 Me H 4-Cl-C6H4 H 2,6-Cl2-4-CF3 O 3-216 80 Me H 8) H 3-CF3 O 3-217 70 Me H 4-F-C6H4 H 3-CF3 O 3-218 Me H 4-F-C6H4 H 2,6-Cl2-4-CF3 O 3-219 72 Me H 2,4-Cl2-C6H3 H 3-CF3 O 3-220 Me H 2,4-Cl2-C6H3 H 2,6-Cl2-4-CF3 O 3-221 Me H 2,4-Cl2-C6H3 H 2-Cl-6-F-4-CF3 O 3-222 75 Me H 2,6-Cl2-C6H3 H 3-CF3 O 3-223 Me H 2,6-Cl2-C6H3 H 2,6-Cl2-4-CF3 O 3-224 Me H 2,6-Cl2-C6H3 H 2,Cl-6-F-4-CF3 O 3-225 Me H 3,4-Cl2-C6H3 H 3-CF3 O 3-226 Me H 3,4-Cl2-C6H3 H 2,6-Cl2-4-CF3 O 3-227 Me H 3,4-Cl2-C6H3 H 2,Cl-6-F-4-CF3 O 3-228 74 Me H 3,4-Cl2-C6H3 H 3-CF3 O 3-229 73 Me H 2,4-F2-C6H3 H 3-CF3 O 3-230 79 Me H 2,3,4-Cl3-C6H2 H 3-CF3 O 3-231 Me H 3-CF3-C6H4 H 2,6-Cl2-4-CF3 O 3-232 Me H 3-CF3-C6H3 H 2-Cl-6-F-4-CF3 O 3-233 71 Me H 4-CF3-C6H4 H 3-CF3 O 3-234 Me H 4-CF3-C6H4 H 2,6-Cl2-4-CF3 O 3-235 Me H 4-CF3-C6H3 H 2-Cl-6-F-4-CF3 O 3-236 70 Me H 3-NO2-C6H4 H 3-CF3 O 3-237 Me H 3-NO2-C6H4 H 2,6-Cl2-4-CF3 O 3-238 Me H 3-NO2-C6H4 H 2-Cl-6-F-4-CF3 O 3-239 Me H 4-NO2-C6H4 H 2,6-Cl2-4-CF3 O 3-240 81 Me H C6H5CH2 H 3-CF3 O 3-241 115 Me H C6H5CH2 H 2,6-Cl2-4-CF3 O 3-242 Me H C6H5CH2 H 2-Cl-6-F-4-CF3 O 3-243 Me H 3-Cl-C6H4CH2 H 2,6-Cl2-4-CF3 O 3-244 Me H 3-Cl-C6H4CH2 H 2-Cl-6-F-4-CF3 O 3-245 Me H 4-Cl-C6H4CH2 H 2,6-Cl2-4-CF3 O 3-246 Me H 4-Cl-C6H4CH2 H 2-Cl-6-F-4-CF3 O 3-247 82 Me H C6H5C(CH3)H H 3-CF3 O 3-248 Me Cl H H 2,6-Cl2-4-CF3 O 3-249 247 Me Cl Me H 2,6-Cl2-4-CF3 O 3-250 Me Cl Me H 2-Cl-6-F-4-CF3 O 3-251 245 Me Cl Me H 4-NO2-2-CF3 O 3-252 244 Me Cl Me H 2-NO2-4-CF3 O 3-253 248 Me Cl Et H 2,6-Cl2-4-CF3 O 3-254 Me Cl Et H 2,-6-Cl2-4-CF3 S 3-255 Me Cl Et H 2-Cl-6-F-4-CF3 O 3-256 Me Cl Et H 2-Cl-6-F-4-CF3 S 3-257 Me Cl Pr H 2,6-Cl2-4-CF3 O 3-258 Me Cl Pr H 2-Cl-6-F-4-CF3 O 3-259 249 Me Cl i-Pr H 2,6-Cl2-4-CF3 O 3-260 Me Cl i-Pr H 2-Cl-6-F-4-CF3 O 3-261 243 Me Cl i-Pr H 2-NO2-4-CF3 O 3-262 Me Cl Bu H 2,6-Cl2-4-CF3 O 3-263 Me Cl Bu H 2-Cl-6-F-4-CF3 O 3-264 Me Cl s-Bu H 2,6-Cl2-4-CF3 O 3-265 Me Cl s-Bu H 2-Cl-6-F-4-CF3 O 3-266 246 Me Cl Hex H 2,6-Cl2-4-CF3 O 3-267 Me Cl Hex H 2-Cl-6-F-4-CF3 O 3-268 Me Cl c-Pr H 2,6-Cl2-4-CF3 O 3-269 Me Cl c-Pr H 2,Cl-6-F-4-CF3 O 3-270 160 Me Cl ClC2H4 H 2,6-Cl2-4-CF3 O 3-271 Me Cl ClC2H4 H 2-Cl-6-F-4-CF3 O 3-272 Me Cl MeOCH2 H 2,6-Cl2-4-CF3 O 3-273 Me Cl MeOCH2 H 2-Cl-6-F-4-CF3 O 3-274 Me Cl MeOCH2CH2 H 2,6-Cl2-4-CF3 O 3-275 Me Cl MeOCH2CH2 H 2-Cl-6-F-4-CF3 O 3-276 Me Br H H 2,6-Cl2-4-CF3 O 3-277 Me Br H H 2,6-Cl2-4-CF3 S 3-278 Me Br H H 2-Cl-6-F-4-CF3 O 3-279 Me Br H H 2-Cl-6-F-4-CF3 S 3-280 Me Br Me H 2,6-Cl2-4-CF3 O 3-281 Me Br Me H 2-Cl-6-F-4-CF3 O 3-282 Me Br Et H 2,6-Cl2-4-CF3 O 3-283 Me Br Et H 2-Cl-6-F-4-CF3 O 3-284 Me Br Pr H 2,6-Cl2-4-CF3 O 3-285 Me Br Pr H 2-Cl-6-F-4-CF3 O 3-286 Me Me H H 2,6-Cl2-4-CF3 O 3-287 Me Me H H 2,6-Cl2-4-CF3 S 3-288 Me Me H H 2-Cl-6-F-4-CF3 O 3-289 Me Me H H 2-Cl-6-F-4-CF3 S 3-290 Me Me Me H 2,6-Cl2-4-CF3 O 3-291 Me Me Me H 2,6-Cl24-CF3 S 3-292 Me Me Me H 2-Cl-6-F-4-CF3 O 3-293 Me Me Me H 2-Cl-6-F-4-CF3 S 3-294 152 Me Me Me H 4-NO2-2-CF3 O 3-295 151 Me Me Me H 2-NO2-4-CF3 O 3-296 Me Me Et H 2,6-Cl2-4-CF3 O 3-297 Me Me Et H 2,6-Cl2-4-CF3 S 3-298 Me Me Et H 2-Cl-6-F-4-CF3 O 3-299 Me Me Et H 2-Cl-6-F-4-CF3 S 3-300 Me Me Et H 4-NO2-2-CF3 O 3-301 Me Me Et H 2-NO2-4-CF3 O 3-302 Me Me Pr H 2,6-Cl2-4-CF3 O 3-303 Me Me Pr H 2,6-Cl2-4-CF3 S 3-304 Me Me Pr H 2-Cl-6-F-4-CF3 O 3-305 Me Me Pr H 2-Cl-6-F-4-CF3 S 3-306 Me Me i-Pr H 2,6-Cl2-4-CF3 O 3-307 Me Me i-Pr H 2,6-Cl2-4-CF3 S 3-308 Me Me i-Pr H 2-Cl-6-F-4-CF3 O 3-309 Me Me i-Pr H 2-Cl-6-F-4-CF3 S 3-310 Me Et H H 2,6-Cl2-4-CF3 O 3-311 Me Et H H 2-Cl-6-F-4-CF3 O 3-312 Me Et Me H 2,6-Cl2-4-CF3 O 3-313 Me Et Me H 2,6-Cl2-4-CF3 S 3-314 Me Et Me H 2-Cl-6-F-4-CF3 O 3-315 Me Et Me H 2-Cl-6-F-4-CF3 S 3-316 157 Me Et Me H 4-NO2-2-CF3 O 3-317 156 Me Et Me H 2-NO2-4-CF3 O 3-318 169 Me Et Et H 2,6-Cl2-4-CF3 O 3-319 227 Me H —CH2CH2CH2CH2— 2,6-Cl24-CF3 O 3-320 Me H —CH2CH2CH2CH2— 2-Cl-6-F-4-CF3 O 3-321 228 Me —CH2C (CH2) OC (CH2) CH2— 2,6-Cl2-4-CF3 O 3-322 Me H —CH2C (CH2) OC (CH2) CH2— 2-Cl-6-F-4-CF3 O 3-323 Et H H H 2,6-Cl2-4-CF3 O 3-324 Et H H H 2-Cl-6-F-4-CF3 O 3-325 Et H Me H 2,6-Cl2-4-CF3 O 3-323 Et H Me H 2-Cl-6-F-4-CF3 O 3-327 162 Et H Me H 4-NO2-2-CF3 O 3-328 Et H Me H 2-Cl-6-F-4-CF3 O 3-329 161 Et H Me H 2-NO2-4-CF3 O 3-330 163 Et H Et H 2,6-Cl2-4-CF3 O 3-331 Et H Et H 2,6-Cl2-4-CF3 S 3-332 Et H Et H 2-Cl-6-F-4-CF3 O 3-333 Et H Et H 2-Cl-6-F-4-CF3 S 3-334 Et H Pr H 2,6-Cl2-4-CF3 O 3-335 Et H Pr H 2-Cl-6-F-4-CF3 O 3-336 Pr H H H 2,6-Cl2-4-CF3 O 3-337 Pr H H H 2-Cl-6-F-4-CF3 O 3-338 Pr H Me H 2,6-Cl2-4-CF3 O 3-339 Pr H Me H 2-Cl-6-F-4-CF3 O 3-340 Pr H Et H 2,6-Cl2-4-CF3 O 3-341 i-Pr H H H 2,6-Cl2-4-CF3 O 3-342 i-Pr H H H 2-Cl-6-F-4-CF3 O 3-343 i-Pr H Me H 2,6-Cl2-4-CF3 O 3-344 i-Pr H Me H 2-Cl-6-F-4-CF3 O 3-345 167 i-Pr H Et H 2,6-Cl2-4-CF3 0 3-346 i-Pr H Et H 2-Cl-6-F-4-CF3 O 3-347 s-Bu H Et H 2,6-Cl2-4-CF3 O 3-348 s-Bu H Et H 2-Cl-6-F-4-CF3 O 3-349 168 t-Bu H Et H 2,6-Cl2-4-CF3 O 3-350 t-Bu H Et H 2-Cl-6-F-4-CF3 O 3-351 Hex H Et H 2,6-Cl2-4-CF3 O 3-352 Hex H Et H 2-Cl-6-F-4-CF3 O 3-353 166 c-Pr H Et H 2,6-Cl2-4-CF3 O 3-354 c-Pr H Et H 2-Cl-6-F-4-CF3 O 3-355 c-Hex H Et H 2,6-Cl2-4-CF3 O 3-356 c-Hex H Et H 2-Cl-6-F-4-CF3 O 3-357 MeOCH2 H H H 2,6-Cl2-4-CF3 O 3-358 MeOCH2 H H H 2-Cl-6-F-4-CF3 O 3-359 MeOCH2 H Me H 2,6-Cl2-4-CF3 O 3-360 MeOCH2 H Me H 2-Cl-6-F-4-CF3 O 3-361 170 MeOCH2 H Et H 2,6-Cl2-4-CF3 O 3-362 MeOCH2 H Et H 2-Cl-6-F-4-CF3 O 3-363 CF3 H H H 2,6-Cl2-4-CF3 O 3-364 CF3 H H H 2-Cl-6-F-4-CF3 O 3-365 CF3 H Me H 2,6-Cl2-4-CF3 O 3-366 CF3 H Me H 2-Cl-6-F-4-CF3 O 3-367 172 CF3 H Me H 2-NO2-4-CF3 O 3-368 CF3 H Et H 2,6-Cl2-4-CF3 O 3-369 CF3 H Et H 2-Cl-6-F-4-CF3 O 3-37O 241 CF3 H i-Pr i-Pr 2-NO2-4-CF3 O 3-371 250 CCl3 H Pr H 2,6-Cl2-4-CF3 O 3-372 249 CCl3 H i-Pr H 2,6-Cl2-4-CF3 O 3-373 171 EtO2CH2 H Et H 2,6-Cl2-4-CF3 O 3-374 EtO2CH2 H Et H 2-Cl-6-F-4-CF3 O 3-375 175 MeO2C H Et H 2,6-Cl2-4-CF3 O 3-373 MeO2C H Et H 2-Cl-6-F-4-CF3 O 3-377 3-Cl-C6H5 H Et H 2,6-Cl2-4-CF3 O 3-378 3-Cl-C6H5 H Et H 2-Cl-6-F-4-CF3 O 3-379 176 4-Cl-C6H5 H Et H 2,6-Cl2-4-CF3 O 3-380 4-Cl-C6H5 H Et H 2-Cl-6-F-4-CF3 O 3-381 4-Br-C6H5 H Et H 2,6-Cl2-4-CF3 O 3-382 4-Br-C6H5 H Et H 2-Cl-6-F-4-CF3 O TABLE 4 Synthesis of 3-(substituted Pyridyloxy) pyrazole derivatives Compound Example No.", "No.", "R1 R2 R3 R4 Xn Y 4-1 Me H H H 3,5-Cl2 O 4-2 225 Me H H H 3-Cl-5-CF3 O 4-3 Me H H H 3-Cl-5-CF3 S 4-4 Me H H H 5-CF3 O 4-5 Me H Me H 3,5-Cl2 O 4-6 133 Me H Me H 3-Cl-5-CF3 O 4-7 187 Me H Me H 3-Cl-5-CF3 S 4-8 Me H Me H 5-CF3 O 4-9 Me H Me H 3,5-(NO2)2 O 4-10 237 Me H Me Me 3-Cl-5-CF3 O 4-11 265 Me H Me MeOCH2 3-Cl-5-CF3 0 4-12 Me H Me MeOCH2 3-Cl-S-CF3 O 4-13 131 Me H Et H 4-Me-6-NO2 0 4-14 130 Me H Et H 3-NO2-6-MeO O 4-15 129 Me H Et H 3,5-Cl2 O 4-16 134 Me H Et H 3-Cl-5-CF3 O 4-17 188 Me H Et H 3-Cl-5-CF3 S 4-18 Me H Et H 5-CF3 O 4-19 Me H Et H 3,5-(NO2)2 O 4-20 Me H Et Me 3-Cl-S-CF3 O 4-21 235 Me H Et Et 3-Cl-S-CF3 O 4-22 Me H Et MeOCH2 3-Cl-S-CF3 O 4-23 135 Me H Pr H 3-Cl-S-CF3 O 4-24 189 Me H Pr H 3-Cl-S-CF3 S 4-25 132 Me H i-Pr H 3-Cl-S-CF3 O 4-26 190 Me H i-Pr H 3-Cl-S-CF3 S 4-27 236 Me H i-Pr i-Pr 3-Cl-S-CF3 O 4-28 233 Me H Eu H 3-Cl-S-CF3 C 4-29 191 Me H Bu H 3-Cl-S-CF3 S 4-30 218 Me H s-Bu H 3-Cl-S-CF3 O 4-31 220 Me H i-Bu H 3-Cl-S-CF3 O 4-32 136 Me H t-Bu H 3-Cl-S-CF3 O 4-33 137 Me H Pent H 3-Cl-S-CF3 O 4-34 Me H 2-Pent H 3-Cl-S-CF3 O 4-35 138 Me H Hex H 3-Cl-S-CF3 O 4-36 139 Me H Hep H 3-Cl-S-CF3 O 4-37 140 Me H Oct H 3-Cl-S-CF3 O 4-38 141 Me H Dod H 3-Cl-S-CF3 O 4-39 219 Me H c-Pr H 3-Cl-S-CF3 O 4-40 142 Me H c-Hex H 3-Cl-S-CF3 O 4-41 197 Me H H2C = CH H 3-Cl-S-CF3 S 4-42 143 Me H H2C = CHCH2 H 3-Cl-S-CF3 O 4-43 192 Me H H2C = CHCH2 H 3-Cl-5-CF3 S 4-44 Me H HC ≡ CCH2 H 3-Cl-5-CF3 O 4-45 Me H CF3CH2 H 3-Cl-5-CF3 O 4-46 144 Me H 2-ClO2H4 EL 3-Cl-5-CF3 O 4-47 Me H 2-BrC2H4 H 3-Cl-5-CF3 O 4-48 Me H 2-FO2H4 H 3-Cl-S-CF3 O 4-49 Me H -HOC2H4 H 3-Cl-5-CF3 O 4-50 221 Me H MeO H 3-Cl-5-CF3 O 4-51 222 Me H EtC H 3-Cl-5-CF3 O 4-52 Me H H2O = CHCH2O H 3-Cl-5-CF3 O 4-53 Me H C6H5CH2O H 3-Cl-5-CF3 O 4-54 193 Me H MeOCH2 H 3-Cl-5-CF3 S 4-55 194 Me H MeOCH2CH2 H 3-Cl-5-CF3 S 4-56 195 Me H (CH3)2CH(CO2Me)H H 3-Cl-5-CF3 S 4-57 145 Me H C6H5 H 3-Cl-5-CF3 O 4-58 146 Me H 3-Cl-C6H4 H 3-Cl-5-CF3 O 4-59 Me H 4-Cl-C6H4 H 3-Cl-5-CF3 O 4-60 Me H 2,4-Cl2-C6H3 H 3-Cl-5-CF3 O 4-61 147 Me H 3,4-Cl2-C6H3 H 3-Cl-5-CF3 O 4-62 148 Me H 3-CF3-C6H4 H 3-Cl-5-CF3 O 4-63 149 Me H 4-CF3-C6H4 H 3-Cl-5-CF3 O 4-64 150 Me H 3-NO2-C6H4 H 3-Cl-5-CF3 O 4-65 196 Me H 4-Cl-C6H4CH2 H 3-Cl-5-CF3 S 4-66 252 Me Cl Me H 3-Cl-5-CF3 O 4-67 251 Me Cl Et H 3-Cl-5-CF3 O 4-68 253 Me Cl Pr H 3-Cl-5-CF3 O 4-69 254 Me Cl i-Pr H 3-Cl-5-CF3 O 4-70 255 Me Cl Pent H 3-Cl-5-CF3 O 4-71 256 Me Cl Hex H 3-Cl-5-CF3 O 4-72 257 Me Cl Hep H 3-Cl-5-CF3 O 4-73 258 Me Cl Oct H 3-Cl-5-CF3 O 4-74 259 Me Cl Dod H 3-Cl-5-CF3 O 4-75 260 Me Cl c-Hex H 3-Cl-5-CF3 O 4-76 Me Cl HC ≡ CCH2 H 3-Cl-5-CF3 O 4-77 261 Me Cl ClC2H4 H 3-Cl-5-CF3 O 4-78 263 Me Br Et H 3-Cl-5-CF3 O 4-79 Me Me H H 3-Cl-5-CF3 O 4-80 154 Me Me Me H 3-Cl-5-CF3 O 4-81 155 Me Me Et H 3-Cl-5-CF3 O 4-82 153 Me Me i-Pr H 3-Cl-5-CF3 O 4-83 Me Et H H 3-Cl-5-CF3 O 4-84 158 Me Et Me H 3-Cl-5-CF3 O 4-85 159 Me Et Et H 3-Cl-5-CF3 O 4-86 Et H H H 3-Cl-5-CF3 O 4-87 164 Et H Me H 3-Cl-5-CF3 O 4-88 165 Et H Et H 3-Cl-5-CF3 O 4-89 262 Et Cl Me H 3-Cl-5-CF3 O 4-90 CF3 H H H 3-Cl-5-CF3 O 4-91 176 CF3 H Me H 3-Cl-5-CF3 O 4-92 238 CF3 H Me Me 3-Cl-S-CF3 O 4-93 173 CF3 H Et H 3-Cl-5-CF3 O 4-94 239 CF3 H Et Et 3-Cl-5-CF3 O 4-95 240 CF3 H i-Pr i-Pr 3-Cl-5-CF3 O When the compound of the present invention is used as a herbicide, it can be used as it is.", "It can be also used in the form of a herbicide containing one or more adjuvants in combination.", "Generally, as adjuvants, various carriers, extenders, solvents, surfactants and stabilizers are incorporated, and the compound of the present invention is preferably formed into preparations in the form of a wettable powder, an emulsifiable concentrate, a dust, granules, a flowable agent or the like.", "The solvent as one adjuvant in the herbicide containing the compound of the present invention as an active ingredient is properly selected from water, alcohols, ketones, ethers, aliphatic and aromatic hydrocarbons, halogenated hydrocarbons, acid amides, esters or nitriles.", "One of these solvents may be used, or a mixture of two or more solvents of these may be used.", "The extender is selected from mineral powders including clays such as kaolin and bentonite, talcs such as talc and pyrophyllite, oxides such as diatomite and white carbon or plant powders such as soybean powder and CMC.", "Further, a surfactant may be used as a spreading agent, a dispersant, an emulsifier or a penetrant.", "The surfactant includes, for example, nonionic surfactants, cationic surfactants and amphoteric surfactants.", "These surfactants are used alone or as a mixture of at least two members of these depending upon an end in use.", "The method of use of a herbicide containing the compound of the present invention as an active ingredient includes soil treatment, water surface treatment, foliar treatment, and the herbicide can produce an excellent effect when applied before and during germination.", "Further, the herbicide containing the compound of the present invention as an active ingredient may contain other active components such as other herbicide, an insecticide, a fungicide, a plant growth regulator, etc., in combination, or may be used in combination with these.", "The present invention will be explained further in detail with reference to Preparation Examples of herbicides containing the compound of the present invention as an active ingredient and Examples of testing herbicides for herbicidal efficacy.", "In addition, “part” stands for “part by weight”.", "Preparation Example 1 Emulsifiable Concentrate 20 Parts of a compound of the present invention, 35 parts of xylene, 40 parts of cyclohexanone and 5 parts of Sorpol 900A (supplied by Toho Chemical Co., Ltd.) were homogeneously mixed, to give an emulsifiable concentrate.", "Preparation Example 2 Wettable Powder 50 Parts of a compound of the present invention, 25 parts of diatomite, 22 parts of clay and 3 parts of Lunox 1000C (supplied by Toho Chemical Co., Ltd.) were homogenously mixed and pulverized, to give a wettable powder.", "Preparation Example 3 Granules A mixture containing 5 parts of a compound of the present invention, 35 parts of bentonite, 55 parts of talc, 5 parts of sodium ligninsulfonate was homogenously mixed and pulverized, and then water was added.", "The mixture was kneaded and granulated with an extrusion granulator, and the granulated product was dried and particle-size-adjusted, to give granules.", "The compound of the present invention was tested for herbicidal efficacy according to those methods shown in the following Test Examples with regard to preparations prepared according to the above-shown methods.", "The herbicidal efficacy thereof on test weeds and their phytotoxicity to test crops were evaluated on the basis of five ratings in which 1 shows no influence and 5 shows complete die.", "Test Example 1 Herbicidal Efficacy Test by Pre-Emergence Treatment Under Submergence Condition A 1/10,000-are pot was filled with paddy field soil, and, after plowing, seeds of Echinochloa oryzicola, Cyperus difformis, Monochoria vaginalis, Scirpus juncoides, Eleocharis acicularis and other annual broadleaf weeds such as Lindernia procumbens, Rotala indica and Elatine triandra were sown.", "Rice (oryza sativa) seedlings (cultiver “Koshihikari”) at a 2.5-leaf stage were transplanted and kept under a submergence condition.", "After one day, the wettable powder or emulsifiable concentrate of the compound of the present invention, prepared according to Preparation Example, was diluted and dropped on a water surface at a predetermined dose.", "On the 15th day after the treatment, the herbicidal efficacy on the test weeds and the injury to the rice were investigated on the basis of the ratings of 1 to 5, and Table 5 shows the results.", "TABLE 5 Herbicidal efficacy test by pre-emergence treatment under submergence condition Comp'd Dose Herbicidal efficacy Injury No.", "kg/ha Eo Cd Blw Mv Sj Ea Os 3-3 1 5 5 5 5 5 5 1.5 0.5 5 5 5 5 5 5 1.5 3-14 1 5 5 5 5 5 5 2.5 0.5 5 5 5 5 5 5 2.3 3-15 1 5 5 5 5 5 5 1.5 0.5 5 5 5 5 5 5 1.3 3-44 1 5 5 5 5 5 5 3.5 0.5 5 5 5 5 5 5 2.5 3-45 1 5 5 5 5 5 5 1.7 0.5 5 5 5 5 4 5 1.5 3-74 1 5 5 5 5 5 5 1.8 0.5 5 5 5 5 5 5 1.8 3-89 1 5 5 5 5 5 5 1.5 0.5 5 5 5 5 5 5 1.3 3-130 1 5 5 5 5 5 5 1.8 0.5 5 5 5 5 5 5 1.5 3-143 1 5 5 5 5 5 5 1.5 0.5 5 5 5 5 4 5 1.3 3-146 1 5 5 5 5 5 5 1 0.5 5 5 5 5 5 5 1 3-158 1 5 5 5 5 5 5 2 0.5 5 5 5 5 5 5 1.5 3-169 1 5 5 5 5 4.2 5 1 0.5 5 5 5 5 4 5 1 3-183 1 5 5 5 5 5 5 2 0.5 5 5 5 5 5 5 2 3-249 1 5 5 5 5 5 5 1.5 0.5 5 5 5 5 5 5 1.2 3-253 1 5 5 5 5 5 5 1.6 0.5 5 5 5 5 5 5 1.4 3-330 1 5 5 5 5 5 5 1.5 0.5 5 5 5 5 5 5 1.2 4-6 1 5 5 5 5 5 5 1.4 0.5 4.8 5 5 5 4.6 5 1.2 4-16 1 5 5 5 5 5 5 2.5 0.5 5 5 5 5 4.8 5 2 4-17 1 5 5 5 5 4.8 5 1.8 0.5 5 5 4.8 5 4.5 5 1.5 4-23 1 5 5 5 5 5 5 1.3 0.5 5 5 4.8 5 4.9 5 1.1 Eo: Echinochloa oryzicola, Cd: Cyperus difformis, Blw: anuual broadleaf weeds, Mv: Monochoria vaginalis, Sj: Scirpus juncoides, Ea: Eleocharis acicularis, Os: Oryza sativa Test Example 2 Herbicidal Efficacy Test by Soil Treatment Before Germination Under Upland Field Condition A vat having an area of 5×10 cm2 and a depth of 5 cm was filled with upland field soil, and seeds of Chenopodium album, Echinochloa crus-galli, Amaranthus viridis, Digitaria ciliaris and sweet corn (Zea mays) were sown thereon and covered with soil by a depth of 0.5 cm.", "Next day, the wettable powder or emulsifiable concentrate of the compound of the present invention, prepared according to Preparation Example, was diluted and sprayed uniformly onto the covering soil at a predetermined dose.", "On the 21st day after the treatment, the herbicidal efficacy on the test weeds and the injury to the corn were investigated on the basis of the ratings of 1 to 5, and Table 6 shows the results.", "TABLE 6 Herbicidal efficacy test by soil treatment before germination under upland field condition Comp'd Dose Herbicidal activity Injury No.", "kg/ha Ca Ec Av Dc Zm 3-3 1 5 3.5 5 4.5 1.5 3-14 1 5 4.9 5 5 — 3-44 1 5 5 5 5 3 3-45 1 5 5 5 5 2 3-74 1 5 4.9 5 5 1 3-183 1 5 3 5 4 1 3-249 1 5 4.5 5 5 1.2 3-253 1 5 4 5 5 1.5 3-330 1 5 3.5 5 5 1.5 Ca: Chenopodium album, Ec: Echinochloa crus-galli, Av: Amaranthus viridis, Dc: Digitaria ciliaris, Zm: Zea mays.", "Test Example 3 Herbicidal Efficacy Test by Foliar Treatment After Germination Under Upland Field Condition A vat having an area of 5×10 cm2 and a depth of 5 cm was filled with upland field soil, and seeds of Chenopodium album, Echinochloa crus-galli, Amaranthus viridis and Abutilon theophrasti were sown thereon and covered with soil by a depth of 0.5 cm.", "Water was sprayed as required to grow them for 14 days.", "The wettable powder or emulsifiable concentrate of the compound of the present invention, prepared according to Preparation Example, was diluted and sprayed uniformly to shoot of the plants at a dose of 1,000 liters/ha.", "On the 14th day after the treatment, the herbicidal efficacy on the test weeds were investigated on the basis of the ratings of 1 to 5, and Table 7 shows the results.", "TABLE 7 Herbicidal efficacy test by foliar treatment after germination under upland field condition Comp'd Dose Herbicidal activity No.", "kg/ha Ca Ec Av Dc 3-3 1 5 4.5 5 5 3-14 1 5 5 5 5 3-15 1 5 5 5 5 3-44 1 5 5 5 5 3-45 1 5 5 5 5 3-67 1 5 4.9 5 5 3-74 1 5 4.9 5 5 3-82 1 5 3.8 5 5 3-89 1 5 5 5 5 3-130 1 5 5 5 5 3-158 1 5 5 5 5 3-183 1 5 5 5 5 3-249 1 5 5 5 5 3-253 1 5 5 5 5 3-330 1 5 5 5 5 4-30 1 5 5 5 5 Ca: Chenopodium album, Ec: Echinochloa crus-galli, Av: Amaranthus viridis, Dc: Digitaria ciliaris.", "INDUSTRIAL UTILITY The pyrazole derivative of the present invention exhibits excellent herbicidal activity at a low dosage without causing phytotoxicity on crops and has utility as a herbicide." ] ]	Patent_10468527
[ [ "Composite structures of membranes that are selectively permeable to hydrogen and combustible gas processors using same", "The invention relates to a composite structure consisting of a relatively long filtration bar comprising, from the outside, an ultra-thin layer (26) that is selectively permeable to hydrogen and made from palladium or silver alloy.", "Said layer is disposed on a permeable, rigid, refractory substrate consisting of a more or less solid body (30) that is covered with an intermediary thin layer (28) having a relatively smooth surface.", "The body (30) and the intermediary layer (28) are made respectively by sintering with fine and ultra-fine Inconel grains.", "A rigid metallic axial structure (32) is embedded in the body (30).", "Veinlets (31), which are made in the body (30) through the destruction of thermo-destructible wires during sintering, increase the permeability of the substrate.", "The invention is particularly applicable to hydrogen-producing combustible gas processors." ], [ "1.Improved composite membrane structure selectively permeable to hydrogen (10-24-40) constituted by a continuous filtering film (12-26-42) measuring several microns thick and made of palladium or a palladium-based alloy deposited on a rigid porous refractory substrate (1-30-46) connected to a pipe (20-34-52) for collecting the extracted hydrogen, characterised in that: said substrate is a permeable sintered metallic body (16-30-46) provided with good mechanical resistance or open pores measuring between several microns and about ten microns; a thin metallic film (14-28-44) known as the intermediate film with a thickness of between twenty and fifty microns and containing open pores smaller than one micron is deposited on the body of the substrate; the body of the substrate and the intermediate film are made of a metal or alloy having within the range of use temperatures and pressures of the structure, heat expansion coefficients in the presence of hydrogen which are both compatible with those of palladium; the material constituting the intermediate film has within said range a satisfactory chemical stability with regard to the filtering film and the body of the substrate.", "2.Membrane structure selectively permeable to hydrogen according to claim 1, characterised in that the heat expansion coefficient of the materials constituting the body of the substrate and the intermediate film is lower than or at the most slightly greater than that of the material constituting the filtering film.", "3.Membrane structure selectively permeable to hydrogen according to claim 2, characterised in that: the ultra-thin filtering film (12-26-42) is made of a palladium-based alloy including silver and/or nickel; the sintered body (16-30-46) of said substrate is embodied from a relatively fine powder with appropriate size grading formed from a nickel-based super-alloy including chromium and iron and in particular from an Inconel 600 powder; the intermediate film (14-28-44) is embodied from an ultra-fine powder with suitable size grading of nickel or a super-alloy similar to the one used for the body of the substrate; the filtering film is fixed by microwelds to the tops of the surface grains of the intermediate film.", "4.Membrane structure selectively permeable to hydrogen according to one of the preceding claims, characterised in that the body (16-34-46) of said substrate comprises small veins (17-31-47) adapted to improve its permeability.", "5.Membrane structure selectively permeable to hydrogen according to one of the preceding claims, characterised in that said substrate (16-30) is a cylindrical rod (10-24) provided at one extremity with a collar (20-34) for evacuating the extracted hydrogen and, if appropriate, equipped with a vector gas feed collar (22-36) at the other extremity.", "6.Membrane structure according to claim 5, characterised in that said cylindrical rod (24a-b-c) is relatively long and comprises a rigid axial metallic reinforcement (32a-b-c) adapted to give it good mechanical resistance.", "7.Membrane structure selectively permeable to hydrogen, characterised in that it is in the form of a grid, preferably square and relatively large, constituted by a relatively large number of rods according to claim 6, said rods being placed a small distance from one another and mounted fixed on two hollow transversal beams (71-73) respectively allocated to the collection of the extracted hydrogen and vector gas feeding.", "8.Membrane structure according to claim 7, characterised in that said relatively long rod (24c) is of the glove-finger type with vector gas injection, its axial reinforcement (32c) is hollow and allocated to said injection and opens into a cavity (38) provided in a cupel (40) welded to said other extremity.", "9.Membrane structure selectively permeable to hydrogen according to one of claims 1 to 4, characterised in that said substrate, which includes a porous body (46) and an intermediate film (44), is a sealed plate fitted in a metallic border (48a-b) provided with an orifice connected to a pipe (52a-b) for evacuating the extracted hydrogen and, if appropriate, to another orifice opposite the first connected to a vector gas feeding pipe (50a-b).", "10.Membrane structure according to claim 9, characterised in that said plate has relatively large dimensions, said substrate being provided with a rigid metallic internal reinforcement (49) adapted to give it good mechanical resistance.", "11.Combustible gas processor (54-65-91) in which a cold plasma reaction chamber (58-66-92) fed in suitable conditions by a conditioning cell (88) supplying a primary mixture of said gas, water vapour and air provides a secondary mixture formed of hydrogen and carbon dioxide and carbon monoxide, characterised in that: said chamber (58-66-92) comprises several membrane structures selectively permeable to hydrogen (64-68-94) according to one of claims 1 to 10 and connected to a common hydrogen collector (76), said structures having a given shape and dimensions; placed close to these structures is/are one or several electrodes provided with a nonconducting refractory sheath (60-62, 68a-b, 96a-b) having a high dielectric coefficient and an adapted shape and dimensions enabling them to effectively cooperate with said structures so as to constitute a unit intended to produce in response to an appropriate electric power source (84) barrier electric discharges generating said cold plasma in the spaces separating the electrodes-and structures.", "12.Combustible gas processor according to claim 11, characterised in that in the reaction chamber (65-92) each unit formed by an insulated electrode (78a-c or 96a-b) associated with a filtering structure (68a-b or 94) is followed by a unit formed by an insulated electrode (78b-d) and a basket (80a-b or 98a-b) containing catalyst granules for high temperature “water-gas shift” reaction.", "13.Gas processor of the type including a reaction chamber provided with a chemical catalyst specific to the reaction concerned, characterised in that membrane structures selectively permeable to hydrogen according to one of claims 1 to 10 are placed close to said catalyst.", "14.Combustible gas processor according to claim 11 or 12, characterised in that: the filtering structures (68a-b-c-d) and the insulated electrodes (78a-b-c-d) have the shape of long pencils (24a-b) assembled in grids of given dimensions, in particular square; said grids are constituted by a relatively large number of pencils almost touching fixed to two projecting beams (71-73) which are either neutral gas injection and hydrogen evacuation pipes for the filtering structures (68) or electric insulated linking conductors (79a-b) for the insulated electrodes (78), said beams (71-73) being adapted to be mounted secured to a frame installed in the reaction chamber.", "15.Hydrogen purification device (100), characterised in that it includes: a filtering chamber (102) constituted by a refractory casing (106) lagged by a nonconducting coating (144) containing one or several membranes (1081 .", ".", ".", "n) selectively permeable to hydrogen, said device being of the type defined by one of claims 1 to 10, said membranes being connected to two common pipes (110-122) respectively allocated to vector gas feeding and collection of the extracted hydrogen; heating (118-120-126), temperature adjustment (140-142-138) and compression (130) means adapted to provide the flow of hydrogen to be purified with a temperature and pressure respectively situated inside pressure and temperature ranges corresponding to the best possible functioning of the membranes (108).", "16.Hydrogen purification device according to claim 15, characterised in that: said heating means include a boiler (116) and a burner (118) associated with high thermic conduction heating pipes (120) passing through the boiler (116); the boiler (116) is fed with excess pressure by the hydrogen to be purified and communicates with the filtering chamber (102) via a perforated wall (122); the burner (118) is fed by the residual hydrogen evacuated downstream of the filtering chamber (102) by a collecting pipe (136) and by the compressed air provided by a compressor (138) whose flow is dependent on a regulation device (142) controlled by a signal delivered by a thermo couple (140) placed in the filtering chamber (102); the chimney (126) of the burner (118) has a ring-shaped section, surrounds the filtering chamber (102) and is lagged by the nonconducting coating (144)." ], [ "The invention concerns improvements to composite structures with membranes selectively permeable to hydrogen and able to be used in combustible gas processors so as to produce pure hydrogen.", "It also concerns improvements made to these processors owing to the use of the embodied composite structures.", "Generally speaking, so as to obtain a high gas flow through a selective filtering membrane, it is necessary to simultaneously satisfy the following four conditions: the material making up the membrane needs to be extremely selective and highly permeable to the gas to be extracted; the membrane also needs to be as thin as possible, the filtered gas flow being an inverse function of its thickness; the difference of partial pressures of the gas to be removed needs to be as high as possible between the upstream and downstream portions of the membrane, the effectiveness of filtering being directly dependent on this difference of pressures the surface of the membrane needs to be as large as possible.", "Moreover, it is known that in the particular case of combustible gas processors, the temperature in the reaction chamber is high (generally between 300 and 600° C.), and that owing to this the only material really effective for embodying a membrane and intended to be worked inside this range of temperatures is palladium or a palladium-based alloy, said material being required for clear economic reasons for reducing the total quantity.", "In addition, in the case of easily transportable pure hydrogen generators sought in the motor car industry for producing cars with electric traction equipped with combustible batteries of the proton exchanger membranes type (PEM), it is essential for the required electric powers of between 50 and 100 kW that the total volume occupied by the membrane composite structures, and thus by the reaction chamber where they are installed, is as reduced as possible.", "So as to embody membranes as thin as possible yet relatively large, various researchers have suggested producing composite structures constituted by a thin film of palladium or a palladium alloy laid on a permeable rigid substrate resisting to the pressure of the environment.", "The patent U.S. Pat.", "No.", "2,958,391 granted to A. J Derosset describes a composite structure with membranes selectively permeable to hydrogen, said structure including a thin film of palladium or a palladium alloy laid directly on a porous sintered metal substrate in the form of a plate or elongated cylinder.", "In principle, only the hydrogen filters through the membrane formed by the thin film and penetrates into the permeable porous substrate connected to a collecting pipe.", "This type of structure is clearly advantageous when the filtering film is sufficiently thick so as to be really effective and when the sintered substrate possesses sufficient mechanical resistance despite its porosity for satisfying the four conditions mentioned earlier.", "However, this type of structure has certain defects.", "The first is the risk of allowing micro-holes to be formed in the filtering thin film owing to the relatively significant surface roughness of the wall of the substrate.", "This surface roughness results from the relatively large size of the metal grains used required by the minimum sought-after permeability for the porous substrate.", "The origin of the second risk lies in the fact that, so as to constitute the substrate, the document does not provide selecting a metal having a heat expansion coefficient compatible with the relatively low heat expansion coefficient (namely 11.8 10−6/° C. for palladium) of the filtering film.", "This metal is required so as to allow full selectivity of the permeability of this film, by provoking microcracks due to prejudicial differential expansions.", "In addition, where the gas mixture including the hydrogen to be filtered is subjected to high pressure and a high temperature, the six conditions mentioned above are insufficient for a structure including a filtering film laid on a substrate.", "In fact, it is also essential that at the high temperatures and pressures in question (generally 300 to 600° C. and 5 to 15 bars), the metal of the filtering film does not diffuse inside the metal of the substrate which would significantly reduce the selective permeability of the membrane with respect to the hydrogen.", "This also means that it is essential that the two metals in contact are chemically stable with respect to each other at the temperatures and pressures concerned.", "The patent U.S. Pat.", "No.", "5,498,278 granted to D. J. Edlund proposes a solution to these various problems.", "Here, the composite structure described includes three elements: (1) a flexible porous intermediate film, non-sintered, textured and heat and chemically stable, is placed between (2) a thin metal film selectively permeable to hydrogen, such as palladium or a palladium, silver and/or nickel alloy, and (3) a permeable rigid substrate.", "The intermediate film in question is a woven or non-woven film made for example of aluminium, silicon, glass or carbon fibres.", "It totally separates the external filtering film from the internal permeable rigid substrate and renders them totally independent of each other.", "Any consideration of direct compatibility, especially chemical or thermic, between the material of this filtering film and that of the substrate is in principle eliminated as virtually useless owing to the presence of this particular intermediate film serving as a barrier.", "Thus, the substrate could be more of less any and for example made of full metal or dense ceramic material rendered permeable by cuts or perforations.", "In this structure and according to the document, the filtering film and the flexible intermediate film possess maximum efficiency when they comprise microwaves in two orthogonal directions enabling them to operate as micro-blowers adapted to absorb any differential movement with respect to the substrate.", "However, these arrangements have one major drawback which is a direct consequence of the non-metallic nature of the intermediate film.", "Indeed, this renders impossible any genuine weld of the metallic filtering film and the intermediate film which is not so.", "The reciprocal fixing of these two films having different natures can only be a sort of glueing with relative stability and effectiveness.", "In these circumstances, at the end of a relatively short period of use comprising successive periods of functioning and stoppage, the filtering film, which undergoes relatively significant heat contractions and expansions and with regard to those (virtually nul) of the intermediate film, shall inevitably come away from its support and shall quickly become fragile, then cracked and finally non-operative.", "An identical situation would occur if the body of the substrate and the intermediate film or either or both were made of a ceramic material, namely being fragile and brittle.", "The first object of the invention is to embody a composite structure with a membrane selectively permeable to hydrogen and including a thin filtering film placed on a rigid porous substrate which retains its constituent qualities at high pressures and temperatures in the presence of a gas mixture including hydrogen.", "The second object of the invention is to embody these membranes and substrates which, in the presence of hydrogen, are chemically as well as thermically compatible to one another.", "The third object of the invention is to embody these membrane composite structures which possess large surfaces, small individual volumes and shapes enabling them to be easily adapted to their particular conditions of use.", "The fourth object of the invention concerns various types of combustible gas processors adapted to optimise their functioning via the usage of these membrane structures.", "According to the invention, an improved membrane composite structure, selectively permeable to hydrogen and constituted by an ultra-thin filtering film with a thickness of several microns and made of palladium or a palladium-based alloy placed on a rigid refractory porous substrate connected to a pipe for collecting the extracted hydrogen, is characterised in that: said substrate includes a sintered metallic body provided with relatively good mechanical resistance and open pores measuring from few microns up to ten microns; a thin sintered metallic film, known as an intermediate film with a thickness measuring between twenty and fifty microns and containing open pores smaller than one micron, is placed on the body of said substrate; the body of the substrate and the intermediate film are made of an alloy or alloys provided with coefficients of heat elongation and expansion in the presehce of hydrogen rendering them both compatible with those of the palladium; the material constituting the intermediate film possesses within the range of the use temperatures of the structure a chemical stability which is satisfactory with regard to the filtering film and the body of the substrate.", "According to one additional characteristic, the heat expansion coefficients of the materials constituting the body of the substrate and the intermediate film are lower or at the most slightly higher than that of the material constituting the filtering film.", "According to a set of additional characteristics: the ultra-thin filtering film is made of a palladium-based alloy including silver and/or nickel; the body of the substrate is embodied from a relatively fine powder having suitable size grading formed from a super-alloy having an extremely low coefficient of heat expansion and preferably being a nickel, chromium or iron-based; the intermediate film is embodied from an ultra-fine powder with suitable size grading made of nickel or a super-alloy similar to the one used for the body of the substrate; said filtering film is fixed by micro-welds to the surface grains of said intermediate film.", "By means of these arrangements, the filtering film is placed on a thin intermediate film exhibiting extremely small surface roughness constituting a suitable support.", "This support is indeed a particularly smooth surface which in fact is made up of rounded micro-grains having different shapes and sizes juxtaposed irregularly and welded together during a sintering operation (agglomeration of powders, especially metallic subjected to a suitable pressure and temperature).", "For this intermediate film to contain open pores, that is to say pores communicating with one another and measuring at the most one micron, the powder grains used need to have an appropriate size grading, that is to say they are about three to five times the maximum size of these pores.", "The sintering of grains situated outside this range can indeed result in embodying pores with the sought-after size, but more of less closed and therefore relatively impermeable.", "So as to place an ultra-thin metallic film on a metallic support, several methods known for their effectiveness are available and in particular vacuum vaporisation, electrolysis, “electroless” and PVD (Physical Vapour Deposition).", "In the case where this support is sintered, these techniques make it possible to embody immediate, effective and stable micro-welds for the ultra-thin film on the tops of the surface micro-grains of the support.", "By using grains measuring several microns, a particularly smooth intermediate film is obtained which directly makes it possible to embody a continuous ultra-thin filtering film with approximately no micro-hole and being between two and five microns thick.", "Locally, this thickness shall be (1) close to the size of the interval existing between the tops of two juxtaposed top micro-grains of the intermediate film, and (2) larger than the average depth of the micro-gaps separating these tops.", "This possibility of embodying a thin filtering film means leading to an acceptable cost for the palladium required for the production of a composite structure conforming to the invention, namely a weight of one quarter of a gram and a price in January 2001 of about three US dollars for a square decimetre of two microns thick.", "As regards the compatibility in the presence of hydrogen the palladium and alloys constituting the body of the substrate and the intermediate film, the following shall be noted.", "In accordance with the work entitled “Topics in Applied Physics (Volume 28)”—Hydrogen in Metals I, page 56, the maximum value of the ratio C═H/M of the densities of atomic hydrogen and palladium in the context of the use of the membrane (typically 10 bars and 300° C.) is assessed at C=2 10−3, which results in a linear expansion of 10−4.Values of the same order are calculated for the silver and the nickel.", "This results in firstly that the constituents of the composite structures according to the invention are fully compatible with one another in their normal sphere of use, and secondly that these coefficients are lower than the heat expansion of the palladium (about 4.10−3) during the rise in temperature of these structures in the reaction chamber mentioned earlier.", "As regards what provides the heat compatibility of the palladium and of the metal of the substrate, it shall be observed that it assumes particular importance during the rise in temperature of the processing chamber (from the 20° C. of the ambient environment to the 350 or 400° C. reference point).", "According to the invention, the heat expansions of the body of the substrate and the intermediate film are lower than or at the most slightly greater than the heat expansion of the filtering film.", "This is essential so that the micro-elements of the filtering film delimited by the tops of the micro-grains to which they are welded are unable to undergo any tensile stress likely to provoke micro-cracks of this film.", "In the case of the palladium and Inconel 600 (nickel, chromium and iron-based super-alloy), the heat expansion coefficients are 11.8 10−6/° C. for the first and 11.5 10−6/° C. for the second, this explaining the preferred choice of this super-alloy for embodying the composite structure according to the invention.", "On the other hand, if it is desired to use stainless steel 304 (quoted in a large number of publications as being a normal component of the substrate of the composite structures concerned) for embodying a structure according to the invention, its heat coefficient of expansion of 17.4 10−6/° C., considerably greater than that of the filtering film, would prove to be unsuitable for the embodiment of composite structures intended to be used in a processing chamber of the type mentioned above.", "In fact in this case, the micro-elements of the filtering film would be subjected to a traction stress during the rise in temperature of the processing chamber and as a result could exhibit micro-cracks likely in the long run to considerably reduce the selective permeability of the filtering film with respect to the hydrogen.", "In addition to the thermal compatibility of the palladium and Inconel mentioned above, their relative chemical compatibility, that is their negligible speed of intermetallic diffusion within the range of the pressures and usual operating temperatures of the composite structures for the selective filtering of hydrogen according to the invention, is also ensured.", "Thus results from the experimentally observed properties of the Inconel, the nickel and certain nickel super-alloys including chromium and iron.", "According to one first embodiment of a composite structure according to the invention, said substrate is a cylindrical rod provided at one extremity with a collar for removing the extracted hydrogen and, if appropriate, equipped with a vector gas feed collar at the other extremity, said collars and their welding being made of metals compatible with the material of the substrate and stable with regard to hydrogen.", "According to a second embodiment according to the invention, said substrate is a sealed plate fixed in a metallic border compatible with the material of the substrate and provided with an orifice connected to said evacuation pipe and, if appropriate, fitted with another orifice opposite the first and connected to a vector gas feed pipe.", "According to a first important application according to the invention above, a combustible gas processor, in which a cold plasma reaction chamber fed in adequate conditions by a primary mixture of said gas, water vapour and air, produces a secondary mixture formed of hydrogen and carbon dioxide and monoxide, characterised in that: said chamber comprises several said membrane structures selectively permeable to hydrogen and having a given shape and dimensions and connected to a common pipe for collecting the hydrogen; placed immediately close to these structures are electrodes provided with a refractory non-conducting sheath with a high dielectric coefficient, said electrodes having an appropriate shape and dimensions enabling them to effectively cooperate with said structures so as to constitute a unit adapted to produce in response to an appropriate electric feeding, barrier electric discharges generating said cold plasma in the spaces separating the electrodes and the structures.", "By means of these arrangements, it is possible to embody improved membrane structures selectively permeable to hydrogen and comprising an adequate substrate enabling them to simultaneously satisfy all the specifications which concern them.", "As for the shapes to be given to these structures, it shall be hereby noted that long and thin rods equipped with an adequate axial reinforcement make it possible to embody structures having the shape of relatively large and particularly advantageous grids.", "Indeed, these grids can be easily associated with insulated electrodes having the same shape and dimensions so as to optimise the cold plasma reaction chamber of a combustible gas processor in accordance with the instructions of the international patent application WO 98/28223 filed by one of the inventors of the present invention.", "The characteristics and advantages of the invention shall appear more precisely from a reading of the following description of embodiments given by way of non-restrictive examples with reference to the accompanying drawings on which: FIGS.", "1a-b-c represent cross sectional views of two structures with the shape of rods, respectively short and long, structures in the shape of plates, of a filtering membrane according to the invention; FIGS.", "2a-b represent longitudinal cutaway views of two short rods according to the invention; FIGS.", "3a-b-c represent longitudinal cutaway views of three long rods according to the invention FIGS.", "4a-b represent front views of two structures in the shape of plates, respectively circular and square, of two filtering membranes according to the invention; FIG.", "5 represents the diagram of a cross sectional view of the reaction chamber of a combustible gas processor producing hydrogen and equipped with structures of filtering membranes in the shape of short rods according to the invention associated with a non-conducting electrode having the same shape; FIG.", "6 represents the diagram of a longitudinal cutaway view of the reaction chamber of a combustible gas processor producing hydrogen with an improved yield and equipped with the filtering structures in the shape of long rods according to the invention associated with non-conducting electrodes having the same shape and with baskets containing a suitable catalyst; FIG.", "7 represents a set of filtering structures or non-conducting electrodes used several times in the reaction chamber of FIG.", "6; FIG.", "8 represents the diagram of a longitudinal cutaway view of a reaction chamber similar to that of FIG.", "6 and equipped with filtering structures in the shape of rectangular plates associated with non-conducting electrodes and baskets containing a suitable catalyst, also in the shape of rectangular plates; FIGS.", "9a-b represent the diagram and a partial cutaway view of a hydrogen purification device and especially for purifying the hydrogen generally available on the market.", "According to the units of FIGS.", "1a and 2a-b, 1b and 3a-b-c, 1c and 4a-b, various membrane structures selectively permeable to hydrogen (with the dimensions given hereafter by way of non-restrictive examples) are represented and in particular the structures 10a-b in the shape of short rods (diameter 20 mm and length 120 mm), the structures 24a-b-c in the shape of long rods (diameter 8 mm and length 400 mm) and the structures 40a-b in the shape of circular or elliptic plates 40a (diameter between 100 and 200 mm) or rectangular or square plates 40b (side between 100 and 400 mm) having a thickness of between 3 and 8 mm.", "According to the figures shown above, these various structures all include starting form the outside (1) an ultra-thin filtering film 12, 26 or 42, (2) a thin intermediate film 14, 28 or 44, and (3) a rigid sintered substrate 16, 30 or 46 (FIG.", "1a-b-c).", "The ultra-thin filtering film is made of a palladium (70 to 80%) and silver (20 to 30%) alloy and its thickness is between 3 and 4 microns.", "The thin intermediate film is embodied from an ultra-fine powder having suitable size grading (range of between 3 and 5 microns for example) made of nickel or a nickel-based super-alloy.", "It measures up to fifty microns in thickness and contains relatively small open pores smaller than one micron providing it with a particularly smooth surface but with reduced porosity and permeability.", "The rigid and sintered body of the substrate is embodied from a relatively fine powder with suitable size grading (range of between 30 and 50 microns for example) made of Inconel 600.It contains relatively large open pores measuring from few microns to about ten microns providing it with relatively large porosity and permeability, but also as mentioned earlier a relatively significant surface roughness.", "It shall be noted that the composition given above for the filtering film 12-26-42 has been given by way of example and that a palladium-based alloy including silver and/or nickel can also be suitable.", "Inconel 600 is a stainless steel super-alloy including 75% nickel, 15.5% chromium and 8% iron to which small proportions of carbon, manganese and silicon are added.", "The melting temperature of the Inconel is 1,370° C., its module of elasticity 200 GPa and its limit of elasticity on traction is 800 MPa.", "By way of fair comparison, it shall be noted that the values of the three preceding characteristics of Inconel 600 are approximately equal to or slightly greater than those of a material used by several of the inventors of the patents mentioned above, namely type 304 stainless steel.", "In fact, the preferred choice of Inconel to constitute the substrate 16-30-46 is determined by its coefficient of heat expansion extremely close to that of palladium, whereas the coefficient of heat expansion of the steel in question is relatively far from this.", "As seen earlier, this coefficient renders this steel unsuitable for a usage in certain treatment chambers.", "On the other hand, Inconel and nickel-based alloys known for their low coefficient of heat expansion are suitable for all types of combustible gas treatment chambers.", "According to FIG.", "1b, a structure 24 with the shape of a long rod comprises an axial reinforcement 32 made of Inconel 600.According to FIG.", "1c, a plate structure 40 comprises a metallic border 48, also made of Inconel 600.According to FIGS.", "2a-b and 3a-b-c, the short rod structures 10a-b or long ones 24a-b-c all comprise at one extremity a collar 20a-b or 34a-b-c and preferably made of Inconel 600 welded to the thin intermediate film 14a-b or 28a-b-c by a compatible welding.", "These collars 20a or 34a of the membrane structures 10a-24a with the shape of a glove finger (one ultra-thin filtering film 13 or 27 is placed on the extremity section) are intended to provide both removal of the extracted hydrogen and the securing of these structures to a collecting pipe.", "The short rod 10b or long rod 24b structures comprise a collar 22 or 36 fixed to the other extremity in the place of the ultra-thin film 13 or 27.These collars 22-36 are provided to inject a neutral vector gas (nitrogen for example) into the substrate 16 or 30 so as to drive the produced hydrogen as it is gradually extracted.", "According to FIGS.", "3a-b-c, the long rod structures 24a-b-c all comprise a rigid metallic axial reinforcement 32a-b-c made of Inconel 600 which gives them the shape of pencils.", "The reinforcements 32a-b are full rods 2 mm in diameter and the reinforcement 32c is a hollow rod with external and internal diameters of 3 and 1.5 mm respectively.", "The two glove finger-shaped membrane structures 24a and 24c differ from each other by the fact that the hollow axial reinforcement 32c opens into a cavity 38 provided in a boat 40 welded to the other extremity of the long rod 24c in the place of the ultra-thin film 27 of the pencil 24a so as to allow the use of a vector gas of the hydrogen extracted by a glove finger-shaped membrane.", "The reinforcements 32a-b-c are also made of Inconel 600, a material with advantageous mechanical characteristics adapted to provide the permeable porous bodies of the long pencil structures 24a-b-c incorporating them with a sufficient solidity and stiffness to enable them to be easily manipulated and a good resistance to the impacts inevitably sustained during the time they are used in combustible gas processors mounted on vehicles.", "The axial reinforcements 32a-b-c of the pencils 24a-b-c project from the fixing collars 34a-b and 34c-36 so as to be able to be welded to the hydrogen collecting pipe for the first two and to the vector gas injector pipe for the last two.", "The aim of this arrangement is to improve the effectiveness of the fixings of the structures.", "According to FIGS.", "4a-b, the circular 40a and rectangular 40b plate-shaped structures respectively comprise metallic borders 48a-b in which the substrates 45a-b are seal-fixed according to the invention.", "The cross sectional view of these structures embodied along the lines A-A′ or B-B′ is shown on FIG.", "1c.", "It shall be noted that in the case of large square or circular plates (for example with a diameter or side exceeding about 10 cm), two plies formed of several relatively fine rigid metallic rods could before embodiment of the substrate 46a-b be placed in the shape of a cross in the borders 48a-b and welded to the latter so as to play the same role as the axial reinforcements 32a-b-c of the long rods mentioned earlier.", "In the case of large relatively elongated elliptic or rectangular plates, only one of these plies, such as 49 on FIG.", "4b, connecting the central portions of their closest border sections could suffice.", "The borders 48a-b each comprise two opposing orifices connected to pipes 50a-52a and 50b-52b respectively allocated to one vector gas injection and the removal of the extracted hydrogen.", "In the absence of any vector gas, the pipes 50a-b could be eliminated.", "The substrates 16, 30 or 46 are embodied by means of a suitable sintering (see above) adapted to the sought-after porosity and permeability in moulds having adequate shapes of relatively fine Inconel 600 grains with calibrated size grading and adapted following this operation to generate rigid bodies with significant permeability and porosity containing open pores measuring up to ten microns.", "These moulds with adequate shapes shall be two half-cylinders for the rod substrates and two trays for the plates, one of these trays comprising a recess having a shape identical to the outer shape of the border 48 and a depth identical to its thickness.", "So as to improve the permeability of these substrates, fine wires made of a heat-destructible material are previously placed in the form of several films in the metallic powder mass poured into the mould prior to sintering.", "During this sintering operation, the wires in question are destroyed and a network of micro-channels or small veins, such as 17, 31 or 47, is established which appears at points on FIGS.", "1a-b-c.", "The small veins 17a-b and 47b represented by the dots on FIGS.", "2a and 4b are longitudinal (those of the rods of FIGS.", "2b and 3a-b-c have been omitted) and the small veins 47a of FIG.", "4a follow curved lines connecting the orifices 50a-52a of the border 48a.", "The thin intermediate film 14 is embodied by depositing on the body 16 a suitable gel containing an ultra-fine metallic powder with calibrated size-grading and made of nickel or a nickel-based super-alloy (which could be Inconel 600) formed of small grains adapted to generate following an adequate sintering operation open pores smaller than one micron and communicating with one another.", "The porosity and permeability of the thin intermediate film 14 (which measures 30 to 50 microns thick) are relatively slight but its surface roughness gives its external surface a particularly smooth condition which is fully suitable as a support for the depositing of an ultra-thin filtering film 2 to 4 microns thick.", "This ultra-thin filtering film 12 can with reference to the documents published earlier be embodied via a depositing of the palladium and silver alloy carried out by means of any method adapted for this purpose and known for its effectiveness, especially one of those methods mentioned earlier.", "By means of the presence of the extremely smooth intermediate film 14, the ultra-thin filtering film 12 is almost perfectly continuous, that is to say almost without any micro-holes or micro-cracks,which provides it with an almost full impermeability to any gas but hydrogen, the rate of the filtered pure hydrogen impurities being able to drop down to about 5 ppm when the thickness of the filtering film is between 4 and 5 microns.", "By means of the nature (Inconel 600) of the metallic grains used to embody the porous body 16 and the intermediate film 14, the coefficients of heat expansion of these two components and of the filtering film 12 made of palladium or a palladium/silver alloy of the structures 10a-b, 24a-b-c and 40a-b are approximately identical.", "This results in minimising as much as possible the problems of differential expansion between the components of the filtering structures according to the invention in the reaction chambers of the combustible gas processors operating at temperatures able to vary between 300 and 600° C. Moreover, it shall be noted that this range of temperatures is the one in which selective permeability is maximum with regard to the hydrogen of an ultra-thin film made of a palladium and silver alloy and that this selective permeability rapidly reduces to temperatures lower than the bottom threshold of this range.", "In these reaction chambers, the total pressure can reach between 12 and 15 bars with a partial hydrogen pressure of between 3 and 6 bars.", "Downstream of the ultra-thin filtering film 12, the hydrogen pressure is by almost 2 bars lower than the partial pressure of the hydrogen existing upstream.", "Because of this, the ultra-thin membrane 12 can be subjected to a total differential pressure greater than 10 bars.", "By means of the approximately full porous body 16 and the thin intermediate film 14, which constitute the substrate of the membrane 12 and give this substrate a high mechanical resistance, the pressure exerted downstream of this membrane is, indeed, without causing any damage for the ultra-thin film 12, applied on compression to the substrate 16 which constitutes a continuous support for the entire surface of the membrane.", "Because of this, the ultra-thin filtering membrane structures according to the invention can have both large surfaces and reduced individual spatial requirements whilst being adapted to withstand high temperatures and pressures.", "FIG.", "5 represents the cross section of a cold plasma reaction chamber 54 of a combustible gas processor, namely a hydrogen generator, of the type described in the international patent application WO 98/28223 mentioned earlier.", "According to FIG.", "5, a cylindrical casing with an internal diameter of between 56 and 70 mm and provided with high heat insulation and adapted to withstand internal relatively high pressure and temperature (10 to 15 bars and 300 to 600° C.) surrounds a reaction chamber 58.Installed in this chamber 58 is an axial cylindrical electrode 60 with a length of 200 mm and provided with a non-conducting sheath 62 with a high dielectric coefficient and made of a ceramic material 3 mm thick and giving this insulated electrode an external diameter of 20 mm.", "Placed fully around this insulated electrode 60-62 a small distance away (3 mm for example) are six short cylindrical rod-shaped structures 641 .", ".", ".", "6 conforming to one of the models described on FIGS.", "2a-b.", "These rods have the same dimensions as the electrode.", "FIGS.", "6-8 represent the diagrams of longitudinal views of two combustible gas processors whose hydrogen productivity is considerably improved by the use of membrane structures selectively permeable to the hydrogen according to the invention and associated with insulated electrodes and baskets containing a suitable catalyst.", "The dimensions of these various elements constituting the reaction chambers of these processors are clearly given hereafter solely by way of non-restrictive examples.", "According to FIG.", "6, the reaction chamber 65 of a processor and intended to be installed on a motor vehicle possesses a cylindrical casing 66 with a diameter and length of 50 cm and conforms to the specifications of the casing 56 of FIG.", "5.Placed after one another in this chamber 65 are twenty sets (only four, namely 68a-b-c-d are represented) of the membrane structures selectively permeable to hydrogen according to the invention.", "These sets of structures 68 have the shape of square grids with a side measuring 300 mm and a thickness of 8 mm.", "They are constituted by long pencil-shaped membranes of the type with two collars described on FIG.", "3b and whose two extremities are welded to two hollow beams 70a-b-c-d and 72a-b-c-d allocated to the vector gas injection for the first ones and to the collecting of hydrogen for the second ones.", "The hollow beams 70 and 72 project from the grid and are respectively connected to two pipes 74 and 76 which ensure feeding with vector gas (nitrogen for example) for the first and removal of the hydrogen produced in the chamber for the second.", "The difference between the pencils of the structures 68 is small (<1 mm) so that the surface filtering the hydrogen of each grid is slightly less than three times the surface of the square it forms.", "These grids 68a-b-c-d are rigidly fixed to a frame (not shown) installed in the chamber 65 and are separated from one another by gaps of 12 mm.", "Installed under the grids 68a-c filtering the hydrogen and fixed to the same frame are electrodes 78a-b, also in the shape of grids externally identical to the grids 68a-c.", "These grids of electrodes are made up of long pencils similar to those of the grids 68a-c and comprise one axial electrode and one non-conducting sheath respectively in accordance with the constituents 60-62 of the insulated electrode shown on FIG.", "5.The pencils of the grids 68a-b and 78a-b are placed zigzag and the size of the free spaces separating them are at least 2 mm.", "FIG.", "7 represents the outer aspect of the structure grids filtering the hydrogen and of the grids of electrodes mentioned above.", "The square grids of the processor of FIG.", "6 measure 30 cm sideways and each include thirty-four pencils measuring 28 cm long and 8 mm in diameter and spaced from one another by 0.8 mm.", "The extremities of these pencils are fixed to tow projecting beams 71 and 73 measuring 36 cm long and 1 cm in diameter.", "In the case of a grid of filtering structures, these beams 71-73 are pipes allocated respectively to vector gas injection and removal of the extracted hydrogen.", "In the case of a grid of insulated electrodes, the beams 71-73 are insulated electric conductors ensuring feeding of these electrodes, one extremity of one of these conductors being adapted so as to establish a link with a generator.", "This grid of electrodes could first of all constitute a bare unit to which a ceramic moulding from a casting is then applied during a sintering operation.", "In addition to their respective individual functions, these beams 71-73 also ensure fixing of the two types of grids concerned to the frame mentioned earlier.", "Installed under the grids 68b-d and fixed to the frame mentioned earlier are two square metal baskets 80a-b having a rigid border and measuring 300 mm sideways like the filtering grids 68a-b-c-d but with a thickness of 10 mm so that the differences separating these baskets and grids are about 1 mm.", "These baskets 80a-b contain a known type of catalyst formed of ceramic granules coated with a mixture of iron and chromium oxides which is a specific of the “water-gas shift” reaction within the range of temperatures of 300 to 550° C. (which corresponds to the maximum effectiveness range of the filtering structures according to the invention).", "This reaction shall be presented hereafter.", "As for the baskets, in view of their constitution, these are fully permeable to the gases.", "According to the international patent application WO 98/28223 mentioned above, the electrodes 78a-b are connected by electric high insulation conductors 82 to a generator 84 delivering an extremely high alternative voltage (10 to 20 k-V) at a high frequency (for example 1 MHz) pulsed with a period of for example 1 ms.", "Pipes 861 .", ".", ".", "6 installed at the outlet of a gas conditioning cell 88 ensure the feeding with gas for treating the reaction chamber 65 and to this effect project onto orifices regularly distributed at the bottom of the casing 66 of this chamber.", "By means of the shape of the grids of the two electrodes 78a-b and the four filtering structures 68a-b-c-d and the high permeability of the baskets of granules 82a-b, the various gas mixtures circulating in the reaction chamber 65 do so in the best possible conditions.", "The gases provided by the pipes 86 constitute a suitable first mixture of combustible gases (hydrocarbon or alcohol in particular), water vapour and air.", "This suitable mixture is embodied in the conditioning cell 88 which each receives the three gases concerned so as to be agitated, heated and compressed and then finally delivered with relative flows and adequate partial pressures at a total pressure of between 10 and 15 bars and a temperature of between 300 and 500° C. as required by the reaction chamber 65 so as to operate in conditions providing as best as possible the sought-after results.", "A canalisation 90 ensures evacuation of the carbon dioxide produced in the chamber 65.According to the FIG.", "8 shows the reaction chamber 92 of a processor 91 using other filtering structures according to the invention.", "The description of this chamber 92 shall only concern what distinguishes it from the chamber 66 of the processor 65 of FIG.", "6.Placed in this chamber 92 are two groups with intervals of 12 mm, each group including twenty filtering structures according to the invention (only four structures 94a-b-c-d being shown) in the form of large rectangular plates (measuring 30×20 cm for example) with a thickness of 8 mm conforming to the one described on FIG.", "4b.", "Placed alternatively between two filtering plates 94 are two insulated electrodes, such as 96a-b, in the form of rectangular plates measuring 15×20 cm and being 8 mm thick and two rectangular baskets with a perforated rigid border, such as 98a-b, also measuring 15×20 cm but with a thickness of 11 mm and filled with catalyst granules identical to the preceding ones.", "So as to increase the effectiveness of conversions made in this reaction chamber, the direction of circulation of the vector gas in the filtering structures 94a-b-c-d and that of the reactive mixture injected into the chamber shall be inverted in relation to each other.", "All of these forty sets of plates have a square cross section measuring 40 cm sideways with a length of 60 cm.", "A cylindrical casing 93 with a diameter of 60 cm and 80 cm long shall be suitable for said sets.", "The total surface of these membrane structures brought together is 960 dm2.The feedings and evacuations concerning this chamber 92 of FIG.", "8 are, like all those of the chamber 58 of FIG.", "5, identical to those of the chamber 65 of FIG.", "6.It shall be noted that the plates described placed in the direction of circulation of the gases can on the other hand be placed perpendicular to this direction with passages alternatively placed at either of their extremities.", "FIG.", "9a represents the diagram of a longitudinal section of a hydrogen purification device, and FIG.", "9b is a cross sectional view of its heating chamber.", "The hydrogen to be purified is in particular normal industrial hydrogen (containing about 10−4 impurities) with the aim of reducing this proportion of impurities to about 5 ppm.", "This device 100 mainly includes a filtering chamber 102 and a heating chamber 104.The filtering chamber 102 comprises a stainless steel cylindrical casing 106 containing a relatively large number of membranes 1081 .", ".", ".", "n selectively permeable to hydrogen in the form of square grids measuring 30 cm sideways according to FIG.", "7.The number of grid membranes is, along with the pressure, one of the parameters determined by the flow of the pure hydrogen to be obtained.", "Each of the grid membranes 108 is connected to two pipes 110 and 112 respectively allocated to vector gas feeding and the collection of the extracted pure hydrogen, said pipes traversing the downstream bottom 114 of the filtering chamber 102.The heating chamber 104 includes a boiler 116 and a burner 118 placed upstream of several pipes 120 with high heat conduction which traverse the boiler 116 (see FIG.", "9b) and open into a cavity 122 communicating with a chimney 124 via a perforated partition 126.The chimney 124 constitutes a pipe with a ring-shaped cross section which surrounds the wall of the filtering chamber 102.The boiler 116 is separated from the filtering chamber 102 by a party-wall 128 provided with perforations, adapted to evenly distribute the flow of hydrogen to be purified entering the chamber 102.The boiler 116 is fed with hydrogen to be purified by a compressor 130 which generates an excess pressure of between 4 and 10 bars and opens into a distribution box 132 communicating with the boiler 116 by a perforated wall 134.Close to the downstream bottom 114 of the filtering chamber 102, a pipe 136 is connected for collecting the residual hydrogen and ends at the entrance of the burner 118 which is moreover fed with compressed air by a compressor 138.Placed at the center of the filtering chamber 102 is a thermo couple 140 connected (by means, not shown) to a regulation device 142 adapted to produce a signal for controlling the flow from the air compressor 138.The unit formed by the filtering chamber 102, the heating chamber 104 and the ring-shaped chimney 124 is heat insulated with the aid of a non-conducting sheath 144 with rockwool.", "With reference to FIGS.", "5, 6 and 8, it shall be noted that the aim of conditions essential to correctly feed the described reaction chambers 58-65-92 with the primary mixture, in this case of a methane gas to be converted into hydrogen, is to enable these reaction chambers to carry out as best as possible the conversions defined by the following chemical equations: CH4+O2→CO2+2 H2→ strong exothermic reaction (1) CH4+2 H2O→CO2+4 H2→ strong endothermic reaction (2) CH4+H20→CO=3 H2→ moderate endothermic reaction (3) CO+H2O→CO2+H2→ moderate exothermic reaction (4) In the case of the reforming of a primary gas mixture containing an alcohol or a hydrocarbon, other than methane, similar equations can be written.", "Carrying out as best as possible the above conversions signifies in particular to ensure that the quantity of heat consumed by the highly endothermic reaction (2) is approximately equal to or slightly lower than the quantity of heat freed by the highly exothermic partial oxidation reaction (1).", "A good combination of theory and experimentation makes it possible to attain this objective.", "The same also applies for the two moderately exothermic (4) and endothermic (3) reactions.", "The three conversions defined by the equations (1)-(2)-(3) are carried out fully in the reaction chamber 58 (FIG.", "5) and in the sections of the chambers 65 and 92 (FIG.", "6 and 8) occupied by the filtering structures 68a-b or the sections of the plates 94a-b-c-d associated with the insulated electrodes 78a-b or 96a-b.", "The conversion according to the equation (4) above, known as “water-gas shift”, is ensured by the catalyst action effected by the granules coated with a mixture of iron and chromium oxides of the baskets 80a-b and 98a-b of FIGS.", "6 and 8.The portions of the filtering plates 94 placed on both sides 0.5 mm from the baskets 98a-b directly cooperate with the catalyst contained in these baskets.", "With reference to FIGS.", "5, 6 and 8 and the instructions of the international patent application WO 98/28223 mentioned earlier, by means of the presence of the high dielectric coefficient nonconducting sheaths of the electrodes 60-78-96 installed immediately close to the hydrogen filtering structures 64-68-96, an extremely short HF electric field (from 3 to 4 microseconds) is uniformly created periodically (1,000 Hz) in the free spaces traversed by the primary mixture in question which separate these sheaths from these filtering structures.", "This field generates barrier electric discharges in the spaces concerned which create a cold plasma (small population of extremely high energy electrons embedded in a medium passive at moderated initial temperature) which plays the same role as an appropriate chemical catalyst.", "A series of reactions defined by the equations (1)-(2)-(3) above then occurs whose period of persistence is in particular greater than the period of the barrier electric discharge which started it.", "A secondary mixture formed of hydrogen and residual gases, mainly including carbon monoxide (a poison for PEM type combustible batteries) and carbon dioxide, is produced on this occasion.", "The structures 64 of membranes permeable to the hydrogen of the reaction chamber 58 (FIG.", "5) extracted the hydrogen as it was gradually produced.", "This makes it possible for the three reactions (1), (2) and (3) concerned to be effected as best as possible in a particularly reduced required space.", "But this chamber 58, whose architecture exactly takes up the information of the patent application quoted as a reference, also produces the mixture of residual gases mentioned earlier which, despite the capacity of producing the hydrogen it still possesses is, according to this information, is only recycled as heat in the boiler of the conditioning cell associated with the reaction chamber.", "In the reaction chambers 65 and 92 of FIGS.", "6 and 8, this particular capacity possessed by carbon monoxide is collected effectively with an excellent yield (via the elimination of the inverse reaction and displacement of the chemical balance in the direction of a more complete reaction).", "This is effected by means of the immediate presence downstream of the insulated electrodes 78a-b and 94a-b of the baskets 80a-b and 98a-b filled with catalysing granules of the “water-gas shift” reaction according to the equation (4) mentioned above and installed immediately close to the filtering membrane structures 68a-b and 94a-b.", "It shall be noted that it is easy to install in the reaction chamber 54 of FIG.", "5 structures with rods twice longer than those described, and, above the insulated electrode 60-62, a basket of the same shape containing a suitable catalyst so as to obtain the same results as those provided by the chambers 65 and 92.In these circumstances, starting from a suitable combustible gas, water vapour and air primary mixture, the processor reaction chambers 65 to 92 with relatively reduced spatial requirement, improved by the use of the membrane structures according to the invention at high pressure and a high temperature alternatively associated with insulated electrodes and baskets with an adequate catalyst, produce in separate evacuation canalisations, such as 76 and 90, with considerable effectiveness and thus a high yield, basically almost pure hydrogen and carbon dioxide (a residue of three components of the initial primary mixture is however generally added to the latter).", "The origin of this high yield resides in the fact that the carbon monoxide present in the secondary mixture produced by the pair formed by the insulated electrode 78a-b or 96a-b and the filtering structure 68a-c or 94 (first section) is immediately treated by the pair formed by the catalyst basket 80a-b or 98a-b and the filtering structure 68b-d or 94 (second section) which follows it along the path of the reactive gas mixtures circulating in the reaction chamber.", "The hydrogen produced in the reaction chambers 58-65-92 is generally intended to be used for feeding the PEM type combustible batteries mentioned earlier.", "Experience has shown that, so as to feed such a battery delivering 100 electric W, it is necessary to have available about 1 dm2 membranes selectively permeable to the hydrogen.", "As a result, with the six short rod membranes 20 cm long and a diameter of 2 cm, the reaction chamber 58, whose internal diameter measures up to 12 cm at the most and having a length of 30 cm, is able to provide a sufficient quantity of hydrogen for feeding a PEM battery producing 750 W which allows advantageous applications in a large number of fields, especially in the leisure activities industry.", "Similarly, with the grid membrane structures 68a-b-c-d of FIG.", "6 which each have a total filtering membrane surface area of about 26 dm2, it is possible to produce a reaction chamber with a diameter and length of 50 cm containing ten electrode/structure pairs and ten basket/structure pairs, said chamber being able to produce a hydrogen flow able to feed a PEM battery providing 52 kW, which corresponds to the electric power required by the motor vehicle industry for feeding its future average power reduced pollution vehicles.", "Similar considerations can clearly be applied to the reaction chamber 92 of FIG.", "8 which, with a reduced spatial requirement, comprise plate filtering structures able to feed a PEM battery delivering 96 kW.", "With reference to FIGS.", "9a-b, the hydrogen to be purified, which is cold and at low pressure in the compressor 130, is injected compressed into the distribution box 132 and into the boiler 116.Throughout the start of the phase for putting the purification device 100 into action, this boiler 116 is itself cold so that all the hydrogen injected by the compressor 130 passes through the filtering chamber 102 and then the recovery pipe 136 so as to end up at the entrance of the burner 118.This is because the grid membranes 1081 .", ".", ".", "n selectively permeable to hydrogen and occupying the major portion of the filtering chamber 102 are cold and are thus unable to operate, their operating temperatures being between 300 and 550° C. In the burner 118, the injected hydrogen is mixed with the compressed air provided by the compressor 138 and the mixture is immediately ignited.", "The flames traverse the heating pipes 120 and the combustion gases are evacuated by the ring-shaped chimney 124.The hydrogen to be purified circulates in the boiler 116 sweeping the hot walls of the heating pipes 120.During this passage, it rapidly heats up and then penetrates the filtering chamber 102 by traversing the perforations of the party wall 128.It is then at a temperature inside a range enabling the membranes 108 to function correctly.", "Moreover, the filtering chamber 102 is heated by the ring-shaped chimney 124 lagged by the insulating sheath 144 surrounding it.", "In addition, the thermo couple 140, which sends a signal to the device 142 for controlling the flow of the air compressor 138, makes it possible to adjust to an optimum value the temperature of the filtering chamber 102.As soon as this is done, pure hydrogen is extracted by the membranes 108 operating in the best possible conditions and then is evacuated by the collecting pipe 112 driven by the vector gas introduced into the pipe 110.Any resultant residual hydrogen possesses a coefficient of impurities much higher than that of the-hydrogen to be purified initially injected but having a pressure lower than by at least one or two bars than that of the latter.", "This residual hydrogen is evacuated by the recovery pipe 136 and injected into the burner 118 where it is used as indicated earlier.", "Accordingly, a normal industrial hydrogen purification device is embodied able to have available pure hydrogen (impurity rate lower than 10 ppm) enabling it to be used to feed PEM type combustible batteries in the best possible conditions.", "In the case where the period for needing this extremely pure hydrogen is relatively short (for example several hours), the hydrogen purification device described on FIGS.", "9a-b can be significantly simplified.", "By way of example, the filtering chamber shall contain a single membrane of the short rod type described on FIG.", "2b, and the heating chamber shall contain an electric heating resistor fed by a current adjusted according to the temperature of the filtering chamber measured by a thermo couple.", "The heat insulation or lagging sheath shall be retained but the chimney shall of course be eliminated, as well as the pipe for collecting effluent from the filtering chamber.", "After a short period of usage, the hydrogen with a relatively high rate of impurities, which shall then be located in the filtering chamber, shall be evacuated via action of an appropriate valve.", "In the case where the irreducible impurity rate of the extremely pure hydrogen produced by a purification device according to FIG.", "9 or by a processor according to FIGS.", "5, 6, 8 would mainly originate from the carbon monoxide and because of this would be regarded as still being too much for feeding a PEM type combustible battery, the purified flow of hydrogen could be introduced into an additional treatment chamber functioning at temperatures of between 200 and 250° C. embodying the elimination of the CO by a well-known suitable catalyst in the industry concerned.", "This operation can make use of several types of catalysts and in particular make use of the ruthenium deposited on aluminium granules.", "This is a reverse transformation (methanation) of the one defined by the chemical equation (3) mentioned above, that is to say: CO+3H2→CH4+H2O.", "It adds to the previously pure hydrogen obtained a mixture of methane and water vapour fully supported by the batteries concerned.", "The invention is not limited to the embodiments described above.", "In this respect, it shall be noted that short or long rod structures, with or without a glove-finger shape, are able inside the same reaction chamber to be associated with insulated electrodes and catalyst baskets in the form of plates.", "As for the dimensions of these various structures, they shall mainly be determined by considerations of the resistance of materials, having regard to the maximum amplitude of the impacts it could be exposed to during their use.", "Moreover, it shall be noted that Inconel 600 shown above could be preferably replaced by other Inconel grades or even by certain types of Hastelloy.", "These Hastelloy types are also nickel-based super-alloys incorporating chromium and iron whose mechanical characteristics and chemical stability at high temperature are close to those of stainless steel and the heat expansion coefficient of at least one of them lower than that of palladium, namely 11.3 10−6/° C. The hydrogen filtering membrane structures according to the invention are not exclusively intended for combustible gas processors producing pure hydrogen.", "In fact, these structures shall advantageously be used in combustible gas processors carrying out the following reactions: the catalytic vapour-reforming of hydro carbons or alcohols, the dehydrogenation of ethane, the dehydrogenation of propane, the dehydrogenation of cyclohexane, the dehydrogenation of ethylbenzene, the conversion of carbon monoxide by the “water-gas shift” mentioned earlier.", "The above conversion reactions are carried out in the presence of their specific catalysts which are well-known in the industries concerned.", "In the case of the conversion of carbon monoxide according to the chemical equation (4) mentioned above, it is to be noted that a simple modification of the architecture of the reaction chamber 65 of the processor is able to embody this conversion.", "This modification shall firstly consist of replacing the insulated electrodes 78a-b by catalyst baskets identical to those with the reference 80a-b, and secondly of eliminating the electric generator 84 and of replacing the feeding with CH4 of the conditioning cell 88 by CO or by any synthetic gas rich in CO and H2.In the case where the temperature on the reaction chamber would be within a range of between 150 and 300° C., the catalyst used would be a mixture of copper and zinc oxides.", "However, in this case the effectiveness of membrane structures provided with a palladium or palladium/silver alloy filtering film would no longer be maximum.", "So as to adapt the reaction chamber 92 to the treatment of carbon monoxide, catalyst baskets as long and wide as the filtering structures 94a-b-c-d (or even bulk catalyst granules) shall be inserted between these structures, the direction of circulation of the vector gas in these structures being opposite the direction of the current of reactive gases circulating in the chamber.", "For these reactions, the benefit of the hydrogen filtering membrane structures according to the invention and likely to be subjected to high temperatures and pressures is as follows: elimination of inverse reactions and movement of the chemical equilibrium in the direction of a more complete reaction of conversion from CO into CO2 and thus a significant increase of the yield of the methods implemented in these processors.", "This effect increases with the pressure and when the temperature of the reactive mixture is situated inside the range of the optimum functioning temperatures of the filtering films of the structures according to the invention.", "All the above confirms the advantage for various industries concerning membrane structures according to the invention selectively permeable to hydrogen, said structures possessing an effective filtering film which is both ultra-thin and relatively extensive, occupies a small individual space and which functions without suffering damage at high pressure and operates best at high temperatures." ] ]	Patent_10468552
[ [ "Isocyanate composition and its use in the preparation of expanded polyurethane with improved physico-mechanical properties", "Isocyanate compositions with isocyanate functionality between 2.2 and 2.9 which include: a) 20 to 80% by weight of the reaction product of methylene diphenyl isocyanate (MDI) with an ethylene oxide (EO)/propylene oxide (PO) polyether polyol of functionality 2 to 8, an average molecular weight of 200 to 6000, and an ethylene oxide content of 20 to 90% having a free NCO group content of 26 to 33% by weight; and 20 to 80% by weight of an MDI polymer." ], [ "1.An isocyanate composition having an isocyanate functionality of 2.2 to 2.9 which comprises: a) 20 to 80% by weight of the reaction product of methylene diphenyl isocyanate (MDI) with at least one polyether polyol comprising ethylene oxide (EO) and propylene oxide (PO) with a functionality of 2 to 8, an average molecular weight of 200 to 6000 and an ethylene oxide content of 20 to 90% by weight and in which said reaction product has a free NCO group content of 26 to 33% by weight; and b) 10 to 80% by weight of a polymeric methylene diphenyl isocyanate having a general formula (I): where Φ represents a phenyl group and n is a whole number greater than or equal to 1.2.An isocyanate composition according to claim 1 which comprises 30 to 70% by weight of component a) wherein the at least one polyether polyol has an average molecular weight of 400 to 6000 and 10 to 70% by weight of component b) and 5 to 30% by weight of modified methylene diphenyl isocyanate uretonimine.", "3.An isocyanate composition according to claim 1 in which the at least one polyether polyol comprises a mixture comprising a first polyether polyol having an average molecular weight of 1000 to 6000 and a second polyether polyol having an average molecular weight of less than 1000, wherein the first and second polyols, independently comprise ethylene oxide and propylene oxide with a functionality of 2 to 8, and an ethylene oxide content of 20 to 90% by weight and the second polyether polyol is present at a concentration of less than 50% by weight relative to the first polyol.", "4.An isocyanate composition according to claim 1 in which the methylene diphenyl isocyanate comprises a mixture of the 4,4′ and 2,4′ MDI isomers, in which the 2,4′ isomer concentration is from 20% to 30% based on the total amount of MDI.", "5.An isocyanate composition according to claim 1 in which the at least one polyether polyol comprises a polyether diol.", "6.An isocyanate composition according to claim 5 in which the polyether diol has an ethylene oxide content of 50 to 75% or 80% and especially 70 to 80%.", "7.An isocyanate composition according to claim 1 in which component a) comprises the reaction product of MDI comprising 20 to 30% of the 2,4′-MDI isomer and a polyether diol polyether diol having an ethylene oxide content of 70 to 80% in which the reaction product has a free NCO group content of 29 to 33%.", "8.An isocyanate composition according to claim 1 in which the methylene diphenyl isocyanate and the polymeric methylene diphenyl isocyanate are both reacted with the at least one polyether polyol.", "9.A process for the preparation of a flexible expanded polyurethane which comprises reacting together: i) an isocyanate composition having an isocyanate functionality of 2.2 to 2.9 which comprises: a) 20 to 80% by weight of the reaction product of methylene diphenyl isocyanate (MDI) with at least one polyether polyol comprising ethylene oxide (EO) and propylene oxide (PO) with a functionality of 2 to 8, an average molecular weight of 200 to 6000 and an ethylene oxide content of 20 to 90% by weight in which said reaction product has a free NCO group content of 26 to 33%; and b) 20 to 80% by weight of a polymeric methylene diphenyl isocyanate having a general formula (I): where Φ represents a phenyl group and n is a whole number greater than or equal to 1; and ii) a polyol component comprising at least one polyol, with a functionality of 2 to 8 and an equivalent weight of 200 to 2000 and water.", "10.A process according to claim 9 in which the isocyanate composition is as defined in claim 1.11.A process according to claim 9 in which the isocyanate composition comprises a reaction product of MDI with 20 to 30% of the 2,4′-MDI isomer and a polyether diol having an ethylene oxide content of 70 to 80% in which the reaction product has a free NCO group content of 29 to 33%.", "12.A process according to claim 9 in which, in i), the methylene diphenyl isocyanate and the polymeric methylene diphenyl isocyanate are both reacted with the at least one polyether polyol.", "13.A process according to claim 12 in which at least one polyol and the polyol to be reacted with the isocyanate composition are the same and optionally the MDI and polymeric MDI are reacted with the polyether polyol to produce the expanded polyurethane in a single step.", "14.A process according to claim 9 in which the water is between 3 and 6 parts by weight to 100 parts of the polyol component.", "15.Use of an isocyanate composition according to claim 1 in the preparation of an expanded polyurethane having a density up to 50 Kg/m3, a bearing capacity greater than 40 N, according to ISO 2439-97, a % thickness loss of less than 5% and a % compression resistance loss of less than 16% when tested under Peugeot Test Method D42.1047-84." ], [ "The invention concerns certain isocyanate compositions and their use in the preparation of flexible expanded polyurethanes which have improved physico-mechanical properties.", "More specifically, the invention concerns certain methylene diphenyl isocyanate (MDI)-based isocyanate compositions and their use in the preparation of flexible expanded polyurethanes which have improved physico-mechanical properties.", "The term “flexible expanded polyurethanes which have improved physico-mechanical properties”, as used in this description and in the claims refers to expanded polyurethanes or polyurethane foams including those suitable for use for slabs, in molding (cold and hot) and for integral skin with a density preferably up to 50 kg/m3 or, more preferably, between 25 and 50 kg/m3, a compression resistance at 40% deflection, measured according to DIN-EN-ISO 3386-98, suitably greater than 3 kPa and optimally, a permanent deformation, or compression set tested according to ISO 1856-80 of lower than about 15% and preferably lower than 10%.", "In certain fields, for example for furniture and in the automotive industry, it is desirable that products made from flexible expanded polyurethanes or polyurethane foams, both slab and molded, have good comfort properties and physico-mechanical properties.", "In general, attainment of such properties does not require particular steps to be taken for high-density (≧55 kg/m3) foams, whereas medium to low density (25 to 45 kg/m3) foams typically require the use of secondary expanding agents combined with a primary expanding agent for example water in the expansion stage, in order to overcome processability problems, especially in the case of lower-density products.", "Halogenated hydrocarbons have been used for many years as secondary expanding agents, in particular chlorofluoroalkanes such as FREON 11 (trichlorofluoromethane), because of their ease of availability, their compatibility with polyurethane reagents and because of their properties as expanding agents.", "However, with the phase out of chlorofluoro alkanes, following the 1987 Montreal Protocol which sought to limit the use and production of products implicated in the depletion of the stratospheric ozone layer, other procedures for obtaining low-density polyurethane foams with good physico-mechanical properties with water as the only expanding agents have been developed, for instance as described in EP-A-486,034.Other expanding agents are used in EP 477,920.The Applicant has now found certain MDI-based isocyanate compositions which provide medium-low density expanded polyurethanes, having excellent comfort and physico-mechanical properties, using only water as the expanding agent.", "In addition, the isocyanate compositions of the invention are surprisingly stable and have an excellent “shelf-life”.", "The invention provides an isocyanate composition having an isocyanate functionality of 2.2 to 2.9 which comprises: a) 20 to 80% by weight, preferably 40 to 60%, of the reaction product of methylene diphenyl isocyanate (MDI) with at least one polyether polyol comprising ethylene oxide (EO) and propylene oxide (PO) with a functionality of 2 to 8, an average molecular weight of 200 to 6000, preferably 500 to 2500, and an ethylene oxide content of 20 to 90% by weight, preferably 50 to 75% or 80% and especially 70 to 80% and in which said reaction product has a free NCO group content of 26 to 33% by weight and preferably 29 to 33%; and b) 10 to 80% by weight, preferably 20 to 80%, more preferably 40 to 60% and especially 40 to 50% of a polymeric methylene diphenyl isocyanate having a general formula (I): where Φ represents a phenyl group and n is a whole number greater than or equal to 1.More specifically, the invention provides an isocyanate composition having an isocyanate functionality of 2.2 to 2.9 which comprises, and preferably consists essentially of; a) 30 to 70% by weight, preferably 40 to 60% of the reaction product of methylene diphenyl isocyanate with at least one polyether polyol comprising ethylene oxide (EO) and propylene oxide (PO) with a functionality of 2 to 8, an average molecular weight of 400 to 6000, preferably 600 to 2500, and an ethylene oxide content of 20 to 90% by weight, preferably 50 to 75% or 80% and especially 70 to 80% and in which said reaction product has a free NCO group content of 26 to 33% by weight and preferably 29 to 33%; b) 10 to 70% by weight of a polymeric methylene diphenyl isocyanate having a general formula (I): where Φ represents a phenyl group and n is a whole number greater than or equal to 1; and c) 5 to 30% by weight, preferably 10 to 20% of uretonimine modified methylene diphenylisocyanate.", "In a preferred embodiment, the invention provides an isocyanate composition having an isocyanate functionality of 2.2 to 2.9, which comprises, and preferably consists essentially of; a) 20 to 80% by weight, preferably 40 to 60%, of the reaction product of MDI with a mixture comprising a first polyether polyol having an average molecular weight of 1000 to 6000, preferably 1500 to 2500, and a second polyether polyol having an average molecular weight of less than 1000, wherein the first and second polyols, independently comprise ethylene oxide and propylene oxide with a functionality of 2 to 8, and an ethylene oxide content of 20 to 90% by weight, preferably 50 to 75% or 80% and especially 70 to 80% and the second polyether polyol is present at a concentration of less than 50% by weight relative to the first polyol and in which said reaction product has a free NCO group content of 26 to 33% by weight and preferably 29 to 33%; and b) 20 to 80% by weight, preferably 40 to 60%, of a polymeric methylene diphenyl isocyanate having a general formula (I): where Φ represents a phenyl group and n is a whole number greater than or equal to 1.In another embodiment, the polyol to be reacted with the methylene diphenyl isocyanate may be reacted with the MDI and the polymeric MDI of formula (I) together to produce an isocyanate composition.", "The invention further provides an isocyanate composition having an isocyanate functionality of 2.2 to 2.9 which comprises the reaction product obtained by reacting a mixture of methylene diphenyl isocyanate (MDI) comprising 20 to 30% 2,4′-methylene diphenyl isocyanate based on the total amount of MDI and a polymeric methylene diphenyl isocyanate having a general formula (I): where Φ represents a phenyl group and n is a whole number greater than or equal to 1 with at least one polyether polyol comprising ethylene oxide (EO) and propylene oxide (PO) with a functionality of 2 to 8, an average molecular weight of 200 to 6000, preferably 500 to 2500, and an ethylene oxide content of 20 to 90% by weight, preferably 50 to 75% or 80% and especially 70 to 80%.", "Suitable polymeric MDI's, according to the subject invention include polymethylene polyphenyl polyisocyanates with average functionality of 2.6 to 2.8; said products are available under various names such as “TEDIMON 31” (Enichem S.p.A.), “SUPRASEC DNR” (Huntsman), “VORANATE M-220” (Dow) and DESMODUR 44 V20 (Bayer).", "Uretonimine MDI-modified is a reaction product of methylenediphenyl isocyanate with an excess of a carbodiimide derivative.", "Preferably, MDI used in the preparation of isocyanate prepolymer (a) comprises a mixture of the 4,4′ and 2,4′ isomers, in which the 2,4′ isomer concentration is from 10 to 60% by weight, preferably from 18 to 50% and especially from 20% to 30% based on the total amount of MDI.", "The polyether polyol employed to produce the reaction product with MDI and optionally polymeric MDI to produce an isocyanate composition according to the invention suitably has a hydroxyl functionality of 2 to 8.Polyether diols, that is polyether polyols, having a functionality of 2 may be expected to impart good elongation properties to polyurethane foams produced therefrom due to the absence of cross-linking associated with polyether polyols having a functionality of 3 or higher but also poor compression set and dynamic fatigue characteristics, for example a high level of % thickness loss and compression load loss when tested under Peugeot test method D42.1047-84.It has been found that polyisocyanate compositions according to the invention in which the polyether polyol has a functionality of 2 provide good elongation properties but surprisingly exhibit excellent dynamic fatigue properties as well.", "Accordingly, a further preferred embodiment of the invention provides an isocyanate composition having an isocyanate functionality of 2.2 to 2.9, which comprises, and preferably consists essentially of; a) 20 to 80% by weight, preferably 40 to 60% of the reaction product of methylene diphenyl isocyanate with at least one polyether polyol comprising a polyether diol (having a functionality of 2) which comprises ethylene oxide (EO) and propylene oxide (PO), an average molecular weight of 400 to 6000, preferably 600 to 2500, and an ethylene oxide content of 20 to 90% by weight, preferably 50 to 75% or 80% and especially 70 to 80% and in which said reaction product has a free NCO group content of 26 to 33% by weight and preferably 29 to 33%; and b) 20 to 80% by weight, preferably 40 to 60%, of a polymeric methylene diphenyl isocyanate having a general formula (I): where Φ represents a phenyl group and n is a whole number greater than or equal to 1.Desirably the polyether diol in this embodiment comprises ethylene oxide (EO) and propylene oxide (PO), an average molecular weight of 400 to 6000, preferably 600 to 2500.Preferably the polyether diol has an ethylene oxide content of 50 to 75% or 80% and especially 70 to 80%.", "Suitably MDI used in combination with the polyether diol comprises a mixture of the 4,4′ and 2,4′ isomers, in which the isomer 2,4′ concentration is from 18 to 50% and especially from 20% to 30% based on the total amount of MDI.", "Polyisocyanate compositions comprising a reaction product of MDI with 20 to 30% of the 2,4′-MDI isomer and a polyether diol as described herein and wherein the reaction product has a free NCO group content of 29 to 33% are especially preferred.", "Optionally the MDI may be mixed with a polymeric MDI of formula I as described herein prior to reaction with the polyether diol to form the reaction product.", "A further aspect of the invention provides a process for the preparation of a flexible expanded polyurethane with improved physico-mechanical properties which comprises reacting together: i) an isocyanate composition having an isocyanate functionality of 2.2 to 2.9 which comprises: a) 20 to 80% by weight, preferably 40 to 60%, of the reaction product of methylene diphenyl isocyanate (MDI) with at least one polyether polyol comprising ethylene oxide (EO) and propylene oxide (PO) with a functionality of 2 to 8, an average molecular weight of 200 to 6000, preferably 500 to 2500, and an ethylene oxide content of 20 to 90% by weight, preferably 50 to 75% or 80% and especially 70 to 80%, and in which said reaction product has a free NCO group content of 26 to 33% and preferably 29 to 33% by weight; and b) 20 to 80% by weight, preferably 40 to 60%, of a polymeric methylene diphenyl isocyanate having a general formula (I): where Φ represents a phenyl group and n is a whole number greater than or equal to 1; and ii) a polyol component comprising at least one polyol, with a functionality of 2 to 8 and an equivalent weight of 200 to 2000 and water.", "Preferably, the expanded polyurethane foam is prepared using an isocyanate composition preferred herein, in particular, a polyisocyanate composition comprising a reaction product of MDI with 20 to 30% of the 2,4′-MDI isomer and a polyether diol as described herein and wherein the reaction product has a free NCO group content of 29 to 33%.", "Optionally the MDI may be mixed with a polymeric MDI of formula I as described herein prior to reaction with the polyether diol to form the reaction product.", "Polyurethanes produced employing a preferred isocyanate composition suitably exhibit a % thickness loss of less than 5% and preferably less than 3% and a % compression resistance loss of less than 16% when tested under Peugeot Test Method D42.1047-84.The at least one polyol and the polyol to be reacted with the isocyanate composition may be the same.", "Optionally the MDI and the polymeric MDI are reacted with the polyol polyether to produce the expanded polyurethane in a single step.", "The invention further provides, use of an isocyanate composition according to any one of claims in the preparation of an expanded polyurethane having a density up to 50 Kg/m3, a bearing capacity greater than 40 N, preferably greater than 200N and more preferably from 80 to 400 N according to ISO 2439-97, a % thickness loss of less than 5% and a % compression resistance loss of less than 16% when tested under Peugeot Test Method D42.1047-84.The polyol used in the preparation of flexible expanded polyurethanes according to the process may be selected from polyether polyols, polyether polyols containing ester groups, polyether polyols containing amine groups, polyester polyols, and the like.", "Preferred polyols include polyether polyols obtained through condensation of olefinic oxides having from 2 to 6 carbon atoms on (starter) compounds having at least two atoms of active hydrogen.", "The preferred olefinic oxides are ethylene oxide (EO) and propylene oxide (PO), and compounds which may provide EO or PO units in the polyether polyol.", "Suitable starter compounds include glycols, triols, tetrols, amines, alkanolamines, polyamines and the like and mixtures thereof.", "In a preferred embodiment, the polyether polyol suitably comprises ethylene oxide and/or propylene oxide and the starter is selected from a glycol, for example dipropylene glycol, a triol for example glycerin and trimethylolpropane, a tetrol for example pentaerythritol, a diamine for example ethylene diamine, an aromatic amine for example ortho-toluene diamine, an alkanol amine for example triethanolamine, and a polyfunctional hydroxyl alkane for example xylitol, arabitol, sorbitol and mannitol.", "The polyol may be used as is, or may contain solid particles, preferably polymeric particles.", "The particles are suitably in dispersion or partially linked to polyol chains, with dimensions under 20 micrometers.", "Polymers especially suitable for this purpose include polyacrylonitrile, polystyrene, polyvinyl chloride, copolymers comprising any of these polymers, and, urea-based polymers.", "Said solid particles may be prepared for polymerization in situ in the polyol or be prepared separately and later added to the polyol.", "The polyol compound may also include one or more additives commonly used in the preparation of expanded polyurethanes as amine catalysts, such as triethylendiamine, and/or metallic such as stannous octoate, cell regulators, thermo-oxidation stabilizers, pigments and the like.", "Details on polyurethane polymerization are described in “Saunders & Frisch—Polyurethanes, Chemistry and Technology” Interscience, New York, 1964.In the production of an expanded polyurethane according to the process of this invention, the expanding agent suitably comprises water.", "Water may be used alone or combined with secondary expanding agents other than the chlorofluoro alkanes and preferably water is present at a higher level than any other expanding agents.", "Water has a critical function in the preparation of expanded polyurethanes since through it urea bonds are formed, associated to the development of carbon dioxide which triggers the polyurethane resin expansion/swelling process thus obtaining flexible expanded polyurethanes.", "Suitably water is present in an amount from 3 to 6 parts by weight to 100 parts of polyol compound.", "Carbon dioxide is suitably used to expand polyurethane resin, preferably as a primary agent developed in situ by reacting water and the polyisocyanate NCO groups.", "In the preparation of reduced-density expanded polyurethanes, for example, having a density equal to or lower than 25 Kg/m3, the expanding function of carbon dioxide from water alone may not be sufficient to reach the desired density without incurring problems (burning or “scorching”) due to the exothermal reaction of water with the diisocyanate groups.", "For this reason, a secondary expanding agent in addition to water may be used.", "Suitable secondary expanding agents include air, liquid or gaseous CO2, nitrogen, alkane hydrofluorides with low or zero ozone depletion potential, hydrocarbons for example n-pentane, i-pentane, and cyclopentane, dimethyl carbonate and mixtures thereof.", "Whilst the primary expanding agent in the polymerization mass is preferably generated in situ, external introduction of the primary and/or secondary expanding agent may also be employed, for example injection.", "The flexible expanded polyurethanes obtained according to the present process suitably have a density of 25 to 50 Kg/m3 or lower at core, and a bearing capacity (as per ISO 2439 norm) greater than 40 N and preferably 80 to 400 N. These polyurethanes advantageously do not exhibit thermo-oxidation degradation phenomena, such as scorching, and also possess excellent mechanical properties, such as elongation at breakage, permanent deformation, compression resistance and air permeability.", "Due to these characteristics, the foams derived from the subject invention may beneficially be used in various fields including in the furniture and/or decorating sector and the transportation and/or automotive industries which typically require materials with the above-mentioned properties.", "The invention is illustrated by the following non-limiting examples.", "EXAMPLE 1 An isocyanate compound is prepared by reacting 42 parts by weight of a mixture of 4,4′-MDI/2,4′-MDI in the ratio of 80/20; 14.0 parts by weight of a mixture of 4,4′-MDI/2,4′-MDI in the ratio of 50/50, with an ethylene oxide-based polyether polyol and propylene oxide with an average molecular weight of 2500 in which the EO/PO ratio is 75/25 (EniChem's Nixolen VS 40).", "At the end of the reaction performed at 70° C. for approximately 2 hours, a prepolymer is obtained with a 30.1 percentage of free NCO.", "40 parts by weight of Polymeric MDI (TEDIMON 31) are then added to the prepolymer until a 30.5 percentage of free NCO is obtained.", "EXAMPLE 2 An isocyanate compound is prepared by reacting 55 parts by weight of a 4,4′-MDI/2,4′-MDI mixture in the ratio of 80/20; 8 parts by weight of a 4,4′-MDI/2,4′-MDI mixture in the ratio of 50/50, with Nixolen VS 40 and an ethylene oxide-based polyether polyol with an average molecular weight of 600 (Enichem's Priowax 600).", "At the end of the reaction, performed at 70° C. for approximately 2 hours a prepolymer is obtained to which Polymeric MDI (TEDIMON 31) is then added until a percentage of 30.4 free NCO is obtained.", "EXAMPLE 3 An isocyanate compound is prepared by reacting 50 parts by weight of a 4,4′-MDI/2,4′-MDI mixture in 80/20 ratio; 10 parts by weight of a 4,4′-MDI/2,4′-MDI mixture in a 50/50 ratio, 10 parts of uretonimine modified MDI (TEDIMON 318 by the Applicant) with an ethylene oxide-based polyether polyol and propylene oxide with an average molecular weight of 4000 in which the EO/PO ratio is 20/80 (TERCAROL 838).", "At the end of the reaction, performed at 70° C. for approximately 2 hours, a prepolymer is obtained with a 29.9 percentage of free NCO.", "Polymeric MDI (TEDIMON 31) is then added to the prepolymer until a 30.5 percentage of free NCO is obtained.", "EXAMPLES 4 to 6 The compounds in Examples 1 to 3 were used for the preparation of flexible expanded polyurethanes combined with the polyol components listed in the Table below.", "The same Table shows the physico-mechanical properties of the foams thus obtained.", "The dynamic fatigue properties of the foams tested in Examples 4 to 6 were measured according to Peugeot test method D42.1047-84.The 50% permanent deformation data, (compression set) was measured according to ISO 1856-80.The compression resistance data or compression force deflection was measured according to DIN EN ISO 3386-1-98.The bearing capacity or indentation force deflection was measured according to ISO 2439-97 and the foam density was measured according to DIN EN ISO 845-95.TABLE Example 4 5 6 Tercarol 427 100 100 100 Tercarol 241 1.5 1.5 1.5 XD7436 2.0 2.0 2.0 DEOA 0.5 0.5 0.5 Water, pp 3.7 3.7 3.7 NIAX A 107, pp 0.2 0.2 0.2 NIAX A 310, pp 0.2 0.2 0.2 POLYCAT 77, pp 0.15 0.15 0.15 NIAX L 3410, pp 1.0 1.0 1.0 ISOCYANATE Example 1 95 — — (index) ISOCYANATE Example 2 — 95 — (index) ISOCYANATE Example 3 — — 95 (index) Density, kg/m3 43 44.5 45 Perm.", "Deformation 50%, 4.5 4.8 5.5 % (compression set) Comp resistance.", "40%, 7.5 7.2 6.4 kPa Bearing capacity 40%, N 320 294 233 Dynamic Fatigue % Thickness loss 2.1 2.2 1.9 % Compression 11.5 13.3 15.2 resistance loss TERCAROL ® 241 - Polyether polyol PM 4000 with functionality = 3 TERCAROL ® 427 - Polyether polyol PM 6000 with functionality = 3 XD 7436 - crosslinker NIAX A 107 - Witco Corporation aminated Catalyst NIAX A 310 - Witco Corporation aminated Catalyst NIAX L 3410 - Witco Corporation silicone Tensoactive POLYCAT 77 - Air Product aminated Catalyst The polyurethanes of Examples 4 to 6 were produced using isocyanate compositions in which the prepolymer was produced using a diol, that is having a functionality of 2.Expanded polyurethane foams produced from an isocyanate composition having a diol component in which there is essentially no cross linking provides excellent elongation properties but surprisingly, as the data in the Table illustrates, also provides excellent compression set and dynamic fatigue properties when measured under the stringent Peugeot Dynamic Fatigue test (200,000 cycles between 25 and 75% deflection at 3 Hz.", "Measurements 30 minutes after fatigue completion)." ] ]	Patent_10468556
[ [ "Optical pickup apparatus", "An optical pickup apparatus for detecting a symmetric S-shaped signal to obtain a focus-error signal by an SSD method for stability in operation is provided.", "In an optical pickup apparatus for irradiating laser light onto an optical recording medium and for directing the light reflected from at least the optical recording medium to a light-receiving device section through a diffraction element to detect a focus-error signal by a spot size detection method using diffracted light caused by the diffraction element, the position of the light-receiving device section is set to the position offset closer to the diffraction element from the focal position of the 0 order light passing through the diffraction element." ], [ "1.An optical pickup apparatus which irradiates laser light onto an optical recording medium and directs light reflected from at least the optical recording medium to a light-receiving device section through a diffraction element and detects a focus-error signal by a spot size detection method using diffracted light caused by the diffraction element, wherein the position of the light-receiving device section is set to a position offset closer to the diffraction element from a focal position of 0 order light passing through the diffraction element.", "2.An optical pickup apparatus according to claim 1, wherein the position of the light-receiving device section is offset closer to the diffraction element from the focal position of the 0 order light passing through the diffraction element as long as the following relation is substantially satisfied: (NA[+1]/NA[−1])<(D2/D1)≦(NA[+1]/NA[−1])2 where NA[+1] indicates a numerical aperture of +1 order light diffracted by the diffraction element; NA[−1] indicates a numerical aperture of −1 order light diffracted by the diffraction element; D1 indicates a distance from a focal position of the +1 order light diffracted by the diffraction element to the light-receiving device section; and D2 indicates a distance from a focal position of the −1 order light diffracted by the diffraction element to the light-receiving device section.", "3.An optical pickup apparatus according to claim 1, wherein the position of the light-receiving device section is offset closer to the diffraction element from the focal position of the 0 order light passing through the diffraction element so that the following relation is substantially satisfied: (D2/D1)=(NA[+1]/NA[−1])2 where NA[+1] indicates a numerical aperture of +1 order light diffracted by the diffraction element; NA[−1] indicates a numerical aperture of −1 order light diffracted by the diffraction element; D1 indicates a distance from a focal position of the +1 order light diffracted by the diffraction element to the light-receiving device section; and D2 indicates a distance from a focal position of the −1 order light diffracted by the diffraction element to the light-receiving device section.", "4.An optical pickup apparatus according to claim 1, wherein the light-receiving device section includes a light-receiving device corresponding to +1 order light diffracted by the diffraction element, and a light-receiving device corresponding to −1 order light diffracted by the diffraction element, each light receiving device being divided into three or five light-receiving regions, and a width of a center light-receiving region of the light-receiving device corresponding to the +1 order diffracted light is different in size from a width of a center light-receiving region of the light-receiving device corresponding to the −1 order diffracted light in order so as to compensate for a deviation between a focal position on the optical recording medium and a position of the an origin of the focus-error signal.", "5.An optical pickup apparatus according to claim 4, wherein a ratio of the width (s1) of the center light-receiving region of the light-receiving device corresponding to the +1 order diffracted light to the width (s2) of the center light-receiving region of the light-receiving device corresponding to the −1 order diffracted light is substantially set as follows: s1:s2=(D1/NA[−1]):(D2/NA[+1]) where NA[+1] indicates a numerical aperture of the +1 order light diffracted by the diffraction element; NA[−1] indicates a numerical aperture of the −1 order light diffracted by the diffraction element; D1 indicates a distance from a focal position of the +1 order light diffracted by the diffraction element to the light-receiving device section; and D2 indicates a distance from a focal position of the −1 order light diffracted by the diffraction element to the light-receiving device section.", "6.An optical pickup apparatus according to claim 4, wherein a ratio of the width (s1) of the center light-receiving region of the light-receiving device corresponding to the +1 order diffracted light to the width (s2) of the center light-receiving region of the light-receiving device corresponding to the −1 order diffracted light is substantially set as follows: s1:s2=NA[−1]:NA[+1] where NA[+1] indicates a numerical aperture of the +1 order light diffracted by the diffraction element; and NA[−1] indicates a numerical aperture of the −1 order light diffracted by the diffraction element." ], [ "<SOH> BACKGROUND ART <EOH>Optical pickup apparatuses for detecting a focus-error signal by a spot size detection method (SSD method) using diffracted light caused by a diffraction element are known.", "Such an optical pickup apparatus is described with reference to FIGS.", "6 through 8 .", "FIG.", "6 illustrates an example structure of an optical pickup apparatus for detecting a focus-error signal by an SSD method.", "A laser beam emitted from a laser light source 21 such as a laser diode reaches an objective lens 23 through a beam splitter 22 , and is irradiated via the objective lens 23 onto an information recording surface of an optical recording medium 10 such as an optical disc.", "The light reflected from the optical recording medium 10 returns to the beam splitter 22 through the objective lens 23 , the optical path thereof being refracted by the beam splitter 22 , and is directed to a diffraction element 24 .", "The reflected light is divided by the diffraction element 24 into the 0 order light passing therethrough, and +1 order light (diffracted light) and −1 order light (diffracted light) diffracted by the diffraction element 24 .", "The 0 order light, the +1 order light, and the −1 order light reach a light-receiving device section 25 .", "In the light-receiving device section 25 , for example, light-receiving patterns shown in FIG.", "8 are formed.", "A light-receiving device 31 has a light-receiving region E corresponding to the 0 order light.", "A light-receiving device 32 has three divided light-receiving regions A, S 1 , and B, and corresponds to the +1 order light.", "A light-receiving device 33 also has three divided light-receiving regions C, S 2 , and D, and corresponds to the −1 order light.", "Each of the light-receiving regions E, A, S 1 , B, C, S 2 , and D of the light-receiving devices 31 , 32 , and 33 outputs an electrical signal having a current level corresponding to the light intensity of the incident light.", "The electrical signal output from each of the light-receiving devices 31 , 32 , and 33 is supplied to a matrix amp (not shown) for processing, such as current-to-voltage conversion, amplification, and matrix calculation, thereby generating a required signal.", "That is, a playback signal, focus-error signal, tracking error signal, etc., corresponding to the information recorded in the optical recording medium 10 are generated.", "The objective lens 23 is held by a two-axis mechanism (not shown) having a focus coil and a tracking coil so as to be displaceable in the near-and-apart direction with respect to the optical recording medium 10 (focusing direction) and in the direction transverse to the track orientation of the optical recording medium (tracking direction).", "A focus drive signal is generated by a servo circuit (not shown) based on the focus-error signal to drive the focus coil of the two-axis mechanism, so that the objective lens 23 is driven in the focusing direction so as to be focused with respect to the optical recording medium 10 .", "A tracking drive signal is further generated by the servo circuit based on the tracking error signal to drive the tracking coil of the two-axis mechanism, so that the objective lens 23 is driven in the tracking direction so as to track with respect to the optical recording medium 10 .", "In the SSD method, the focus-error signal is generated according to the spot size of the diffracted light.", "In the focused state shown in FIG.", "8 ( a ), the spot size of the +1 order light incident on the light-receiving device 32 is equivalent to the spot size of the −1 order light incident on the light-receiving device 33 .", "On the other hand, in the defocused state where the objective lens 23 is too close to or too far from the optical recording medium 10 , as shown in FIGS.", "8 ( b ) and 8 ( c ), the spot size of the +1 order light incident on the light-receiving device 32 is different from the spot size of the −1 order light incident on the light-receiving device 33 .", "Accordingly, by comparing the spot sizes on the light-receiving devices 32 and 33 , the focus-error signal can be generated.", "More specifically, the focus-error signal is generated by, in the subsequent matrix amp, calculating (A+B+S 2 )−(C+D+S 1 ) on the outputs of the light-receiving regions A, S 1 , B, C, S 2 , and D. In general, when the objective lens 23 moves from the position most distant from the optical recording medium 10 to the position closest thereto, as known in the art, in the focus-error signal, a so-called S-shaped curve shown in FIG.", "7 is observed in the vicinity of the focused position.", "A substantially linear region from peak P 1 to peak P 2 in the curve corresponds to a so-called in-focus region.", "In basic operation, when the objective lens 23 is positioned within the in-focus region, a focus servo controls the position of the objective lens 23 to be brought to the position of the origin of the S-shaped curve (i.e., the position where focus error=0) based on the focus-error signal.", "As shown in FIG.", "7 , it is assumed herein that the distance of the in-focus region of the S-shaped signal is indicated by d. In other words, “d” is defined as the displacement distance of the optical recording medium (the distance by which the optical recording medium changes with respect to the position of the objective lens) when the S-shaped signal varies from the peak P 1 to the peak P 2 .", "Furthermore, one-side in-focus regions d 1 and d 2 of the S-shaped curve with respect to the origin of the S-shaped curve are defined as the displacement distances of the optical recording medium when the S-shaped signal goes from the origin of the S-shaped curve to the peaks P 1 and P 2 of the S-shaped curve, respectively.", "Then, the following equation holds true: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "d=d 1 +d 2 Formula (1) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "The origin of the S-shaped curve coincides with the focal position on the optical recording medium.", "This relationship is established, in the standard SSD method, when the diffracted light (the +1 order light and the −1 order light) diffracted by the diffraction element 24 has the same spot diameter r, as shown in FIG.", "8 ( a ), resulting in substantial coincidence with the focal position of the 0 order light (strictly speaking, however, it is shifted towards the diffraction element 24 by L·cos θ, where θ denotes the angle of diffraction and L denotes the distance between the diffraction element 24 and the light-receiving device section 25 ).", "It is assumed herein that the NA (numerical aperture) of the objective lens 23 is indicated by NA[L].", "It is further assumed that the NA of the 0 order light in the light focused at the light-receiving device section 25 which passes through the diffraction element 24 is indicated by NA[0].", "It is still further assumed that the NAs of the +1 order light and the −1 order light diffracted by the diffraction element 24 are indicated by NA[+1]′ and NA[−1]′, respectively.", "It is also assumed that the NAs are so small that the following approximation applies: NA=sin θ=tan θ=θ.", "Then, the following relationship is obtained: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "NA[−1]′<NA[+1]′ Formula (2) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "Thus, the following relationship holds true: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "NA[− 1 ]′+NA[+ 1]′=2 ·NA[L] Formula (3) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "As shown in FIG.", "6 , the distances from the position of the light-receiving device section 25 (the focal position of the 0 order light) to the focal positions of the diffracted light (the +1 order light and the −1 order light) diffracted by the diffraction element 24 are indicated by D11 and D12, respectively.", "Then, the above-noted one-side in-focus regions d 1 and d 2 of the S-shaped curve can be approximated as follows: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "d 1={(½)· D 11·( NA[+ 1]′) 2 }/( NA[L ]) 2 Formula (4) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "d 2={(½)· D 12·( NA[− 1]′) 2 }/( NA[L ]) 2 Formula (5) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "Since the spot diameters r of the diffracted light on the light-receiving devices 32 and 33 on the origin of the S-shaped curve are the same, the following equation is obtained: r ⁢ / ⁢ 2 = ⁢ D11 · NA ⁡ [ + 1 ] ′ = ⁢ D12 · NA ⁡ [ - 1 ] ′ Formula ⁢ ⁢ ( 6 ) Therefore, the following relationship holds true from Formula (6): in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "D 11 /D 12 =NA[− 1 ]′/NA[+ 1]′ Formula (7) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "If NA[0]=NA[+1]′=NA[−1]′ can be approximated, D11 is equal to D12, and the following equation is obtained from Formulas (4) and (5): in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "d 1 /d 2=1 Formula (8) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "Thus, the following relationship is obtained between the diffracted light (the +1 order light and the −1 order light) and the 0 order light: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "NA[+ 1 ]′=L /( L−D 11)· NA[ 0] Formula (9) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "NA[− 1 ]′=L /( L+D 12)· NA[ 0] Formula (10) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "If the distance L between the diffraction element 24 and the light-receiving device section 25 is sufficiently large, or if the distances D11 and D12 are sufficiently small, Formula (8) holds true.", "If the above-noted approximation does not apply, however, the relationship NA[0]=NA[+1]′=NA[−1]′ does not hold true, and Formula (11) rather than Formula (8) is obtained: d1 / d2 = ⁢ ( NA ⁡ [ + 1 ] ′ ) 2 / ( NA ⁡ [ - 1 ] ′ ) 2 · ( D11 ⁢ / ⁢ D12 ) = ⁢ NA ⁡ [ + 1 ] ′ / NA ⁡ [ - 1 ] ′ Formula ⁢ ⁢ ( 11 ) In this case, an asymmetric in-focus region of the S-shaped curve is exhibited.", "That is, the in-focus region shown in FIG.", "7 is exhibited.", "An asymmetric S-shaped curve means instability in gain of a focus servo signal or an asymmetric focus margin, and is disadvantageous in view of the stability in recording to and playback from an optical recording medium.", "In a device supporting a high-density recording medium, the objective lens 23 has a high NA.", "In order to accomplish the same in-focus region d of the S-shaped curve as that described above, as is understood from Formulas 4 and 5, the focal change distance D11 (D12) with respect to the diffraction element 24 must increase as the numerical aperture (NA[L]) of the objective lens 23 increases.", "This further results in a greater amount of change in the NA of the diffracted light diffracted by the diffraction element 24 than that of the 0 order light, as is given by Formulas 9 and 10.In an optical pickup apparatus which includes the objective lens 23 having a high NA, therefore, a more asymmetric in-focus region of the S-shaped curve is exhibited.", "Furthermore, desirably, the distance L from the diffraction element 24 to the light-receiving device section 25 should be reduced in order to reduce the size of the optical pickup apparatus.", "However, as is understood from Formulas 9 and 10, as the distance L from the diffraction element 24 to the light-receiving device section 25 becomes shorter, the amount of change in the NA of the diffracted light diffracted by the diffraction element 24 becomes greater than that of the 0 order light.", "Thus, this case also results in a more asymmetric in-focus region of the S-shaped curve.", "As described above, an optical pickup apparatus which obtains a focus-error signal by the SSD method using a diffraction element has a problem of such an asymmetric in-focus region of the S-shaped curve.", "A noticeably asymmetric in-focus region of the S-shaped curve is exhibited, resulting in a large problem, particularly in an optical pickup apparatus which includes the objective lens 23 having a high NA and in which the distance L from the diffraction element 24 to the light-receiving device section 25 is small, that is, a compact optical pickup apparatus used for high-density optical recording media." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 is a diagram illustrating the structure of an optical pickup apparatus according to an embodiment of the present invention.", "FIG.", "2 is a diagram illustrating a focus-error signal obtained by the optical pickup apparatus of the embodiment.", "FIG.", "3 is a diagram illustrating a light-receiving device in a light-receiving device section of the optical pickup apparatus of the embodiment.", "FIG.", "4 is a diagram illustrating a modification of the light-receiving device section of the embodiment.", "FIG.", "5 is a diagram illustrating a modification of the light-receiving device section of the embodiment.", "FIG.", "6 is a diagram illustrating the structure of an optical pickup apparatus of the related art.", "FIG.", "7 is a diagram illustrating a focus-error signal obtained by the optical pickup apparatus of the related art.", "FIG.", "8 is a diagram illustrating a light-receiving device in a light-receiving device section of the optical pickup apparatus of the related art.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to an optical pickup apparatus for detecting a focus-error signal by a spot size detection (SSD) method using diffracted light caused by a diffraction element.", "BACKGROUND ART Optical pickup apparatuses for detecting a focus-error signal by a spot size detection method (SSD method) using diffracted light caused by a diffraction element are known.", "Such an optical pickup apparatus is described with reference to FIGS.", "6 through 8.FIG.", "6 illustrates an example structure of an optical pickup apparatus for detecting a focus-error signal by an SSD method.", "A laser beam emitted from a laser light source 21 such as a laser diode reaches an objective lens 23 through a beam splitter 22, and is irradiated via the objective lens 23 onto an information recording surface of an optical recording medium 10 such as an optical disc.", "The light reflected from the optical recording medium 10 returns to the beam splitter 22 through the objective lens 23, the optical path thereof being refracted by the beam splitter 22, and is directed to a diffraction element 24.The reflected light is divided by the diffraction element 24 into the 0 order light passing therethrough, and +1 order light (diffracted light) and −1 order light (diffracted light) diffracted by the diffraction element 24.The 0 order light, the +1 order light, and the −1 order light reach a light-receiving device section 25.In the light-receiving device section 25, for example, light-receiving patterns shown in FIG.", "8 are formed.", "A light-receiving device 31 has a light-receiving region E corresponding to the 0 order light.", "A light-receiving device 32 has three divided light-receiving regions A, S1, and B, and corresponds to the +1 order light.", "A light-receiving device 33 also has three divided light-receiving regions C, S2, and D, and corresponds to the −1 order light.", "Each of the light-receiving regions E, A, S1, B, C, S2, and D of the light-receiving devices 31, 32, and 33 outputs an electrical signal having a current level corresponding to the light intensity of the incident light.", "The electrical signal output from each of the light-receiving devices 31, 32, and 33 is supplied to a matrix amp (not shown) for processing, such as current-to-voltage conversion, amplification, and matrix calculation, thereby generating a required signal.", "That is, a playback signal, focus-error signal, tracking error signal, etc., corresponding to the information recorded in the optical recording medium 10 are generated.", "The objective lens 23 is held by a two-axis mechanism (not shown) having a focus coil and a tracking coil so as to be displaceable in the near-and-apart direction with respect to the optical recording medium 10 (focusing direction) and in the direction transverse to the track orientation of the optical recording medium (tracking direction).", "A focus drive signal is generated by a servo circuit (not shown) based on the focus-error signal to drive the focus coil of the two-axis mechanism, so that the objective lens 23 is driven in the focusing direction so as to be focused with respect to the optical recording medium 10.A tracking drive signal is further generated by the servo circuit based on the tracking error signal to drive the tracking coil of the two-axis mechanism, so that the objective lens 23 is driven in the tracking direction so as to track with respect to the optical recording medium 10.In the SSD method, the focus-error signal is generated according to the spot size of the diffracted light.", "In the focused state shown in FIG.", "8(a), the spot size of the +1 order light incident on the light-receiving device 32 is equivalent to the spot size of the −1 order light incident on the light-receiving device 33.On the other hand, in the defocused state where the objective lens 23 is too close to or too far from the optical recording medium 10, as shown in FIGS.", "8(b) and 8(c), the spot size of the +1 order light incident on the light-receiving device 32 is different from the spot size of the −1 order light incident on the light-receiving device 33.Accordingly, by comparing the spot sizes on the light-receiving devices 32 and 33, the focus-error signal can be generated.", "More specifically, the focus-error signal is generated by, in the subsequent matrix amp, calculating (A+B+S2)−(C+D+S1) on the outputs of the light-receiving regions A, S1, B, C, S2, and D. In general, when the objective lens 23 moves from the position most distant from the optical recording medium 10 to the position closest thereto, as known in the art, in the focus-error signal, a so-called S-shaped curve shown in FIG.", "7 is observed in the vicinity of the focused position.", "A substantially linear region from peak P1 to peak P2 in the curve corresponds to a so-called in-focus region.", "In basic operation, when the objective lens 23 is positioned within the in-focus region, a focus servo controls the position of the objective lens 23 to be brought to the position of the origin of the S-shaped curve (i.e., the position where focus error=0) based on the focus-error signal.", "As shown in FIG.", "7, it is assumed herein that the distance of the in-focus region of the S-shaped signal is indicated by d. In other words, “d” is defined as the displacement distance of the optical recording medium (the distance by which the optical recording medium changes with respect to the position of the objective lens) when the S-shaped signal varies from the peak P1 to the peak P2.Furthermore, one-side in-focus regions d1 and d2 of the S-shaped curve with respect to the origin of the S-shaped curve are defined as the displacement distances of the optical recording medium when the S-shaped signal goes from the origin of the S-shaped curve to the peaks P1 and P2 of the S-shaped curve, respectively.", "Then, the following equation holds true: d=d1+d2 Formula (1) The origin of the S-shaped curve coincides with the focal position on the optical recording medium.", "This relationship is established, in the standard SSD method, when the diffracted light (the +1 order light and the −1 order light) diffracted by the diffraction element 24 has the same spot diameter r, as shown in FIG.", "8(a), resulting in substantial coincidence with the focal position of the 0 order light (strictly speaking, however, it is shifted towards the diffraction element 24 by L·cos θ, where θ denotes the angle of diffraction and L denotes the distance between the diffraction element 24 and the light-receiving device section 25).", "It is assumed herein that the NA (numerical aperture) of the objective lens 23 is indicated by NA[L].", "It is further assumed that the NA of the 0 order light in the light focused at the light-receiving device section 25 which passes through the diffraction element 24 is indicated by NA[0].", "It is still further assumed that the NAs of the +1 order light and the −1 order light diffracted by the diffraction element 24 are indicated by NA[+1]′ and NA[−1]′, respectively.", "It is also assumed that the NAs are so small that the following approximation applies: NA=sin θ=tan θ=θ.", "Then, the following relationship is obtained: NA[−1]′<NA[+1]′ Formula (2) Thus, the following relationship holds true: NA[−1]′+NA[+1]′=2·NA[L] Formula (3) As shown in FIG.", "6, the distances from the position of the light-receiving device section 25 (the focal position of the 0 order light) to the focal positions of the diffracted light (the +1 order light and the −1 order light) diffracted by the diffraction element 24 are indicated by D11 and D12, respectively.", "Then, the above-noted one-side in-focus regions d1 and d2 of the S-shaped curve can be approximated as follows: d1={(½)·D11·(NA[+1]′)2}/(NA[L])2 Formula (4) d2={(½)·D12·(NA[−1]′)2}/(NA[L])2 Formula (5) Since the spot diameters r of the diffracted light on the light-receiving devices 32 and 33 on the origin of the S-shaped curve are the same, the following equation is obtained: r ⁢ / ⁢ 2 = ⁢ D11 · NA ⁡ [ + 1 ] ′ = ⁢ D12 · NA ⁡ [ - 1 ] ′ Formula ⁢ ⁢ ( 6 ) Therefore, the following relationship holds true from Formula (6): D11/D12=NA[−1]′/NA[+1]′ Formula (7) If NA[0]=NA[+1]′=NA[−1]′ can be approximated, D11 is equal to D12, and the following equation is obtained from Formulas (4) and (5): d1/d2=1 Formula (8) Thus, the following relationship is obtained between the diffracted light (the +1 order light and the −1 order light) and the 0 order light: NA[+1]′=L/(L−D11)·NA[0] Formula (9) NA[−1]′=L/(L+D12)·NA[0] Formula (10) If the distance L between the diffraction element 24 and the light-receiving device section 25 is sufficiently large, or if the distances D11 and D12 are sufficiently small, Formula (8) holds true.", "If the above-noted approximation does not apply, however, the relationship NA[0]=NA[+1]′=NA[−1]′ does not hold true, and Formula (11) rather than Formula (8) is obtained: d1 / d2 = ⁢ ( NA ⁡ [ + 1 ] ′ ) 2 / ( NA ⁡ [ - 1 ] ′ ) 2 · ( D11 ⁢ / ⁢ D12 ) = ⁢ NA ⁡ [ + 1 ] ′ / NA ⁡ [ - 1 ] ′ Formula ⁢ ⁢ ( 11 ) In this case, an asymmetric in-focus region of the S-shaped curve is exhibited.", "That is, the in-focus region shown in FIG.", "7 is exhibited.", "An asymmetric S-shaped curve means instability in gain of a focus servo signal or an asymmetric focus margin, and is disadvantageous in view of the stability in recording to and playback from an optical recording medium.", "In a device supporting a high-density recording medium, the objective lens 23 has a high NA.", "In order to accomplish the same in-focus region d of the S-shaped curve as that described above, as is understood from Formulas 4 and 5, the focal change distance D11 (D12) with respect to the diffraction element 24 must increase as the numerical aperture (NA[L]) of the objective lens 23 increases.", "This further results in a greater amount of change in the NA of the diffracted light diffracted by the diffraction element 24 than that of the 0 order light, as is given by Formulas 9 and 10.In an optical pickup apparatus which includes the objective lens 23 having a high NA, therefore, a more asymmetric in-focus region of the S-shaped curve is exhibited.", "Furthermore, desirably, the distance L from the diffraction element 24 to the light-receiving device section 25 should be reduced in order to reduce the size of the optical pickup apparatus.", "However, as is understood from Formulas 9 and 10, as the distance L from the diffraction element 24 to the light-receiving device section 25 becomes shorter, the amount of change in the NA of the diffracted light diffracted by the diffraction element 24 becomes greater than that of the 0 order light.", "Thus, this case also results in a more asymmetric in-focus region of the S-shaped curve.", "As described above, an optical pickup apparatus which obtains a focus-error signal by the SSD method using a diffraction element has a problem of such an asymmetric in-focus region of the S-shaped curve.", "A noticeably asymmetric in-focus region of the S-shaped curve is exhibited, resulting in a large problem, particularly in an optical pickup apparatus which includes the objective lens 23 having a high NA and in which the distance L from the diffraction element 24 to the light-receiving device section 25 is small, that is, a compact optical pickup apparatus used for high-density optical recording media.", "DISCLOSURE OF INVENTION In view of such a problem, an object of the present invention is to provide an optical pickup apparatus which obtains a focus-error signal by the SSD method, in which a focus-error signal exhibiting a symmetric S-shaped curve can be detected.", "To this end, according to the present invention, in an optical pickup apparatus for irradiating laser light onto an optical recording medium and for directing the light reflected from at least the optical recording medium to a light-receiving device section through a diffraction element to detect a focus-error signal by a spot size detection method using diffracted light caused by the diffraction element, the position of the light-receiving device section is set to the position offset closer to the diffraction element from the focal position of the 0 order light passing through the diffraction element.", "Particularly, the position of the light-receiving device section is offset closer to the diffraction element from the focal position of the 0 order light passing through the diffraction element as long as the following relation is satisfied: (NA[+1]/NA[−1])<(D2/D1)<(NA[+1]/NA[−1])2 Alternatively, the position of the light-receiving device section is offset closer to the diffraction element from the focusing position of the 0 order light passing through the diffraction element so that the following relation is substantially satisfied: (D2/D1)=(NA[+1]/NA[−1])2 In these cases, NA[+1], NA[−1], D1, and D2 are defined as follows: NA[+1] indicates the numerical aperture of the +1 order light diffracted by the diffraction element; NA[−1] indicates the numerical aperture of the −1 order light diffracted by the diffraction element; D1 indicates the distance from the focal position of the +1 order light diffracted by the diffraction element to the light-receiving device section; and D2 indicates the distance from the focal position of the −1 order light diffracted by the diffraction element to the light-receiving device section.", "The light-receiving device section includes a light-receiving device corresponding to the +1 order diffracted light, and a light-receiving device corresponding to the −1 order diffracted light, each being divided into three or five light-receiving regions, and the width of the center light-receiving region of the light-receiving device corresponding to the +1 order diffracted light is different in size from the width of the center light-receiving region of the light-receiving device corresponding to the −1 order diffracted light in order to compensate for a deviation between the focal position on the optical recording medium and the position of the origin of the focus-error signal.", "Particularly, the ratio of the width (s1) of the center light-receiving region of the light-receiving device corresponding to the +1 order diffracted light to the width (s2) of the center light-receiving region of the light-receiving device corresponding to the −1 order diffracted light is substantially set as follows: s1:s2=(D1/NA[−1]):(D2/NA[+1]) Alternatively, the ratio of the width (s1) of the center light-receiving region of the light-receiving device corresponding to the +1 order diffracted light to the width (s2) of the center light-receiving region of the light-receiving device corresponding to the −1 order diffracted light is substantially set as follows: s1:s2=NA[−1]:NA[+1] According to the present invention, therefore, the position of the light-receiving device section is offset closer to the diffraction element from the focal position of the 0 order light passing through the diffracted light, thereby correcting for an asymmetric shape of the focus-error signal (S-shaped signal).", "Furthermore, a deviation between the focal position with respect to the optical recording medium and the origin of the S-shaped curve of the focus-error signal when the light-receiving device section is offset is overcome by making the width of the center light-receiving region of three or five divided light-receiving regions of the light-receiving device corresponding to the +1 order diffracted light different in size from that of the light-receiving device corresponding to the −1 order diffracted light.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a diagram illustrating the structure of an optical pickup apparatus according to an embodiment of the present invention.", "FIG.", "2 is a diagram illustrating a focus-error signal obtained by the optical pickup apparatus of the embodiment.", "FIG.", "3 is a diagram illustrating a light-receiving device in a light-receiving device section of the optical pickup apparatus of the embodiment.", "FIG.", "4 is a diagram illustrating a modification of the light-receiving device section of the embodiment.", "FIG.", "5 is a diagram illustrating a modification of the light-receiving device section of the embodiment.", "FIG.", "6 is a diagram illustrating the structure of an optical pickup apparatus of the related art.", "FIG.", "7 is a diagram illustrating a focus-error signal obtained by the optical pickup apparatus of the related art.", "FIG.", "8 is a diagram illustrating a light-receiving device in a light-receiving device section of the optical pickup apparatus of the related art.", "BEST MODE FOR CARRYING OUT THE INVENTION An optical pickup apparatus of an embodiment of the present invention is described below.", "FIG.", "1 illustrates an example structure of an optical pickup apparatus of an embodiment of the present invention for detecting a focus-error signal by an SSD method.", "A laser beam emitted from a laser light source 1 such as a laser diode reaches an objective lens 3 through a beam splitter 2, and is irradiated via the objective lens 3 onto an information recording surface of an optical recording medium 10 such as an optical disc.", "The light reflected from the optical recording medium 10 returns to the beam splitter 2 through the objective lens 3, the optical path thereof being slit by the beam splitter 2, and is directed to a diffraction element 4.The reflected light is divided by the diffraction element 4 into the 0 order light passing therethrough, +1 order light (diffracted light) and −1 order light (diffracted light) diffracted by the diffraction element 4.The 0 order light, the +1 order light, and the −1 order light reach a light-receiving device section 5.Particularly in the optical pickup apparatus of this embodiment, the light-receiving device section 5 is positioned so as to be offset closer to the diffraction element 4 than the related art unit shown in FIG.", "6, as is described below.", "In the light-receiving device section 5, for example, light-receiving patterns shown in FIG.", "3 are formed.", "A light-receiving device 11 has a light-receiving region E corresponding to the 0 order light.", "A light-receiving device 12 has three divided light-receiving regions A, S1, and B, and corresponds to the +1 order light.", "A light-receiving device 13 also has three divided light-receiving regions C, S2, and D, and corresponds to the −1 order light.", "Each of the light-receiving regions E, A, S1, B, C, S2, and D of the light-receiving devices 11, 12, and 13 outputs an electrical signal having a current level corresponding to the light intensity of the incident light.", "The electrical signal output from each of the light-receiving devices 11, 12, and 13 is supplied to a matrix amp (not shown) for processing, such as current-to-voltage conversion, amplification, and matrix calculation, thereby generating a required signal.", "That is, a playback signal, focus-error signal, tracking error signal, etc., corresponding to the information recorded in the optical recording medium 10 are generated.", "The objective lens 3 is held by a two-axis mechanism (not shown) having a focus coil and a tracking coil so as to be displaceable in the near-and-apart direction with respect to the optical recording medium 10 (focusing direction) and in the direction transverse to the track orientation of the optical recording medium (tracking direction).", "A focus drive signal is generated by a servo circuit (not shown) based on the focus-error signal to drive the focus coil of the two-axis mechanism, so that the objective lens 3 is driven in the focusing direction so as to be focused with respect to the optical recording medium 10.A tracking drive signal is further generated by the servo circuit based on the tracking error signal to drive the tracking coil of the two-axis mechanism, so that the objective lens 3 is driven in the tracking direction so as to track with respect to the optical recording medium 10.In the SSD method, the focus-error signal is generated according to the spot size of the diffracted light, and, as is similar to the related art, the focus-error signal is generated by, in the subsequent matrix amp, calculating (A+B+S2)−(C+D+S1) on the outputs of the light-receiving regions A, S1, B, C, S2, and D. That is, the principle is also used in which the spot size of the +1 order light incident on the light-receiving device 12 and the spot size of the −1 order light incident on the light-receiving device 13, which vary depending upon the focusing conditions (focused/defocused), are utilized.", "In this example, however, because of offset of the light-receiving device section 5, the spot size of the +1 order light incident on the light-receiving device 12 is not equivalent to the spot size of the −1 order light incident on the light-receiving device 13 in the focused state.", "Thus, as shown in FIG.", "3, the light-receiving device 12 and the light-receiving device 13 have different sizes.", "More specifically, the light-receiving regions S1 and S2 each constituting the center region of the three divided light-receiving regions are designed so as to have different widths in the division direction.", "This meaning is also described below.", "In this example, the focus-error signal exhibiting an S-shaped curve shown in FIG.", "2 is observed.", "As is understood from comparison between FIG.", "2 and FIG.", "7, in this example, a symmetric S-shaped curve (symmetric S-curve in-focus region) is obtained.", "As is similar to the case shown in FIG.", "7, it is assumed that the distance of the in-focus region of the S-shaped signal is indicated by d. In other words, “d” is defined as the displacement distance of the optical recording medium (the distance by which the optical recording medium changes with respect to the position of the objective lens) when the S-shaped signal varies from the peak P1 to the peak P2.In this example shown in FIG.", "2 where the symmetry of the S-curve in-focus region is improved, one-side in-focus regions of the S-shaped curve with respect to the origin of the S-shaped curve are equivalent, and are indicated by dθ.", "Thus, d0θ=d/2 is obtained.", "A description is made below with terminology defined as follows: NA[+1] indicates the NA of the +1 order light caused by the diffraction element 4; NA[−1] indicates the NA of the −1 order light caused by the diffraction element 4; NA[0] indicates the NA of the 0 order light passing through the diffraction element 4; NA[L] indicates the NA of the objective lens 3; D1 indicates the distance from the focal position of the +1 order light diffracted by the diffraction element 4 to the light-receiving device section 5; D2 indicates the distance from the focal position of the −1 order light diffracted by the diffraction element 4 to the light-receiving device section 5; s1 indicates the width of the center light-receiving region S1 of the light-receiving device 12 corresponding to the +1 order light; s2 indicates the width of the center light-receiving region S2 of the light-receiving device 13 corresponding to the −1 order light; r1 indicates the spot diameter of the +1 order light reaching the light-receiving device 12; and r2 indicates the spot diameter of the −1 order light reaching the light-receiving device 13.In order to exhibit a symmetric S-curve in-focus region, there is a need for design in which each one-side in-focus region d0 of the S-shaped curve is equal to d/2, as shown in FIG.", "2.When the diffracted light (the +1 order light and the −1 order light) is converted by the diffraction element 4 to NA[+1] and NA[−1], respectively, the focal change distances D1 and D2 of the diffracted light required for achieving the one-side in-focus region d0(=d/2) of the S-shaped curve are given by the following formulas: D1=2d0·(NA[L])2/(NA[+1])2} Formula (12) D2=2d0·(NA[L])2/(NA[−1])2} Formula (13) From the above relationship, the region is determined as follows: d0 = ⁢ ( 1 / 2 ) · D1 · ( NA ⁡ [ + 1 ] ) 2 ⁢ ( NA ⁡ [ L ] ) 2 = ⁢ ( 1 / 2 ) · D2 · ( NA ⁡ [ - 1 ] ) 2 ⁢ ( NA ⁡ [ L ] ) 2 ⁣ Formula ⁢ ⁢ ( 14 ) The ratio of the focal change distances D1 to D2 is determined as follows: D1/D2=(NA[−1]/NA[+1])2 Formula (15) In the related art, as described in Formula (7), the focal change distance ratio (D11/D12 ratio) corresponding to the D1/D2 ratio is given by NA[−1]′/NA[+1]′; whereas, in this example, the light-receiving device section 5 is corrected (offset) so as to be closer to the diffraction element 4 from to the focal position of the 0 order light so that the position relationship expressed by Formula (15) is established, thereby improving the symmetry of the S-curve in-focus region.", "Thus, while the position relationship of the related art is expressed by D1/D2=NA[−1]/NA[+1], the position of the light-receiving device section 5 is offset closer to the diffraction element 4 from the original focal position of the 0 order light as long as the following relationship holds true: NA[+1]/NA[−1]<(D2/D1)≦(NA[+1]/NA[−1])2 This allows the one-side S-curve in-focus regions of the S-shaped signal to become closer to d0 and d0 shown in FIG.", "2 than d1 and d2 shown in FIG.", "7.This means that the symmetry of the S-curve in-focus region is improved.", "Since Formula (15), i.e., D1/D2=(NA[−1]/NA[+1])2 is derived based on dθ=d/2, the D1/D2 ratio is substantially equal to the position relationship (NA[−1]/NA[+1])2 (where the light-receiving device section 5 is offset), thereby optimizing the symmetry of the S-curve in-focus region.", "The improvement in symmetry of the S-curve in-focus region achieves stable gain of the focus servo signal or a symmetric focus margin, and therefore achieves stable recording to and playback from the optical recording medium.", "As described above, a noticeably asymmetric S-curve in-focus region is exhibited particularly in an optical pickup apparatus supporting high-density recording media or a compact optical pickup apparatus.", "By offsetting the light-receiving device section 5 for improvement in symmetry, an optical pickup apparatus suitable for such purposes can be realized.", "The spot diameters r1 and r2 of the diffracted light (the +1 order light and the −1 order light) reaching the light-receiving devices 12 and 13 when focused with respect to the optical recording medium 10 are determined as follows, respectively: r1=2·D1·NA[+1] Formula (16) r2=2·D2·NA[−1] Formula (17) In the standard SSD method, the diffracted light (the +1 order light and the −1 order light) diffracted by the diffraction element 4 has the same spot size on the light-receiving devices 12 and 13 when focused with respect to the optical recording medium 10, and the following relationship is obtained: r1/r2=1 Formula (18) Thus, the diffracted light is divided at the same ratio by the light-receiving devices for signal calculation according to the SSD method, so that the origin of the S-shaped curve coincides with the focal position on the optical recording medium 10.In this example, on the other hand, when the optical recording medium 10 is located at the focal position, the spot diameter ratio of the diffracted light diffracted by the diffraction element 4 on the light-receiving devices 12 and 13 is given as follows from Formulas (16) and (17), and (12) and (13): r1 / r2 = ⁢ D1 · NA ⁡ [ + 1 ] ⁢ / ⁢ D2 · NA ⁡ [ - 1 ] = ⁢ NA ⁡ [ - 1 ] ⁢ / ⁢ NA ⁡ [ + 1 ] Formula ⁢ ⁢ ( 19 ) In this case, there occurs a deviation between the focal position on the optical recording medium and the origin of the S-shaped curve if the light is divided on the light-receiving devices 12 and 13 according to the standard method.", "In this example, therefore, as shown in FIG.", "3, the width ratio s1/s2 of the center light-receiving regions S1 and S2 of the light-receiving devices 12 and 13 which the diffracted light reaches is defined as follows: s1/s2=NA[−1]/NA[+1] Formula (20) or s1/s2=D1·NA[+1]/D2·NA[−1] Formula (21) In FIG.", "3, the 0, +1, and −1 order light spots are indicated by round hatched portions on the light-receiving devices 11, 12, and 13, respectively.", "Since the position of the light-receiving device section 5 is offset closer to the diffraction element 4 from the focal position of the 0 order light, in the focused state, the spot sizes of the diffracted light irradiated onto the light-receiving devices 12 and 13 differ from each other, as shown in FIG.", "3.Thus, if the light-receiving devices 32 and 33 shown in FIG.", "8 are used, the focus-error signal obtained by calculating (A+B+S2)−(C+D+S1) is not at the zero level (origin of the S-shaped curve) in the focused state.", "Since the ratio of the division-direction widths s1 to s2 of the center light-receiving regions S1 and S2 of the three divided regions of the light-receiving devices 12 and 13 shown in FIG.", "3 is designed herein so as to substantially satisfy Formula (20) or (21), the focus-error signal obtained by calculating (A+B+S2)−(C+D+S1) is at the zero level (origin of the S-shaped curve) in the focused state.", "In other words, the light intensities of the light detected at the light-receiving regions S1 and S2 are equivalent even if the spot sizes differ from each other.", "By designing the light-receiving devices 12 and 13 in this fashion, the focus-error signal can be obtained by calculating (A+B+S2)−(C+D+S1) according to the standard SSD method although the detected spot sizes of the diffracted light appear to differ from each other in the focused state.", "Thus, there is no change in design of circuits such as a subsequent matrix amp.", "FIG.", "4 shows a modification example of the light-receiving devices of the light-receiving device section 5.This example indicates that the light-receiving device 11 corresponding to the 0 order light is divided into four detector portions.", "As described above, the light-receiving device section 5 is offset closer to the diffraction element 4 from the focal position of the 0 order light.", "This also means that the 0 order light incident on the light-receiving device 11 for the 0 order light has a larger spot size.", "Thus, even in the case where the light-receiving device 11 is a four-division light-receiving device having light-receiving regions E1, E2, E3, and E4, suitable light detection can be performed in the light-receiving regions E1, E2, E3, and E4.Since the light-receiving device 11 is a four-division light-receiving device, a so-called push-pull signal or tracking error signal can be detected by the light-receiving device 11 for the 0 order light.", "The push-pull signal is detected as wobble information when the optical recording medium 10 is, for example, an optical disc having wobbled grooves formed therein.", "In some cases, the tracking error signal may be generated from the push-pull signal.", "Such a push-pull signal is obtained by, for example, calculating (E1+E4)−(E2+E3) on the output of the four-division light-receiving device 11.The four-division light-receiving device 11 can also be used to obtain a tracking error signal by a so-called DPD (Differential Phase Detection).", "In this case, a signal (E1+E3) and a signal (E2+E4) are obtained from the output of the light-receiving device 11.The phase error between the signal (E1+E3) and the signal (E2+E4) is detected to generate a tracking error signal as a value corresponding to the phase error.", "FIG.", "5 shows an example where each of the light-receiving devices 12 and 13 of the light-receiving device section 5 is divided into five detector portions.", "In this case, the light-receiving device 12 has five light-receiving regions F, A, S1, B, and G. The light-receiving device 13 has five light-receiving regions H, D, S2, C, and K. In the SSD method, in some cases, the five-division light-receiving devices 12 and 13 may be used to obtain a focus-error signal.", "In such cases, the focus-error signal is obtained by calculating {(S1+F+G)+C+D}−{(S2+H+K)+A+B} on the outputs of the light-receiving devices 12 and 13.In such cases, also, since the width ratio s1/s2 of the center light-receiving regions S1 and S2 is designed so as to substantially satisfy above-described Formula (20) or (21) according to the offset of the light-receiving device section 5, the focus-error signal obtained by calculating {(S1+F+G)+C+D}−{(S2+H+K)+A+B} is at the zero level (origin of the S-shaped curve) in the focused state.", "Originally, such a five-division detector is used for the purpose of reduction of fake portions F of the focus-error signal shown in FIG.", "2.However, an asymmetric S-shaped curve as exhibited in the related art prevents a reduction of one fake portion.", "In this example, on the other hand, the light-receiving device section 5 is offset so as to exhibit a symmetric S-shaped curve, thereby, advantageously, increasing the fake reduction effect by using the five-division detector.", "The optical pickup apparatus of an embodiment has been described; however, various modifications may be made to the configuration of the optical pickup apparatus, particularly to the type or number of optical devices, etc.", "The optical pickup apparatus of the present invention may be incorporated into a recording device and playback device compatible with various optical recording media such as optical discs, magneto-optical discs, and optical cards.", "Such an optical pickup apparatus is suitable particularly for incorporation into a device supporting high-density optical recording media.", "As is understood from the foregoing description, according to the optical pickup apparatus of the present invention, the light-receiving device section is located at the position offset a predetermined amount closer to the diffraction element from the focal position of the 0 order light, thereby improving the S-shaped signal symmetry of a focus-error signal detected by the SSD method.", "This results in stable gain of the focus-error signal and stable recording and playback operation of the optical recording medium.", "The S-shaped signal having symmetry allows for a symmetric focus margin.", "Thus, the overall focus margin increases, thereby also achieving stable recording and playback operation of the optical recording medium.", "Another advantage is that the distance between the diffraction element and the light-receiving device section can consequently be reduced.", "Therefore, desirably, the size of the optical pickup apparatus is reduced.", "The asymmetry of the S-shaped signal is noticeable particularly in an optical pickup apparatus supporting high-density optical recording media using a high-NA objective lens or a compact optical pickup apparatus.", "However, the asymmetry of the S-shaped signal in such a case can be overcome by the optical pickup apparatus of the present invention, thus, desirably, achieving an optical pickup apparatus for the purpose of high-density recording and playback.", "The width of the center light-receiving region of the light-receiving device corresponding to the +1 order diffracted light is different in size from the width of the center light-receiving region of the light-receiving device corresponding to the −1 order diffracted light in order to compensate for a deviation between the focal position on the optical recording medium and the position of the origin of the focus-error signal.", "There arises still another advantage in that a deviation caused between the focal position with respect to the optical recording medium and the origin of the S-shaped curve of the focus-error signal even when the light-receiving device section is offset can be overcome, and the focus-error signal can be obtained using a similar calculation method to the standard method, whereby there is no need for a configuration modification of a subsequent calculation circuit." ] ]	Patent_10468588
[ [ "Blaster lights used for signalling and/or marking purposes", "Blister lights are used for signaling and/or marking purposes.", "The blister lights include a housing which is embedded in a traffic area, e.g.", "a street, an airport runway for taking off and for landing, or the like.", "It further includes a housing cover by which the housing embedded in the traffic area can be closed on its upper side, the cover being detachably connected to the housing and including at least one light outlet.", "It additionally includes an illuminating element which is arranged in the housing and by which the light can radiate through the light outlet of the housing cover.", "In order to enable the illuminating element to have higher current densities and thus to enable the blister lights to have a higher light output capacity, a thermoconducting bridge is formed between illuminating element and the housing cover.", "By this, the thermal energy produced by the illuminating element can be conducted to the housing cover From these, the thermal energy is guided to the traffic area via the housing which is embedded therein." ], [ "1.A blister light, for at least one of signaling and marking purposes, comprising: a housing, embeddable in a traffic area; a housing cover, by which the housing is closable at its upper side, the cover being detachably connected to the housing and including at least one light exit opening; and a luminous device, arranged in the housing, adapted to emit light through the light exit opening in the housing cover; and a thermoconducting bridge, formed between the luminous device and the housing cover, adapted to conduct thermal energy produced by the luminous device to the housing cover.", "2.The blister light as claimed in claim 1, wherein the luminous device includes at least one power LED.", "3.The blister light as claimed in claim 1, wherein the luminous device includes at most six power LEDs per light exit opening.", "4.The blister light as claimed in claim 2, wherein a collimator element is assigned to the at least one power LED.", "5.The blister light as claimed in claim 2, wherein the at least one power LED is seated with its carrier plate on a base plate fitted on at least one cover inner part connected to an underside of the housing cover, and forms, with said cover inner part, the thermoconducting bridge to the housing, cover.", "6.The blister light as claimed in claim 5, wherein the base plate is connected to the underside of the housing cover via two cover inner parts, arranged on both sides of the carrier plate.", "7.The blister light as claimed in claim 6, wherein the two cover inner parts are arranged on the end sections of the base plate.", "8.The blister light as claimed in claim 5, wherein each cover inner part bears against the base plate in two dimensions on one side, and against the underside of the housing cover in two dimensions on the other side.", "9.The blister light as claimed in claim 5, wherein the mutually assigned bearing surfaces on the side of the cover inner part and of the base plate, and the mutually assigned bearing surfaces on the side of the cover inner part and of the cover, are formed in a metallically smooth fashion.", "10.The blister light as claimed in claim 1, wherein the mutually assigned bearing surfaces, on the side of the cover and of the housing, are formed in a metallically smooth fashion.", "11.The blister light as claimed in claim 5, wherein the base plate is formed from a material having a high coefficient of thermal conductivity.", "12.The blister light as claimed in claim 5, wherein the carrier plate of the power LED is formed from a material having a high coefficient of thermal conductivity.", "13.The blister light as claimed in claim 5, wherein at least one cover inner part is formed from a material having a high coefficient of thermal conductivity.", "14.The blister light as claimed in claim 3, wherein a collimator element is assigned to at least one power LED.", "15.The blister light as claimed in claim 3, wherein at least one power LED is seated with its carrier plate on a base plate fitted on at least one cover inner part connected to an underside of the housing cover, and forms, with said cover inner part, the thermoconducting bridge to the housing, cover.", "16.The blister light as claimed in claim 4, wherein the at least one power LED is seated with its carrier plate on a base plate fitted on at least one cover inner part connected to an underside of the housing cover, and forms, with said cover inner part, the thermoconducting bridge to the housing, cover.", "17.The blister light as claimed in claim 15, wherein the base plate is connected to the underside of the housing cover via two cover inner parts, arranged on both sides of the carrier plate.", "18.The blister light as claimed in claim 16, wherein the base plate is connected to the underside of the housing cover via two cover inner parts, arranged on both sides of the carrier plate.", "19.The blister light as claimed in claim 6, wherein each cover inner part bears against the base plate in two dimensions on one side, and against the underside of the housing cover in two dimensions on the other side.", "20.The blister light as claimed in claim 7, wherein each cover inner part bears against the base plate in two dimensions on one side, and against the underside of the housing cover in two dimensions on the other side.", "21.The blister light as claimed in claim 6, wherein the mutually assigned bearing surfaces on the side of the cover inner part and of the base plate, and the mutually assigned bearing surfaces on the side of the cover inner part and of the cover, are formed in a metallically smooth fashion.", "22.The blister light as claimed in claim 7, wherein the mutually assigned bearing surfaces on the side of the cover inner part and of the base plate, and the mutually assigned bearing surfaces on the side of the cover inner part and of the cover, are formed in a metallically smooth fashion.", "23.The blister light as claimed in claim 2, wherein the mutually assigned bearing surfaces, on the side of the cover and of the housing, are formed in a metallically smooth fashion.", "24.The blister light as claimed in claim 3, wherein the mutually assigned bearing surfaces, on the side of the cover and of the housing, are formed in a metallically smooth fashion.", "25.The blister light as claimed in claim 5, wherein the base plate is formed from a metal.", "26.The blister light as claimed in claim 5, wherein the carrier plate of the power LED is formed from a metal.", "27.The blister light as claimed in claim 1, wherein the traffic area includes at least one of a road, an airport taxiway, and an airport runway.", "28.The blister light as claimed in claim 1, wherein the thermal energy is dissipatable to the traffic area via the housing, upon the housing being embedded in the traffic area.", "29.A blister light, comprising: a housing, embeddable in a traffic area; a housing cover, detachably connected to the housing and including at least one opening; luminous means, arranged in the housing, for emitting light through an opening in the housing cover; and thermoconducting means, between the luminous means and the housing cover, for conducting thermal energy produced by the luminous means to the housing cover.", "30.The blister light as claimed in claim 29, wherein the thermal energy is dissipatable to the traffic area via the housing, upon the housing being embedded in the traffic area.", "31.The blister light as claimed in claim 29, wherein the luminous means includes at least one power LED.", "32.The blister light as claimed in claim 29, wherein the luminous means includes at most six power LEDs per light exit opening." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>In known blister lights, in which use is also made as luminous device of, inter alia, LEDs (light-emitting diodes), the quantity of heat produced during operation of the luminous device is usually transferred by convection into a gas, which is often air.", "In some known blister lights, the luminous device is assigned a heat sink starting from which the quantity of heat produced during operation of the luminous device is given off to the gas or air by convection.", "Because of the limited spatial conditions prevailing in blister lights, even blister lights equipped with a heat sink with low heat transfer resistance have a total heat transfer resistance of usually more than 30 K/W power loss.", "In the use, known from the prior art, of LEDs mounted on PC plates, a heat transfer resistance of more than 150 K/W results between the barrier layer of the LEDs and the PC plate.", "In some automotive applications, a heat transfer resistance of the order of approximately 70 K/W is achieved.", "These high heat transfer resistances, which accompany maximum temperatures of the barrier layers that are usually of the order of magnitude of a little above 120° C., limit the current density possible and/or permissible during operation of the blister light.", "They thus produce, in a corresponding way, a limitation of the light-emitting power of the LEDs that can be achieved per unit of area of the barrier layer.", "In the case of freely arranged lighting devices, for example traffic lights or the like, this limitation of the emitting power per unit of area is compensated by an increase in the area used for the emission of light and in the number of the light-emitting diodes.", "This enlargement of the light-emitting area is not practicable for blister lights, since blister lights are embedded in the traffic area.", "Further, since the emitting area of a blister light that is available in total for the emission of light is limited by the projection of the blister light above the surface of the traffic area, it is necessary for this projection to be as small as possible owing to a multiplicity of technically and mechanically conditioned requirements placed on the blister light." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of an embodiment of the invention to provide a blister light, in the case of which the light-emitting power can be increased without increasing the light-emitting area, without exceeding permissible barrier surface temperatures of the luminous device or reducing the service life of the luminous device.", "An object may be achieved according to an embodiment of the invention, by virtue of the fact that formed between the luminous device and the housing cover of the blister light is a thermoconducting bridge.", "By this bridge, thermal energy produced by the luminous device can be conducted to the housing cover.", "Starting from this, the thermal energy can be dissipated to the traffic area via the housing embedded in the traffic area.", "Owing to this configuration of the blister light with a thermoconducting bridge connecting the luminous device to the housing cover or the housing, it is possible to optimize the light output power by using a higher current density in the luminous device.", "This occurs since the thermal energy occurring can be given off to the traffic area by creating a thermal dissipation path with a low heat transfer resistance, such that the temperatures at the barrier layers of the luminous device remain in the permissible range, and the service life of the luminous means is not reduced.", "The capacitance of the traffic area for absorbing thermal energy is virtually unlimited, the traffic area having, moreover, a very large surface for emitting heat.", "According to an embodiment of the invention, the traffic area is used like a heat sink of unlimited absorptive capacity by virtue of the fact that the barrier layer of the luminous device is connected to the traffic area via the outlined heat dissipation path with a low heat transfer resistance.", "The heat dissipation from the luminous device, by convection that is substantially less effective by comparison with the outlined heat dissipation path to the traffic area, takes place in the case of the blister light according to an embodiment of the invention only at a negligible order of magnitude.", "Owing to the electric current densities prevailing in the case of the inventive configuration of the blister light, it is particularly expedient when power LEDs, also termed high-intensity LEDs, are used as luminous device.", "The most varied requirements placed on blister lights can be fulfilled by way of such power LEDs, without particular further measures.", "There is a need, moreover, only for a comparatively limited number of power LEDs.", "These emit through a transparent emission window that is implemented by the light exit opening present in the housing cover.", "Owing to the high emitting power of the unit of area of the emitting area that can be realized by means of power LEDs, the requirements placed on blister lights with regard to the emitting power can be fulfilled without the projection of the blister light above the surface of the traffic area being more than 13 mm, smaller projections also being possible.", "The required light-emitting powers can be realized in many instances even with a single power LED provided as a luminous device.", "It is expedient to provide at most six power LEDs per light exit opening as the luminous device.", "In order to optimize still further the light output or the light-emitting power of the blister light according to an embodiment of the invention, it is expedient when a collimator element is assigned to the at least one power LED.", "This collimator element can be used in order to emit the optical radiation directly through the emitting window; alternatively, the light emission can be performed by means of the collimator element after a preceding reflection on a beam-shaping or beam-deflecting mirror.", "The respectively desired optical radiation distribution can be achieved by the use of collimators, lenses, prisms, deffractors and/or mirrors.", "The thermoconducting bridge between the luminous device and the housing cover can be realized in a less complicated way in technical design terms when the at least one power LED is seated with its carrier plate on a base plate that is fitted on at least one cover inner part connected to the underside of the housing cover, and forms with the cover inner part the thermoconducting bridge to the housing cover and thus to the housing.", "The thermoconducting bridge is advantageously formed by the base plate and two cover inner parts which are arranged on both sides of the carrier plate and can be connected to the underside of the housing cover.", "The two cover inner parts can be arranged on the end sections of the base plate, in which case it is advantageous that each cover inner part bears against the base plate in two dimensions on one side, and against the underside of the housing cover in two dimensions on the other side.", "In order to simplify the dissipation of the thermal energy from the at least one power LED to the traffic area, it is expedient when the mutually assigned bearing surfaces on the side of the cover inner part and of the base plate as well as the mutually assigned bearing surfaces on the side of the cover inner part and of the cover are formed in a metallically smooth fashion, so that the heat transfer resistance is as low as possible there.", "Of course, the mutually assigned bearing surfaces on the side of the cover and of the housing should correspondingly also be formed in a metallically smooth fashion.", "Materials with a high coefficient of thermal conductivity, for example metals, apply in particular as material for the base plate on which the power LED is seated with its carrier plate, for the carrier plate and the cover inner parts.", "The thermal energy produced by way of the power LED can be transferred to the traffic area, which serves as a virtually unlimited heat sink, by way of the previously outlined thermoconducting bridge of the blister light according to an embodiment of the invention.", "The total heat transfer resistance of the barrier layer of the power LED for the base plate is at most approximately 11 K/W; a heat transfer resistance which is less than 1.5 K/W must for this purpose be added for the heat dissipation path from the base plate to the traffic area.", "Although the maximum light output power is reached at a current of 350 to 1000 mA in the case of the use of power LEDs as the luminous device, the thermal energy produced in the event of the current densities resulting therefrom can be dissipated immediately in the case of the blister light according to an embodiment of the invention." ], [ "This application is the national phase under 35 U.S.C.", "§ 371 of PCT International Application No.", "PCT/DE02/00597 which has an International filing date of Feb. 19, 2002, which designated the United States of America and which claims priority on German Patent Application numbers DE 101 08 144.8 and 101 49 263.4, respectively filed Feb. 20, 2001 and Oct. 5, 2001, the entire contents of which are hereby incorporated herein by reference.", "FIELD OF THE INVENTION The invention generally relates to a blister light used for signaling and/or marking purposes.", "Preferably, it relates to one including a housing that is embedded in a traffic area, for example a road, an airport taxiway, an airport runway or the like; and a housing cover by which the housing embedded in the traffic area can be closed at its upper side.", "The cover may be detachably connected to the housing and may have at least one light exit opening.", "The light preferably includes a luminous device that is arranged in the housing and by which light can be emitted through the light exit opening in the housing cover.", "BACKGROUND OF THE INVENTION In known blister lights, in which use is also made as luminous device of, inter alia, LEDs (light-emitting diodes), the quantity of heat produced during operation of the luminous device is usually transferred by convection into a gas, which is often air.", "In some known blister lights, the luminous device is assigned a heat sink starting from which the quantity of heat produced during operation of the luminous device is given off to the gas or air by convection.", "Because of the limited spatial conditions prevailing in blister lights, even blister lights equipped with a heat sink with low heat transfer resistance have a total heat transfer resistance of usually more than 30 K/W power loss.", "In the use, known from the prior art, of LEDs mounted on PC plates, a heat transfer resistance of more than 150 K/W results between the barrier layer of the LEDs and the PC plate.", "In some automotive applications, a heat transfer resistance of the order of approximately 70 K/W is achieved.", "These high heat transfer resistances, which accompany maximum temperatures of the barrier layers that are usually of the order of magnitude of a little above 120° C., limit the current density possible and/or permissible during operation of the blister light.", "They thus produce, in a corresponding way, a limitation of the light-emitting power of the LEDs that can be achieved per unit of area of the barrier layer.", "In the case of freely arranged lighting devices, for example traffic lights or the like, this limitation of the emitting power per unit of area is compensated by an increase in the area used for the emission of light and in the number of the light-emitting diodes.", "This enlargement of the light-emitting area is not practicable for blister lights, since blister lights are embedded in the traffic area.", "Further, since the emitting area of a blister light that is available in total for the emission of light is limited by the projection of the blister light above the surface of the traffic area, it is necessary for this projection to be as small as possible owing to a multiplicity of technically and mechanically conditioned requirements placed on the blister light.", "SUMMARY OF THE INVENTION It is an object of an embodiment of the invention to provide a blister light, in the case of which the light-emitting power can be increased without increasing the light-emitting area, without exceeding permissible barrier surface temperatures of the luminous device or reducing the service life of the luminous device.", "An object may be achieved according to an embodiment of the invention, by virtue of the fact that formed between the luminous device and the housing cover of the blister light is a thermoconducting bridge.", "By this bridge, thermal energy produced by the luminous device can be conducted to the housing cover.", "Starting from this, the thermal energy can be dissipated to the traffic area via the housing embedded in the traffic area.", "Owing to this configuration of the blister light with a thermoconducting bridge connecting the luminous device to the housing cover or the housing, it is possible to optimize the light output power by using a higher current density in the luminous device.", "This occurs since the thermal energy occurring can be given off to the traffic area by creating a thermal dissipation path with a low heat transfer resistance, such that the temperatures at the barrier layers of the luminous device remain in the permissible range, and the service life of the luminous means is not reduced.", "The capacitance of the traffic area for absorbing thermal energy is virtually unlimited, the traffic area having, moreover, a very large surface for emitting heat.", "According to an embodiment of the invention, the traffic area is used like a heat sink of unlimited absorptive capacity by virtue of the fact that the barrier layer of the luminous device is connected to the traffic area via the outlined heat dissipation path with a low heat transfer resistance.", "The heat dissipation from the luminous device, by convection that is substantially less effective by comparison with the outlined heat dissipation path to the traffic area, takes place in the case of the blister light according to an embodiment of the invention only at a negligible order of magnitude.", "Owing to the electric current densities prevailing in the case of the inventive configuration of the blister light, it is particularly expedient when power LEDs, also termed high-intensity LEDs, are used as luminous device.", "The most varied requirements placed on blister lights can be fulfilled by way of such power LEDs, without particular further measures.", "There is a need, moreover, only for a comparatively limited number of power LEDs.", "These emit through a transparent emission window that is implemented by the light exit opening present in the housing cover.", "Owing to the high emitting power of the unit of area of the emitting area that can be realized by means of power LEDs, the requirements placed on blister lights with regard to the emitting power can be fulfilled without the projection of the blister light above the surface of the traffic area being more than 13 mm, smaller projections also being possible.", "The required light-emitting powers can be realized in many instances even with a single power LED provided as a luminous device.", "It is expedient to provide at most six power LEDs per light exit opening as the luminous device.", "In order to optimize still further the light output or the light-emitting power of the blister light according to an embodiment of the invention, it is expedient when a collimator element is assigned to the at least one power LED.", "This collimator element can be used in order to emit the optical radiation directly through the emitting window; alternatively, the light emission can be performed by means of the collimator element after a preceding reflection on a beam-shaping or beam-deflecting mirror.", "The respectively desired optical radiation distribution can be achieved by the use of collimators, lenses, prisms, deffractors and/or mirrors.", "The thermoconducting bridge between the luminous device and the housing cover can be realized in a less complicated way in technical design terms when the at least one power LED is seated with its carrier plate on a base plate that is fitted on at least one cover inner part connected to the underside of the housing cover, and forms with the cover inner part the thermoconducting bridge to the housing cover and thus to the housing.", "The thermoconducting bridge is advantageously formed by the base plate and two cover inner parts which are arranged on both sides of the carrier plate and can be connected to the underside of the housing cover.", "The two cover inner parts can be arranged on the end sections of the base plate, in which case it is advantageous that each cover inner part bears against the base plate in two dimensions on one side, and against the underside of the housing cover in two dimensions on the other side.", "In order to simplify the dissipation of the thermal energy from the at least one power LED to the traffic area, it is expedient when the mutually assigned bearing surfaces on the side of the cover inner part and of the base plate as well as the mutually assigned bearing surfaces on the side of the cover inner part and of the cover are formed in a metallically smooth fashion, so that the heat transfer resistance is as low as possible there.", "Of course, the mutually assigned bearing surfaces on the side of the cover and of the housing should correspondingly also be formed in a metallically smooth fashion.", "Materials with a high coefficient of thermal conductivity, for example metals, apply in particular as material for the base plate on which the power LED is seated with its carrier plate, for the carrier plate and the cover inner parts.", "The thermal energy produced by way of the power LED can be transferred to the traffic area, which serves as a virtually unlimited heat sink, by way of the previously outlined thermoconducting bridge of the blister light according to an embodiment of the invention.", "The total heat transfer resistance of the barrier layer of the power LED for the base plate is at most approximately 11 K/W; a heat transfer resistance which is less than 1.5 K/W must for this purpose be added for the heat dissipation path from the base plate to the traffic area.", "Although the maximum light output power is reached at a current of 350 to 1000 mA in the case of the use of power LEDs as the luminous device, the thermal energy produced in the event of the current densities resulting therefrom can be dissipated immediately in the case of the blister light according to an embodiment of the invention.", "BRIEF DESCRIPTION OF THE DRAWINGS The invention is explained in more detail further below with the aid of an embodiment and with reference to the drawings, in which: FIG.", "1 shows a perspective external view of a blister light according to an embodiment of the invention; FIG.", "2 shows a first sectional illustration of the blister light according to an embodiment of the invention; FIG.", "3 shows a second sectional illustration of the blister light according to an embodiment of the invention; FIG.", "4 shows a perspective illustration of the underside of a housing cover of the blister light according to an embodiment of the invention shown in FIGS.", "1 to 3; FIG.", "5 shows a side view of a luminous device of the blister light according to an embodiment of the invention; FIG.", "6 shows a front view of the luminous device shown in FIG.", "5; FIG.", "7 shows a perspective illustration of the luminous device shown in FIGS.", "5 and 6; FIGS.", "8 to 10 show illustrations of the principle of further blister lights according to and embodiment of the invention; and FIG.", "11 shows the characteristic of a power LED by comparison with that of a conventional 5 mm LED.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A blister light 1 according to an embodiment of the invention and shown with the aid of FIGS.", "1 to 4 has a housing 2 that is of approximately cylindrical construction and is embedded in a traffic area 3.The traffic area 3 can be, for example, a road, an airport taxiway, an airport runway or the like.", "The blister light 1 serves for signaling and/or marking purposes.", "On its upper side, the housing 2 of the blister light 1 can be closed by way of a housing cover 4 that can be detachably connected to the housing 2, for example by way of screw connections 5.The housing cover 4 projects above the traffic area only slightly, for example by less than 13 mm.", "The housing 2 is either directly surrounded by the material forming the traffic area 3, or else thermally conducting structural materials such as epoxide- or polyester-filled mortar are provided between the housing 2 and the material forming the actual traffic area 3.A power LED 6 is provided as luminous device inside the housing 2.As shown in FIG.", "11, which shows the light output power as a function of the current intensity for a power LED and for an ordinary 5 mm LED, this power LED 6 has its maximum light output power for a through-current of approximately 700 to 750 mA.", "The power LED 6 is seated on a carrier plate 7 that, for its part, is connected to a base plate 9 by suitable connecting device 8, as can best be seen in FIG.", "5.The carrier plate 7 of the power LED 6, and the base plate 9, bear against one another in two dimensions.", "The total heat transfer resistance between the barrier layer of the power LED 6 up to the base plate 9 is approximately 11 K/W.", "In comparison, the corresponding values of an ordinary 5 mm LED, for which a heat transfer resistance of approximately 220 K/W results, may be gathered from FIG.", "11.The power LED 6 is assigned in the exemplary embodiment shown a collimator element 10 by which the light output of the power LED 6 can be optimized.", "From the collimator element 10, the light emitted by the power LED 6 reaches an optical unit 11 that the emitted light leaves through a light exit opening 12 provided in the housing cover 4.The configuration of the light beam emitted by the blister light 1 is a function, inter alia, of how the optical unit 11 is, for its part, configured, it being possible for said unit to have lenses, prisms, diffractors, mirrors and the like adapted as appropriate depending on the desired nature of the light emission.", "In the region of its radiation exit surface, the collimator element 10 is held via a lens holder 13 that, for its part, is connected by suitable connecting elements 14 to spacers, for example metallic polygonal sleeves 15, which are excellent conductors of heat and project from the base plate 9 on both sides of the carrier plate 7.A shutter or closure 16 is arranged on the side of the lens holder 13 averted from the collimator element 10.The configuration of the module composed of base plate 9, carrier plate 7, power LED 6, polygonal sleeves 15, collimator element 10, lens holder 13 and shutter or closure 16 may best be seen from FIGS.", "5 to 7.As follows most effectively from FIG.", "4, the base plate 9 is fitted at its two lateral end sections on a respective cover inner part 17, 18.The two cover inner parts 17, 18 are formed in the same way and so only the cover inner part 17 is described below.", "As follows best from a combined look at FIGS.", "2 to 4, the cover inner part 17 is connected in two dimensions to the underside 19 of the housing cover 4.The cover inner part 17 correspondingly bears in two dimensions on the end section of the base plate 9.The mutually assigned bearing surfaces on the side of the cover inner part and of the base plate as well as the mutually assigned bearing surfaces on the side of the cover inner part and of the cover are formed in a metallically smooth fashion, in order to ensure undisturbed heat transfer between the base plate 9 and the cover inner part 17, or the cover inner part 17 and the housing cover 4.In the way described above, the base plate 9 and the cover inner part 17 or the cover inner part 18 form a thermoconducting bridge between the power LED or its carrier plate 7 and the housing cover 4.Via a metallically smooth bearing surface 20 on the cover side, the housing cover 4 is connected, for its part, to a bearing surface 21 on the housing side, which is likewise metallically smooth, the result of this being that the heat is output from the housing cover 4 via the housing 2 to the traffic area or the material forming the traffic area.", "Both the carrier plate 7 of the power LED 6, and the base plate 9 and the two cover inner parts 17, 18 are formed from a material with a high coefficient of thermal conductivity such that the thermoconducting bridge, formed by the base plate 9 and the cover inner parts 17, 18, to the housing cover 4.The housing cover 4 and the housing 2 create a heat dissipation path from the power LED 6 up to the traffic area 3.This heat dissipation path has a total heat transfer resistance of approximately 1.5 K/W, the traffic area 3 forming a virtually infinitely large heat sink.", "Thus, in the case of the blister light 1 described above, dissipation of heat is effected by the luminous device or by the power LED 6 virtually without convection, it being the case, rather, that a thermoconducting bridge to the housing cover 4 is created by the cover inner parts 17, 18 and the base plate 9.The result is that comparatively large quantities of heat can be output via the housing cover 4 and the housing 2 to the traffic area 3.This renders possible the light output powers of the power LED 6, and thus emitting powers of the blister light 1 that meet any requirements and in the case of which it is plausible nevertheless to comply at once with the restrictions with regard to the dimensioning of the light exit opening 12 that are prescribed by the slight projection of the housing cover 4 above the upper side of the traffic area 3.FIGS.", "8 and 10 show in plan view further embodiments of blister lights 1 equipped according to the invention with thermoconducting bridges.", "The embodiment in accordance with FIG.", "8 includes a housing cover 4 with two light exit openings 12 arranged symmetrically relative to an axis.", "The embodiment in accordance with FIG.", "10 has a housing cover 4 with two light exit openings 12 whose axes of symmetry enclose an angle.", "FIG.", "9 shows a further embodiment of a blister light 1, formed with thermoconducting bridges according to the invention, with two times four power LEDs.", "The invention being thus described, it will be obvious that the same may be varied in many ways.", "Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims." ] ]	Patent_10468600
[ [ "Solarization stable borosilicate glass and uses thereof", "The invention relates to a borosilicate glass consisting of 0.01 0.05 wt.", "% Fe2O3 and 0.05 0.8 wt.", "% TiO2, which is highly resistant to solarization and which is especially suitable for use as backlight bulbs." ], [ "1.A borosilicate glass of a composition (in weight-% and on an oxide base) of SiO2 70-80 B2O3 13-18 Al2O3 0.5-4 Li2O 0-1 Na2O 2-5 K2O 1-3 MgO 0-1 CaO 0-1 BaO 0-1 Fe2O3 0.01-0.05 TiO2 0.05-08 2.The borosilicate glass in accordance with claim 1, characterized by a composition (in weight-% on and on an oxide base) of SiO2 70-80 B2O3 13-18 Al2O3 0.5-<2 Na2O 2-5 K2O >1-3 MgO 0-1 CaO 0-1 BaO 0-1 Fe2O3 0.01-0.05 TiO2 0.05-08 3.The borosilicate glass in accordance with claim 1, characterized in that it contains at least 0.2 weight-%, preferably at least 0.2 weight-%, of TiO2.4.The borosilicate glass in accordance with at least one of claim 1, characterized in that it additionally contains (in weight-% on and on an oxide base) ZrO2 0-1 SnO2 0-0.5 MnO2 0-0.1 Sb2O3 0-0.5 5.The borosilicate glass in accordance with at least one of claim 1, characterized in that, except for unavoidable impurities, it is free of As2O3 and PbO.", "6.The borosilicate glass in accordance with at least one of claim 1, with a transmission drop at lambda=300 nm of less than 5% following the HOK-4 irradiation for 15 hours of a glass sample of 0.2 mm thickness.", "7.The borosilicate glass in accordance with at least one of claim 1, with a transmission temperature Tg<520° C., with a thermal expansion coefficient α20/300 between 3.7×10−6/K and 4.2×10−6/K, a transmission tau at lambda ≦260 nm of ≦30% (with 0.2 mm sample thickness).", "8.The borosilicate glass in accordance with at least one of claim 1, with a transmission temperature Tg<520° C., with a thermal expansion coefficient α20/300 between 3.7×10−6/K and 4.2×10−6/K, a transmission tau at lambda ≦260 nm of ≦0.7% (with 0.2 mm sample thickness).", "9.Use of the glass in accordance with at least one of claim 1 8 for producing fluorescent tubes for the background illumination of displays.", "10.Use of the glass in accordance with at least one of claim 1 8 for producing fluorescent tubes for brake lights of vehicles.", "11.Use of the glass in accordance with at least one of claim 1 for producing flash tubes.", "12.Use of the glass in accordance with at least one of claim 1 for producing gas discharge lamps." ], [ "The invention relates to a solarization-stable borosilicate glass and uses thereof.", "Special fluorescent tubes, so-called “backlights”, are used for background illumination of, for example, displays of personal computers, laptops, pocket calculators, vehicle navigation systems, for example.", "While conventional fluorescent tubes are made of soft glass, which has a low solarization stability, more solarization-stable glass is needed for backlights whose structure corresponds to that of fluorescent tubes in principle, in order to assure long term functionality.", "Because of the structure of the backlights, the glass used must be capable of being melted together with tungsten.", "To this end it must have a thermal expansion matched to the expansion behavior of W. With the thermal expansion coefficient α20/300 of W of 4.4×10−6/K, glass with α20/300 between 3.7×10−6/K and 4.2×10−6/K is suitable.", "This is also a difference in respects to the said soft glass, which is melted together with Fe—Ni alloys.", "The glass should preferably have a low transformation temperature Tg, i.e.", "Tg<520° C., so that it can be preferably processed at lower temperatures.", "The transmission progression of the glass is essential.", "As high a possible a transparency is demanded in the visible range in order to obtain a high light yield from the lamp, in the UV range a transmission defined in accordance with the purpose is demanded.", "For example, the effects of harmful UV radiation ≦260 nm must be prevented by means of a corresponding lowering of the UV transmission in order not to let plastics, for example in laptops, become yellowed and brittle.", "For this, glass with a UV transmission at lambda ≦260 nm tau of <0.7%, measured at 0.2 mm thick samples, is suitable.", "For flash tubes or gas discharge lamps, transmissions tau at lambda ≦260 nm of ≦30% (with 0.2 mm thick samples) are sufficient.", "The transition from the opaque to the transparent wavelength range should be as short as possible, i.e.", "in this range the transmission curve should extend as steeply as possible.", "The minimum demand made on the transmission in the visible wavelength range is, at tau >400 nm and a sample thickness of 0.2 mm, a transmission of 92%.", "Thus, the requirement is tau (>400 nm; 0.2 mm)≧92%.", "A further essential property of glass for “backlights” is the solarization stability which is required for making possible a long service life of the lamps, i.e.", "as constant as possible a light yield.", "Glass is to be considered “solarization stable” here which, following 15 hours of HOK-4 radiation, i.e.", "a radiation from an Hg high-pressure lamp with a main emission at 365 nm and a radiated strength of 850 μW/cm2 at 200 to 280 nm at a distance of 1 m, shows a transmission drop of less than 5% at 300 nm on a glass sample of 0.2 mm thickness.", "The demands made on glass for flash tubes, gas discharge lamps and backlights are very similar.", "All should have the highest possible solarization stability and have a high transmission in the visible range.", "Various documents are already known in the patent literature describing more or less solarization-stable glass, in particular glass for lamps.", "However, this glass has the most varied disadvantages, in particular a solarization stability which does not meet the present-day high requirements.", "U.S. Pat.", "No.", "5,994,248 describes a headlight lens made of glass of a very broad composition range, part of which is SiO2, and wherein Al2O3, B2O3, earth alkali oxides and alkali oxides, as well as small amounts of iron oxide can be further components.", "However, the K2O portion is only allowed to lie between 0 and 1 weight-%.", "The properties which are essential for backlights, flash tubes and gas discharge lamps, such as solarization stability and a thermal expansion matched to tungsten, are not of importance here.", "DE 195 45 422 A1 relates to a bonded glass for anodic bonding of silicon components with glass components, which has a high content of Li2O and contains high Fe2O3/FeO dopings.", "JP 10-36135 A describes glass for electronic image capturing.", "In this case the lowest possible α-radiation of the glass is essential.", "To this end, in a wide basic glass composition the U, Th, Ra contents are <100 ppb, and the contents of Fe2O3, TiO2, PbO, ZrO are >100 ppm.", "EP 0 735 007 B1 describes a solid lead- and arsenic-free glass with resistance against solarization, containing defined amounts of SnO2 and CeO2, by means of which the solarization stability is increased, but not to a satisfactory degree.", "The same applies to the glass containing CeO2 and Fe2O3 in WO 98/55413.The closest prior art is represented by JP 8-12369 A.", "For UV blocking, the borosilicate glass for gas discharge lamps described therein contains a total of 0.03 to 3 weight-% of at least two of the components V2O5, Fe2O3, TiO2 and CeO2.A high transmission and high solarization stability cannot be accomplished by means of these components with in part large individual proportions, nor by their combination.", "It is therefore the object of the present invention to make available a solarization-stable glass which does not let UV (<260 nm) through and has a high transmission in the visible range, as well as a thermal expansion matched to the expansion behavior of tungsten.", "This object is attained by a borosilicate glass in accordance with the main claim.", "Glass with the desired transmission properties preferably consists of a basic glass system of 70 to 80 weight-% of SiO2, 13 to 18 weight-% of B2O3, 0.5 to 4 weight-%, preferably 0.5 to <2 weight-% of Al2O3, alkali oxides, namely preferably 2 to 5 weight-% of Na2O, and 1 to 3 weight-%, preferably >1 to 3 weight-% of K2O, and 0 to 1 weight-% of Li2O, preferably free of Li2O, and optionally earth alkali oxides, namely preferably 0 to 1 weight-% of MgO, 0 to 1 weight-% of CaO and 0 to 1 weight-% of BaO, preferably free of BaO.", "The simultaneous presence of TiO2 and Fe2O3 in definite proportions, namely 0.01 to 0.05 weight-% of Fe2O3 and 0.05 to 0.8 weight-% of TiO2 is important for the invention.", "The respective minimum proportions of Fe2O3, as well as of TiO2 are necessary for achieving the high degree of solarization stability.", "A TiO2 content of at least 0.1 weight-% is preferred, a content of at least 0.2 weight-% of TiO2 is particularly preferred, and at least 0.4 weight-% of TiO2 are most particularly preferred.", "Because of the simultaneous presence of these two components in the amounts mentioned, the UV edge, i.e.", "the transition between absorption and transmission at the desired wavelength, is maintained.", "Higher Fe2O3 contents than 0.05 weight-% would mean a lowering of the transmission in the range between 350 to approximately 600 nm, which can be blamed on the effects of Fe3+.", "But higher TiO2 contents than 0.8 weight-% would lead to the displacement of the UV edge into the longer wave visible range and therefore to a yellow tint of the glass.", "Furthermore, when increasing both components past the cited highest contents, ilmenite is formed, which leads to a brown coloration of the glass, and therefore to lowering the transmission.", "The glass can contain customary refining agents in customary amounts, for example evaporation refining agents such as Cl and F, but also redox refining agents, which are effective because of their polyvalent cations, for example SnO2 and Sb2O3, which are preferably present in the glass in respectively 0 to 0.5 weight-%.", "An SnO2 content between 0 and 0.2 weight-percent is particularly preferred.", "Except for unavoidable impurities, the glass does not contain As2O3, since As2O3 would have disadvantageous effects on the solarization stability.", "The same applies to PbO.", "Therefore the glass is free of PbO, except for unavoidable impurities.", "The glass can contain up to 0.5 weight-%, preferably up to 0.1 weight-% of MnO2.At this order of magnitude it is used as a refining agent and displaces the UV edge into the long wave range.", "The glass can contain 0 to 1 weight-% of ZrO2.ZrO2 is mainly of advantage for the chemical resistance of the glass.", "Higher ZrO2 contents would have a negative effect on melting, and the processing temperature of the glass would become too high.", "Moreover, there would be the danger of undissolved mixture particles to remain behind.", "It is preferred to omit the addition of ZrO2, so that the glass is free of ZrO2, except for unavoidable impurities in the form of raw materials or vat corrosion.", "Except for unavoidable impurities, the glass is furthermore free of CeO2.This is of great advantage for the transmission properties, because CeO2 has very negative effects on the solarization stability.", "EXEMPLARY EMBODIMENTS Customary raw materials were used for producing the sample glass and the comparison glass, i.e.", "it is not particularly necessary to use expensive low-Fe materials.", "The well homogenized mixture was melted in the laboratory in a Pt-crucible at 1600° C., refined and homogenized.", "The glass was cast thereafter and cooled at 20 k/h.", "Table 1 shows a melting example for a 0.5 l molten mass.", "Oxide Weight-% Raw Material Original Quantity SiO2 74.90 SiO2 670.51 B2O3 16.90 H3BO3 145.08 Al2O3 1.14 Al2O3 7.94 Na2O 3.73 Na2B4O7 101.04 K2O 1.44 K2CO3 18.65 CaO 0.60 CaCO3 0.1727 MgO 0.42 Dolomit 16.38 NaCl 0.45 NaCl 4.01 TiO2 0.40 TiO2 3.36 Fe2O3 0.025 Fe2O3 0.0913 The properties of the glass obtained in this way are shown in Table 2, Example A4.Table 2 shows six examples of glass in accordance with the invention (A1 to A6) with their components (in weight-% on an oxide basis and their essential properties.", "Table 3 shows the compositions and properties of five pieces of comparison glass (V1 to V5).", "0.45 weight-% of NaCl were added to each of the glass mixture.", "Only about 0.29 weight-% of NaCl can be found in the finished glass pieces.", "The following properties are shown in Tables 2 and 3: ▪ the thermal expansion coefficient α20/300 [10−6/K] ▪ the transformation temperature Tg [° C.] ▪ the solarization stability, stated as the difference of transmission at lambda=300 nm between a non-irradiated sample of 0.2 mm thickness, and one after 15 hours of irradiation by a HOK-4 lamp, stated as Delta15 tau (300 nm, 0.2 mm) [%] ▪ the transmission at lambda=>400 nm and a sample thickness of 0.2 mm tau (>400 nm; 0.2 mm) to document the high transmission in the visible range ▪ the transmission at lambda=>260 nm and a sample thickness of 0.2 mm tau (>260 nm; 0.2 mm) to document high transmission in the UV range (UV blockage).", "TABLE 1 Compositions (in weight-% on an oxide basis) and essential properties of glass (A) in accordance with the invention A1 A2 A3 A4 A5 A6 SiO2 75.17 74.57 74.92 74.90 75.13 75.00 B2O3 16.96 16.94 16.90 16.90 16.95 16.92 Al2O3 1.15 1.65 1.14 1.14 1.15 1.15 Na2O 3.74 3.73 3.73 3.73 3.74 3.73 K2O 1.45 1.45 1.44 1.44 1.45 1.44 CaO 0.60 0.60 0.60 0.60 0.60 0.60 MgO 0.42 0.42 0.42 0.42 0.42 0.42 TiO2 0.075 0.200 0.400 0.400 0.100 0.100 SnO2 — — — — — 0.200 MnO2 — — — — 0.025 — Fe2O3 0.013 0.01 0.013 0.025 0.013 0.013 α20/300 [10−6/K] 3.84 3.84 3.88 3.80 3.82 3.88 Ta [° C.] 503 495 511 496 496 504 Δ15τ (300 nm; 0.2 mm) 4.4 2.2 2.1 2.4 3.8 4.3 [%] τ (260 nm, 0.2 mm) [%] 21 8.7 0.6 0.3 15.7 18.7 τ (>400 nm, 0.2 mm) [%] >92 >92 >92 >92 >92 >92 TABLE 2 Compositions (in weight-% on an oxide basis) and essential properties of comparison glass (V) V1 V2 V3 V4 V5 SiO2 75.17 75.22 74.57 75.11 75.22 B2O3 16.96 16.97 16.94 16.95 16.97 Al2O3 1.15 1.15 1.65 1.15 1.15 Na2O 3.74 3.74 3.73 3.74 3.74 K2O 1.45 1.45 1.45 1.45 1.45 CaO 0.60 0.60 0.60 0.60 0.60 MgO 0.42 0.42 0.42 0.42 0.42 NaCl 0.45 0.45 0.45 0.45 0.45 TiO2 0.075 0.034 0.200 0.100 0.0033 CeO2 — — — 0.05 — Fe2O3 0.0084 0.013 0.0084 0.013 0.0084 α20/300 [10−6/K] n.b.", "3.55 n.b.", "n.b.", "n.b.", "Tg [° C.] n.b.", "497 n.b.", "n.b.", "n.b.", "Δ15τ (300 nm; 0.2 mm [%] 5.8 5.5 6.7 5.8 7.8 τ (260 nm: 0.2 mm) 41.3 39.8 9.6 11 65.8 τ (>400 nm; 0.2 mm) >92 >92 >92 >92 >92 The transmission curves tau over lambda (200 to 400 nm) for some exemplary and comparison embodiments before (a), and after (b), irradiation are shown in FIGS.", "1 to 5.In detail: FIG.", "1: A1 and V1, V5, respectively not irradiated and following 100 hours of irradiation by means of an HOK-4 lamp (sample thickness: 0.2 mm) FIG.", "2: A2 and V3, respectively not irradiated and following 100 hours of irradiation by means of an HOK-4 lamp (sample thickness: 0.2 mm) FIG.", "3: A3, A4, respectively not irradiated and following 100 hours of irradiation by means of an HOK-4 lamp (sample thickness: 0.21 mm) FIG.", "4: A5, A6, respectively not irradiated and following 100 hours of irradiation by means of an HOK-4 lamp (sample thickness: 0.21 mm) FIG.", "5: A4 and V4, respectively not irradiated and following 100 hours of irradiation by means of an HOK-4 lamp (sample thickness: 0.2 mm).", "The figures document that the desired transmission progression is achieved by means of the special contents of TiO2 and Fe2O3, which is represented in particular by the comparison with the samples V1, V5, V3, which are low in Fe2O3, or the samples V2, V5, which are low in TiO2.The importance of the lack of CeO2 for the transmission is also made clear.", "The figures, as well as the indication of Delta15 tau (300 nm; 0.2 mm) in the tables make clear the differences in the solarization stability between the glass of the invention and comparison glass.", "The negative effect of CeO2 becomes clear in the comparison between V4 and, for example, A5 or A6, but in particular with A1 (see the table) or A4 (see FIG.", "5).", "The glass in accordance with the invention has a high degree of solarization stability, expressed by Delta15 tau (300 nm; 0.2 mm) of <5%, a high transmission in the visible range (see the progression of transmission), in particular expressed by tau (>400 nm; 0.2 mm)≧92%, and good UV blocking (see the progression of transmission), in particular expressed by tau (≦260 nm; 0.2 mm)≦30%.", "The glass moreover has a transformation temperature Tg<520° C., so that it can be easily worked.", "The glass furthermore has a thermal expansion coefficient α20/300 between 3.7×10−6/K and 4.2×10−6/K.", "It is therefore well matched to the thermal expansion properties of tungsten, i.e.", "it can be melted together with W. With these properties, glass is well suited for producing lamp bulbs for flash tubes and for gas discharge lamps.", "In preferred embodiments with comparatively high TiO2 contents, the glass shows good UV blocking, in particular expressed by tau (≦260 nm; 0.2 mm)≦0.7%.", "Therefore the glass is outstandingly suitable for producing “backlights”, for example for the background lighting of, for example, displays of personal computers, laptops, notebooks, pocket calculators, vehicle navigation systems, scanners, but also of mirrors and pictures.", "In the same way it is well suited for producing brake lights for vehicles.", "The third, additional, brake light in particular can preferably be produced by means of such a special fluorescent tube." ] ]	Patent_10468612
[ [ "Method and device for determining a temperature variable", "A device and method for determining a temperature variable, in particular a temperature variable that characterizes the condition of an exhaust-gas treatment system of a combustion engine, are described.", "The temperature is specified on the basis of variables that characterize the mass flow in the exhaust-gas treatment system, and/or of a second temperature variable that characterizes the temperature upstream from the exhaust-gas treatment system." ], [ "1-5.", "(canceled) 6.A method for determining at least one of a first temperature of an exhaust-gas treatment system of a combustion engine and a second temperature of an exhaust temperature downstream from the exhaust-gas treatment system, comprising: providing a first variable that characterizes an exhaust gas mass flow in the exhaust-gas treatment system; providing a second variable corresponding to a third temperature that characterizes an exhaust temperature upstream from the exhaust-gas treatment system; measuring the first temperature and the second temperature by a temperature sensor; calculating a difference between the first temperature and the second temperature; comparing at least one of the first temperature and the second temperature with a threshold value; and recognizing an error when the threshold value is exceeded.", "7.The method as recited in claim 6, further comprising: determining the first variable on the basis of at least one of an air variable that characterizes a quantity of air fed to the combustion engine and a fuel variable that characterizes a mass of a fuel fed to the combustion engine.", "8.The method as recited in claim 6, wherein: the first temperature and the second temperature are used in a dynamic condition of the combustion engine to control/regulate the exhaust-gas treatment system.", "9.The method as recited in claim 6, wherein: the method is provided for calculating the first temperature of a particle filter contained in the exhaust-gas treatment system.", "10.The method as recited in claim 6, further comprising: measuring the second temperature by another temperature sensor." ], [ "<SOH> BACKGROUND INFORMATION <EOH>To control and/or monitor exhaust-gas treatment systems, exact knowledge of the condition, especially of the flow condition, is necessary.", "This condition is determined in particular by the temperature in the exhaust-gas treatment system.", "However, this temperature is only accurately detectable with very great effort and expense." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Because the temperature variable is specified on the basis of other variables that characterize the condition of the combustion engine and/or of the exhaust-gas treatment system, such as the mass flow in the exhaust-gas treatment system and a second variable that characterizes the temperature upstream from the exhaust-gas treatment system, an exact determination of the mean temperature of the particle filter and/or of the exhaust downstream from the particle filter is possible.", "The procedural method according to the present invention is not limited here to particle filters; it is usable with all exhaust-gas treatment systems.", "This also applies especially to all types of catalytic converters.", "Determination of the temperature variable according to the present invention makes this variable available more quickly.", "Rapid recognition of the condition of the particle filter is therefore possible.", "In particular, the degree of loading of the particle filter, or a defect in the particle filter, is recognizable significantly more quickly.", "Because of the earlier recognition of the condition, more precise control, regulation and/or monitoring of the exhaust-gas treatment system is possible.", "It is especially advantageous when the variable that characterizes the mass flow in the exhaust-gas treatment system is determined on the basis of an air variable that characterizes the quantity of air fed to the combustion engine, and/or a fuel variable which characterizes the mass of fuel fed to the combustion engine.", "That allows this variable to be determined very simply and precisely, without need of an additional sensor.", "The air variable is usually available in the control unit, since it is also used for controlling the combustion engine, especially the quantity of fuel injected and/or the quantity of air.", "This variable is usually measured using a sensor in the intake line of the combustion engine.", "The fuel variable is also a variable that is present in the control unit.", "For example, a variable derived from the desired moment and/or a variable derived from the actuating signal of a quantity-determining actuator may be used here.", "Preferably, the method is used to determine the temperature of the exhaust-gas treatment system and/or the temperature of the gases downstream from the exhaust-gas treatment system.", "The method according to the present invention is preferably suitable for exhaust-gas treatment systems that include at least one particle filter.", "At the same time, additional systems that treat the exhaust gas may be present, in addition to the particle filter.", "It is especially advantageous when the temperatures determined are used only in certain conditions, for example in dynamic conditions, to control and/or regulate the exhaust-gas treatment system.", "It is also advantageous when the temperature determined is used for error monitoring.", "Preferably, there may be provision for the measured values of the temperature to be compared with the calculated values, and for errors to be recognized on the basis of this comparison." ], [ "FIELD OF THE INVENTION The present invention relates to a method and a device for determining a temperature variable.", "BACKGROUND INFORMATION To control and/or monitor exhaust-gas treatment systems, exact knowledge of the condition, especially of the flow condition, is necessary.", "This condition is determined in particular by the temperature in the exhaust-gas treatment system.", "However, this temperature is only accurately detectable with very great effort and expense.", "SUMMARY OF THE INVENTION Because the temperature variable is specified on the basis of other variables that characterize the condition of the combustion engine and/or of the exhaust-gas treatment system, such as the mass flow in the exhaust-gas treatment system and a second variable that characterizes the temperature upstream from the exhaust-gas treatment system, an exact determination of the mean temperature of the particle filter and/or of the exhaust downstream from the particle filter is possible.", "The procedural method according to the present invention is not limited here to particle filters; it is usable with all exhaust-gas treatment systems.", "This also applies especially to all types of catalytic converters.", "Determination of the temperature variable according to the present invention makes this variable available more quickly.", "Rapid recognition of the condition of the particle filter is therefore possible.", "In particular, the degree of loading of the particle filter, or a defect in the particle filter, is recognizable significantly more quickly.", "Because of the earlier recognition of the condition, more precise control, regulation and/or monitoring of the exhaust-gas treatment system is possible.", "It is especially advantageous when the variable that characterizes the mass flow in the exhaust-gas treatment system is determined on the basis of an air variable that characterizes the quantity of air fed to the combustion engine, and/or a fuel variable which characterizes the mass of fuel fed to the combustion engine.", "That allows this variable to be determined very simply and precisely, without need of an additional sensor.", "The air variable is usually available in the control unit, since it is also used for controlling the combustion engine, especially the quantity of fuel injected and/or the quantity of air.", "This variable is usually measured using a sensor in the intake line of the combustion engine.", "The fuel variable is also a variable that is present in the control unit.", "For example, a variable derived from the desired moment and/or a variable derived from the actuating signal of a quantity-determining actuator may be used here.", "Preferably, the method is used to determine the temperature of the exhaust-gas treatment system and/or the temperature of the gases downstream from the exhaust-gas treatment system.", "The method according to the present invention is preferably suitable for exhaust-gas treatment systems that include at least one particle filter.", "At the same time, additional systems that treat the exhaust gas may be present, in addition to the particle filter.", "It is especially advantageous when the temperatures determined are used only in certain conditions, for example in dynamic conditions, to control and/or regulate the exhaust-gas treatment system.", "It is also advantageous when the temperature determined is used for error monitoring.", "Preferably, there may be provision for the measured values of the temperature to be compared with the calculated values, and for errors to be recognized on the basis of this comparison.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 shows a block diagram of an exhaust-gas treatment system.", "FIG.", "2 shows a diagram of the procedure according to the present invention.", "FIG.", "3 shows a flow chart of the procedure.", "DETAILED DESCRIPTION The procedural method according to the present invention is described below on the example of a particle filter.", "The procedural method according to the present invention is not limited here to the application with a particle filter, it is also usable with other exhaust-gas treatment systems, in catalytic converters in particular.", "FIG.", "1 shows the essential elements of an exhaust-gas treatment system of a combustion engine 100.Fresh air is fed to it via a fresh air line 105.The exhaust gases from combustion engine 100 pass through an exhaust line 110 into the environment.", "An exhaust-gas treatment system 115 is positioned in the exhaust line.", "This may be a catalytic converter and/or a particle filter.", "It is also possible for a plurality of catalytic converters for different pollutants, or combinations of at least one catalytic converter and one particle filter to be provided.", "Also provided is a control unit 170, which includes at least one engine control unit 175 and one exhaust-gas treatment control unit 172.Engine control unit 175 applies actuating signals to a fuel metering system 180.Exhaust-gas treatment control unit 172 applies actuating signals to engine control unit 175, and in one embodiment applies them to an actuator 182 which is positioned in the exhaust line upstream from the exhaust-gas treatment system, or in the exhaust-gas treatment system.", "In addition, various sensors may be provided, which supply the exhaust-gas treatment control unit and the engine control unit with signals.", "For example, at least one first sensor 194 is provided, which supplies signals that characterize the condition of the air that is fed to the combustion engine.", "A second sensor 177 supplies signals that characterize the condition of fuel metering system 180.At least one third sensor 191 supplies signals that characterize the condition of the exhaust gas upstream from the exhaust-gas treatment system.", "At least one fourth sensor 193 supplies signals that characterize the condition of exhaust-gas treatment system 115.Furthermore, at least one sensor 192 may supply signals that characterize the condition of the exhaust gases downstream from the exhaust-gas treatment system.", "Preferably, sensors are used that detect temperature values and/or pressure values.", "In the embodiment described below, sensor 191 is constructed as a temperature sensor.", "This sensor supplies a signal TV which characterizes the temperature upstream from the exhaust-gas treatment system.", "Sensor 192 is constructed as a temperature sensor.", "This sensor 192 supplies a signal TN which characterizes the temperature downstream from the exhaust-gas treatment system.", "This signal TN may also be calculated on the basis of other variables.", "The output signals of first sensor 194, third sensor 191, fourth sensor 193 and fifth sensor 192 preferably act on exhaust-gas treatment control unit 172.The output signals of second sensor 177 preferably act on engine control unit 175.Additional sensors that are not shown may also be provided, which characterize a signal referring to the driver's wish or other environmental or engine operating conditions.", "It is especially advantageous when the engine control unit and the exhaust-gas treatment control unit constitute one structural unit.", "However, provision may also be made for them to be constructed as two control units which are spatially separated from each other.", "The method according to the present invention is preferably utilized for controlling combustion engines, in particular for combustion engines having an exhaust-gas treatment system.", "In particular, it may be utilized with exhaust-gas treatment systems in which a catalytic converter and a particle filter are combined.", "It is also utilizable with systems that are equipped only with a catalytic converter.", "Starting from the available sensor signals, engine controller 175 calculates actuating signals for acting on fuel metering system 180.The latter then meters the appropriate quantity of fuel to combustion engine 100.During combustion, particles may develop in the exhaust gas.", "These are captured by the particle filter in exhaust-gas treatment system 115.In the course of operation, corresponding quantities of particles collect in particle filter 115.This results in degradation of the functioning of the particle filter and/or of the combustion engine.", "For that reason there is provision for a regeneration process to be initiated at certain intervals or when the particle filter has reached a certain loading condition.", "This regeneration may also be referred to as special operation.", "The loading condition is recognized for example on the basis of various sensor signals.", "First, for example, the differential pressure between the input and the output of particle filter 115 may be evaluated.", "Second, it is possible to determine the loading condition on the basis of various temperature variables and/or various pressure values.", "Furthermore, additional values may also be drawn upon to calculate or simulate the loading condition.", "A corresponding procedure is known for example from German Patent 199 06 287.If the exhaust-gas treatment control unit recognizes that the particle filter has reached a certain loading condition, the regeneration is initialized.", "A variety of possibilities are available for regenerating the particle filter.", "For one example, there may be provision for certain substances to be fed to the exhaust gas via actuator 182, which then bring about a corresponding reaction in exhaust-gas treatment system 115.These additionally metered substances cause a temperature increase and/or oxidation of the particles in the particle filter, among other things.", "For example, there may be provision for fuel and/or oxidizing agents to be supplied via actuator 182.In one design there may be provision for an appropriate signal to be conveyed to engine control unit 175, and for the latter to carry out a post-injection.", "The post-injection makes it possible to introduce hydrocarbons into the exhaust gas selectively, which contribute to regeneration of exhaust-gas treatment system 115 by increasing the temperature.", "Provision is usually made for the loading condition to be determined on the basis of a variety of variables.", "The various conditions are recognized through comparison with a threshold value, and the regeneration is initiated depending on the recognized loading condition.", "According to the present invention, the dynamic thermal behavior of the particle filter is modeled.", "The model of temperature TF of the particle filter is based mainly on the temperature TV of the exhaust-gas stream upstream from particle filter 115.This temperature is used for a calculation of the thermal balance of the particle filter.", "The balance of the streams of heat flowing in and away produces this correlation: TF=(1/CPF)∫MCPA(TV−TN)dt Here variable CPF characterizes the heat capacity of the filter, and variable CPA represents the specific heat capacity of the exhaust mass flow.", "Exhaust mass flow M will preferably be calculated from other known variables, such as the air mass, which is measured using sensor 194, and fuel mass QK, which is provided by engine control unit 175.Instead of these variables, corresponding substitute variables may also be used, such as the duration of actuation of a quantity-determining actuator, or the actuating signal of a quantity-determining actuator.", "Variable TN corresponds to the temperature downstream from the filter.", "If no sensor 192 is used downstream from the exhaust-gas treatment system to determine temperature TN downstream from the exhaust gas, then temperature TN is preferably determined according to the following formula.", "TN=TFBF+TV*(1−BF) In the first calculation, an initialization value is specified here for temperature TF.", "Preferably, temperature TV measured at power-up or some other temperature value is used as the starting value for this.", "In the next calculation, temperature TF from the preceding calculation step is used.", "This is possible because the temperature of the filter changes significantly more slowly than the time to calculate a program run-through.", "Variable BF is a variable that characterizes the part of the exhaust stream which is involved in the heat exchange with the filter.", "Variable 1−BF characterizes that part of the exhaust stream that passes through the filter without an exchange of heat.", "A corresponding procedure is shown in FIG.", "2.Elements already described in FIG.", "1 are designated with the same reference symbols in FIG.", "2.An integrator which essentially performs an integration of the variables according to the above formula is designated as 200.It is fed the output signal from a multiplication point 210.One of the variables present at the input to the latter is variable M, which characterizes the mass flow of exhaust leaving the combustion engine.", "At the second input of linkage point 210 the output signal from another multiplication point 220 is present, which feeds output signal CPA to a value specification 230.The output signal from a subtraction point 240 is applied to the second input of multiplication point 220.Applied to subtraction point 240 are both output signal TV from sensor 191 and output signal TN from a linkage point 280.Also fed to integrator 200 are output signals dt from a value specification 250, which includes various integrator constants.", "Output signal T0 from a starting value specification 255, in which the starting value of the integration is stored, is also fed to integrator 200.Integrator 200 applies a signal to a division 215, at whose second input output signal CPF from a value specification 260 which supplies signal CPF is present.", "Applied to the output of the division is signal TF, which characterizes the filter temperature.", "This is furnished to a control unit 170, and in particular to exhaust-gas treatment control unit 172.Signal TF also arrives at a multiplication 275, to whose other input signal BF from signal specification 270 is applied.", "The output signal from multiplication 275 acts on addition point 280.Also applied to addition point 280 is the output signal from multiplication 294, which is again applied to output signal TV from sensor 191, and the output signal from a subtraction point 292 which constitutes the difference between a constant value 1 and variable BF.", "The structure illustrated in FIG.", "2 emulates the formulas specified above.", "According to the present invention, temperature TF of the exhaust-gas treatment system or the temperature of the exhaust gases in the exhaust-gas treatment system, as well as temperature TN downstream from the exhaust-gas treatment system, are calculated on the basis of a variable that characterizes the mass flow in the exhaust-gas treatment system, and a second temperature that characterizes the temperature upstream from the exhaust-gas treatment system.", "The mass flow, for its part, is calculated on the basis of the air mass and of the fuel mass that are fed to the combustion engine.", "Instead of the air mass and the fuel mass, other variables that characterize these variables may also be used.", "Instead of temperature TF in the exhaust-gas treatment system and temperature TN downstream from the exhaust-gas treatment system, other temperature variables may also be determined in the corresponding manner.", "According to the present invention, temperature variables TF and/or TN calculated in this way are used to control the exhaust-gas treatment system.", "This makes it possible for example to dispense with sensors 192, 193.It is especially advantageous when the calculated variables are used only in certain operating conditions.", "A corresponding procedure is illustrated in FIG.", "3.In step 300, a check is performed to determine whether an operating condition is present in which it is appropriate to use the calculated variables instead of the measured variables.", "Such operating conditions are present in particular in dynamic conditions, in which certain operating parameters change quickly.", "For example, there may be provision for a check to be performed of whether the change of the driver's wish, of the quantity of fuel injected, the rotational speed and/or the quantity of fresh air is greater than a threshold value.", "If so, i.e., if such a dynamic condition is present, in step 310 the exhaust-gas treatment system is controlled as a function of the calculated temperature.", "If query 300 shows that no such condition is present, then step 310 checks whether the magnitude of the difference between simulated temperatures TN and/or TF and measured temperatures TN and/or TF is smaller than a threshold value.", "If this is true, then the measured temperature is used in step 330 to control the combustion engine and/or the exhaust-gas treatment system.", "If it is not true, i.e., the measured and the simulated temperatures deviate significantly from each other, step 340 recognizes errors.", "In one embodiment according to the present invention, it may also be provided that the measured temperatures are always used for controlling and the simulated ones only for error recognition." ] ]	Patent_10468662
[ [ "Method and system for recording video data", "The invention relates to a recording method whereby a communication link, preferably specifically in one venue, is established in at least one venue between a telephone, particularly the mobile telephone of a user, and a camera system installed in said venue.", "The video data is recorded by the camera system, whereby the user can have an interactive function.", "The invention also relates to a system for recording video data." ], [ "1.Recording method in which a communication link, in particular a communication link specific to a venue or scene, is set up at at least one venue or scene between a telephone, in particular a mobile telephone of a user and a camera system installed at the scene and video data are recorded by means of the camera system with a user as a participant.", "2.Method in accordance with claim 1, wherein the camera system is called via a telephone number specific to the scene by means of the telephone for the setting up of the communication link.", "3.Method in accordance with claim 1 wherein at least one, preferably precisely one, telephone number specific to the scene is displayed on a display device, in particular a placard, a poster or a monitor, installed at the scene with the display device in particular being used as a background during the recording.", "4.Method in accordance with claim 1, wherein a running recording at the scene is displayed visually and/or acoustically, in particular on a display device.", "5.Method in accordance with claim 1, wherein audio data is picked up, and in particular stored, via the telephone and/or in that audio data is picked up, and in particular stored, via a sound recording system installed at the scene.", "6.Method in accordance with claim 1, wherein the camera system operates automatically and in particular in accordance with an at least substantially pre-settable recording procedure which can preferably be influenced via the telephone and/or wherein the camera system is activated, deactivated and/or controlled via a recording unit and/or via the telephone.", "7.Method in accordance with claim 1, wherein the communication link is set up between the telephone and a recording unit serving for the communication with the camera system and in particular for the control of the camera system.", "8.Method in accordance with claim 1, wherein technical recording equipment, in particular for the lighting and/or sound recording, installed at the scene is activated, deactivated and/or controlled subsequent to the setting up of the communication link, in particular via a recording unit and/or via the telephone.", "9.Method in accordance with claim 1, wherein a communications unit, in particular in the form of a speech computer, is used for the communication with the user and/or in that voice messages, in particular information, instructions, explanations and/or direction instructions, are preferably communicated to the user via the telephone by menu control.", "10.Method in accordance with claim 1, wherein user data, in particular relating to the specific user and/or to the respective recording, are communicated to a recording unit, and in particular stored, via the telephone, in particular via a keypad of the telephone and/or in that a distinction is made by means of stored user data and/or pre-settable criteria between at least temporarily blocked users, on the one hand, and users who are to be accepted and who preferably satisfy pre-settable selection criteria, on the other hand.", "11.Method in accordance with claim 1, wherein at least the recorded data including the video data is preferably stored at the scene and/or wherein the recorded data at least including the video data, and in particular recording data stored at the scene, is transmitted via a communication network or a data network, in particular via an existing telephone network, to a place which is preferably spatially separated from the scene, in particular to an Internet server or to a central recording facility and/or wherein the recorded data at least including the video data is broadcast on television and/or made available on the Internet.", "12.Method in accordance with claim 1, wherein a live recording of the scene is made available, in particular on the Internet, at times or at least substantially without interruption, by means of the camera system, with definable recording sections with respect to the time of starting and of ending being stored by means of the telephone.", "13.Method in accordance with claim 1, wherein the video data is subjected to later processing and is in particular combined with recorded audio data, preferably synchronised.", "14.Method in accordance with claim 1, wherein video data are recorded at a plurality of spatially separated scenes and/or wherein the setting up of a communication link is prevented outside of a restricted region containing the respective scene, but no further scene.", "15.Method in accordance with claim 1, wherein a publicly accessible place is selected as a scene, in particular a square or a street; and/or in that a building is selected as the scene, in particular a restaurant, a discotheque, a cinema or an event hall, preferably a building part designed as a stage.", "16.System for the recording of video data at at least one scene, in particular for carrying out the method of claim 1, having at least one camera system installed at the scene to which a communication link preferably a scene-specific communication link can be set up with a telephone located at the scene, in particular a mobile telephone of a user and can be operated with an established communication link for the recording of video data with the user as participant.", "17.System in accordance with claim 16, wherein the camera system can be called up by means of the telephone via at least one telephone number specific to the scene.", "18.System in accordance with claim 16, wherein at least one display device is provided which is installed at the scene on which at least one and preferably precisely one telephone number specific to the scene is displayed or can be displayed, with a placard, a poster or a monitor in particular being provided as the display device and/or in that the display device is formed as a recording background.", "19.System in accordance with claim 16, wherein a display unit installed at the scene is provided with which a running recording can be indicated optically and/or acoustically, in particular in the form of a coloured light signal and/or an illuminated written display, with the display unit preferably being integrated into a device for displaying at least one telephone number specific to the scene.", "20.System in accordance with claim 16, wherein audio data transmitted via the telephone of the user can be recorded, and in particular stored, and/or in that a sound recording system installed at the scene is provided for the recording of in particular storable audio data.", "21.System in accordance with claim 16, wherein the camera system is automatically operatable and in particular in accordance with an at least substantially pre-determinable recording procedure, which can preferably be influenced via the telephone.", "22.System in accordance with claim 16, wherein a recording unit is provided which is designed for communication with the telephone and with the camera system, and in particular for the control of the camera system, with the recording unit in particular being installed at the scene and in particular being arranged in the spatial vicinity of the camera system and preferably being integrated into the camera system.", "23.System in accordance with claim 22, wherein the recording unit includes a communication unit, in particular in the form of a speech computer, for the communication with the user, with speech messages, in particular information, instructions, explanations, and/or direction explanations being conveyable to the user, preferably under menu control, in particular by means of the communication unit via the telephone.", "24.System in accordance with claim 22, wherein the recording unit comprises a control unit, in particular a computer-controlled control unit, for the communication with the camera system.", "25.System in accordance with claim 16, wherein user data, in particular user specific data and/or data relating to the respective recording, can be conveyed to a recording unit and are is in particular storable via the telephone, in particular via a keyboard of the telephone and/or wherein an evaluation unit is provided which is designed to distinguish between at least temporarily blocked users on the one hand and users which are to be allowed and which preferably satisfy pre-determinable selection criteria on the other hand, by comparison of transmitted user data of calling users with stored user data and/or pre-determinable criteria, with the evaluation unit preferably being integrated into the recording unit.", "26.System in accordance with claim 16, wherein a storage device is provided for the storage of recording data including at least the video data, in particular in a databank, with the storage device preferably being installed at the scene and in particular being integrated into the camera system or into a recording unit.", "27.System in accordance with claim 16, wherein the recording data including at least the video data and in particular the recording data stored at the scene can be transmitted via a communication network or data network, in particular via an existing telephone network, to a location spatially separate from the scene, with the recording data in particular being transmittable to an internet server spatially separate from the scene and/or wherein the recording data can be transmitted to a recording center spatially separated from the scene.", "28.System in accordance with claim 16, wherein video data stored in particular in a recording unit and/or on an internet server can be called up in reduced image quality for preview and/or selection purposes.", "29.System in accordance with claim 16, wherein a plurality of camera systems are provided which are associated with various scenes and/or in that a common recording center is associated with a plurality of spatially separated scenes." ], [ "The invention relates to a method and to a system for the recording of video data at at least one venue (or scene), in particular at a plurality of venues (or scenes).", "In particular in the field of television and of the Internet, there is a need for entertainment with individual and spontaneous elements and for possibilities to win over any desired people in a simple manner to participate in the most varied activities, with activities being particularly promising in which “the man or woman on the street” can participate and the viewer is thus entertained by people like him, i.e.", "by people with whom he can identify.", "It is an object of the invention to provide a possibility, which can be realised in a simple manner, for the recording of video data with the participation of, basically, any desired persons.", "This object is satisfied by the features of the independent method claim 1, and in particular in that a communication link and preferably a communication link specific to a venue or scene is set up at at least one venue or scene between a telephone, in particular a mobile telephone, of a user and a camera system installed at the venue or scene and video data are recorded by means of the camera system with a user as a participant.", "Basically, any desired location in the world can be made into a stage for “the man or the woman on the street” with the invention.", "A publicly accessible location is preferably chosen as the venue or scene, e.g.", "a square or a street.", "Alternatively, in accordance with the invention, the venue can be located at a non-public location and be e.g.", "in a building.", "An admission fee can, for example, be charged for the access to the venue.", "In accordance with the invention, the user himself can determine the time at which the video data are recorded in that the user sets up a communication link with e.g.", "his mobile telephone at a pre-determined scene to a camera system associated with the scene.", "In accordance with the invention, the participant thus initiates the recording of personal video data himself on a voluntary basis by telephone.", "The invention advantageously uses an infrastructure present at the scene with the telephone and, in the case of a mobile telephone, an infrastructure present with the user.", "The video data can be presentations or show acts of the user, e.g.", "the singing of a song, pantomime presentations or spoken or sung greetings to friends and acquaintances.", "Furthermore, the user can be prompted to answer quiz questions.", "The user can further use the invention to present himself to a wide public, whereby e.g.", "singles shows can be realised.", "On the other hand, the invention can be used for the carrying out of polls.", "Furthermore, recruitment activities can be carried out by means of the invention.", "The invention can in particular be used for casting purposes.", "In accordance with the invention, the loudspeaker of the telephone can be used for direct communication with the user, which is in particular of advantage if, e.g.", "with quiz games, the communication should take place to the exclusion of surrounding people who should e.g.", "not hear the quiz questions asked.", "In a particularly preferred embodiment of the invention, the camera system is called by the telephone over a telephone number specific to the scene to set up the communication link.", "The user at the scene e.g.", "dials the telephone number specific to the scene using his mobile telephone to set up the communication link.", "The telephone number can generally be any desired one and can e.g.", "be a normal mobile radio network number or a toll number.", "Provision is furthermore preferably made for at least a telephone number specific to the scene to be displayed at a display device installed at the scene.", "In this manner, a normal advertising area can be made into an interactive display device whose installation location can be made into a recording studio by anyone simply by calling the telephone number displayed.", "In accordance with the invention, either precisely one telephone number can be associated with the scene or a plurality of telephone numbers specific to the scene can be provided which make a selection possible for the user from e.g.", "different categories such as “singles show”, “quiz game”, “casting” and “poll”.", "In addition to the telephone number or telephone numbers, instructions can be displayed which explain the purpose which the telephone numbers serve to the viewer located at the scene.", "This information can, for example, be displayed in an advertising environment.", "The display device can e.g.", "be a placard, a poster or a monitor.", "The display device is preferably used as the background during the recording.", "During the recording, the user e.g.", "stands in front of a display device, e.g.", "a placard wall such as is customarily used for advertising purposes, which is designed as a recording background.", "For example, known television studios can be reproduced in a square or in a street with the invention by a corresponding design of the display device and selection of the recording conditions.", "It is further provided in accordance with the invention for an ongoing recording to be displayed visually and/or acoustically at the scene.", "Such an “on air” display preferably takes place on a display device showing the telephone number(s) specific to the scene and serving as the recording background.", "A placard wall e.g.", "thus satisfies an effective additional function.", "The “on air” display can e.g.", "take place by a red light and/or by the wording “on air” lighting up during the recording, whereby a genuine studio atmosphere arises at the scene.", "The display unit installed at the scene creates a particular attraction for the user since he cannot only personally transform the scene into his own studio by initiating a recording by telephone, but can also direct the attention of persons at the scene to the recording activity and thus to his person with the aid of the additional visual and/or acoustic display.", "The display unit does not have to be integrated e.g.", "into a placard wall displaying the telephone number and serving as the recording background and can generally be arranged at any desired position at the scene.", "It is furthermore proposed in accordance with the invention that audio data can be picked up, and in particular stored, over the telephone.", "The sound, in particular the language, of the user for the video data can thus be picked up and, optionally, recorded for later use, without additional technical recording effort utilising the infrastructure present at the scene or with the user.", "Alternatively or additionally, audio data can be recorded via a sound recording system installed at the scene.", "Surrounding sound can hereby be picked up and thus the atmosphere of the scene be captured.", "It is furthermore preferably provided that the camera system operates automatically, and in particular in accordance with an at least substantially pre-settable recording procedure, preferably influenceable over the telephone.", "It is preferred for the camera system to be activated, deactivated and/or controlled via a recording unit and/or over the telephone.", "The camera system is preferably both switched on and also switched off again after the termination of the recording by means of the telephone.", "Everyone can thus more or less become his own studio manager.", "In the case of a permanently running camera system, an additional procedure is started and ended by means of the telephone which in particular includes a transmission and/or recording of the data provided by the camera system not taking place outside the periods started and ended by the user.", "The activation and deactivation of the camera system or of the additional procedures takes place either directly between the telephone and the camera system or via an intermediate station e.g.", "in the form of a recording unit located at the scene or of a recording centre spatially separated from the scene.", "A recording unit is preferably provided which is made for communication with the telephone and with the camera system and in particular for the controlling of the camera system.", "The communication between the telephone and the camera system can hereby be set up via the recording unit.", "The recording unit is preferably installed at the scene and is in particular arranged in spatial proximity to the camera system, preferably integrated into the camera system.", "The recording unit can include a communication unit, in particular in the form of a speech computer, for the communication with the user.", "Voice messages, in particular information, instructions, explanations and/or direction instructions, can be transmitted to the user over the telephone using the communication unit.", "The communication with the user preferably takes place with menu control, with the user being able to be guided through a menu, for example, with the help of the keypad of his mobile telephone.", "The user can be guided directly by means of the voice messages and can, for example, be prompted to look into a camera which is installed, preferably in a hidden or camouflaged manner, at the scene in order to be protected from vandalism.", "Furthermore, game rules can be explained to the user, the user can be informed of any legal conditions which may apply and the user can be advised of the start and of the end of the actual recording.", "Alternatively or additionally to a communication unit, voice messages can be transmitted to the user at least in part by a person acting as a studio manager, as a host or as a director who e.g.", "is located at a recording centre to which the user has previously set up a communication link.", "In accordance with a further preferred embodiment of the invention, in particular user data specific to the user and/or relating to the respective recording are transmitted to a recording unit, and in particular stored, over the telephone, with the keypad of the telephone preferably being used for the inputting of the data.", "The user hereby advantageously himself ensures that user data are collected which are e.g.", "required for the respective recording or for a later evaluation.", "The participants can thus fill a central database with their personal data themselves on a voluntary basis.", "The user data are for example user-specific information such as sex and age or data which relate to the respective recording.", "The user can thus, e.g.", "in the framework of a quiz game or of a poll, choose between different answers to a question in that he presses a respectively corresponding key on the telephone.", "It is furthermore preferably provided in accordance with the invention for a distinction to be made by means of stored user data and/or pre-determinable criteria between at least temporarily blocked users, on the one hand, and users to be accepted, on the other hand.", "The stored user data cannot only be the aforesaid data, but also e.g.", "the telephone number of the telephone used by the user to be called.", "This opens up the possibility e.g.", "of blocking specific mobile telephones directly and so of excluding their owners from taking part.", "The recording system can thereby be protected from troublemakers.", "In addition to this protection from vandalism, such calling users can moreover be identified who satisfy specific pre-settable selection criteria.", "If e.g.", "the telephone numbers of the callers are recorded with the respective time, then it can be ensured with the help of these data that every user is only accepted once within a pre-settable time of, for example, one day or one week.", "The functionality of the system is also ensured hereby.", "Furthermore, the stored user data can also be used for competitions in that e.g.", "the calls of the users are counted and the reaching of a specific number of calls is rewarded.", "As a result, the system security can be increased, the functionality of the system ensured and a plurality of additional use variants can be provided simultaneously by the storage of user data.", "The recording unit can include a control unit, in particular a computer supported control unit, for the communication with the camera system.", "Provision can alternatively or additionally be made for the camera system to be remote controlled e.g.", "by a recording centre spatially separated from the scene.", "This can likewise take place automatically by means of a control unit or manually by a person located at the recording centre.", "In accordance with a further preferred embodiment of the invention, a memory unit is provided, e.g.", "in the form of a computer hard disk, for the storage of recording data including at least the video data.", "The memory device is preferably installed at the scene and is in particular integrated into the camera system or into a recording unit, with the recording data in particular being stored in a database.", "Generally, video data can also be recorded on magnetic tapes such as conventional videocassettes.", "In a further variant of the invention, it is proposed that the recording unit is designed as an Internet server.", "Recording data which include at least the recorded video data can thus be stored at the scene and can be activated over the Internet.", "In accordance with a further preferred embodiment of the invention, technical recording equipment installed at the scene can be activated, deactivated and/or controlled subsequent to the setting up of the communication link, in particular via a recording unit and/or over the telephone.", "The technical recording equipment is, for example, lighting and/or sound recording equipment.", "This user-initiated operation of the recording equipment is in particular of advantage when a building, e.g.", "a restaurant, a discotheque, a cinema or an event hall, is used as the scene.", "The scene can be a part of the building made as a stage.", "In particular when the scene is a building or a building part, a telephone installed at the scene can be provided for the communication.", "Mobile telephones of the users can then be dispensed with.", "Provision is further made in accordance with the invention for recording data including at least the video data, in particular stored at the scene, to be able to be transmitted to a location spatially separated from the scene via a communication network or a data network.", "For this purpose, e.g.", "a recording unit of the camera system is simply connected to a local telephone network e.g.", "via an ISDN, ADSL, HDSL, TDSL or analogue connection.", "It is generally also possible in accordance with the invention to transmit the recorded data over a wireless communications network, in particular over a mobile radio network, from a transmitter unit located at the scene to a receiver unit spatially separated from the scene and e.g.", "located at a recording centre.", "In a variant of the invention, the recording data can be transmitted to an Internet server in order to make the recording data available on the Internet in this manner.", "It is also possible for the recording data to be transmitted to a recording centre spatially separated from the scene.", "The video data can here be subjected e.g.", "to a later processing and can in particular be put together, preferably synchronised, with picked-up audio data.", "Generally, any suitable data transmission method can be used for the transmission.", "In a further variant of the invention, provision is made for recording data at least including the video data to be broadcast on television.", "In this connection, the recordings can each form a programme part which includes e.g.", "a show act or a presentation under the participation of the calling user.", "A television programme can be put together from a plurality of recorded programme parts.", "The invention hereby makes it possible to collect programme pieces recorded during a recording period, e.g.", "during one day, at one or more scenes and to compile a television programme according to specific selection criteria from the collected programme pieces which is broadcast, for example, daily in the evening.", "This television programme can have a great variety with a minimum of production costs and show e.g.", "a singing Chinese person, a South African woman answering quiz questions, a single Swede looking for a partner and an exchange student living in the US who is greeting her parents in Germany in a single show, and indeed in each case at original scenes and without the use of labour intensive and cost intensive recording teams.", "The recorded programme parts can be subjected to a later processing.", "The programme parts can in particular be cut as required, be provided with a commentary and/or have a music soundtrack added.", "It is also alternatively or additionally possible in accordance with the invention to make the recording data available on the Internet.", "Recordings prepared e.g.", "in the framework of quiz shows, singles shows or other entertainment activities can thus be called up by everyone, and indeed either free of charge or at a charge.", "The recordings can also be used, however, for purely non-public purposes e.g.", "within a framework of recruitment, casting or poll activities, with provision being able to be made for only authorised persons to have access to the stored recording data.", "Furthermore, the camera system can be used in the manner of a so-called web cam, in that a live recording of the scene is made available at times or at least substantially without interruption on the Internet by means of the camera system.", "The user can ensure that, by means of the telephone, recording sections which can be defined by him with respect to the starting point and the end point and in which he is himself recorded are stored.", "The user can thus e.g.", "immortalise himself on the Internet, since the stored recorded sections, unlike the live transmission, are not lost.", "In accordance with a particularly preferred embodiment of the invention, provision is made that video data are recorded at a plurality of spatially separated scenes.", "In accordance with the invention, a network of scenes and camera systems spanning the world can generally be used, with only a power supply and a connection possibility to a communication network or a data network, for which a normal telephone line is sufficient, having to be present for the camera systems at the respective scene.", "Spontaneous and individual presentations or other recordings can hereby be collected with participants from all over the world.", "A variety of further application possibilities arises due to the use of a plurality of spatially separated camera systems.", "For instance, rally games an e.g.", "be carried out with the invention in which the participants have to successively visit a plurality of scenes in accordance with a predetermined procedure and e.g.", "to perform specific tasks there.", "The rally can, for example, be watched by everyone via television and/or via the Internet live or e.g.", "as part of subsequently produced reports.", "The distribution of the scenes is preferably limited to a city, to a region or to a country.", "Provision is made in accordance with the invention for the setting up of the communication link being prevented in each case outside a limited zone containing the respective scene, but no further scenes.", "A recording specific to the scene is hereby guaranteed and it is ensured that disturbances by such persons who call up from outside the respective zone under a telephone number associated solely with the respective scene are prevented.", "The limited zone is in particular a mobile radio cell covering the scene.", "The satisfaction of the object underlying the invention also takes place by the features of the independent apparatus claim and in particular takes place by a system for the recording of video data at at least one scene with at least one camera system installed at the scene to which a communication link can be set up, which is preferably specific to the scene, using a telephone located at the scene, in particular a mobile telephone of a user and which can be operated, when the communication link has been established, for the recording of video data with the user as a participant.", "Only a camera system thus has to be made available for each scene by an operator of the system.", "The further infrastructure is already present when the mobile telephones of the users and the respective mobile radio networks are used or a telephone present at the scene is used.", "Otherwise only a telephone has to be made available at the scene.", "Further preferred embodiments of both the recording method in accordance with the invention and of the recording system are set forth in the dependent claims, in the description and in the drawing.", "The invention will be described in the following by way of example with reference to the drawing whose only FIGURE shows a schematic representation of an embodiment of a recording system which is suitable for the carrying out of the recording method in accordance with the invention.", "In the variant of the invention shown, the recordings take place at a variety of individual venues or scenes 11 of which one is shown in the FIGURE by a chain-dotted line.", "Further scenes 11′, 11″, 11′″, etc.", "are indicated in the FIGURE by arrows directed to an Internet server 45 and to a recording centre 17 which will be described in more detail at another point.", "The scene 11 is a publicly accessible location, for example a square or part of a street in a town.", "Non-public scenes and scenes e.g.", "requiring an entrance fee can generally also be considered.", "The scene 11 can also be located in the interior of a building.", "A part of a building designed as a stage can, e.g., serve as the venue 11.A display device 23 is installed, e.g.", "in the form of a placard wall usually used for advertising purposes, at the scene 11.A telephone number 21, e.g.", "of a mobile radio network, which is associated exclusively with the respective scene 11, is displayed on the display device 23.Furthermore, a display unit 24, e.g.", "in the form of a red lamp or of an illuminated message “on air”, is integrated in the display device 23 and can be controlled via a recording unit 31 described in more detail in the following in order to visually display an on-going recording.", "The recording system furthermore includes a camera system 27 installed at the scene 11 which has a camera 39 and a computer controlled recording unit 31 connected to the camera 39 arranged in spatial proximity to the camera 39.The recording unit 31 includes a computer aided control unit 47 for the camera 39, an evaluation unit 37 e.g.", "in the form of a computer hard disk for the storage in particular of recording data and user data as well as a communication unit 25 in the form of a speech computer for the communication with a user 15.The camera system 27 is positioned such that the camera 39 can view the whole scene 11, or at least a part thereof, on the one hand, and the camera system 27 is protected as much as possible from vandalism, on the other hand.", "For this purpose, the camera system 27 can be located, for example, in an apartment from where there is a good view of the scene 11.The camera system 27, in particular the camera 39, is preferably positioned in a hidden or camouflaged manner such that uninitiated persons cannot discover the location of the camera 39.The recording system moreover utilises mobile telephones 13 of users 15 located at the scene 11 as well as the respective mobile radio networks for the carrying out of the recording method in accordance with the invention.", "Generally, a telephone installed at the scene 11 can also be used.", "Mobile telephones 13 can then be dispensed with.", "Any person 15 with a mobile telephone 13 who is located at the scene 11 can have themselves recorded by means of the camera system 27.For this purpose, the user 15 dials the telephone number 21 specific to the scene and displayed on the display unit 23 with his mobile telephone 13.In a preferred variant of the invention, a communication link 19 is hereby set up between the mobile telephone 13 and the local recording unit 31.In accordance with an alternative, which is indicated by broken lines in the FIGURE, the communication between a telephone 13 and the camera system 27 can also take place via a recording centre 17 which can be located at any desired place in the world and which can communicate with the camera system 27 via generally any desired communication network or data network, e.g.", "a normal telephone network.", "The functions of the recording unit 31 can also be carried out in full or in part by the recording centre 17 so that, in this respect, the recording centre 17 will not be dealt with in any more detail in the following.", "The recording system furthermore includes an Internet server 45 to which recorded data, in particular video data fed into the recording unit 31 via the camera 39, can be transmitted.", "The communication between the recording unit 31 and the Internet server 45 spatially separated from the scene 11 takes place via a normal telephone network 35.For this purpose, the recording unit 31 is connected, e.g.", "via a local ISDN connection at the scene 11, to the telephone network 35.Generally other communication networks and basically also wireless communication networks can be used for the data transmission.", "The Internet server 45 can be located at any desired location in the world.", "Irrespective of whether a recording centre 17, which may be present, is used for the control of the procedures at the scene 11 or not, recorded data can alternatively or additionally also be transmitted from the scene 11 to the recording centre 17, where the data can be prepared, processed later and compiled in a suitable manner into a programme, e.g.", "for a television broadcast, or can be evaluated in another manner and for other purposes.", "It is also possible in accordance with the invention that the recording unit 31 is itself made as an Internet server.", "Recorded and stored data can then be called up directly at the recording unit 31.The recording system furthermore includes technical recording equipment, and indeed lighting equipment 29 as well as a sound recording system 30.This recording equipment 29, 30, which is in particular used at a location 11 located in a building, can be activated, deactivated and controlled via the control unit, and indeed via corresponding communication networks 43 which are made, for example, as simple control lines.", "Audio data picked up both with the sound recording system 30 and via the microphones of the mobile telephones 13 can be stored together with video data delivered by the camera 39 in corresponding databases of the memory device 37.Moreover, pre-set selection data as well as user data can be stored in the recording unit 31.Said user data include in particular telephone numbers of telephones 13 of calling users 15 as well as data input by the users 15 themselves over the telephones 13, in particular over the telephone keypads.", "The manner of operation of the system is as follows, for example: A user 15 at the scene 11 dials with his mobile telephone 13 the telephone number 21 which is displayed on the placard wall 23 reproducing the studio of a known television show.", "An automatic check is made in the recording unit 31 with the help of the evaluation unit 49 with reference to stored telephone numbers whether the caller 15 is accepted.", "This is e.g.", "not the case if more than three calls have already been made with the telephone 13 on the same day, or more than ten calls within the last week.", "Persons calling from outside the mobile radio cell covering the scene 11 can also be excluded by applying corresponding selection criteria.", "The communication between the called camera system 2 and both blocked and accepted users 15 takes place through the speech computer 25 of the recording unit 31.If the recording centre 17 is used, then a person working as a studio manager, a host or as a director in the recording centre 17 can also talk to the user 15.The user 15 can, for example, thus be informed that he has the opportunity of a presentation, for example, to sing a song, to answer quiz questions, to introduce himself in the framework of a singles show or to take part in a poll and will thereby be recorded by a camera 39.In the case of recruitment, casting or poll activities, the user 15 can be advised of what is expected of him within the framework of the respective activity.", "If the user 15 is not aware of the existence and/or the location of the camera 39, the user 15 can also be instructed via his mobile telephone 13 to turn in a specific direction to be covered by the camera 39.Moreover, the user 15 can be directed to a specific position, for example a marked position, at the scene 11 via his mobile telephone 13.To satisfy any existing legal requirements or other provisions, the user 15 can moreover be informed of the respective legal background before the start of a recording.", "The user 15 can in particular be asked to declare his agreement to his being recorded and, optionally, to the recording being made accessible to the public and/or used commercially.", "During this preparation phase, the user 15 can be led through a menu by the recording unit 31 in which he can select from proposed menu items via speech control or via the keypad of his telephone 13.The user 15 can also be prompted to transmit specific data such as his sex, his age, his occupation, etc.", "to the recording unit 31 via the keypad of his telephone 13 in order to fill a database of the memory device 37.Any desired information can be translated into keypad inputs in this manner and stored in digital form.", "The input of information or data either by speech or via the telephone keypad is not limited to the preparation for the recording, but can also take place during the actual recording.", "The “on air” or “transmitting” display 24 is already activated automatically by the recording unit 31 directly subsequently to the setting up of the communication link 19 or at the start of the actual recording, whereby a forthcoming or ongoing recording is displayed to persons located at the scene 11.Moreover, the recording equipment 29, 39 is automatically activated by the recording unit 31 to provide an optimum lighting and to pick up surrounding sound.", "It can also be made possible for the user 15 to himself operate the recording equipment 29, 30 installed at the scene 11 and the camera system 37, at least within certain limits, via his telephone 13.The recording system is conceived such that, during the actual recording, the user 15 stands in front of the placard wall 23 which thus forms the recording background.", "The user 15 can immerse himself in the scenario pre-determined by the design of the placard wall 23, that is more less “beam” himself into the scenario, simply by calling the telephone number 21 specific to the scene.", "The start and end of the actual recording can likewise be advised to the user over his telephone 13.Video data recorded with the camera 37 during the recording, audio data picked up with the sound recording system 30 and the telephone 13, user data transmitted via the keypad of the telephone 30 and the telephone numbers of the telephones 13 uses for calls are stored in databases of the memory device 37.These recording data can be transmitted to the Internet server 45 and/or to the optionally present recording centre 17 directly subsequent to the recording via the telephone network 35 or called up at a later time.", "The recording unit 31 can be fitted with a preview function which makes it possible at least for authorised persons to view the stored video data comparatively fast with reduced image quality from any desired location.", "The putting together, in particular the synchronisation of image and sound can take place by suitable processes in the recording unit 31, at the.", "Internet server 45 or in the optionally present recording centre 17.Whereas data recorded for entertainment purposes will be made publicly accessible on the Internet or broadcast on television at no charge or at a fee, data obtained e.g.", "within the framework of poll, recruitment or casting activities can only be accessed by authorised persons, e.g.", "the respective principal.", "A programme can be compiled and broadcast at a later time from a plurality of recordings collected for television in the form e.g.", "of individual programme parts which were recorded during a recording period covering, for example, one or more days at a scene 11 or at a plurality of scenes 11, 11′, 11″, 11′″, etc." ] ]	Patent_10468664
[ [ "Environmental Performance Assessment", "Abstract of the Disclosure The present invention provides a method of assessing the sustainability performance of an entity.", "This is achieved by monitoring the operation of the entity, and using this to determine one or more sustainability indicators, each sustainability indicator being a respective value determined based on the operation of the entity.", "The sustainability indicators are then compared to respective thresholds allowing the sustainability performance to be determined in accordance with the results of the comparison." ], [ "1.(canceled).", "2.(canceled).", "3.(canceled).", "4.(canceled).", "5.(canceled).", "6.(canceled).", "7.(canceled).", "8.(canceled).", "9.(canceled).", "10.(canceled).", "11.(canceled).", "12.(canceled).", "13.(canceled).", "14.(canceled).", "15.(canceled).", "16.(canceled).", "17.(canceled).", "18.(canceled).", "19.(canceled).", "20.(canceled).", "21.(canceled).", "22.(canceled).", "23.(canceled).", "24.(canceled).", "25.(canceled).", "26.(canceled).", "27.(canceled).", "28.(canceled).", "29.(canceled).", "30.(canceled).", "31.(canceled).", "32.(canceled).", "33.(canceled).", "34.", "(new): A method for assessing the sustainability performance of an entity, wherein the method comprises: a) monitoring an operation of the entity; b) selecting at least one sustainability indicator, wherein the sustainability indicator is measure of the operation of the entity in a particular environmental area; c) comparing the selected sustainability indicator to a first threshold, wherein the first threshold is a predetermined value representing a level of efficiency relative to the selected sustainability indicator; and, d) generating an indication of the sustainability performance in accordance with the results of the comparison.", "35.", "(new): The method according to claim 35, wherein the sustainability indicator comprises at least one component value, and wherein the sustainability indicator that includes at least one component value is determined based on a weighted sum of the at least one component values.", "36.", "(new): The method according to claim 35, wherein the sustainability indicator comprises at least one of: a) an energy indicator representing an amount of energy used by the entity; b) a water indicator representing an amount of water used by the entity; and, c) a waste indicator representing an amount of waste generated by the entity.", "37.", "(new): The method according to claim 37, wherein the energy indicator comprises at least one energy component value, and wherein the energy component value represents the amount of energy used from an energy source.", "38.", "(new): The method according to claim 38, wherein the energy indicator is determined by: a) determining an energy component based on the amount of energy used from a source; b) multiplying each energy component by a parameter to determine a modified component, wherein each parameter is predetermined in accordance with the energy source; and, c) summing each of the modified energy components.", "39.", "(new): The method according to claim 35, wherein the sustainability indicator comprises at least one of: a) a social commitment indicator representing an impact of the entity on a local community; b) a resource conservation indicator representing an amount of ecological products used; and, c) a chemical indicator representing an amount of chemicals used.", "40.", "(new): The method according to claim 40, wherein the social commitment indicator is a ratio of a number of employees of the entity living within a predetermined distance of the entity to a total number of employees of the entity.", "41.", "(new): The method according to claim 40, wherein the resource conservation indicator is a ratio of a number of ecolabel products used to a total number of products used.", "42.", "(new): The method according to claim 40, wherein the chemical indicator is a ratio of an amount of biodegradable chemicals used to a total amount of chemicals used.", "43.", "(new): The method according to claim 35, wherein the sustainability indicator further comprises the presence and implementation of an environmental policy.", "44.", "(new): The method according to claim 35, wherein in response to a successful comparison in which the selected sustainability indicator is greater than or equal to the first threshold, the method further comprises: a) comparing the sustainability indicator to a second threshold, wherein the second threshold is a predetermined value representing a level of efficiency greater than the level of efficiency represented by the first threshold; and, b) generating a second indication of the sustainability performance in accordance with the results of the second comparison.", "45.", "(new): The method according to claim 35, wherein the method further comprises determining whether the comparison is successful, wherein a successful comparison is generated by the selected sustainability indicator being greater than or equal to the first threshold.", "46.", "(new): The method according to claim 46, wherein the method further comprises certifying the entity in response to a successful comparison.", "47.", "(new): The method according to claim 44, wherein the method further comprises: a) comparing the selected sustainability indicator to a second threshold, wherein the second threshold is a predetermined value representing a level of efficiency greater than the level of efficiency represented by the first threshold; and, b) determining whether the comparison of the sustainability indicator to the second threshold is successful, wherein a successful comparison is generated by the sustainability indicator being greater than or equal to the second threshold.", "48.", "(new): The method according to claim 35, wherein the first threshold is determined in accordance with at least one of: a) a location of the entity; and, b) a nature of the entity's operation.", "49.", "(new): The method according to claim 35, wherein the first threshold is determined in accordance with an average of the at least one selected sustainability indicator determined for a predetermined sample number of entities.", "50.", "(new): The method according to claim 50, wherein the first threshold is 5% higher than the average of the at least one selected sustainability indicator.", "51.", "(new): The method according to claim 50, wherein the first threshold is 30% higher than the average of the at least one selected sustainability indicator.", "52.", "(new): The method according to claim 35, wherein the monitoring of the entity is performed by the entity or by a member of the entity.", "53.", "(new): The method according to claim 35, wherein the monitoring of the entity is performed by an individual accredited by predetermined standards.", "54.", "(new): The method according to claim 35, wherein the method further comprises generating a report, and wherein the report indicates the sustainability performance of the entity by indicating information comprising the results of the comparison.", "55.", "(new): The method according to claim 55, wherein the report further indicates improvements that could be made to the operation of the entity to thereby enhance the sustainability performance of the entity.", "56.", "(new): The method according to claim 35, wherein the method is performed using a processing system comprising: a) input means adapted to receive the selected sustainability indicator; b) memory means adapted to store data representative of the first threshold; and, c) a processor, wherein the processor is adapted to: i) compare the selected sustainability indicator to the first threshold; and, ii) generate the indication of the sustainability performance in accordance with the results of the comparison.", "57.", "(new): The method according to claim 57, wherein the method further comprises causing the processor to store entity data in the memory means, and wherein the entity data comprises: a) an identity of the entity; and, b) the sustainability indicator.", "58.", "(new): The method according to claim 58, wherein the entity data further comprises: a) a location of the entity; and, b) a nature of the entity's operation.", "59.", "(new): The method according to claim 58, wherein the method further comprises determining the first threshold in accordance with the entity data stored in the store.", "60.", "(new): A system for assessing the sustainability performance of an entity, wherein the system comprises: a) input means adapted to receive at least one sustainability indicator, wherein the sustainability indicator is a measure of the operation of the entity in a particular environmental area; b) memory means adapted to store a first threshold, wherein the first threshold is a predetermined value representing a level of efficiency relative to the sustainability indicator; and, c) a processor, wherein the processor is adapted to: i) compare the sustainability indicator to the first threshold; and, ii) generate an indication of the sustainability performance in accordance with the results of the comparison.", "61.", "(new): The system according to claim 61, wherein the input means comprises a remote processing system coupled to the system via a communications network.", "62.", "(new): The system according to claim 61, wherein the system further comprises a number of interconnected processing systems.", "63.", "(new): A computer program product for assessing the sustainability performance of an entity, wherein the computer program product includes computer executable code which when executed by a suitably programmed processor causes the processor to perform the method of claim 35." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates to a method of assessing the sustainability performance of an entity, and in particular, to a method of certifying entities that attain predetermined sustainability standards." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In a first broad form the present invention provides a method of assessing the sustainability performance of an entity, the method including: a) Monitoring the operation of the entity; b) Determining one or more sustainability indicators, each sustainability indicator being a respective value determined based on the operation of the entity; c) Comparing one or more of the sustainability indicators to respective thresholds; and, d) Generating an indication of the sustainability performance in accordance with the results of the comparison.", "The sustainability indicators generally include at least one of: a) An energy indicator representing the amount of energy used by the entity; b) A water indicator representing the amount of water used by the entity; and, c) A waste indicator representing the amount of waste generated by the entity.", "Other indicators can alternatively be used, and in general the indicators used will depend on the nature of the entity being assessed.", "Typically at least one of the sustainability indicators includes one or more component values, the sustainability indicator being determined based on a weighted sum of the component values.", "This is however not essential, and some indicators may be calculated directly, for example by measurement, or from utility bills, or the like.", "Thus, for example, the energy indicator can be formed from one or more energy component values, each energy component value representing the amount of energy used from a respective energy source.", "In this case, the method of determining the energy indicator typically includes: a) Determining a respective energy component based on the amount of energy used from a respective source; b) Multiplying each energy component by a respective parameter to determine a respective modified component, each parameter being predetermined in accordance with the respective energy source; and, c) Summing each of the modified energy components.", "The sustainability indicators may also include at least one of: a) A social commitment indicator representing the impact of the entity on the local community; b) A resource conservation indicator representing the amount of ecological products used; and, c) A pollution indicator representing the amount of pollution to air, water and land.", "In this case, the social commitment indicator may be a ratio of the number of employees living within a predetermined distance of the entity to the total number of employees.", "Alternatively, the social commitment indicator might be the amount of goods purchased locally as a percentage of total goods purchased.", "The resource conservation indicator is generally a ratio of the number of ecolabel products used to the total number of products used, while the pollution indicator might be a ratio of the amount of biodegradable chemicals used to the amount of non-biodegradable chemicals used.", "The sustainability indicators may also require at least the presence and implementation of a sustainability policy.", "Typically, in response to a successful comparison, the method further includes: a) Comparing one or more of the sustainability indicators to respective second thresholds; and, b) Generating a further indication of the sustainability performance in accordance with the results of the second comparison.", "The method usually includes: a) Comparing each indicator to a respective threshold; and, b) Determining that the entity satisfies minimum requirements in response to a successful comparison for each indicator.", "Typically each sustainability indicator is normalised relative to the size of the operation.", "Thus the sustainability indicators are typically calculated as either a ratio or per unit value, such as per guest at a hotel.", "In this case, the method preferably includes: a) Comparing each indicator to a normalised curve for this indicator; and, b) Recommending improvements for each indicator in terms of this normalised curve.", "The method preferably further includes certifying the entity in response to a successful determination.", "Thus, the entity typically has to satisfy a number of comparisons before it is determined that the entity satisfies minimum sustainability requirements, thereby qualifying for certification.", "However, alternatively, each comparison could be assessed independently, so that separate certification is based on each comparison.", "Alternatively, the indicator values could be combined and the assessment performed on the basis of a single threshold comparison.", "Typically the method includes: a) Comparing each indicator to a respective second threshold; and, b) Determining that the entity satisfies best practice requirements in response to a successful second comparison for each indicator.", "This allows different levels of certification to be provided.", "It will be appreciated that any number of levels of certification may be provided as desired.", "Each threshold is typically determined in accordance with at least one of: a) The entity's location; and, b) The nature of the entity's operation.", "This allows the certification to take into account environmental factors that are location or industry specific.", "For example the impact of electricity generation on the environment will differ depending on how the electricity is generated.", "Accordingly, the effect of using electricity from the National Grid will vary depending on the entity's location.", "The effect of this can be handled by setting thresholds based on factors, such as the location or nature of the entity.", "Each threshold may be determined in accordance with an average of the respective sustainability indicators determined for a sample number of entities, although other techniques, such as studying environmental reports, building environmental impact studies, recommendations from government or other organisations or the like.", "In the case in which averages are used, the threshold can be set 5% higher than the average of the respective sustainability indicators.", "This allows the system to ensure that the entity must be above average to get the minimum level of certification.", "In this case, the second threshold can be 30% higher than the average of the respective sustainability indicators, for example.", "The entity or a member of the entity may perform the monitoring.", "Alternatively, an accredited individual could perform the monitoring.", "However, generally a mixture of the two would be used, allowing for example, the member of the entity to do the initial assessment, with the accredited individual monitoring in future years.", "Typically the method further includes generating a report, the report indicating the sustainability performance of the entity by indicating at least the results of the comparisons.", "The report may also further indicate improvements that could be made to the operation to thereby the sustainability performance of the entity.", "Typically the method is performed using a processing system including at least: a) An input for receiving the one or more sustainability indicators; b) A store for storing the respective thresholds; and, c) A processor, the processor being adapted to: i) Compare the one or more of the sustainability indicators to respective thresholds; and, ii) Generate the indication of the sustainability performance in accordance with the results of the comparison.", "In this case, the method typically further includes causing the processor to store entity data in the store, the entity data representing at least: a) The identity of the entity; and, b) The sustainability indicators.", "The entity data may also further represent at least: c) The location of the entity; and, d) The nature of the entity's operation.", "The method can then include determining the thresholds in accordance with the entity data stored in the store.", "In a second broad form the present invention provides a system for assessing the sustainability performance of an entity, the system including: a) An input for receiving one or more sustainability indicators, each sustainability indicator being a respective value determined based on the operation of the entity; b) A store for storing respective thresholds; and, c) A processor, the processor being adapted to: i) Compare the one or more of the sustainability indicators to the respective thresholds; and, ii) Generate the indication of the sustainability performance in accordance with the results of the comparison.", "In this case, the input can be formed from a remote processing system coupled to the system via a communications network, although other forms of input could also be used.", "The system can also be formed from a number of interconnected processing systems.", "Typically the system is adapted to perform the method of the first broad form of the invention.", "In a third broad form the present invention provides a computer program product for assessing the sustainability performance of an entity, the computer program product including computer executable code which when executed by a suitably programmed processor causes the processor to perform the method of the first broad form of the invention." ], [ "Detailed Description of the Invention BACKGROUND OF THE INVENTION The present invention relates to a method of assessing the sustainability performance of an entity, and in particular, to a method of certifying entities that attain predetermined sustainability standards.", "DESCRIPTION OF THE PRIOR ART The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.", "In recent years there has been a move by many companies to improve their environmental and sustainable policies, thereby making the companies more responsible.", "The driving force behind this is the realisation that many consumers are willing to sacrifice cost savings when purchasing products or services that they perceive to be more environmentally responsible.", "Thus, for example, market surveys in the US have shown that on average, US citizens will pay an additional $19.00 per person for holidays at environmentally friendly locations than other normal locations.", "Accordingly, appreciating this companies have been attempting to make themselves more responsible, for example by changing operation procedures to reduce energy and water consumption, and to produce less waste.", "A major problem with this however is that there is currently no simple way of determining how environmentally friendly an entity, such as a company is.", "In particular, whilst the company may assert that they are environmentally friendly, the consumer themselves have very little evidence of this, and therefore have to rely on trusting the company.", "This situation is detrimental to both customers and companies themselves.", "In particular, customers can be lead into believing that companies are more responsible than is actually the case.", "Similarly, companies that make a large effort to improve their environmental policy have no way of demonstrating this fact absolutely to the customers.", "This leads to the situation where customers who are willing to pay additional funds for environmentally friendly products and services are unable to judge which products and services fulfil their requirements.", "This in turn will reduce the benefit of being responsible to the companies, thereby preventing a wide scale move to environmentally friendly policies.", "SUMMARY OF THE INVENTION In a first broad form the present invention provides a method of assessing the sustainability performance of an entity, the method including: a) Monitoring the operation of the entity; b) Determining one or more sustainability indicators, each sustainability indicator being a respective value determined based on the operation of the entity; c) Comparing one or more of the sustainability indicators to respective thresholds; and, d) Generating an indication of the sustainability performance in accordance with the results of the comparison.", "The sustainability indicators generally include at least one of: a) An energy indicator representing the amount of energy used by the entity; b) A water indicator representing the amount of water used by the entity; and, c) A waste indicator representing the amount of waste generated by the entity.", "Other indicators can alternatively be used, and in general the indicators used will depend on the nature of the entity being assessed.", "Typically at least one of the sustainability indicators includes one or more component values, the sustainability indicator being determined based on a weighted sum of the component values.", "This is however not essential, and some indicators may be calculated directly, for example by measurement, or from utility bills, or the like.", "Thus, for example, the energy indicator can be formed from one or more energy component values, each energy component value representing the amount of energy used from a respective energy source.", "In this case, the method of determining the energy indicator typically includes: a) Determining a respective energy component based on the amount of energy used from a respective source; b) Multiplying each energy component by a respective parameter to determine a respective modified component, each parameter being predetermined in accordance with the respective energy source; and, c) Summing each of the modified energy components.", "The sustainability indicators may also include at least one of: a) A social commitment indicator representing the impact of the entity on the local community; b) A resource conservation indicator representing the amount of ecological products used; and, c) A pollution indicator representing the amount of pollution to air, water and land.", "In this case, the social commitment indicator may be a ratio of the number of employees living within a predetermined distance of the entity to the total number of employees.", "Alternatively, the social commitment indicator might be the amount of goods purchased locally as a percentage of total goods purchased.", "The resource conservation indicator is generally a ratio of the number of ecolabel products used to the total number of products used, while the pollution indicator might be a ratio of the amount of biodegradable chemicals used to the amount of non-biodegradable chemicals used.", "The sustainability indicators may also require at least the presence and implementation of a sustainability policy.", "Typically, in response to a successful comparison, the method further includes: a) Comparing one or more of the sustainability indicators to respective second thresholds; and, b) Generating a further indication of the sustainability performance in accordance with the results of the second comparison.", "The method usually includes: a) Comparing each indicator to a respective threshold; and, b) Determining that the entity satisfies minimum requirements in response to a successful comparison for each indicator.", "Typically each sustainability indicator is normalised relative to the size of the operation.", "Thus the sustainability indicators are typically calculated as either a ratio or per unit value, such as per guest at a hotel.", "In this case, the method preferably includes: a) Comparing each indicator to a normalised curve for this indicator; and, b) Recommending improvements for each indicator in terms of this normalised curve.", "The method preferably further includes certifying the entity in response to a successful determination.", "Thus, the entity typically has to satisfy a number of comparisons before it is determined that the entity satisfies minimum sustainability requirements, thereby qualifying for certification.", "However, alternatively, each comparison could be assessed independently, so that separate certification is based on each comparison.", "Alternatively, the indicator values could be combined and the assessment performed on the basis of a single threshold comparison.", "Typically the method includes: a) Comparing each indicator to a respective second threshold; and, b) Determining that the entity satisfies best practice requirements in response to a successful second comparison for each indicator.", "This allows different levels of certification to be provided.", "It will be appreciated that any number of levels of certification may be provided as desired.", "Each threshold is typically determined in accordance with at least one of: a) The entity's location; and, b) The nature of the entity's operation.", "This allows the certification to take into account environmental factors that are location or industry specific.", "For example the impact of electricity generation on the environment will differ depending on how the electricity is generated.", "Accordingly, the effect of using electricity from the National Grid will vary depending on the entity's location.", "The effect of this can be handled by setting thresholds based on factors, such as the location or nature of the entity.", "Each threshold may be determined in accordance with an average of the respective sustainability indicators determined for a sample number of entities, although other techniques, such as studying environmental reports, building environmental impact studies, recommendations from government or other organisations or the like.", "In the case in which averages are used, the threshold can be set 5% higher than the average of the respective sustainability indicators.", "This allows the system to ensure that the entity must be above average to get the minimum level of certification.", "In this case, the second threshold can be 30% higher than the average of the respective sustainability indicators, for example.", "The entity or a member of the entity may perform the monitoring.", "Alternatively, an accredited individual could perform the monitoring.", "However, generally a mixture of the two would be used, allowing for example, the member of the entity to do the initial assessment, with the accredited individual monitoring in future years.", "Typically the method further includes generating a report, the report indicating the sustainability performance of the entity by indicating at least the results of the comparisons.", "The report may also further indicate improvements that could be made to the operation to thereby the sustainability performance of the entity.", "Typically the method is performed using a processing system including at least: a) An input for receiving the one or more sustainability indicators; b) A store for storing the respective thresholds; and, c) A processor, the processor being adapted to: i) Compare the one or more of the sustainability indicators to respective thresholds; and, ii) Generate the indication of the sustainability performance in accordance with the results of the comparison.", "In this case, the method typically further includes causing the processor to store entity data in the store, the entity data representing at least: a) The identity of the entity; and, b) The sustainability indicators.", "The entity data may also further represent at least: c) The location of the entity; and, d) The nature of the entity's operation.", "The method can then include determining the thresholds in accordance with the entity data stored in the store.", "In a second broad form the present invention provides a system for assessing the sustainability performance of an entity, the system including: a) An input for receiving one or more sustainability indicators, each sustainability indicator being a respective value determined based on the operation of the entity; b) A store for storing respective thresholds; and, c) A processor, the processor being adapted to: i) Compare the one or more of the sustainability indicators to the respective thresholds; and, ii) Generate the indication of the sustainability performance in accordance with the results of the comparison.", "In this case, the input can be formed from a remote processing system coupled to the system via a communications network, although other forms of input could also be used.", "The system can also be formed from a number of interconnected processing systems.", "Typically the system is adapted to perform the method of the first broad form of the invention.", "In a third broad form the present invention provides a computer program product for assessing the sustainability performance of an entity, the computer program product including computer executable code which when executed by a suitably programmed processor causes the processor to perform the method of the first broad form of the invention.", "BRIEF DESCRIPTION OF THE DRAWINGS An example of the present invention will now be described with reference to the accompanying drawings, in which: FIG.", "1 is a schematic diagram of an example of a system for implementing the present invention; FIG.", "2 is a schematic diagram of an example of one of the processing system of FIG.", "1; FIG.", "3 is a schematic diagram of an example of one of the end stations of FIG.", "1; FIG.", "4 is a flow chart of an overview of the process of obtaining certification using the system of FIG.", "1; and, FIG.", "5 is a flow chart of an example of the process of obtaining certification for an entity in the tourist accommodation industry, using the system of FIG.", "1.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An example of the present invention will now be described with reference to FIG.", "1, which shows a system suitable for implementing the present invention.", "As shown, the system includes a base station 1 coupled to a number of end stations 3, via a communications network 2, and/or via a number of local area networks (LANs) 4.The base station 1 is generally formed from one or more processing systems 10 coupled to a data store 11, the data store 11 usually including a database 12, as shown.", "In use, users of the end stations 3 can access services provided by the base station 1.These services generally include allowing entities, such as companies or individuals, to obtain environmental certification, to obtain information relating to certification and improving environmental procedures, as well as allowing third parties to access details of certified entities.", "The system may be implemented using a number of different architectures.", "Thus, in one example, the communications network 2 is the Internet, with the LANs 4 representing private LANs, such internal LANs within a company or the like.", "In this case, the services provided by the base station 1 are generally made accessible via the Internet 2, and accordingly, the processing systems 10 may be capable of generating web-pages or like that can be viewed by the users of the end stations 3.Alternatively, information can be transferred between the end station 3 and the base station 1 using other techniques as represented by the dotted line.", "These other techniques may include transferring data in a hard, or printed format, as well as transferring the data electronically on a physical medium, such as a floppy disk, CD-ROM, or the like, as will be explained in more detail below.", "In any event, the processing systems 10 may be any form of processing system but typically includes a processor 20, a memory 21, an input/output (I/O) device 22 and an interface 23 coupled together via a bus 24, as shown in FIG.", "2.The interface 23, which may be a network interface card, or the like, is used to couple the processing system to the Internet 2.It will therefore be appreciated that the processing system 10 may be formed from any suitable processing system, which is capable of operating applications software to enable the provision of services.", "However, in general the processing system 10 will be formed from a server, such as a network server, web-server, or the like.", "The end stations 3 must generally be capable of receiving and processing data, as well as transferring data to the base station 1 in some cases.", "Accordingly, in this example, as shown in FIG.", "3, the end station 3 is formed from a processing system including a processor 30, a memory 31, an input/output (I/O) device 32 and an interface 33 coupled together via a bus 34.The interface 33, which may be a network interface card, or the like, is used to couple the end station 3 to the Internet 2.It will be therefore be appreciated that the end station 3 may be formed from any suitable processing system, such as a suitably programmed PC, Internet terminal, lap-top, hand-held PC, or the like.", "The end station 3 may also operate applications software to enable web-browsing or the like.", "Alternatively, the end station 3 may be formed from specialised hardware, such as an electronic touch sensitive screen coupled to a suitable processor and memory.", "In addition to this, the end station 3 may be adapted to connect to the Internet 2, or the LANs 4 via wired or wireless connections.", "It is also feasible to provide a direct connection between the base stations 1 and the end stations 3, for example if the system is implemented as a peer-2-peer network.", "In use, the system allows users of the end stations 3 to attempt to obtain environmental certification from the base station 1.The certification may be obtained for one or a group of companies, individuals, service providers, or the like (hereinafter referred to generally as an entity).", "Overview An overview of the process will now be described with reference to FIG.", "4.As shown at step 100, the first stage is for the entity to be monitored to determine a number of sustainability indicators.", "The sustainability indicators are a measure of the operation of the entity in a particular environmental area.", "Thus for example, the sustainability indicators may include an indication of the amount of energy, such as electricity, consumed, or the amount of water consumed by the entity.", "Examples of sustainability indicators are shown in Appendix A.", "In general, the sustainability indicators can be determined relatively easily.", "Thus for example, in the case of the water consumed, this can simply be measured from a water meter.", "In the case of determining the amount of energy consumed, the system allows users to provide information obtained from utility bills, outlining the amount of energy such as electricity, or gas, obtained from different sources.", "This is then added together to determine the corresponding sustainability indicator, as will be explained in more detail below.", "The sustainability indicators are entered into or calculated using the end station 3, before being transferred to the base station 1 at step 110.As mentioned above, this can happen in anyone of a number of ways depending on the architecture of the implementing system.", "Each sustainability indicator is then compared to a predetermined benchmark by the base station 1 at step 120.In general, the benchmark is intended to represent a minimum level of efficiency, or environmentally friendly operation that is required in order to obtain certification.", "As each sustainability indicator is independent, it is generally necessary to provide a distinct benchmark to correspond to each sustainability indicator.", "However, in addition to this, the environmental impact of an entity's operation may vary depending on the region in which the entity is located.", "Thus for example, in a region where fresh water is readily available, the effects of water consumption will not have such a great impact on the environment as in regions where fresh water is typically scarce.", "Accordingly, the predetermined benchmarks may also be location specific.", "Furthermore, it will be appreciated that different industries will require different benchmarks.", "In particular, it will be readily apparent that a chemical processing plant will typically require more energy and water than for example a hotel.", "Accordingly, the benchmarks may be set according to both the entity's type and location.", "In any event, once each sustainability indicator has been compared to a respective benchmark at step 130, it is determined whether each indicator exceeds the respective benchmark.", "If this is the case, the entity is certified as reaching a predetermined standard of sustainability performance at step 140.Otherwise, certification is not granted.", "In any event, whether certification is granted or not the results of the comparison are used to generate a report at step 150.This report can then be used by the entity to improve their sustainability performance subsequently.", "DETAILED DESCRIPTION OF A SPECIFIC EXAMPLE A detailed description of the operation of the system of FIG.", "1 will now be described.", "In this example, the entity will be taken to be involved in the tourist accommodation industry.", "As shown at step 200, the first stage in the process is for the entity to be monitored to determine the sustainability indicators.", "The sustainability indicators used in this particular example, together with examples of additional sustainability indicators used in other industries are shown in appendix A.", "Thus, in the tourist accommodation industry it is necessary for the respective entity to generate sustainability indicators indicating: The presence of a sustainable environmental policy; Energy consumption; Potable water consumption; Solid waste production; Social commitment; Resource conservation; Cleaning chemicals used; One optional indicator chosen from list; and, One agreed indicator nominated by entity operator.", "The exact manner in which each sustainability indicator is calculated will depend on the nature of the indicator itself.", "However, in general the sustainability indicators are determined by measuring the absolute usage for the entity and then normalising this value, such that the values for different sized entities may be directly compared to the same benchmark, as will be described in more detail below.", "Thus, for example, in the case of water consumption, the indicator requires the calculation of the water consumed per guest night or per roof area.", "This can easily be calculated by determining the total amount of water used during a predetermined time period for the entire entity.", "This value is then normalised by dividing by the number of guests staying per night, or the total roof area, as appropriate.", "Similarly, the solid waste production indicator has the calculation of the volume of waste per guest night or per area under roof.", "Again, this will require that the amount of solid waste produced be measured.", "The social commitment indicator involves determining the number of employees living within twenty kilometres and setting this as a ratio with respect of the total number of employees.", "The resource conservation indicator is an indication of the ratio of Ecolabel products purchased and normal products purchased, whilst the cleaning chemicals used is an indication of the ratio of the biodegradable chemicals to normal chemicals used.", "The only major variations on this are the presence of a sustainable environment policy indictor, which is a yes/no indication of whether an environmental policy is in place, and the energy consumption indictor.", "The energy consumption indicator is generally formed from a sum of component values, with each component value representing the energy obtained from a respective source.", "The reason for this is that energy generated from different sources will generally be in different units.", "In order to calculate the energy consumption indicator, the user provides the component values to applications software executed by the end station 3.The applications software multiplies each entered component value by a respective parameter to ensure that each of the component values are converted into the same energy unit.", "These modified components are then added to calculate a total value, which is then normalised to determine the energy consumption indicator.", "The parameters used are based on the calorific values of the various fuels and on the different units used to measure energy or amount of fuel consumed.", "The applications software can also be adapted to use the energy component values to determine the environmental impact caused as a result of the entity's energy consumption, such as for example, to determine the amount of carbon dioxide generated.", "In this case, the calculation would again sum the component values after they have multiplied by a respective parameter.", "In this instance however, the environmental impact will depend not only on the amount of energy used, but also on the manner in which it is generated.", "Accordingly, the parameters are determined based on the environmental impact of the manner in which the energy is generated.", "Thus, for example, energy obtained from the national grid will typically come from exhaustible energy supplies, such as oil, coal or natural gas.", "As this form of energy generation typically has a large environment impact, for example by generating large amounts of CO.sub.2, the parameter will generally have a high value.", "In contrast, electricity obtained for example from solar power has a lower environment impact, for example as it generates less CO.sub.2, and will therefore have a much lower parameter.", "Once all the sustainability indicators have been determined these are then entered onto the user's end station 3 at step 210.The sustainability indicators are transferred to the base station 1 at step 220, together with entity data indicating at least the nature and location of the entity.", "In the current example of tourist accommodation, the entity data would indicate tourist accommodation and the address at which the accommodation was located.", "The manner in which the data is transferred from the end station 3 to the base station 1 can vary.", "For example, the end station 3 may be accessing a website generated by the end station 1.The website includes appropriate fields allowing the user to enter the data in their respective one of the fields, thereby transferring the data to the base station 1.Alternatively, the data could be transferred electronically in the form of an e-mail, or in the form of a file, transferred using an FTP or the like.", "It is also possible for the user to transfer the information in a hard format, such as via fax, post or the like.", "This could be achieved by causing the end station 3 to print out each sustainability indicator together with the entity data on a sheet of paper which can then be forwarded to the base station 1 for manual input.", "Once the base station 1 has received the sustainability indicators and the entity data, these are typically stored in the database 12 at step 230 for future reference, as will be described in more detail below.", "In any event, at step 230 the processing system 10 operates to determine appropriate benchmarks from the database 12 in accordance with the entity data.", "As mentioned above the benchmarks used will depend on both the location and the nature of the entity.", "Accordingly, the database 12 will typically store a lookup table (LUT) which indicates for each type of entity and each entity location the respective benchmarks that should be used.", "At step 240 the processing system 10 compares each sustainability indicator to a respective first benchmark.", "The first benchmark generally indicates the minimum value of sustainability indicator that is acceptable.", "Thus, if the value of the environment locator exceeds the benchmark, this indicates that the environment policy in this particular area is unacceptable.", "Thus for example, if the energy exceeds a predetermined value, this indicates that the entity is using more energy than is environmentally desirable.", "Accordingly, in this instance the comparison would fail.", "At step 260 the processing system 10 determines if each comparison is successful.", "If not the processing system 10 uses the results to generate a report at step 270.An example report for this example is shown in Appendix C. Otherwise the processor moves on to step 280 and indicates that the entity has been awarded base line certification.", "The processing system 10 then moves on to step 290 to compare each sustainability indicator to a respective second benchmark.", "The second benchmark defines stricter criteria then the first benchmark.", "Thus, for example, the second benchmark requires that the entity uses even less water per guest night in order for a successful comparison to be obtained.", "Again, the processor determines at step 300 whether each comparison has been successful.", "If not, the processor proceeds to step 270 to generate a report.", "However, if the each comparison is successful the processing system 10 moves on to step 310 to award the entity a best practice certification before issuing the report at step 270.The processing system 10 can optionally generate a case study at step 320 as will be described in more detail below.", "Once generated the report is forwarded to the entity at step 330.As shown in Appendix C, the report will generally indicate the particular sustainability indicators that are determined for the entity, together with an indication of whether each sustainability indicator satisfies both the first and second benchmark values.", "In the event that any certification has been awarded, an indication of this will also be included in the report.", "Benchmark Calculation Benchmarks can be calculated in a number of different ways and this will typically depend on the nature of the benchmark itself.", "Typically, for example, the benchmarks are calculated by taking into account a number of different environmental and social performance areas.", "These typically include areas such as greenhouse gas generation, energy management, air quality, fresh water resources, waste water management, waste minimisation, social and cultural impact, land use management, ecosystem conservation.", "As shown in appendix B for example, the energy consumption will generally have an impact on a greenhouse gas production, energy management, air quality, waste minimisation and ecosystem conservation.", "Accordingly, it is preferable for each of these respective areas to be taken into account when generating the benchmarks.", "However, it is also possible to take into account additional information, such as environmental or governmental legislation.", "Similarly, in the case of holiday accommodation for example, certain building standards have also been issued which can be used in the calculation of the benchmarks.", "Accordingly, the calculation of the benchmarks is to a large extent a subjective procedure that will require the constant review of existing environmental policies.", "However, in addition to generating the benchmarks by considering the above factors it is also possible to generate the benchmarks based on the operation of other entities of an equivalent type and location.", "An example of the manner in which this is achieved is to determine sustainability indicators for a large number of entities.", "The value of the determined sustainability indicators are then averaged with the first benchmark being set 5% offset to the average value.", "Thus, in order to gain a base line certification, a company must be operating with at least a 5% greater efficiency than average.", "Similarly, the best practice certification can be based on at least a 30% higher efficiency than the determined average.", "In this instance, when the system is initially configured the benchmarks will have to be determined manually for example by consideration of environmental factors.", "However, once a suitable number of entities have submitted sustainability indicators, this will allow averages to be generated which can then be used to either create new benchmarks or modify existing benchmarks.", "Accordingly, it is advantageous for each of the sustainability indicators to be stored within the database 12.The processing system 10 can then monitor the number of sustainability indicators stored for a given entity type and location and then use this to generate an average when a suitable number is available.", "Additional Features A number of additional features are also available within the present invention.", "Reports The reports can be adapted to indicate improvements that have been achieved, either in comparison to previous years, or in comparison to the benchmarks.", "These improvements can be compared to a normalised curve to show the improvement relative to other entities.", "The generated reports can be tailored to provide the entity with an indication as to areas in which their environmental policy could be improved.", "Thus for example, if the energy sustainability indicator is particularly high, the report could advise the entity to not only attempt to reduce the amount of energy used, but also to obtain more energy from environmentally friendly sources.", "It will be appreciated that the comments produced may be produced manually by having an operative of the base station 1 examine each report and then provide appropriate comments.", "Alternatively however each comment could be a selected standard comment that is downloaded from the database 1 based on differences between the sustainability indicators and the benchmarks.", "Thus, for example, a large difference between the sustainability indicators and the benchmarks may indicate that a large amount of work is required by the entity.", "As a result, the processing system 10 could supply comments addressing the major forms of environmental policy that generally result in such poor performance.", "As an alternative however the operatives of the base station 1 may actually visit the entity and perform a review of the entity's procedures to provide more tailored advice.", "Auditing The above description indicates merely that the sustainability indicators are determined by monitoring the entity.", "However, this could be performed either by the entity, or in the case of a company a member of the company, or by an operative of the base station 1.In the first scenario it is theoretically possible for the entity to manipulate the energy indicator values to ensure that at least base line certification is achieved, for example.", "However, it will typically be difficult for an entity to manipulate figures successfully on a first attempt, primarily because they will be unaware of the benchmark values.", "However, having submitted sustainability indicators once, the entity may be able to guess approximate values for the benchmarks and thereby manipulate sustainability indicators in future years.", "In order to avoid this, the entity can be provided with a certification for one year on the basis of sustainability indicators provided by the entity itself.", "In this instance, to maintain the certification after one year, the entity must submit to an audit in which an operative of the base station 1 will audit the entity and review how the sustainability indicators are determined.", "The operative will perform a detailed review of the entity's operation and determine sustainability indicators based thereon, which are then applied to the method outlined above.", "This ensures that sustainability indicator values are not manipulated by the entity thereby allowing the system to ensure that the certification is only granted when certain levels of environmental procedures are virtually achieved.", "It will be appreciated that the use of an audit can be repeated at predetermined intervals as required.", "Costs Significant cost savings can be achieved by improving the efficiency of a businesses operation, which often ties in well with improving the sustainability performance.", "Thus for example, a reduction in the energy consumption of a company can lead to significant cost savings.", "Accordingly, the present invention can be adapted to provide the user with cost indications.", "Cost indications can be achieved using a look-up table that stores a base cost per unit for each sustainability indicator.", "The system can multiply each sustainability indicator by an appropriate base cost per unit, to estimate a respective cost for the entity.", "Thus, for example, in the case of the energy consumption indicator, the LUT will store an indication of the average cost of one unit of the environmental energy indicator and allow this to calculate a total cost of obtaining the energy for the entity.", "The entity can then be provided with evaluations, such as the reduction in costs that would be obtained if the entity were to meet either the first or second benchmarks.", "In addition to this, once the entity has submitted sustainability indicators over at least two years, the system can be adapted to estimate the cost saving that has been achieved by any improvements in sustainability performance from year to year.", "Case Study In order to encourage other entities to participate in the scheme, when an entity obtains a best practice certification, the base station 1 can operate to generate a case study outlining how this has been achieved.", "In particular, an operative of the base station 1 will visit the entity and assess which factors have had a major impact on obtaining the improved sustainability performance, as well as any impact this may have had on costs, or the like.", "The case study can then be published by the base station 1, for example as a web page, allowing users of the end stations 3 to access the case study and view the facts of the environmental improvement.", "Certification Search The base station 1 will typically publish a list of all entities involved in the project that have achieved at least base line or best practice certification.", "Details of these entities will be searchable in accordance with the entity type and location, allowing consumers such as holidaymakers or the like to perform searches to locate entities that have satisfied the predetermined environmental requirements.", "This allows the consumers to be certain that the products and services they are obtaining come from environmentally friendly sources.", "Architecture The present invention can be implemented using a number of different architectures.", "The architecture described above with respect to FIG.", "1 is particularly advantageous for a number of reasons.", "Firstly, the benchmarks are stored centrally on the database 12.Accordingly, as benchmarks are updated, these only need to be updated at the central location 12.Similarly, the sustainability indicators for each entity are stored centrally at the database 12.This allows benchmarks to be calculated in the manner described above.", "In addition to this, it allows the sustainability indicators obtained over previous years to be compared to current sustainability indicators thereby determining if improvements in performance have been obtained.", "Finally, this form of configuration allows the environmental data and entity data of different companies to be retained secret whilst still allowing the data to be used by the base station 1 in calculations, such as determining benchmarks.", "This allows the benchmarks to be retained secret, thereby avoiding the situation in which entities attempt to manipulate figures to thereby ensure they achieve certification.", "However, this is not essential for operation of the present invention.", "Accordingly for example, each end station could be provided with application software that includes an indication of the benchmarks.", "In this case, it would not be necessary to actually use the base station 1 at all.", "Instead, each entity could simply enter data using their appropriate application software to allow the sustainability indicators to be compared to their respective benchmarks, on the end station 3 itself.", "This would thereby obviate the need for the base station 1.However, it will be appreciated that the certification could not be controlled in this instance.", "Accordingly, the end station 3 could be adapted to generate an indication of whether the comparisons were successful and certification has been achieved.", "In this case, an indication that certification has been achieved could be transferred to a base station 1 allowing the certification to be controlled centrally.", "In this case however there is a risk that the benchmarks would become public information in order to avoid this, it would generally be necessary to ensure that the benchmarks are stored in an encrypted fashion on the end station 3.Furthermore, the transfer indication of certification would also have to be transferred in an encrypted manner to prevent individuals attempted to duplicate the indication and thereby obtain certification fraudulently.", "Finally, it will be appreciated that the method could be implemented by hand.", "However, this would not feasibly allow the benchmarks to be calculated and nor would it allow the certification to be provided in an automated fashion.", "Persons skilled in the art will appreciate that numerous variations and modifications will become apparent.", "All such variations and modifications which become apparent to persons skilled in the art, should be considered to fall within the spirit and scope that the invention broadly appearing before described.", "APPENDIX A Examples of some of the sustainability indicators that may be used are set out below.", "The example below is described with respect to the tourist accommodation industry.", "Accommodation operations can be built and run to cater for different primary markets (e.g.", "short/long stay, conventions, sports activities etc.).", "As a consequence, certain indicators, such as Energy Consumption and Potable Water Consumption, take into account the primary market and functions of the operation being benchmarked.", "Therefore if the operation: has less than 40% of floor space under roof set aside for nonaccommodation purposes (e.g.", "for offices, bars, leisure areas), these indicators will be assessed on a per guest night basis.", "has more than 40% of floor space under roof set aside for non-accommodation purposes (e.g.", "there are large sporting or convention facilities), then these indicators can be assessed on a total area under roof (m2) basis.", "Sustainability Policy Objective: Produce a clear and straightforward written policy that addresses key sustainability issues identified in predetermined standards.", "The Sustainability Policy is an operation's statement with respect to its assessment, control and where appropriate, continual improvement, of environmental and local social impacts.", "The areas that need to be covered are included in the predetermined standards.", "The base station may provide a suitable policy statement that can be adopted by operations.", "Indicator measure: A Sustainability Policy has been produced, endorsed by the operation's executive officer responsible for the entity's environmental program.", "Energy Consumption Objective: Minimize overall energy consumption.", "Significant levels of energy can be consumed by infrastructure (e.g.", "buildings, recreational facilities) and transport facilities (including customer transfer, maintenance and on-site vehicles).", "An overall reduction in energy consumed will have a positive impact on operational costs and can have major environmental benefits, primarily through conservation of natural resources and lowering associated greenhouse gas emissions.", "Energy can be consumed from a variety of sources (e.g.", "grid electricity, natural gas, gasoline, diesel) and total usage is assessed on a standard energy unit basis (Gigajoules, (if).", "Electricity consumption is often quoted in kilowatt-hours (kWh) and in the case of other sources, such as diesel, petroleum, liquefied petroleum gas (LPG) and natural gas, by volume.", "All can be readily converted to joules using GREEN GLOBE supplied conversion factors.", "Indicator measure: Total energy consumption (03) pal Guest nights pa or Area under roof (an') Greenhouse gas reductions: Reduction in emissions from energy production and distribution.", "The long-term solution to reducing energy consumption and greenhouse gas production is to introduce more efficient, less non-renewable energy intensive equipment and procedures.", "However, application of this \"cleaner production\" or \"ecoefficiency\" approach will take time.", "Additionally many operations in the Travel & Tourism industry are already energy efficient and/or further significant reductions in energy from non-renewable sources may not, for operational and commercial reasons, be feasible.", "There may be a case, therefore, for looking for alternative strategies.", "One potential option is involvement in carbon sequestration as an immediate move towards off setting greenhouse gas production.", "Participation in such schemes can be promoted as an Operation Selected Indicator.", "Many operations are making significant efforts to utilize energy from renewable sources (e.g.", "wind, solar, hydro), thereby conserving resources and minimizing greenhouse gas emissions.", "This can be also recognized through adoption of an Operation Selected Indicator that highlights the amount of renewable energy consumed pa. Potable Water Consumption Objective: Minimize consumption of potable water.", "Potable water resources can be consumed not only by drinking, but through other activities such as washing (personal and laundry), recreational facilities, gardens and cleaning of surfaces.", "Many Travel & Tourism operations are also located in regions where access to fresh water is a concern.", "Actions leading to an overall reduction in water usage (from lowering demand and/or increasing reuse and recycle) will be a significant contribution to the local environment and the long-term sustainability of the operation.", "The indicator monitors the overall efficiency of potable water usage with a view to promoting reduction without compromising the operation.", "Indicator measure: Water consumed (kL) pa / Guest nights pa or Area under roof (m2) Greenhouse gas reductions: Reduction in emissions from energy required for potable water treatment, distribution and disposal.", "Solid Waste Production Objective: Reduce the amount of solid waste generated.", "Used or waste materials sent to landfills represent a loss of resources, and their replacement will increase greenhouse gases from production and transport of their replacements.", "The first step for the operation should be to look to reduce quantities of materials consumed (including packaging), to then consider reuse, or if not possible, recycle.", "As part of the Sustainability Policy, consideration should be given to the options that have the best local environmental impact.", "For example, recycling may not always be feasible (e.g.", "no local facility) and on-site waste to energy systems may be a better route, obtaining both energy and a reduction in the volume of waste disposed (measured either as uncompacted, or mechanically compacted, material).", "Indicator measure: Volume of waste landfilled (m3) pa / Guest nights pa or Area under roof (m2) Greenhouse gas reductions: Reduction in emissions from energy required for material production, and subsequent waste transposition and disposal.", "Social Commitment Objective: Develop and maintain positive, productive and sustainable contributions to the local community.", "A key issue in achieving sustainability is to consider the social as well as environmental impact of the operation with local communities.", "Respecting, where appropriate, local traditions and customs, and purchasing where possible local goods and services are positive contributions that can be made, and should be incorporated into the operation's Sustainability Policy.", "Other considerations should include active participation in local communities and organizations.", "The indicator to monitor is the number of owners, managers and/or employees that have a primary address close to where they are based within the operation is used (for remote operations, such as on small non-populated islands, the nearest permanent township can be used instead of the operation).", "This encourages local employment and minimizes environmental impacts due to personnel transportation.", "Indicator measure: Employees with theft primary address within 20 km of the operation/Total employees Greenhouse gas reductions: Reduction in emissions from transport energy consumption.", "Resource Conservation Objective: Reduce consumption of natural resources and the impact on ecosystem biodiversity.", "An active policy of purchasing supplies of materials from sources using environmentally sound ingredients and processes can be a major contribution to resource conservation and biodiversity (i.e.", "through less impact on the balance of the local ecosystem).", "The type of paper used by the operation (e.g.", "for promotional material, stationary, toilets etc.)", "is a high profile example where significant worthwhile reductions in environmental impacts can be achieved.", "A strategy of internal reuse and recycle where possible, coupled with the use of products proven to be environmentally friendly (such as those carrying credible ecolabels) should be adopted.", "For paper, ecolabels are likely to signify avoidance of chlorine-based bleaches, use of biodegradable inks and dyes, and use of wood from sustainable plantations.", "Indicator measure: Ecolabel paper purchased (kg) pa/ Total paper purchased (kg) pa Greenhouse gas reductions: Reduction in emissions associated with virgin raw material consumption.", "Cleaning Chemicals Used Objective: Reduce chemicals discharged into the environment.", "The active (non-water) chemical ingredients of cleaning products (e.g.", "detergents, soaps, shampoos etc.)", "can end up in both wastewater (from toilets, washbasins, kitchens etc.)", "and stormwater systems (from cleaning bays, roofs, windows, car parks etc.).", "They are potential source of contamination of natural water bodies in terms of toxicity and disturbance of the natural balance of ecosystems (e.g.", "phosphates from detergents are known to contribute to eutrophication).", "Along with an overall reduction in the gross amount of chemicals consumed per annum, increased use of ecolabeled biodegradable cleaning products would be a significant step towards overall reduction in chemical contamination of the environment.", "Chemical usage is based on the relative amount of biodegradable chemical constituents in all solids and solutions used for cleaning.", "Measure: Biodegradable cleaning chemicals used (kg) pa/Total cleaning chemicals used (kg) pa Greenhouse gas reductions: Reduction in emissions from energy required for chemical production and water contamination treatment.", "OPTIONAL SUSTALINABILITY INDICATORS In addition to the indicators outlined above, a number of additional indicators can also be used.", "These may for example result in a higher level of certification if these sustainability indicators are accepted in addition to the sustainability indicators outlined above.", "Operation Selected Indicator Objective: Positive commitment to the local environment, society and economy.", "Although optional, the operation can select an indicator form a list provided by the base station.", "The indicator should be considered particularly relevant to the operation and its environmental and/or social impact, and worthy of promotion.", "This may be operation or locality specific and should reflect a commitment to improving local issues.", "Examples of possible indicators that can be selected are listed below: Operation Selected Indicator measures: 002 sequested (tonnes) pa Total CO2 generated (tonnes) pa Renewable energy consumption pal Total energy consumption pa Number of environmentally accredited operators and suppliers dealt with pa Total number of operators and suppliers dealt with pa Monetary contributions made to sponsor conservation projects pa / Net turnover of operation pa Area used for habitat conservation (ha) / Total property area (ha) Value of consumable products purchased produced locally (within country) pa Total value of consumable products purchased pa Monetary contributions made to sponsor local community activities pa/ Net turnover of operation pa Operation Specified Indicator Objective: Positive commitment to the local environment, society and economy Although again optional, the operation can specify an indicator that does not appear in the list associated with the Operation Selected Indicator.", "Again this should be considered particularly relevant to its operation and its environmental impact, and worthy of promotion.", "It should reflect the operation's commitment to improving local issues (e.g.", "water quality, endangered species, habitat preservation, cultural heritage, community development etc.).", "This indicator can be in addition to, or instead of, an Operation Selected Indicator.", "ALTERNATIVE EXAMPLES It will be appreciated that the sustainability indicators will be adapted depending on the industry to which the entity belongs.", "Accordingly, the indicator measures will typically vary to allow an appropriate value to he determined.", "Furthermore, additional sustainability indicators can be defined and combined with those outlined above in a variety of ways to service the needs of different industries, such as: Transport industries — including, airline industry, airports, bus, car and rail companies, or the like.", "Community Agriculture Service industries — including restaurants, marinas, trailer parks, golf courses and the like.", "Examples of some additional sustainability indicators suitable for use with different industries are set out below.", "Stormwater Management Objective: High quality of surface water discharged off-site.", "The operation will occupy significant tracts of land over which a range of activities occur, some of which have direct impacts on stormwater quality, including oil spillages, oil leaks, application of chemicals (e.g.", "cleaning and pesticide) and the disturbance of vegetated areas.", "Chemical and sediment runoff due to natural precipitation and hosing down activities (such as surface cleaning) should end-up in a stormwater management system, which will in turn be discharged off-site, often directly to natural watercourses (after traps), including aquifers, rather than into sewage treatment systems.", "The requirement is to firstly have stormwater management and to then monitor the effectiveness of the operations on-site control (through contamination avoidance and treatment).", "The goal is to maintain discharged water at an acceptable level, which minimizes environmental impact (including seepage into aquifers).", "The indicator is the ratio of stormwater samples passing local regulatory standards to the number of samples taken.", "Noise Nuisance Objective: Minimize social disturbance from aircraft noise.", "In recognition of the significant social nuisance value of noise, many operations now levy runway use charges against the noise associated with the aircraft.", "For example, the operation can differentiate between Chapter 2 and Chapter 3 planes by a significant difference in fees.", "Other issues that need to be considered we flight path keeping in terms of following preferential minimized noise impact routes and adherence to any flight curfews.", "To gauge the overall success of noise minimization measures, the number of complaints and proven infringements to local regulations, to the number of plane departures is monitored.", "Vehicle Management Objective Encourage operation of vehicles performing to maximum efficiency.", "The type of vehicle (size, engine capacity etc) is likely to be dictated by the local market, but the operation can still contribute to minimizing fuel consumption and associated emissions through ensuring regular maintenance as per the manufacturer's schedule.", "The indicator is the ratio of tested exhaust emissions that pass local regulatory standards to the number of services carried out, Exhaust emissions are a good guide to the efficiency of combustion, and hence fuel consumption and level of harmful exhaust gases.", "Air Quality Objective: Improve air quality through reducing local emissions from energy consumption.", "Gasses other than CO2 and particulates are discharged into the air generated when fossil fuels are burnt to produce energy, agricultural crop residues are burnt, from industrial stack emissions etc.", "These include various nitrogen oxides (NOx), which can promote smog, which in turn can lead to respiratory problems.", "Sulphur dioxide (primarily from burning Sulphur containing coal or oil at power stations) can cause acid rain and particulates less than 10 gm in diameter (PM10), lung and asthmatic problems.", "In terms of fuels, these impacts can be reduced by moving to alternatives (e.g.", "renewable or LPG) and more efficient, cleaner burning processes (including better exhaust gas cleaning, particularly at power stations).", "By using the same energy balance produced to assess the community's Energy Consumption, and a knowledge of the relative distribution of fuels among vehicle types (including aircraft and boats), using emission factors supplied by GREEN GLOBE, an estimate the amount of nitrogen oxides (NOx), sulphur dioxide (SO2) and particulates (PM10) produced can be made.", "In addition to the direct burning of fuels, assessment of the impact of any crop burning and factory emissions (typically from stacks) also need to be made, as they can also have a significant impact.", "Resource Conservation Objective: Reduction in consumption of natural resources and impact on ecosystem biodiversity.", "An active policy of careful consumption (e.g.", "minimizing wasteful practices) and purchasing supplies of materials from sources where they have been produced using environmentally sound ingredients and processes can be a major contributions by to resource conservation and biodiversity (i.e.", "through less impact on the balance of the local ecosystem).", "It is recognized, however, that obtaining such detailed information across the entire community can be extremely difficult.", "As a consequence, this indicator, unlike the others, focuses on the lead agency by assessing its consumption and purchasing policies.", "The intention is that they are in an excellent position to \"lead-by-example\".", "The quantity of paper used by the community's lead agency (e.g.", "for promotional material, stationary, toilets etc.)", "is a high profile example where significant worthwhile reduction in environmental impacts can be achieved, and a good demonstration example set.", "A strategy of internal reuse and recycle where possible, coupled to use of products that are proven to be environmentally friendly, such as those carrying credible ecolabels, should be adopted.", "The use of materials that bear ecolabels is likely to signify avoidance of non-biodegradable chemicals which can cause significant harm to the local ecosystem.", "As a consequence, two primary uses of chemicals by the lead agency, cleaning agents and pesticides are assessed in terms of their \"ecosensitivity\".", "Biodiversity Objective: Conserve native habitats and biodiversity The loss of biodiversity as a result of habitat destruction, resource depletion and pollution is a significant environmental problem, but an area's biodiversity can be extremely hard to quantify due to difficulties in obtaining credible data (e.g.", "the number of species present in an area, the size of an area's gene pool etc.", "), which in turn can make benchmarking performance problematic.", "The indicator relates to the relationship between habitat and biodiversity conservation.", "The measure is based on the percentage of land set aside for native or regenerated native vegetation and designated for conservation.", "This provides a comparable quantified indication of the area of native habitat in a community and reflects the measures being taken by the destination to preserve these habitats and their associated biodiversity.", "As this measure also encourages re-vegetation programs, it can provide additional benefits through carbon sequestration.", "Waterways Quality Objective: Improve the quality of surface water, groundwater and aquatic habitats (including the sea).", "The application of chemicals (e.g.", "biocides and fertilizers) to the land, and the discharge of effluents and sediments to water bodies can lead to the degradation of natural water resources.", "In order to assess the both the level of care taken to minimize these impacts on water resources and the subsequent monitoring of performance, the indicator is the proportion of all water samples taken in the area and analyzed that pass relevant statutory water standards.", "Travel & Tourism Objective: Assess the contribution that the local Travel & Tourism industry is making to protect the community's environment and resources.", "The prime focus of the system is to encourage the Travel & Tourism industry to make, and benefit from, worthwhile improvements in key environmental and social performance areas.", "The involvement of individual travel & tourism operations in credible environmental accreditation schemes is used, therefore, as a reflection of the level of commitment made by the local industry to the community's environment.", "Chemicals Land Applied Objective: Reduce artificial and non-biodegradable chemicals in the environment.", "Operations with large land tracts are typically high users of active chemicals (e.g.", "artificial fertilizers, herbicides and insecticides).", "Long-term application of these chemicals can lead to pollution of soils, surface water and groundwater, which can adversely affect the balance of ecosystems.", "A reduction in artificial fertilizers can be achieved by greater use of ecolabeled biodegradable products and alternative organic options, such as wastewater sludges and composted green waste.", "Artificial pesticide application can be also reduced by introducing integrated pest management programs.", "These programs develop locality specific solutions and can include practices such as using grass species suited to the locality, use of micro-organisms to fight pests and avoiding over-application of chemicals.", "Chemical usage is based on the relative amount of biodegradable chemical constituents in all solids and solutions applied to the land.", "APPENDIX B An example of the environmental effects which are considered when determining the sustainability indicators outlined above are set out below.", "Environmental & Social Performance Areas Waste- Eco- Green- Energy Fresh water Waste Social & Land use system house manage- Air water manage- minimi- cultural manage- con- gases ment quality resources ment sation impact ment servation Benchmarking (Earth Check ™) Indicators & Measures Sustainability Policy X X X X X X X X X Policy in place Energy Consumption: Energy consumed/ X X X X X Guest night or area under roof Potable Water Consumption: Water consumed/ X X X X X X Guest night or area under roof Solid Waste Production: Volume of waste/ X X X X Guest night or area under roof Social Commitment: Employees living within X X X X X 20 km/Total employees Resource Conservation: Ecolabel products X X X X X X X X purchased/Products purchased Cleaning Chemicals Used: Biodegradables X X X X X X used/Total chemicals used Optional Indicators & Measures Operation Selected Indicator: Measure selected from a predetermined list Operation Specified Indicator: Agreed measure put forward by the operation APPENDIX C An example of an assessment report issued by a certification body called GREEN GLOBE is set out below.", "Overview This assessment was undertaken against benchmarking indicators for Accommodation developed for GREEN GLOBE.", "The indicators have been carefully selected to improve environmental and social performance in key performance areas such as the reduction of energy use and the reduction of waste and potable water use.", "The indicators are practical and customized for each sector.", "As a standard policy, GREEN GLOBE will continuously improve its indicators over time, but any changes will be introduced with plenty of warning to assist customers.", "The following indicators apply to annual benchmarking of Accommodation in the GREEN GLOBE programme.", "Sustainability policy: Policy in place Energy consumption: Energy consumed/guest night or area under roof Potable water consumption: Water consumed/guest night or area under roof Solid waste production: Volume of waste/guest night or area under roof Social commitment: Employees living within 20 kms/total employees Resource conservation: Ecolabel products purchased/products purchased Cleaning chemicals used: Biodegradables used/total chemicals used Optional indicators are also provided for customers.", "They are opportunities for customizing the Benchmarking for your individual operation.", "Optional indicators are encouraged, are recognized by GREEN GLOBE, but are not used for the final Benchmarking evaluation.", "The data for the list of sustainability indicators set out above has been compiled by (name) in the format provided by GREEN GLOBE and has been submitted for assessment.", "In order to meet the annual benchmarking requirements of GREEN GLOBE and have the right to use the GREEN GLOBE logo, all benchmarks should be at Baseline or better.", "Baseline performance is calculated at 5% above per capita average performance for a nation.", "Best practice is 30% above national average performance.", "In addition, GREEN GLOBE has sector specific Benchmarking information that it will continue to upgrade.", "Such information will help to refine the assessment of baseline and best practice over time.", "Such information will be made available in GREEN GLOBE published reports in the future.", "THE BENCHMARKS 1.Sustainability policy: In Place 2.Energy consumption: ……………… GJ pa/guest night pa or area under roof Environmental & Social Performance Areas Waste- Eco- Green- Energy Fresh water Waste Social & Land use system house manage- Air water manage- minimi- cultural manage- con- gases ment quality resources ment sation impact ment servation Benchmarking (Earth Check ™) Indicators & Measures Sustainability Policy X X X X X X X X X Policy in place Energy Consumption: Energy consumed/ X X X X X Guest night or area under roof Potable Water Consumption: Water consumed/ X X X X X X Guest night or area under roof Solid Waste Production: Volume of waste/ X X X X Guest night or area under roof Social Commitment: Employees living within X X X X X 20 km/Total employees Resource Conservation: Ecolabel products X X X X X X X X purchased/Products purchased Cleaning Chemicals Used: Biodegradables X X X X X X used/Total chemicals used Optional Indicators & Measures Operation Selected Indicator: Measure selected from a predetermined list Operation Specified Indicator: Agreed measure put forward by the operation 3.Potable water consumption: ……….. Id.", "pa/guest night pa or area under roof Environmental & Social Performance Areas Waste- Eco- Green- Energy Fresh water Waste Social & Land use system house manage- Air water manage- minimi- cultural manage- con- gases ment quality resources ment sation impact ment servation Benchmarking (Earth Check ™) Indicators & Measures Sustainability Policy X X X X X X X X X Policy in place Energy Consumption: Energy consumed/ X X X X X Guest night or area under roof Potable Water Consumption: Water consumed/ X X X X X X Guest night or area under roof Solid Waste Production: Volume of waste/ X X X X Guest night or area under roof Social Commitment: Employees living within X X X X X 20 km/Total employees Resource Conservation: Ecolabel products X X X X X X X X purchased/Products purchased Cleaning Chemicals Used: Biodegradables X X X X X X used/Total chemicals used Optional Indicators & Measures Operation Selected Indicator: Measure selected from a predetermined list Operation Specified Indicator: Agreed measure put forward by the operation 4.Solid waste production: ………….. m3 pa/guest night pa or area under roof Environmental & Social Performance Areas Waste- Eco- Green- Energy Fresh water Waste Social & Land use system house manage- Air water manage- minimi- cultural manage- con- gases ment quality resources ment sation impact ment servation Benchmarking (Earth Check ™) Indicators & Measures Sustainability Policy X X X X X X X X X Policy in place Energy Consumption: Energy consumed/ X X X X X Guest night or area under roof Potable Water Consumption: Water consumed/ X X X X X X Guest night or area under roof Solid Waste Production: Volume of waste/ X X X X Guest night or area under roof Social Commitment: Employees living within X X X X X 20 km/Total employees Resource Conservation: Ecolabel products X X X X X X X X purchased/Products purchased Cleaning Chemicals Used: Biodegradables X X X X X X used/Total chemicals used Optional Indicators & Measures Operation Selected Indicator: Measure selected from a predetermined list Operation Specified Indicator: Agreed measure put forward by the operation 5.Social commitment: ……………… .", "Employees with primary address within 20km of operation/total employees Environmental & Social Performance Areas Waste- Eco- Green- Energy Fresh water Waste Social & Land use system house manage- Air water manage- minimi- cultural manage- con- gases ment quality resources ment sation impact ment servation Benchmarking (Earth Check ™) Indicators & Measures Sustainability Policy X X X X X X X X X Policy in place Energy Consumption: Energy consumed/ X X X X X Guest night or area under roof Potable Water Consumption: Water consumed/ X X X X X X Guest night or area under roof Solid Waste Production: Volume of waste/ X X X X Guest night or area under roof Social Commitment: Employees living within X X X X X 20 km/Total employees Resource Conservation: Ecolabel products X X X X X X X X purchased/Products purchased Cleaning Chemicals Used: Biodegradables X X X X X X used/Total chemicals used Optional Indicators & Measures Operation Selected Indicator: Measure selected from a predetermined list Operation Specified Indicator: Agreed measure put forward by the operation 6.Resource Conservation: …………… Ecolabel paper purchased (kg) pa/total paper purchased (kg) pa Environmental & Social Performance Areas Waste- Eco- Green- Energy Fresh water Waste Social & Land use system house manage- Air water manage- minimi- cultural manage- con- gases ment quality resources ment sation impact ment servation Benchmarking (Earth Check ™) Indicators & Measures Sustainability Policy X X X X X X X X X Policy in place Energy Consumption: Energy consumed/ X X X X X Guest night or area under roof Potable Water Consumption: Water consumed/ X X X X X X Guest night or area under roof Solid Waste Production: Volume of waste/ X X X X Guest night or area under roof Social Commitment: Employees living within X X X X X 20 km/Total employees Resource Conservation: Ecolabel products X X X X X X X X purchased/Products purchased Cleaning Chemicals Used: Biodegradables X X X X X X used/Total chemicals used Optional Indicators & Measures Operation Selected Indicator: Measure selected from a predetermined list Operation Specified Indicator: Agreed measure put forward by the operation 7.Cleaning chemicals used: …………... Biodegradable cleaning chemicals used pa/total cleaning chemicals used (kg) pa Environmental & Social Performance Areas Waste- Eco- Green- Energy Fresh water Waste Social & Land use system house manage- Air water manage- minimi- cultural manage- con- gases ment quality resources ment sation impact ment servation Benchmarking (Earth Check ™) Indicators & Measures Sustainability Policy X X X X X X X X X Policy in place Energy Consumption: Energy consumed/ X X X X X Guest night or area under roof Potable Water Consumption: Water consumed/ X X X X X X Guest night or area under roof Solid Waste Production: Volume of waste/ X X X X Guest night or area under roof Social Commitment: Employees living within X X X X X 20 km/Total employees Resource Conservation: Ecolabel products X X X X X X X X purchased/Products purchased Cleaning Chemicals Used: Biodegradables X X X X X X used/Total chemicals used Optional Indicators & Measures Operation Selected Indicator: Measure selected from a predetermined list Operation Specified Indicator: Agreed measure put forward by the operation 8.Optional Indicators: 9.CEO Endorsement of information: In Place Conclusions and Recommendation \"Accommodation Name\" has passed the GREEN GLOBE requirements to become recognized as GREEN GLOBE Accommodation for the next 12 months.", "Retention of this status in 12 months is dependent on (text as appropriate for each individual case.)" ] ]	Patent_10468668
[ [ "Water-soluble glass as corrosion protector in dishwashing machines", "A zinc-containing, water-soluble glass composition comprising from 41 to 54 mole % of P2O5, 10 to 30 mole % of alkali oxides, up to 5 mole % of SO3 and up to 25 mole % of ZnO." ], [ "1.A zinc-containing, water-soluble glass composition comprising: from 41 to 54 mole % of P2O5, 10 to 30 mole % of alkali oxides, up to 5 mole % of SO3 and up to 25 mole % of ZnO.", "2.The composition according to claim 1 characterised in that not more than 40 mole % of the total amount of alkali oxides in the composition is constituted by one or more members of the group consisting of Li2O and Na2O.", "3.The composition according to claim 1 characterised in that the composition additionally comprises at least one alkaline-earth oxide with a total amount of alkaline-earth oxides of less than 20 mole %.", "4.The composition according to claim 1 characterised in that the composition additionally comprises at least one oxide of antimony or arsenic present in the composition in an amount of of less than 5 mole %.", "5.The composition according to claim 1 characterised in that the composition additionally comprises at least one oxide of an element from the group consisting of silicium, germanium, tin and lead present in the composition in an amount of with a total amount of such oxides of less than 10 mole %.", "6.The composition according to claim 1 characterised in that the composition it additionally comprises at least one oxide of an element selected from the group consisting of silicon, aluminium and boron present in the composition in an amount of from 0.1 to 10 mole.", "7.The composition according to claim 1 characterised in that the composition does not comprise more than 0.5 mole-% of oxides of elements from the group IIIb of the Periodic System of Elements.", "8.The composition according to claim 1 characterised in that the composition comprises from 41 to 54 mole % of P2O5, from 10 to 30 mole % of alkali oxides, up to 5 mole % of SO3, up to 25 mole % of ZnO, less than 5 mole % alkaline-earth oxides, and from 0.3 to 3 mole % of oxides of elements selected from the group consisting of silicon, aluminium and boron.", "9.The composition according to claim 1 characterised in that the composition is present in the form of a transparent shaped body.", "10.The composition according to claim 9 characterised in that the shaped body is manufactured by continuous glass manufacturing processes.", "11.The composition according to claim 1 any of claims 1 to 8 characterised in that the composition it is present in a comminuted form.", "12.The composition according to claim 11 characterised in that the composition is manufactured by the breaking of thin glass plates.", "13.The composition according to claim 11 characterised in that the composition is manufactured by a process which includes a milling step.", "14.The composition according to claim 13, characterised in that the milled glass has an average particle size of not more than 500 microns.", "15.A process for the inhibition of corrosion of glassware in an automatic dishwashing process which comprises the step of: contacting said glassware with a zinc-containing, water-soluble glass composition according to claim 1.16.A process for the inhibition of corrosion of glassware in an automatic dishwashing process characterised by: providing a zinc-containing, water-soluble glass composition according to claim 1 at an appropriate place within an automatic dishwashing machine wherein the said composition is accessible to the wash liquor and/or rinse water.", "17.A process for the inhibition of corrosion of glassware in an automatic dishwashing process characterised by: contacting the glassware, in an automatic dishwashing machine, with wash liquor and/or rinse water containing an effective amount of a composition according to claim 1.18.A process for the inhibition of corrosion of glassware in an automatic dishwashing process characterised by: providing a zinc-containing, water-soluble glass composition according to claim 9 at an appropriate place within an automatic dishwashing machine wherein the said composition is accessible to the wash liquor and/or rinse water.", "19.A process for the inhibition of corrosion of glassware in an automatic dishwashing process characterised by: contacting the glassware, in an automatic dishwashing machine, with wash liquor and/or rinse water containing an effective amount of a composition according to claim 11." ], [ "The invention is related to a novel zinc-containing, water-soluble glass composition, the use thereof for inhibition of corrosion of glassware in an automatic dishwashing process and related processes.", "Corrosion of glassware in cleaning and/or rinsing cycles of an automatic dishwashing machine is a well-known problem.", "It is believed that corrosion of glassware can be seen as two separate phenomena.", "On the one hand, corrosion is obviously caused by the leakage of minerals from the glass composition, accompanied by the hydrolysis of the silicate network.", "On the other hand, deposition of silicate material on the glassware may take place.", "Those phenomena will result, after a certain number of cleaning cycles, to damages on the glassware, such as turbidity, scratches, streaks and the like.", "It is known that silicate compounds could be active against leakage of minerals from glass compositions.", "However, deposition of silicate material on the surface of glassware would be increased.", "There are different approaches in the prior art proposed for the solution of above identified problems.", "One approach is the use of zinc, either in metallic form (U.S. Pat.", "No.", "3,677,820) or in the form of zinc compounds.", "The use of soluble zinc salts for inhibition of corrosion of glassware in automatic dishwashing processes is, for example, disclosed in U.S. Pat.", "No.", "3,255,117.There are, however, various disadvantages of the use of soluble zinc salts, in particular formation of precipitates of insoluble zinc salts with other ions in the wash liquor or rinse water.", "Thus, the use of insoluble zinc compounds for the inhibition of corrosion of glassware in automatic dishwashing processes has been proposed in European Patent Application EP 0 383 480 A1, EP 0 383 482 A1 and EP 0 387 997 A1.More particularly insoluble zinc salts such as zinc silicate, zinc carbonate, zinc oxide, basic zinc carbonate, zinc hydroxide, zinc oxalate, zinc monophosphate and zinc pyrophosphate have been proposed.", "With these prior art compositions, it is disadvantageous that, due to the low solubility, or even insolubility, of the zinc compounds, it is difficult, if not impossible, to ensure an continuously sufficient amount of active corrosion inhibiting agent in the wash liquor or rinse water.", "Moreover, due to the high specific density of above mentioned insoluble zinc salts, separation problems of powder mixtures or deposition problems with liquid mixtures have occurred.", "WO 97/11151 discloses glassy particles containing agents useful for laundry and cleaning products.", "Amongst others, material care agents are disclosed such as usual corrosion inhibitors such as paraffin oil, benzotriazole, and the like.", "The agents are encapsulated in glassy particles derived from at least partially water-soluble hydroxylic compounds such as sucrose, glucose and maltodextrin.", "No zinc-containing water-soluble glass compositions are disclosed therein.", "WO 00/39259 discloses the use of water-soluble glasses as corrosion protection for glassware.", "This water-soluble glass composition comprises at least one compound, which in cleaning and/or rinsing cycles of a dishwashing machine releases a corrosion inhibiting agent.", "The solubility of the glass is defined by a mass loss of at least 0.5 mg under specified conditions.", "Amongst others, zinc-containing glasses are disclosed.", "In preferred embodiments, the glass building component is preferably phosphorous pentoxide and additionally comprises at least one alkali oxide.", "The examples disclosed therein are characterised by a content of SO3 of around 20%.", "However, the glass composition disclosed in WO 00/39259 which is based on ZnO (which is presently believed to be a very effective glassware-protecting agent) turned out to be unsatisfactory for manufacture of a consumer appealing product.", "In fact, although it is possible, under certain conditions, to manufacture a transparent glass from the composition, this glass looses its transparency rapidly after some dishwashing cycles developing an unappealing appearance.", "Related compositions obtained by variation of the components resulted in problems of producing a transparent glass in a continuous manufacturing process, which is required for production of large amounts of the glass product.", "In such processes, the raw materials for glasses are molten in a furnace at temperatures where they form a liquid glass melt with viscosities from 1 to 1000 dPas (100 dPas characterises the well know “Melting Point”) Afterwards the melt is continuously slowly cooled down and remains during a long period working temperature range where it shows a viscosity of 104 to 108 dPas.", "This is called the processing range of glass, determined by the “Working Point”, where the liquid glass shows a viscosity of 104 dPas and the “Littleton Point”, 107,6 dPas, where the shape of glass is formed by pressing or/and blowing.", "Finally, the composition reaches the glass transition temperature range (Tg) where its viscosity increases becoming a solid material.", "In this range (“Annealing Point”, viscosity of 1013 dPas and “Strain Point” 1014,6 dPas) tensions could be minimised due to annealing.", "The time during which the glass remains within the working temperature range facilitates devitrification of the glass formulation.", "In non-continuous processes (such as those employed in the manufacture of optical glasses or glasses used as fillers for plastics), the glass is quickly cooled down after it comes out of the furnace and therefore devitrification does usually not occur.", "The formulation of the glass is, however, much more critical, when manufacturing transparent glasses in a continuous process with above described prolonged cooling down periods.", "Thus, it is an object of the present invention to provide for a zinc-containing, water-soluble glass composition for use as a corrosion inhibiting product in an automatic dishwashing process which allows production of transparent glass in a continuous manufacturing process which glass maintains its transparency upon dissolution over a sufficient number of dishwashing cycles.", "This object is solved by a zinc-containing, water-soluble glass composition comprising from 41 to 54 mole % of P2O5, 10 to 30 mole % of alkali oxides, up to 5 mole % of SO3 and up t 25 mole % of ZnO.", "In a preferred embodiment of the inventive glass composition not more than 40 mole %, preferably not more than 20 mole%, most preferably not more than 1 Omole % of the total amount of alkali oxides in the glass formulation is constituted by one or more members of the group consisting of Li2O and Na2O.", "Preferably, the inventive composition additionally comprises at least one alkaline-earth oxide with a total amount of alkaline-earth oxides of less than 20 mole %, preferably less than 10 mole % and, most preferably less than 5 mole %.", "Also preferably, the inventive composition additionally comprises at least one oxide of antimony or arsenic with a total amount of such oxides of less than 5 mole %, preferably less than 3 mole %, most preferably less than 1 mole %.", "The inventive composition may comprise oxides of an element from the group consisting of silicium, germanium, tin and lead with a total amount of such oxides of less than 10 mole %, preferably less than 5 mole %, most preferably less than 3 mole %, wherein no single of such oxides is present in an amount exceeding 5 mole %, more preferably 2 mole %.", "Even more preferably, the inventive composition additionally comprises at least one oxide of an element selected from the group consisting of aluminium and boron with a total amount of such oxides of from 0.1 to 10 mole %, preferably from 0.2 to 5 mole %, most preferably from 0.3 to 3 mole %.", "It is also preferred that the glass compositions according to the present invention do not comprise more than 0.5 mole % of oxides of elements from the group IIIb of the Periodic System of Elements (i.e.", "the group comprising Scandium, Yttrium, the Lanthanide series and the Actinide series).", "The presently most preferred composition according to the invention consists of from 41 to 54 mole % of P2O5, from 10 to 30 mole % of alkali oxides, up to 5 mole % of SO3, up to 25 mole % of ZnO, less than 5 mole % of alkaline-earth oxides, and from 0.3 to 3 mole % of oxides of elements selected from the group consisting of silicon, aluminium and boron.", "In the most preferred embodiment of the invention, the composition is present in the form of a transparent shaped body, preferably manufactured by continuous glass manufacturing processes like casting, pressing or blowing.", "In an alternative, the composition is present in a comminuted form, preferably either manufactured by breaking of thin glass plates or by milling, wherein the milled glass most preferably has an average particle size of not more than 500 microns.", "The invention is specifically related to the use of the inventive glass composition for inhibition of corrosion of glassware in an automatic dishwashing process, particularly to the use of a transparent glass composition, which remains transparent upon dissolution.", "Thus, the invention is also related to processes for inhibition of corrosion of glassware in an automatic dishwashing process, either characterised by contacting the glassware, in an automatic dishwashing machine, with wash liquor and/or rinse water containing an effective amount of the inventive composition, or by providing the composition, in particular in the form of a shaped body, such as a glass block drop casted and pressed in a continuous manufacturing process, at an appropriate place within an automatic dishwashing machine being accessible for the wash liquor and/or rinse water.", "Very surprisingly, only the specific selection of components in their indicated ranges simultaneously fulfils the requirements of releasing ZnO during the dishwashing cycles in an amount enough to ensure glassware corrosion protection, providing for a dissolution rate of the glass enabling to use a block of reasonable weight for a reasonable number of washing cycles (for example, 40 g for 60 cycles), and allowing manufacture of a transparent glass block in an continuous manufacturing process which glass block does not loose its transparency during a sufficient number of dishwashing cycles.", "Although a multitude of glass formulations is known from the prior art, we are presently not aware of any such composition with this specific choice of components in the specified ranges.", "In particular, above mentioned advantageous features enabling the desired use of the glass composition for manufacturing a consumer appealing glass product for inhibition of corrosion of glassware in automatic dishwashing has neither been anticipated nor obvious for someone skilled in the art from the prior art documents related to glass formulations.", "The compositions as exemplified in the following Table have been produced in a continuous commercial glass pressing processing as outlined hereinabove.", "It is easily possible to manufacture shaped bodies of transparent glass with a weight of about 40 g. When used an automatic dishwashing machine, the glass block was completely dissolved after several dishwashing cycles.", "For example 1 a glass block with the dimensions (1×5×3) cm3 was completely dissolved after 60 cycles in the dishwasher, if you choose the conditions described in prEN 12875-1 with Calgonit Powerball Tab as detergent.", "No loss of transparency of the glass block was observed over this time period.", "The glass corrosion inhibiting activity of these glass blocks were tested according the methods as described in detail in WO 00/39259.The results were found to be at least equal, in case of the composition according to Example 1 even significantly better.", "TABLE Component Exp.", "1 [mole %] Exp.", "2 [mole %] Exp.", "3 [mole %] P2O5 50 45 43 Na2O 1 0 0 Li2O 0 3 0 K2O 27.5 25 29 ZnO 14.5 15 19 CaO 3 5 0 SO3 0 4.5 3 Sb2O3 0 0.5 0 SiO2 2 0 3 Al2O3 0.5 0 1 B2O3 1.5 2 2 Total 100 100 100 The features disclosed in the foregoing description, and the claims may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof." ] ]	Patent_10468669
[ [ "Location system and communication", "A location system comprises a plurality of transponders whose locations are detectable by a base system.", "The base system interrogates (51-55) the transponders one at a time in accordance with a schedule of consecutive time slots.", "In response to a priority request received (53) from one of the transponders, the base system interrupts the schedule and interrogates substantially immediately (56,57,55) the signalling transponder so as to determine its location with minimal latency." ], [ "1.-18.", "(canceled) 19.A location system comprising a plurality of devices, each of whose locations is detectable, and a base system for detecting and registering the locations of the devices, the base system being arranged to interrogate the locations of the devices one at a time in a sequence of time slots so that the location of each device is repeatedly interrogated and to interrogate the location of any one of at least some of the devices substantially immediately in response to a radio signal from that same device, each of the devices comprising a transponder arranged to transmit an ultrasonic signal in response to receipt of a polling signal from the base system.", "20.A location system as claimed in claim 19, in which the base system is arranged to interrogate the location of that same device in the first time slot to start after receipt of the signal from that same device.", "21.A location system as claimed in claim 19, in which the signal from that same device is a manually actuable signal.", "22.A location system as claimed in claim 19, in which the base system comprises a plurality of fixed ultrasonic transducers and a processor for determining the position of each device from the propagation times of ultrasonic energy from the device to each of at least some of the transducers.", "23.A location system as claimed in claim 19, in which the polling signal is an electromagnetic signal.", "24.A location system as claimed in claim 23, in which the electromagnetic signal is a radio signal.", "25.A location system as claimed in claim 19, in which the location of each device is detectable anywhere within a predetermined region of space.", "26.A location system comprising a plurality of devices, each of whose locations is detectable, and a base system for detecting and registering the locations of the devices, the base system being arranged to interrogate the locations of the devices one at a time in a sequence of time slots so that the location of each device is repeatedly interrogated and to interrogate the location of any one of at least some of the devices substantially immediately in response to a signal from that same device, the sequence of time slots being delayed by a time slot following the or each substantially immediate interrogation.", "27.A location system as claimed in claim 26, in which the base system is arranged to interrogate the location of that same device in the first time slot to start after receipt of the signal from that same device.", "28.A location system as claimed in claim 26, in which the signal from that same device is a manually actuable signal.", "29.A location system as claimed in claim 26, in which the signal from that same device is an electromagnetic signal.", "30.A location system as claimed in claim 29, in which the electromagnetic signal is a radio signal.", "31.A location system as claimed in claim 26, in which each of the devices comprises a transponder arranged to transmit a response to receipt of a polling signal from the base system.", "32.A location system as claimed in claim 31, in which the response is an ultrasonic signal.", "33.A detection system as claimed in claim 32, in which the base system comprises a plurality of fixed ultrasonic transducers and a processor for determining the position of each device from the propagation times of ultrasonic energy from the device to each of at least some of the transducers.", "34.A location system as claimed in claim 31, in which the polling signal is an electromagnetic signal.", "35.A location system as claimed in claim 34, in which the electromagnetic signal is a radio signal.", "36.A location system as claimed in claim 26, in which the location of each device is detectable anywhere within a predetermined region of space.", "37.A location system comprising a plurality of devices, each of whose locations is detectable, and a base system for detecting and registering the locations of the devices, the base system being arranged to interrogate the locations of the devices one at a time in a sequence of time slots so that the location of each device is repeatedly interrogated and to interrogate the location of any one of at least some of the devices substantially immediately in response to a signal from that same device, the or each substantially immediate interrogation replacing interrogation of another of the devices which would have been interrogated in the absence of the signal from that same device.", "38.A location system as claimed in claim 37, in which the base system is arranged to interrogate the location of that same device in the first time slot to start after receipt of the signal from that same device.", "39.A location system as claimed in claim 37, in which the signal from that same device is a manually actuable signal.", "40.A location system as claimed in claim 37, in which the signal from that same device is an electromagnetic signal.", "41.A location system as claimed in claim 40, in which the electromagnetic signal is a radio signal.", "42.A location system as claimed in claim 37, in which each of the devices comprises a transponder arranged to transmit a response to receipt of a polling signal from the base system.", "43.A location system as claimed in claim 42, in which the response is an ultrasonic signal.", "44.A detection system as claimed in claim 43, in which the base system comprises a plurality of fixed ultrasonic transducers and a processor for determining the position of each device from the propagation times of ultrasonic energy from the device to each of at least some of the transducers.", "45.A location system as claimed in claim 42, in which the polling signal is an electromagnetic signal.", "46.A location system as claimed in claim 45, in which the electromagnetic signal is a radio signal.", "47.A location system as claimed in claim 37, in which the location of each device is detectable anywhere within a predetermined region of space.", "48.An asynchronous time-division multiplex communication system comprising a scheduler for scheduling a plurality of messages for transmission and for transmitting substantially immediately a priority message in response to a request generated externally of the communication system.", "49.A communication system as claimed in claim 48, in which the scheduler is arranged to transmit the priority message in a first time slot beginning after receipt of the request.", "50.A communication system as claimed in claim 48, in which the priority message is generated externally of the communication system.", "51.A communication system as claimed in claim 48, in which each message comprises a data packet having a header containing destination information." ], [ "The present invention relates to a location system and to a time-division multiplex communication system.", "GB 2320089A, the contents of which are incorporated herein by reference, discloses a location system which comprises a base system and a plurality of transponders.", "The transponders comprise devices which may be carried by personnel or fixed to objects within an environment, such as an office.", "Each transponder is allocated a unique address and comprises a radio receiver for receiving polling signals from the base system.", "When the transponder receives a polling signal containing its address, it responds by emitting a burst of ultrasonic energy.", "The base system comprises a plurality of acousto-electric transducers distributed around and fixed in the environment.", "When any of the transducers receives an ultrasonic signal from a transponder, this is sent to a processor of the base system for determining and registering the location of the transponder currently being polled.", "The processor determines the position of each transponder on the basis of the time taken for the ultrasonic energy to propagate from the transponder to the transducers which detected the ultrasonic energy.", "The base system polls the transponders in accordance with a sequence with each transponder being polled in a time slot of finite duration.", "The polling signal is propagated electromagnetically and its propagation time from the base system to each transponder is so short in comparison with the speed of propagation of ultrasonic energy that it can be ignored.", "Each time slot must be sufficiently long to allow for the ultrasonic energy to propagate to all of the transducers within range and for the ultrasonic energy to decay below a threshold so as to avoid confusion with detection of the next ultrasonic burst.", "In a typical system, 40 milliseconds is sufficient to avoid false detection so that each time slot is 40 milliseconds in duration and hence the transducers can be polled at a maximum rate of 25 per second.", "There is thus a finite limit to the rate at which any transducer can be polled.", "Transducers which are attached to inanimate objects, such as furniture, computers and the like, are unlikely to be moved frequently or rapidly.", "Accordingly, it is unnecessary, and would therefore be wasteful of resources, to poll such transducers at a relatively high rate.", "However, transducers worn by personnel are likely to move more often so that it is necessary to poll such transducers more frequently in order to track the location of personnel.", "Thus, whereas it may be desirable to track transducers worn by personnel at a rate which is typically once per second, transducers attached to inanimate objects may be polled much less frequently, for example at intervals of tens of seconds.", "GB 2320089 discloses a scheduling technique based on assigning to each transponder a location quality of service (LQoS) parameter or value which indicates how frequently the transponder should be polled.", "FIG.", "1 of the accompanying drawings illustrates a simplified polling schedule for a small location system comprising a base system and four transponders.", "A first transponder (Bat 1) is given an LQoS of half, which indicates that Bat 1 should be polled or located in every second time slot.", "Another transponder (Bat 2) is assigned an LQoS of a quarter indicating that it should be polled and located in every fourth time slot.", "The other two transponders have a default value, in this case one eighth, of LQoS so as to fill the schedule.", "The polling schedule polls the transducers in the order “Bat 1, Bat 2, Bat 1, Bat 3, Bat 1, Bat 2, Bat 1, Bat 4” and repeats this sequence continuously unless and until there is a change in the LQoS value assigned to any of the transducers.", "It would be possible to exceed the capacity of such a system if too many transducers were assigned too high values of LQoS.", "In order to avoid such a situation, which might give rise to one or more of the transducers never being polled, the LQoS values are scaled so as to ensure that the polling capacity of the system is never exceeded.", "Such a scheduling technique works well for many applications, particularly for those which require a steady stream of location updates for transducers over a long period of time.", "An example of such an application is telephone-call-forwarding, which needs to know where each person wearing a transducer is located to a sufficient resolution such that telephone calls for each person can be directed to the nearest telephone.", "Such a system needs to know where each person is at any time throughout the day and polling each transducer worn by personnel every second provides sufficient location information for the telephone-call-forwarding application to work satisfactorily.", "There are some applications were such a polling sequence does not provide adequate information for correct or convenient running of the application.", "For example, GB 2 360 356, the contents of which are incorporated herein by reference, discloses an arrangement for providing, among other things, “virtual buttons”.", "In a typical example of such an application, a region of space is allocated as a virtual light switch for controlling room lighting.", "Each transponder has a manually operated button which causes the transponder to transmit a radio message to the base system.", "A transponder may be placed in the region of space for controlling the room lighting and its button actuated so as, for example, to change the state of the room lighting by toggling between on and off conditions.", "Unlike applications which require a stream of location measurements, for example a few seconds apart, such virtual button applications require infrequent locations with a very low latency after the transponder button has been actuated so that the system can respond sufficiently quickly for the particular application being controlled.", "This may be achieved by assigning to transponders which may control virtual buttons a very high LQoS value so that such transponders would be located shortly after their buttons had been actuated.", "However, such a schedule would have the disadvantage that large amounts of location resources would be continuously allocated to such transponders in case they might actually be used to control a virtual button at some point in time.", "Also, assigning high LQoS values to a substantial number of transponders would result in scaling down of all of the transponder LQoS values so as to avoid exceeding the capacity of the location system.", "According to a first aspect of the invention, there is provided a location system comprising a plurality of devices, each of whose locations is detectable, and a base system for detecting and registering the locations of the devices, the base system being arranged to interrogate the locations of the devices one at a time in a sequence of time slots so that the location of each device is repeatedly interrogated and to interrogate the location of any one of at least some of the devices substantially immediately in response to a signal from the one device.", "The base system may be arranged to interrogate the location of the one device in the first time slot to start after receipt of the signal from the one device.", "The signal from the one device may be a manually actuable signal.", "The signal from the one device may be an electromagnetic signal, such as a radio signal.", "Each of the devices may comprise a transponder arranged to transmit a response to receipt of a polling signal from the base system.", "The response may be an ultrasonic signal.", "The base system may comprise a plurality of fixed ultrasonic transducers and a processor for determining the position of each device from the propagation times of ultrasonic energy from the device to each of at least some of the transducers.", "The polling signal may be an electromagnetic signal, such as a radio signal.", "The location of each device may be detectable anywhere within a predetermined region of space.", "The sequence of time slots may be delayed by a time slot following the or each substantially immediate interrogation.", "As an alternative, the or each substantially immediate interrogation may replace interrogation of another of the devices which would have been interrogated in the absence of the signal from the one device.", "According to a second aspect of the invention, there is provided a time-division multiplex communication system comprising a scheduler for scheduling a plurality of messages for transmission and for transmitting substantially immediately a priority message in response to a request generated externally of the communication system.", "The scheduler may be arranged to transmit the priority message in a first time slot beginning after receipt of the request.", "The priority message may be generated externally of the communication system.", "The communication system may comprise an asynchronous system.", "Each message may comprise a data packet having a header containing destination information.", "It is thus possible to provide a location system which is capable of locating a device with low latency when a signal requesting such a location update is received from the device.", "Although the updating may be performed in the next time slot following receipt of the request, this may not always be necessary and it may be sufficient to locate the device within a few time slots of receipt of the signal from the device.", "This low latency updating of location measurement has little substantial effect on locating other devices which have not requested an immediate update and so provides minimum interference with scheduling of those devices which have not requested an immediate update.", "The present invention may therefore be used in combination with the techniques disclosed in GB 2320089A so as to optimise the use of the location resources available in any system while also providing the facility for a low latency location update.", "Such an arrangement is suitable for applications requiring a rapid response, for example such as the virtual button techniques disclosed in GB 2 360 356.When any of the devices is used to actuate a virtual button, it is possible to arrange for such actuation to occur with a very small or imperceptible latency.", "For example, when changing the state of room lighting, it would generally be considered undesirable or at least inconvenient to have to wait a few seconds for the room lighting status to be changed.", "Such problems can be substantially overcome or greatly eliminated by means of the present technique.", "The present invention will be further described, by way of example, with reference to the accompanying drawings, in which: FIG.", "1 illustrates a known schedule for polling transponders in a known type of location system; FIG.", "2 illustrates a location system constituting an embodiment of the invention deployed in an office environment; FIG.", "3 is a block schematic diagram of a transponder of the location system of FIG.", "2; FIG.", "4 is a block schematic diagram of a base system of the location system of FIG.", "2; FIG.", "5 is a flow diagram illustrating a first mode of operation of the location system of FIG.", "2; FIG.", "6 illustrates a polling schedule performed by the first mode of operation; FIG.", "7; is a flow diagram illustrating a second mode of operation of the location system of FIG.", "2; and FIG.", "8 illustrates a polling schedule performed by the second mode of operation.", "FIG.", "2 illustrates a room 1 forming part of an office environment or other environment provided with a transponder location system.", "This is a typical example of a three dimensional space about which personnel may move relatively freely, for example about the room 1 and from room to room of the whole environment.", "For the sake of simplicity, only a single room of the environment is illustrated.", "The room contains furniture illustrated as tables 2 and 3, a chair 4 and a filing cabinet 5.Freedom of movement of personnel is limited only by the need to avoid such obstacles and the walls defining the room 1.The room is also provided with fixtures and fittings exemplified by a door 6 and overhead lighting in the form of individual lights 7 and 8.The room also contains part of a transponder location system.", "This system comprises a computer 9 connected by any suitable communication link, such as wiring, to ceiling-mounted transducers 10 to 13 which convert ultrasonic acoustic energy into corresponding electrical signals and supply these signals to the computer 9.The system comprises a plurality of transponders which are actuated in turn by the computer 9 over a radio communication link and which respond by emitting a pulse of ultrasonic energy.", "For example, transponders 14 and 15 are located on and associated with the computer 9 and another computer or terminal 16 forming part of a local area network served by the computer 9.The locations of such transponders 14 and 15 do not, in general, vary frequently with time.", "However, these transponders serve to determine the locations of the apparatuses to which they are attached.", "Other transponders, such as that illustrated at 17, are carried by personnel such as a user 18.The positions of these transducers 17 thus vary with time according to the location of each user within the room 1.FIG.", "2 illustrates a conventional fixed light switch 19 disposed adjacent the opening for the door 6 and arranged to switch on and off the overhead lighting comprising the lights 7 and 8.FIG.", "1 also illustrates a region 20 which is selected and registered with the base system as a virtual button for controlling the room lighting in accordance with the techniques disclosed in GB 2 360 356.Thus, when the user 18 places the transponder 17 at or in the region 20 and manually actuates the transponder 17, the base system toggles the state of the room lighting provided by the lights 7 and 8.As shown in FIG.", "3, the transponder 17 comprises a radio aerial 30 connected to and shared by a radio receiver 31 and a radio transmitter 32.The aerial 30 receives transponder polling signals from the base system and these are processed by the radio receiver 31 to supply the data carried by the radio signals to a data decoder 33 which extracts transponder addresses from the signal.", "An address comparator 34 compares each address with the address of the particular transponder stored in an address memory 35.When the received address is that of the transponder, the comparator 34 actuates a signal generator 36 which energises an ultrasonic transducer 37 to emit a pulse or burst of ultrasonic energy.", "The transponder 17 comprises a manually operable push button switch 38 which, when manually actuated, causes a signal generator 39 to generate a signal based on the address of the transponder contained in the memory 35.The generated signal is supplied to the radio transmitter 32 which transmits, via the aerial 30, a priority request to the base system.", "FIG.", "4 shows in more detail the base system comprising the computer 9 for controlling the lights 7, 8 and the transducers 10 to 13.The base system comprises a radio aerial 40 connected to and shared by a radio receiver 41 and a radio transmitter 42.FIG.", "4 illustrates schematically part of the structure and operation of the computer 9 for handling the priority requests from the transponders 17.The output of the radio receiver 41 is supplied to a data decoder 43 which decodes data transmitted from the transponders and supplies transponder addresses from received signals to an address comparator 44.When the address comparator 44 receives a transponder address, it compares this with the addresses of some or all of the transponders registered in an address memory 45 of a random access memory (RAM) 46 of the computer 9.If a match is found, a block 47 instructs a priority request to a scheduler 48 which, as described in more detail hereinafter, responds by interrupting the usual schedule and requesting immediate polling to determine the location of the transponder making the priority request.", "A signal generator 49 generates the polling signal for the transponder and this is transmitted by the transmitter 42 via the aerial 40.In the absence of priority requests from any of the transponders, the base system operates in accordance with the system disclosed in GB 2320089A to poll the transponder in accordance with a schedule of the type illustrated in FIG.", "1 with the frequency of polling of each transponder being determined by a location quality of service parameter value.", "The base system is also arranged, in this example, to control the lights 7,8 by means of an appropriate driver 50 in accordance with the virtual button technique as disclosed in GB 2 360 356.One mode of operation of the location system is illustrated in FIG.", "5 in the form of a flow diagram which illustrates how the base system shown in FIG.", "4 responds to a priority request.", "A timer is reset at a step 51 and a step 52 selects the next address from the schedule for polling the next transducer in accordance with the “old schedule” illustrated in FIG.", "6, which is identical to the schedule illustrated in FIG.", "1.A step 53 determines whether a priority request has been received and, if not, a step 54 determines whether the timer has timed out.", "The steps 53 and 54 are repeated until either a priority request is received or the timer times out if no priority request is received in the time slot.", "When the timer times out, the address selected from the “old schedule” is transmitted in a step 55.The reset timer step 51 is then repeated and this cycle of operation is repeated until a priority request is received.", "When, for example, the user 18 places his transponder 17 at the region 20 which has been registered as a virtual button for controlling the room lighting and the user 18 actuates the transponder 17 by actuating the switch 38, the signal generator 39 generates a priority request signal based on the address stored in the memory 35 and comprising the address identifying the particular transponder 17 in the polling schedule.", "The priority request signal is transmitted by the transmitter 32 and the aerial 30 to the aerial 40 of the base system.", "This is detected by the receiver 41, decoded by the decoder 43 and the resulting address is compared by the address comparator 44 with each of the addresses stored in the memory 45 and representing transponders which are permitted to operate the room lighting.", "For example, all of the transponders may be permitted to affect the room lighting or only some of the transponders, such as those carried by personnel, may be registered in the memory 45 for this purpose.", "When the step 53 detects that a priority request has been received, a step 56 selects the address of the signalling transponder which made the priority request, for example by retrieving the relevant address from the memory 45 and supplying it to the scheduler 48.A step 57 effectively causes the base system to wait until the present time slot has been completed and then causes, in a step 58, the polling signal for the transponder which made the priority request to be inserted into the schedule so as to create a “new schedule” as shown in FIG.", "6.For the four transponder or “Bat” system whose scheduling is illustrated in FIG.", "1, it is assumed that Bat 3 has made the priority request.", "Thus, in the next available time slot, the location polling signal for Bat 3 is transmitted.", "The old schedule is therefore effectively interrupted in order to form location detection of Bat 3 as quickly as possible.", "When the Bat 3 address has been transmitted, a step 59 resets the timer and a step 60 restores the address which was selected in the preceding step 52.Control returns to the step 53 so that, in the absence of any further priority requests at least for time being, the previous or old schedule is restored to the new schedule but with each Bat or transponder being polled one time slot later than it would have been according to the old schedule.", "This one time slot delay is illustrated for Bats 2 and 3 in FIG.", "6.The base system thus detects the location of the transponder 17 substantially immediately after receiving a request from the transponder corresponding to actuation of the switch 38 by the user 18.The base system responds to this by toggling the state of the lights 7, 8 between the on and off conditions.", "Thus, the user 18 can operate the room lighting with a minimum or imperceptible latency.", "Although operation has been described for a system which responds to a priority request by polling the requesting transponder in the first time slot to start after receipt of the request, this may not be essential depending on the specific application of the system.", "If it necessary or convenient for the polling of the signalling transponder to be delayed by one or more time slots, this may provide a sufficiently low latency for some applications.", "Thus, it is merely required that the base system response to the priority request be sufficiently rapid to provide a sufficiently low latency response for the particular application being controlled by a virtual button or the like.", "FIG.", "7 illustrates a simplified mode of operation as compared with the mode illustrated in FIG.", "5.As shown in FIG.", "7, the steps 51 to 57 are the same as the corresponding steps in FIG.", "5.However, after the step 57, control returns to the step 55.The effect of this is illustrated in FIG.", "8, which shows the corresponding “old schedule”, (which is the same as illustrated in FIG.", "1), the resulting “new schedule” following receipt of a priority request from Bat 3, and the subsequent scheduling of the Bats 2 and 3.In this case, when a priority request is received from Bat 3, it is polled in the next time slot by the steps 56, 57 and 55.Instead of then returning to the old schedule but delayed by a time slot, polling of the Bat 4 which was selected in the preceding step 52 is replaced by the immediate polling of the Bat 3, after which the new schedule is identical to the old schedule unless and until a further priority request is received.", "Such a simplified schedule may be performed in cases where it is acceptable for polling of any of the transponders to be delayed by one complete polling cycle for the transponder.", "However, if it is desirable or essential for such delays to be avoided, then the schedule illustrated in FIG.", "6 performed by the mode of operation illustrated in FIG.", "5 should be used.", "The location system described hereinbefore may be considered as a specific and non-obvious application of a more general technique which may be employed in any time division multiplex system, such as an asynchronous system.", "In a typical asynchronous system, data to be transmitted are formed into data payloads of data packets, which are assembled with headers containing destination address data before transmission over any type of network or communication system.", "A schedule for transmission of such packets is created, for example merely by transmitting such packets in the order of receipt of the payload data.", "When such packets are received at any point within the network, they may be consumed or forwarded as appropriate.", "For example, each application running on a computer of the network checks the header of each packet which it receives to determine whether the packet is intended for that application.", "If so, the data payload is read into the application, which constitutes its destination.", "If the header in the address does not correspond to that destination, then the packet is ignored or may be forwarded through the network.", "In applications where it is required to respond to a stimulus, which for example may be external to the communication system or network, scheduling of transmission may be interrupted in the manner described hereinbefore so as to insert a packet into the order of transmission at the earliest opportunity.", "For example, when data payloads of complete packets have been scheduled for transmission in succeeding time slots, receipt of an external stimulus can cause a packet to be formed and transmitted substantially immediately, for example in the next time slot, so as to provide a low latency response to such a stimulus.", "In general, a mode of operation corresponding to that illustrated in FIGS.", "5 and 6 is necessary to ensure that no packets are lost because of the interruption in the normal or old schedule.", "A particular example of such a communication system is a system which delivers share price information from a base station to pagers owned by subscribers over an asynchronous time division multiplex radio channel.", "Each pager has a unique address and each time slot contains the address of a pager and the current share price information for the associated subscriber.", "Day traders can pay a higher subscription so as to be informed more often of their current share prices and a scheduling process allocates time slots on the radio channel to investors based on the level of subscription paid by such investors so as to ensure fair allocation of the system resources.", "It is possible to offer a premium service, which may not be available for everyone but which may be made available to nervous small-scale traders who wish to take advantage of a stock-crash feature and for which they pay a small additional fee.", "Thus, if the value of a stock in an investor's portfolio falls to less than, for example, half of what they paid for it, the scheduler allocates the next available time slot to that investor so that they may be informed of a market catastrophe with minimal latency, thus allowing the investor to sell the investment immediately.", "On Jul.", "14, 2004, European counsel forwarded to the undersigned a Declaration executed on Jul.", "5, 2004 by a second inventor, Stephen Hodges.", "That additional signed document is attached herewith.", "As stated in the original Petition, Mr. Hodges was contacted on the fourth attempt, but had not, as of the time of filing of the Petition, responded with an executed declaration.", "As recounted in the original Petition, attempts to contact the other three inventors have been altogether unsuccessful.", "The declarations executed by Messrs. Ward and Hodges include signature blocks for all the inventors of record, and have been executed by the signing inventors, leaving the signature blocks of the nonsigning inventors blank.", "The undersigned therefore respectfully submits that Messrs. Ward and Hodges have made declarations on their own behalves and on behalf of the nonsigning inventors.", "CONCLUSION In light of the above additional facts, together with those facts set forth in the original Petition, the undersigned respectfully requests that the Petition under 37 C.F.R.", "§ 1.47(a) be granted and that the submitted Declarations be accepted as fulfilling all statutory requirements.", "The undersigned hereby declares that all statements made herein of my own knowledge are true and that all statements made on information and belief are believed to be true; and further that these statements are made with the knowledge that willful false statements and the like so made are punishable by fine or imprisonment, or both, under 18 U.S.C.", "§ 1001 and that such willful false statements may jeopardize the validity of the application or any patent issued thereon." ] ]	Patent_10468690
[ [ "Anticancer agent", "An anticancer agent containing platinum preparation, humic substances, water and sodium chloride.", "Potassium tetrachloroplatinate is used as a platinum compound and lignohumic acid ammonium salts are used as humic substances taken in the following ratio of components per 1 ml of solution: lignohumic acid ammonium salts 0.18-0.22 mg potassium tetrachloroplatinate 0.020-0.040 mg sodium chloride isotonic solution 0.97-0.99 ml distilled water up to 1.0 ml" ], [ "1.An anticancer agent incorporating a platinum compound and sodium chloride, characterized in that the agent contains additional humic substances and water, using sodium chloride as a Na salt.", "2.The anticancer agent according to claim 1, wherein potassium tetrachloroplatinate is used as a platinum compound and lignohumic acid ammonium salts are used as humic substances taken in the following ratio of components: lignohumic acid ammonium salts 0.18-0.22 mg potassium tetrachloroplatinate 0.020-0.040 mg sodium chloride isotonic solution 0.97-0.99 ml distilled water up to 1.0 ml." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Antineoplastic agents that incorporate platinum complexes attract specialists' attention featuring strong anti-tumor action, which causes violation of the mechanism of malignant cells gene action.", "Platinum preparations have a wide range of antineoplastic administration.", "Cis-dichlorodiammineplatinum (DDP), being the most thoroughly investigated agent of this anti-tumor group of agents, is active against tumors of various origins: spontaneous and inoculated ones, as well as those induced by viruses and chemical carcinogens.", "The DDP major drawback is connected with its high toxicity, which causes malfunction of kidneys, red marrow and the digestive tract.", "A certain antineoplastic agent (A, RU 2086261) is known to contain a platinum complex in the form of cis-diaminodichloro-trans-dihydroxyplatinum (IV) (oxoplatinum) at a rate of 10-25% and sodium bicarbonate and sodium alginete—at respective rates of 25-55% and 40-60%.", "This agent has been designed for oral administration in pills with the total weight of 0.35-0.60 g each.", "The therapeutic dose of the agent in question has 20 to 150 mg of platinum content.", "The medicine is active in treatment of a relatively wide range of malignant diseases and can be characterized by inhibition of metastases growth and zero nephrotoxic properties.", "Sodium bicarbonate, a component of the medicinal preparation, acts as a loosening agent with respect to the pill.", "When it gets into the gastric juice acid medium, the juice undergoes neutralization and carbon dioxide evolves.", "Gastric juice neutralization prevents substitution of oxoplatinum hydroxyl groups for chloro ligands to be accompanied with tetrachloride generation.", "Resulting in sodium chloride generation, interaction between hydrochloric acid sodium bicarbonate contributes to stabilization of chlorine ions in cis-diaminodichloro-trans-dihydroxyplatinum (IV).", "Selection of sodium alginete as a filling agent is connected with its high binding properties and with the fact that alginic acid generated due to its interaction with hydrochloric acid has good compatibility with living tissues.", "Thus, oxoplatinum acts as the only effective antineoplastic substance in the agent under consideration, which accounts for the necessity of relatively high dosage concerning its administration and increases the probability of complications associated with the toxicity of platinum preparations.", "In the course of research investigation of various platinum complexes on animals and clinical trials of DDP and some of its analogues a variety of biological features characteristic of platinum complexes were discovered: anti-tumor activity and respective side effects.", "It has been shown that insignificant changes in the molecular structure of the complex are capable of causing drastic changes in the above features with respect to bioactivity, including anti-tumor activity.", "Existing relationship between the complex structure and its anti-tumor activity encourages the search for new platinum-containing agents featuring both high activity and low toxicity." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The aim of the present invention is to create such an anticancer agent which would incorporate platinum compounds of relatively low concentration and bioactive agents, and therefore would ensure both reduced toxicity of the agent and enhanced effectiveness of treatment of malignant diseases.", "The set task is solved in such a manner that the medicinal preparation contains additional humic substances and water along with platinum compounds and Na salts, sodium chloride being used as a Na salt.", "The proposed anticancer agent is a combined preparation containing both inorganic substances (platinum compounds) and organic bioactive components (humic substances).", "Platinum compounds feature marked anti-tumor activity.", "Acting as the preparation organic components, humic substances represent a large group of natural compounds.", "Their origin is connected with the hydrolytic decay of wood lignin.", "Entering into the composition of humic substances, humic acids are active components of some pharmaceuticals related to a biogenic stimulants group (e.g.", "Humisol, Befungin, and the like).", "The sodium chloride aqueous solution ensures isotonicity of the preparation medium with the medium of a living body, which enables intramuscular administration of the agent, for example.", "In the course of the research it has been discovered that the complex medicinal preparation containing the platinum preparation and humic substances demonstrates a synergetic increase in activity (shown in an example below).", "It is advisable to use lignohumic acid ammonium salts as humic substances and potassium tetrachloroplatinate as a platinum compound; it is expedient to use a Na salt in the form of sodium chloride isotonic solution in the following ratio of components per 1 ml of solution: lignohumic acid ammonium salts 0.18-0.22 mg potassium tetrachloroplatinate 0.020-0.040 mg sodium chloride isotonic solution 0.97-0.99 ml distilled water up to 1.0 ml The ratio of the proposed preparation components was chosen experimentally on grounds of maximum effectiveness under low toxicity conditions.", "Platinum content in a therapeutic dose amounts to 0.008-0.0010 mg, being significantly lower than in any of the known medicinal preparations.", "Therefore, toxic action of the proposed preparation should be many times as low.", "It has been experimentally demonstrated that with the decreasing proportion of lignohumic acid salts the preparation effectiveness reduces due to the lower stability of the platinum complex; a therapeutic effect does not intensify with the increasing proportion of lignohumic acid salts.", "Thus, the upper limit of the lignohumic acid salts content ensures the preparation maximum effectiveness.", "A reduction in the potassium tetrachloroplatinate content causes a decrease in the therapeutic effect.", "Where the potassium tetrachloroplatinate content exceeds the specified amount, the compound stability occurs, toxicity grows and the therapeutic effect decreases.", "The sodium chloride solution ensures isotonicity of the preparation medium with the medium of a living body, which enables intramuscular administration of the agent, for example.", "The amount of the sodium chloride solution is determined on the basis of conformity between the preparation osmotic pressure and the osmotic pressure of blood plasma.", "Water is used as a dissolving agent.", "The lignohumic acid salts entering into the composition of the proposed agent can be generated by liquid-phase oxidation of hydrolytic lignin with molecular oxygen in the alkaline medium (RU, A, 93037252).", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates generally to the filed of pharmacology and more particularly to anticancer agents containing platinum preparations.", "BACKGROUND OF THE INVENTION Antineoplastic agents that incorporate platinum complexes attract specialists' attention featuring strong anti-tumor action, which causes violation of the mechanism of malignant cells gene action.", "Platinum preparations have a wide range of antineoplastic administration.", "Cis-dichlorodiammineplatinum (DDP), being the most thoroughly investigated agent of this anti-tumor group of agents, is active against tumors of various origins: spontaneous and inoculated ones, as well as those induced by viruses and chemical carcinogens.", "The DDP major drawback is connected with its high toxicity, which causes malfunction of kidneys, red marrow and the digestive tract.", "A certain antineoplastic agent (A, RU 2086261) is known to contain a platinum complex in the form of cis-diaminodichloro-trans-dihydroxyplatinum (IV) (oxoplatinum) at a rate of 10-25% and sodium bicarbonate and sodium alginete—at respective rates of 25-55% and 40-60%.", "This agent has been designed for oral administration in pills with the total weight of 0.35-0.60 g each.", "The therapeutic dose of the agent in question has 20 to 150 mg of platinum content.", "The medicine is active in treatment of a relatively wide range of malignant diseases and can be characterized by inhibition of metastases growth and zero nephrotoxic properties.", "Sodium bicarbonate, a component of the medicinal preparation, acts as a loosening agent with respect to the pill.", "When it gets into the gastric juice acid medium, the juice undergoes neutralization and carbon dioxide evolves.", "Gastric juice neutralization prevents substitution of oxoplatinum hydroxyl groups for chloro ligands to be accompanied with tetrachloride generation.", "Resulting in sodium chloride generation, interaction between hydrochloric acid sodium bicarbonate contributes to stabilization of chlorine ions in cis-diaminodichloro-trans-dihydroxyplatinum (IV).", "Selection of sodium alginete as a filling agent is connected with its high binding properties and with the fact that alginic acid generated due to its interaction with hydrochloric acid has good compatibility with living tissues.", "Thus, oxoplatinum acts as the only effective antineoplastic substance in the agent under consideration, which accounts for the necessity of relatively high dosage concerning its administration and increases the probability of complications associated with the toxicity of platinum preparations.", "In the course of research investigation of various platinum complexes on animals and clinical trials of DDP and some of its analogues a variety of biological features characteristic of platinum complexes were discovered: anti-tumor activity and respective side effects.", "It has been shown that insignificant changes in the molecular structure of the complex are capable of causing drastic changes in the above features with respect to bioactivity, including anti-tumor activity.", "Existing relationship between the complex structure and its anti-tumor activity encourages the search for new platinum-containing agents featuring both high activity and low toxicity.", "SUMMARY OF THE INVENTION The aim of the present invention is to create such an anticancer agent which would incorporate platinum compounds of relatively low concentration and bioactive agents, and therefore would ensure both reduced toxicity of the agent and enhanced effectiveness of treatment of malignant diseases.", "The set task is solved in such a manner that the medicinal preparation contains additional humic substances and water along with platinum compounds and Na salts, sodium chloride being used as a Na salt.", "The proposed anticancer agent is a combined preparation containing both inorganic substances (platinum compounds) and organic bioactive components (humic substances).", "Platinum compounds feature marked anti-tumor activity.", "Acting as the preparation organic components, humic substances represent a large group of natural compounds.", "Their origin is connected with the hydrolytic decay of wood lignin.", "Entering into the composition of humic substances, humic acids are active components of some pharmaceuticals related to a biogenic stimulants group (e.g.", "Humisol, Befungin, and the like).", "The sodium chloride aqueous solution ensures isotonicity of the preparation medium with the medium of a living body, which enables intramuscular administration of the agent, for example.", "In the course of the research it has been discovered that the complex medicinal preparation containing the platinum preparation and humic substances demonstrates a synergetic increase in activity (shown in an example below).", "It is advisable to use lignohumic acid ammonium salts as humic substances and potassium tetrachloroplatinate as a platinum compound; it is expedient to use a Na salt in the form of sodium chloride isotonic solution in the following ratio of components per 1 ml of solution: lignohumic acid ammonium salts 0.18-0.22 mg potassium tetrachloroplatinate 0.020-0.040 mg sodium chloride isotonic solution 0.97-0.99 ml distilled water up to 1.0 ml The ratio of the proposed preparation components was chosen experimentally on grounds of maximum effectiveness under low toxicity conditions.", "Platinum content in a therapeutic dose amounts to 0.008-0.0010 mg, being significantly lower than in any of the known medicinal preparations.", "Therefore, toxic action of the proposed preparation should be many times as low.", "It has been experimentally demonstrated that with the decreasing proportion of lignohumic acid salts the preparation effectiveness reduces due to the lower stability of the platinum complex; a therapeutic effect does not intensify with the increasing proportion of lignohumic acid salts.", "Thus, the upper limit of the lignohumic acid salts content ensures the preparation maximum effectiveness.", "A reduction in the potassium tetrachloroplatinate content causes a decrease in the therapeutic effect.", "Where the potassium tetrachloroplatinate content exceeds the specified amount, the compound stability occurs, toxicity grows and the therapeutic effect decreases.", "The sodium chloride solution ensures isotonicity of the preparation medium with the medium of a living body, which enables intramuscular administration of the agent, for example.", "The amount of the sodium chloride solution is determined on the basis of conformity between the preparation osmotic pressure and the osmotic pressure of blood plasma.", "Water is used as a dissolving agent.", "The lignohumic acid salts entering into the composition of the proposed agent can be generated by liquid-phase oxidation of hydrolytic lignin with molecular oxygen in the alkaline medium (RU, A, 93037252).", "DETAILED DESCRIPTION The impact mechanism of the proposed anticancer agent is described as follows.", "Upon intramuscular administration the agent penetrates into blood and has an effect on hypothalamus, since a rise in endorphins concentration is detected in blood, cerebral tissues and cerebrospinal fluid as early as 20 minutes after the agent administration.", "Particularly high is the concentration of β-endorphin, a universal homeostasis regulator.", "A rise in the β-endorphin concentration in blood causes inhibition of tumor growth due to the impact on certain specific targets of malignant cell membranes.", "The endorphin increased concentration retains within several hours, resulting in a longer period of impact on the tumor.", "In addition, the proposed preparation is capable of inducing the ionophore property, or Kraun effect, which facilitates the permeability of biological membranes to platinum ions.", "This allows to use relatively low platinum concentration.", "The same effect ensures penetration of humic substances into the cell, which rapidly accumulate in the tumoral tissue.", "Having a great number of covalent active bonds, the administered solution reacts with the nucleophilic centers of protein molecules, starting the bifunctional alkylation process in the tumoral tissue nucleic acids.", "This results in long-term violation of DNA and RNA synthesis, as well as in the blocking of the mitotic cycle and enzyme systems of malignant cells, causing their destruction.", "The preparation anti-tumor action was tested on female white rats.", "EXAMPLE 1 The PA-23 rhabdomyosarcoma cell suspension was inoculated in the caudal vein of all rats, being generally used as a model for investigating the ability of such preparations to inhibit stroma development, which is characteristic of most malignant tumors.", "All the animals were divided into 5 groups.", "On the following day after the inoculation a physiological solution was intra-abdominal injected into the rats of Group 1 (0.5 ml per animal); 0.1 ml of the preparation—into the rats of Group 2, 0.3 ml of the preparation—into the rats of Group 3; 0.5 ml of the preparation—into the rats of Group 4; and 0.8 ml of the preparation—into the rats of Group 5.Prior to administration the preparation had been diluted with a physiological solution in the ratio of 1 to 10.Subsequent administration of the preparation (as a placebo control) was carried out every third day.", "On the 20 th day following the tumoral inoculation the rats were destroyed.", "Their bodies were dissected, the number of metastases in their lungs calculated and their respective weight determined by means of analytical balance weighing.", "The Student's t-test was applied to the statistical treatment of the experimental results as shown in Table 1.TABLE 1 Sodium chloride isotonic Metastasizing and solution Anticancer preparation, ml metastases growth Group 1 0.1 0.3 0.5 0.8 indicators (Control) Group 2 Group 3 Group 4 Group 5 Number of rats per 19 20 16 17 19 group Number of 595 616 413 529 579 metastases Average number of 31.3 ± 6.8 30.8 ± 25.8 ± 31.1 ± 30.5 ± 9.5 metastases per rat 9.3 4.5 9.4 Average weight of 50.7 ± 2.6 37.5 ± 42.8 ± 39.0 ± 37.2 ± 3.3 1 metastasis, mg, 3.1 2.9 3.4 % of the 100 74.0 84.4 76.9 73.4 Control Group The above data indicates that the preparation did not have any effect on the frequency of metastases formation in lungs—their number was virtually equal in all of the groups.", "At the same time the preparation had a marked impact on the metastases growth—in all pilot groups the average weight of metastases was less than that of the control group and the percentage of the metastases growth inhibition was within the range of 15 to 26%.", "Thus, the preparation represses the ability of malignant cells to develop stroma with blood vessels, which subsequently results in destruction of the malignant cells themselves.", "At the same time an increase in the preparation dosage did not have any essential influence upon the end result.", "EXAMPLE 2 As in Example 1, the PA-23 rhabdomyosarcomas were used as a model.", "Tumoral inoculation was performed in a similar way.", "The laboratory animals were also divided into groups.", "The sodium chloride isotonic solution was injected into Group 1 (control), potassium tetrachloroplatinate in the sodium chloride isotonic solution—into Group 2, ammonium lignohumic acid salts in the sodium chloride isotonic solution were injected into Group 3, injections of the proposed preparation diluted with a physiological solution in the ratio of 1 to 10 were given to Group 4.The results are shown in Table 2.TABLE 2 Sodium Potassium chloride tetrachloro- Lignohumic Metastasizing and solution platinate acid salts Anticancer metastases growth (Control) solution solution preparation indicators 0.3 ml 0.003 mg/ml 0.020 mg/ml 0.3 ml Number of rats 16 16 17 18 per group Number of 547 531 539 506 metastases Average number 34.2 ± 6.4 33.2 ± 6.2 31.9 ± 6.5 28.1 ± 5.6 of metastases per rat Average weight 48.5 ± 2.8 42.9 ± 4.3 37.8 ± 5.2* 28.4 ± 3.6* of 1 metastasis, mg % of the Control 100 88.5 77.9 58.6 Group *The difference is significant with respect to the Control Group (probability less than 0.05).", "As follows from Table 2, compared to the Control Group, the average weight of 1 metastasis constitutes 58.6% with the proposed preparation applied, whereas these figures constitute 88.5 and 77.9% respectively with the use of its separate components (potassium tetrachloroplatinate and the lignohumic acid salts solution).", "The figures demonstrate the synergetic effect of the activity related to the preparation components.", "Thus, the proposed anticancer agent has a marked antineoplastic action which destroys the mechanism of malignant cells gene action and finally leads to the cells destruction." ] ]	Patent_10468724
[ [ "Method for producing hydrogen gas", "A monometal (1) is contacted with deuterated acidic water solution (2) in which at least some of hydrogen atoms contained in acidic water solution are substituted for deuterium atoms, thereby to generate hydrogen gas.", "With this, a great amount of hydrogen gas can be generated in a short period of time." ], [ "1.A method for producing hydrogen gas, comprising the step of contacting a monometal with deuterated acidic water solution in which at least some of hydrogen atoms contained in acidic water solution are substituted for deuterium atoms, thereby to generate hydrogen gas.", "2.The method for producing hydrogen according to claim 1, wherein said acidic water solution contains at least one kind of acid selected from the group consisting of citric acid, glycine, cinnamic acid, succinic acid, salicylic acid, formic acid, glutamic acid, ascorbic acid, oxalic acid, tartaric acid, lactic acid, acetic acid, sulfuric acid, hydrochloric acid, and nitric acid." ], [ "<SOH> BACKGROUND ART <EOH>Hydrogen gas can be generated by e.g.", "immersing metal in acidic water solution.", "The sole conventional concept was generating hydrogen gas by immersing metal in acidic water solution comprising normal water and an acid component.", "With the above-described conventional method, however, the generation rate of hydrogen is low and it is not possible to generate a large amount of hydrogen gas in a short period of time.", "Hence, this method requires room for improvement, if it is to be put into actual use for supplying various kinds of hydrogen gas.", "The present invention has been made in view of the above-described state of the art.", "The primary object of the invention is to provide a method for producing hydrogen gas which method allows a great amount of hydrogen gas to be generated in a short period of time." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 is an explanatory view in partial section showing an example of contacting condition between a deuterated acidic water solution and a monometal, FIG.", "2 is a graph showing changes over time in the hydrogen generation amount in comparison between a case using the deuterated sulfuric acid water solution and Mg metal and a further case using standard sulfuric acid water solution for industrial use and the Mg metal, FIG.", "3 is a graph showing hydrogen gas generation amounts per a predetermined unit time in comparison between the case using the deuterated sulfuric acid water solution and Mg metal and the further case using standard sulfuric acid water solution for industrial use and the Mg metal, FIG.", "4 is a graph showing hydrogen gas generation amounts per a predetermined unit time in comparison between the case using the deuterated sulfuric acid water solution and Mg metal and a further case using the deuterated sulfuric acid water solution and Zn metal, FIG.", "5 is an explanatory view in partial section showing an example of contacting condition between a deuterated acidic water solution and a monometal relating to a further embodiment, FIG.", "6 is a graph showing the hydrogen generation amount relating to a still further embodiment, FIG.", "7 is a graph showing pH variation in the deuterated sulfuric acid water solution relating to a still further embodiment, and FIG.", "8 a graph showing the hydrogen generation amount relating to a still further embodiment.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to a method for producing hydrogen gas.", "BACKGROUND ART Hydrogen gas can be generated by e.g.", "immersing metal in acidic water solution.", "The sole conventional concept was generating hydrogen gas by immersing metal in acidic water solution comprising normal water and an acid component.", "With the above-described conventional method, however, the generation rate of hydrogen is low and it is not possible to generate a large amount of hydrogen gas in a short period of time.", "Hence, this method requires room for improvement, if it is to be put into actual use for supplying various kinds of hydrogen gas.", "The present invention has been made in view of the above-described state of the art.", "The primary object of the invention is to provide a method for producing hydrogen gas which method allows a great amount of hydrogen gas to be generated in a short period of time.", "DISCLOSURE OF THE INVENTION According to a method relating to claim 1, as illustrated in FIG.", "1, the method comprises the step of contacting a monometal 1 with deuterated acidic water solution 2 in which at least some of hydrogen atoms contained in acidic water solution are substituted for deuterium atoms, thereby to generate hydrogen gas.", "With the above method relating to claim 1, the monometal is contacted with the deuterated acidic water solution in which at least some of hydrogen atoms contained in acidic water solution are substituted for deuterium atoms.", "Then, the deuterated component contained in the deuterated acidic water solution activates the oxidation reaction of the monometal exposed to this deuterated acidic water solution, thus promoting the generation of hydrogen gas due to this oxidation reaction.", "As a result, a great amount of hydrogen gas can be generated in a short period of time.", "According to a presumable theory, at the portion of the monometal contacting the deuterated acidic water solution, the monometal is dissolved by the deuterated acidic water solution, while the reaction for generation of the hydrogen gas from the monometal portion contacting the deuterated acidic water solution is activated by the deuterated component, whereby the generation of the hydrogen gas is further promoted, thereby generating a great amount of hydrogen gas in a short period of time.", "Incidentally, it is needless to say that the deuterated acidic water solution in which at least some of the hydrogen atoms contained in acidic water solution are substituted for deuterium atoms contains a greater amount of deuterium atoms than naturally present.", "According to a method relating to claim 2, in the method of claim 1, said acidic water solution contains at least one kind of acid selected from the group consisting of citric acid, glycine, cinnamic acid, succinic acid, salicylic acid, formic acid, glutamic acid, ascorbic acid, oxalic acid, tartaric acid, lactic acid, acetic acid, sulfuric acid, hydrochloric acid, and nitric acid.", "With the above method relating to claim 2, by using, as the acidic water solution, a relatively easily available acidic solution of e.g.", "citric acid, glycine, cinnamic acid, succinic acid, salicylic acid, formic acid, glutamic acid, ascorbic acid, oxalic acid, tartaric acid, lactic acid, acetic acid, sulfuric acid, hydrochloric acid, and nitric acid, the hydrogen gas can be produced easily as well as economically.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is an explanatory view in partial section showing an example of contacting condition between a deuterated acidic water solution and a monometal, FIG.", "2 is a graph showing changes over time in the hydrogen generation amount in comparison between a case using the deuterated sulfuric acid water solution and Mg metal and a further case using standard sulfuric acid water solution for industrial use and the Mg metal, FIG.", "3 is a graph showing hydrogen gas generation amounts per a predetermined unit time in comparison between the case using the deuterated sulfuric acid water solution and Mg metal and the further case using standard sulfuric acid water solution for industrial use and the Mg metal, FIG.", "4 is a graph showing hydrogen gas generation amounts per a predetermined unit time in comparison between the case using the deuterated sulfuric acid water solution and Mg metal and a further case using the deuterated sulfuric acid water solution and Zn metal, FIG.", "5 is an explanatory view in partial section showing an example of contacting condition between a deuterated acidic water solution and a monometal relating to a further embodiment, FIG.", "6 is a graph showing the hydrogen generation amount relating to a still further embodiment, FIG.", "7 is a graph showing pH variation in the deuterated sulfuric acid water solution relating to a still further embodiment, and FIG.", "8 a graph showing the hydrogen generation amount relating to a still further embodiment.", "BEST MODE OF EMBODYING THE INVENTION Next, an embodiment of a method for producing hydrogen gas relating to the present invention will be described.", "As an example of deuterated acidic water solution, there was employed deuterated sulfuric acid water solution in which some of hydrogen atoms contained in sulfuric acid water solution were substituted for deuterium atoms.", "And, as an example of monometal, magnesium metal (to be referred to as “Mg metal” hereinafter) 1 was employed.", "As an example, as illustrated in FIG.", "1, into 500 ml of the deuterated sulfuric acid water solution 2 (pH=0.4), the Mg metal (a plate-like metal piece having a surface area of about 15.7 cm2) 1 was immersed to generate hydrogen gas.", "In this, FIG.", "2 shows the relationship between the total amount (yield) of the hydrogen gas generated and the time lapsed and FIG.", "3 shows the generation amount of hydrogen gas during period of 16 minutes after the immersion of the Mg metal.", "For comparison, these figures also show the results of a further case in which Mg metal (a plate-like metal piece having a surface area of about 16.0 cm2) was immersed in standard sulfuric acid water solution for industrial use (pH=0.4).", "As can be understood from FIGS.", "2 and 3, compared with the case of immersing the piece in the standard sulfuric acid water solution for industrial use, the generation rate of the hydrogen gas was higher from an early stage of the process in the case of immersing the piece in the deuterated sulfuric acid water solution.", "Specifically, within the initial 16 minutes period, as much as about 225 ml of hydrogen gas could be generated in the case of using the deuterated sulfuric acid water solution, compared with the case of using the standard industrial sulfuric acid water solution which yielded only about 184 ml.", "Hence, it was confirmed that the use of the deuterated sulfuric acid water solution contributed to increase in the generation amount of the hydrogen gas at the early stage.", "Incidentally, the deuterated sulfuric acid water solution can be prepared by appropriately using any of a variety of methods such as incorporating such substance as heavy water, deuterium sulfate, deuterium sulfide, etc.", "so that at least some of the hydrogen atoms contained in the deuterated sulfuric acid water solution are substituted for deuterium atoms and the resultant solution contains a greater amount of deuterium atoms than naturally present.", "As described above, with the hydrogen gas producing method relating to the present invention, it becomes possible to generate a great amount of hydrogen gas in a short period of time.", "Accordingly, if this method is used in a hydrogen gas driven apparatus, a hydrogen self-contained turbo charger, an electric car or a cogeneration system utilizing such hydrogen self-contained turbo charger etc., the start-up performance of the apparatus or the machine will be improved.", "In these manners, this method can be put into actual use.", "Other Embodiments Next, other embodiments of the invention will be described.", "<1> The monometal 1 is not limited to the Mg metal described in the foregoing embodiment.", "It can be instead any metal having a higher ionization tendency than hydrogen (H) (e.g.", "potassium (K) metal, sodium (Na) metal, aluminum (Al) metal, manganese (Mn) metal, zinc (Zn) metal, chromium (Cr) metal, cadmium (Cd) metal, iron (Fe) metal, cobalt (Co) metal, nickel (Ni) metal, tin (Sn) metal, lead (Pb) metal) or can be an alloy of such metals having higher ionization tendency than hydrogen.", "Incidentally, for an example, FIG.", "4 shows the hydrogen generation amounts per a predetermined unit time (min.)", "in comparison between the case in which Mg metal was immersed in the deuterated sulfuric acid water solution and a further case in which Zn metal was immersed in the deuterated sulfuric acid water solution.", "In the case of using Mg metal, almost 5.6 times of amount of hydrogen gas could be obtained, compared with the case using Zn metal.", "For instance, in terms of the mass (for a same surface area) of the metal required for generating 1000 ml of hydrogen gas case, only ⅓ of Mg metal is required relative to the Zn metal required therefor.", "Hence, if Mg metal is used, it is possible to e.g.", "reduce the weight of the apparatus employed for implementing the hydrogen gas producing method of the invention.", "<2> In the foregoing embodiment, for the generation of hydrogen gas by contacting the deuterated acidic water solution with the monometal, there was described as an example the case in which the monometal is immersed in the deuterated acidic water solution.", "Needless to say, the method is not limited to such construction shown in FIG.", "1 for immersing the entire monometal in the deuterated acidic water solution.", "Instead, at least a portion of the monometal may be immersed in the deuterated acidic water solution.", "Further, the method is not limited either to such construction involving immersion of the monometal in the deuterated acidic water solution.", "Alternately, the required contact between the deuterated acidic water solution and the monometal for hydrogen gas generation can be realized also by spraying the deuterated acidic water solution against the monometal or dripping the deuterated acidic water solution onto the monometal or even by causing the deuterated acidic water solution to flow over the monometal.", "<3> Further, as shown in FIG.", "5(a), different kinds of metals 1A, 1B of differing ionization tendencies may be provided and these different kinds of metals 1A, 1B may be immersed in the solution, with the metals being electrically coupled with each other.", "Such different kinds of metals 1A, 1B can be appropriately selected from those metals having a higher ionization tendency than hydrogen (H) (K, Na, Mg, Al, Mn, Zn, Cr, Cd, Fe, Co, Ni, Sn, Pb).", "And, it is preferred that the different kinds of metals having a greater difference in the ionization tendency should be used in combination.", "For, this results in a greater potential difference, thus further promoting generation of hydrogen gas.", "Incidentally, FIG.", "5(a) shows a case in which the different kinds of metals 1A, 1B are coupled via a conducting wire.", "However, for establishing electrical coupling between the different kinds of metals, other methods are also possible, such as directly bonding the different metals to each other or any other appropriate method may be employed for obtaining the conduction between the different kinds of metals.", "As an example of the deuterated acidic water solution, there was prepared deuterated sulfuric acid water solution in which some of hydrogen atoms contained in sulfuric acid solution were substituted for deuterium atoms.", "And, into 500 ml of this solution, a bonded assembly of a copper metal piece and a zinc metal piece having dimensions of: 50 mm length×50 mm width×1 mm thickness was immersed thereby to generate hydrogen gas.", "FIG.", "6 shows the relationship between the amount of hydrogen gas generated and the time lapsed in this experiment.", "For comparison, the figure also shows the result of a further case in which the same assembly was immersed in a standard sulfuric acid water solution.", "In this, the temperature in the water solution was from 15 to 20° C. FIG.", "6(a) shows the relationship between the total generation amount of hydrogen gas and the time.", "As may be understood from this FIG.", "6(a), the generation rate of hydrogen gas was higher in the case of immersion in the deuterated sulfuric acid water solution than the case of immersion in the standard sulfuric acid water solution.", "For more detailed understanding, FIG.", "6(b) shows the hydrogen gas generation amount per unit time.", "From this FIG.", "6(b), it is understood that during the early period after the immersion in the water solution, the immersion in the deuterated sulfuric acid water solution generated about 1.5 times amount of hydrogen gas relative to the immersion in the standard sulfuric acid water solution.", "FIG.", "7 shows the determination of pH variation in the water solution in the above experiment.", "From FIG.", "6 and FIG.", "7, it is understood that the hydrogen gas was generated rapidly under the condition of pH 1.0 or lower.", "Further, in the time period under such condition (˜about 400 (min)), the deuterated sulfuric acid water solution showed similar pH variation to that of the standard sulfuric acid water solution.", "Then, it is understood and confirmed that for a same condition, the deuterated sulfuric acid water solution could generate a greater amount of hydrogen gas in a shorter period of time than the standard sulfuric acid water solution.", "Incidentally, FIG.", "8 shows a difference in the hydrogen generation amounts between a case immersing an assembly of copper and zinc (for both copper and zinc, a metal piece having dimensions of: 50 mm length×50 mm width and 1 mm thickness) and a further case immersing only zinc (two pieces of the metal pieces having the dimensions of 50 mm length×50 mm width and 1 mm thickness).", "From this FIG.", "8, it is understood that the combination of different kinds of metals having differing ionization tendencies can yield a greater amount of hydrogen gas.", "<4> Further, as the monometal, a plurality of pieces of monometal 1 can be prepared and these maybe immersed in the deuterated acidic water solution 2 with establishing a potential difference between the plurality of monometal 1 pieces by means of an external power source (see FIG.", "5(b)).", "With this, the monometal 1 piece having the lower electric potential will act as an anode, thereby to electrochemically promote the oxidation reaction, so that a greater amount of hydrogen gas can be generated in an even shorter period of time.", "Further alternately, one monometal piece and another metal may be immersed in the deuterated acidic water solution with a potential difference between the monometal 1 and the other metal by means of an external power source so that the monometal 1 has the lower electric potential.", "Such other metal can be appropriately selected from those which are conductive to the monometal which is to be provided with the lower electric potential by the external power source.", "<5> In the foregoing embodiment, the acidic water solution comprises the sulfuric acidic water solution.", "Instead, any inorganic acid such as hydrochloric acid, and nitric acid, etc or any organic acid such as citric acid, glycine, cinnamic acid, succinic acid, salicylic acid, formic acid, glutamic acid, ascorbic acid, oxalic acid, tartaric acid, lactic acid, acetic acid, etc.", "may be employed.", "With the present invention, by its deuteration, even an organic acid having a relatively low acidity can generate a sufficient amount of hydrogen gas.", "Further, two kinds or more of such inorganic or organic acids can be used.", "Or, an inorganic acid and an organic acid may be used in combination.", "INDUSTRIAL APPLICABILITY With the hydrogen gas producing method relating to the present invention, it becomes possible to generate a great amount of hydrogen gas in a short period of time.", "Accordingly, if this method is used in a hydrogen gas driven apparatus, a hydrogen self-contained turbo charger, an electric car or a cogeneration system utilizing such hydrogen self-contained turbo charger or for supply of hydrogen gas for a fuel cell, the start-up performance of the apparatus or the machine will be improved.", "In these manners, this method can be put into actual use." ] ]	Patent_10468750
[ [ "Enanitomers of unsaturated alkyllysophosphonocholines and use as anti-neoplastics", "Unsaturated alkyllysophosphonocholines compounds of formula (I) or (II): or pharmaceutically-acceptable salts, prodrugs or isomers thereof.", "The compounds of the invention have anti-neoplastic activity, and accordingly have utility in treating cancer and related diseases.", "The invention also provides enantiomers of these compounds, as well as synthetic methods for producing an enantiomer, substantially free of the other enantiomer.", "Also disclosed are pharmaceutical compositions, as well methods for treating cancer with the pharmaceutical compositions." ], [ "1.A compound of the formula I, or a pharmaceutically acceptable salt, prodrug or isomer thereof: wherein X1 is H, R1, R2, OR2, NR1R2, or S(O)aR2, where a is an integer selected from 0, 1, 2, or 3; X2 is H, R3, R4, OR4, NR3R4, or S(O)aR4, where a is an integer selected from 0, 1, 2, or 3; X3 is (CH2)b, where b is an integer selected from 0, 1, 2, 3, or 4; X4 is (CH2)c, where c is an integer selected from 0, 1, 2, 3, or 4; R1 is a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R2 is H, a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R3 is a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R4 is H, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R5 is (CH2)m where m is an integer selected from 0, 1, 2, 3, or 4; Z is H, X5 is N or As; R6 and R7 are each independently hydrogen, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; and R8 is hydrogen, a straight-chain alkyl group having 1 to 3 carbon atoms or a branched or cyclic alkyl group having 3 carbon atoms.", "2.The compound of claim 1 further comprising a pharmaceutically acceptable anion.", "3.The compound of claim 1, wherein R1 and R2 are each independently a straight-chain or branched alkyl having 16 to 20 carbon atoms, or a straight-chain or a branched alkenyl group having 16 to 20 carbon atoms.", "4.The compound of claim 3, wherein R1 and R2 are each independently a straight-chain or branched alkyl having 18 carbon atoms, or a straight-chain or a branched alkenyl group having 18 carbon atoms.", "5.The compound of claim 1, wherein X1 is OR2 or NHR2, and wherein R2 is a straight-chain or branched alkyl having 16 to 20 carbon atoms, or a straight-chain or a branched alkenyl group having 16 to 20 carbon atoms.", "6.The compound of claim 5, wherein X1 is OR2 or NHR2, and wherein R2 is a straight-chain or branched alkyl having 18 carbon atoms, or a straight-chain or a branched alkenyl group having 18 carbon atoms.", "7.The compound of claim 1, wherein X1 or X2 is —SR2.8.The compound of claim 1, wherein X_or X2 is —S(═O)R2.9.The compound of claim 1, wherein X1 or X2 is —S(═O)2R2.10.The compound of claim 1, wherein X1 or X2 is —S(═O)2OR2.11.The compound of claim 1, wherein X1 or X2 is —OS(═O)2R2.12.The compound of claim 1 wherein X1 or X2 is —OCH3.13.The compound of claim 1, wherein X3, X4, and R5 are each independently a direct link, a —CH2— group, or a —CH2CH2— group.", "14.The compound of claim 1, wherein Z is 15.The compound of claim 1, wherein Z is 16.The compound of claim 1, wherein X1 is O, X2 is O, X4 is O and b is 0.17.The compound of claim 1, wherein the compound is optically active, and substantially free of the R enantiomer.", "18.The compound of claim 1, wherein the compound is optically active and substantially free of the S enantiomer.", "19.The compound of claim 1, wherein the compound is 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the R enantiomer.", "20.The compound of claim 1, wherein the compound is 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the S enantiomer.", "21.A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of claim 1.22.A method of treating a mammal afflicted with a cancer which comprises administering to the mammal a therapeutically effective amount of the pharmaceutical composition of claim 21, comprising from about 0.1 to about 1000 mg of the compound of claim 1 per kg of the body weight of the mammal per day.", "23.A method of claim 22, wherein the cancer is selected from the group consisting of lung cancers, brain cancers, colon cancers, ovarian cancers, breast cancers, leukemias, lymphomas, sarcomas, and carcinomas.", "24.The method of claim 22, comprising administering to the mammal an additional biologically active agent.", "25.The method of claim 24, wherein the additional biologically active agent is selected from the group consisting of antineoplastic agents, antimicrobial agents, and hematopoietic cell growth stimulating agents.", "26.A compound of formula II, or a pharmaceutically acceptable salt, prodrug or isomer thereof: wherein X1 is H, R1, R2, OR2, NR1R2, or S(O)aR2, where a is an integer selected from 0, 1, 2, or 3; X2 is H, R3, R4, OR4, NR3R4, or S(O)aR4, where a is an integer selected from 0, 1, 2, or 3; X3 is (CH2)b, where b is an integer selected from 0, 1, 2, 3, or 4; X4 is (CH2)c, where c is an integer selected from 0, 1, 2, 3, or 4; R1 is a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R2 is H, a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R3 is a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R4 is H, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R5 is (CH2)m where m is an integer selected from 0, 1, 2, 3, or 4; Z is H, X5 is N or As; R6 and R7 are each independently hydrogen, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R8 is hydrogen, a straight-chain alkyl group having 1 to 3 carbon atoms or a branched or cyclic alkyl group having 3 carbon atoms; and R9 is a straight-chain alkyl group having 1 to 3 carbon atoms or a branched or cyclic alkyl group having 3 carbon atoms.", "27.The compound of claim 26 further comprising a pharmaceutically acceptable anion.", "28.The compound of claim 26, wherein R1 and R2 are each independently a straight-chain or branched alkyl having 16 to 20 carbon atoms, or a straight-chain or a branched alkenyl group having 16 to 20 carbon atoms.", "29.The compound of claim 28, wherein R1 and R2 are each independently a straight-chain or branched alkyl having 18 carbon atoms, or a straight-chain or a branched alkenyl group having 18 carbon atoms.", "30.The compound of claim 26, wherein X1 is OR2 or NHR2, and wherein R2 is a straight-chain or branched alkyl having 16 to 20 carbon atoms, or a straight-chain or a branched alkenyl group having 16 to 20 carbon atoms.", "31.The compound of claim 30, wherein X1 is OR2 or NHR2, and wherein R2 is a straight-chain or branched alkyl having 18 carbon atoms, or a straight-chain or a branched alkenyl group having 18 carbon atoms.", "32.The compound of claim 26, wherein X1 or X2 is —SR2.33.The compound of claim 26, wherein X1 or X2 is —S(═O)R2.34.The compound of claim 26, wherein X1 or X2 is —S(═O)2R2.35.The compound of claim 26, wherein X1 or X2 is —S(═O)2OR2.36.The compound of claim 26, wherein X1 or X2 is —OS(═O)2R2.37.The compound of claim 26, wherein X2 is —OCH3.38.The compound of claim 26, wherein X3, X4, and R5 are each independently a direct link, a —CH2— group, or a —CH2CH2— group.", "39.The compound of claim 26, wherein Z is 40.The compound of claim 26, wherein Z is 41.The compound of claim 26, wherein X1 is O, X2 is O, X4 is O and b is 0.42.The compound of claim 26, wherein the compound is optically active, and substantially free of the R enantiomer.", "43.The compound of claim 26, wherein the compound is optically active and substantially free of the S enantiomer.", "44.A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of claim 43.45.A method of treating a mammal afflicted with a cancer which comprises administering to the mammal a therapeutically effective amount of the pharmaceutical composition of claim 44, comprising from about 0.1 to about 1000 mg of the compound of claim 1 per kg of the body weight of the mammal per day.", "46.A method of claim 45, wherein the cancer is selected from the group consisting of lung cancers, brain cancers, colon cancers, ovarian cancers, breast cancers, leukemias, lymphomas, sarcomas, and carcinomas.", "47.The method of claim 46, comprising administering to the mammal an additional biologically active agent.", "48.The method of claim 47, wherein the additional biologically active agent is selected from the group consisting of antineoplastic agents, antimicrobial agents, and hematopoietic cell growth stimulating agents.", "49.A method for making 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the (R) enantiomer, comprising: (i) oxidizing 1-O-hexadecyl-2-O-methyl-sn-glycerol under Swern oxidation conditions sufficient to produce (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde; (ii) reacting the (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde with tetraisopropyl methylenediphosphonate under conditions sufficient to produce diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate; (iii) hydrolyzing the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate under conditions sufficient to produce the 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonic acid; and (iv) reacting the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate with choline tosylate under conditions sufficient to produce 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate.", "50.A method for making 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the (S) enantiomer, comprising: (i) oxidizing 3-O-hexadecyl-2-O-methyl-sn-glycerol under Swern oxidation conditions sufficient to produce (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde; (ii) reacting the (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde with tetraisopropyl methylenediphosphonate under conditions sufficient to produce diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate; (iii) hydrolyzing the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate under conditions sufficient to produce the 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonic acid; and (iv) reacting the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate with choline tosylate under conditions sufficient to produce 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention provides novel unsaturated alkyllysophosphonocholines compounds and enantiomers thereof, as well as pharmaceutical compositions thereof, and methods for treating cancer.", "Synthetic methods for synthesizing enantiomers of the compounds is also provided.", "2.State of the Art Alkyllysophospholipids (ALPs) and alkylphosphocholines (APCs) represent subclasses of potential antitumor agents collectively known as antitumor ether lipids (AELs).", "They do not interact with cellular DNA and are therefore not mutagenic (Berdel, W. E. (1991) Br.", "J Cancer, 64, 208-211; Lohmeyer, M., and Bittman, R. (1994) Drugs of the Future 19, 1021-1037).", "The antitumor activities of these compounds, which are based on lysophosphatidylcholine, are now well established; the prototype of the alkyllysophospholipids (ALPs), 1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OCH 3 ), and other ether-linked phosphocholine analogues are in clinical trials (Lohmeyer, M., and Bittman, R. (1994) Drugs of the Future 19, 1021-1037, Houlihan, W. J., Lohmeyer, M., Workman, P., and Cheon, S. H. (1995) Med Res.", "Rev.", "15, 157-223; Principe, P., and Braquet, P. (1995) Rev.", "Oncol.", "Hematol.", "18, 155-178).", "ALPs also appear to inhibit the proliferation of tumor cells without affecting the growth of normal cells (Berdel, W. E., Andreesen, R., and Munder, P. G. (1985) in Phospholipids and Cellular Regulation, Vol 2.Kuo, J (ed).", "CRC Press: Boca Raton, pp.", "41-73).", "While the mechanism of inhibition of cell proliferation has yet to be resolved, various hypotheses have been proposed.", "In some cells, ALPs and APCs appear to induce apoptosis as a consequence of inhibition of phosphatidylcholine synthesis (Boggs, K. P., Rock, C. O., and Jackowski, S. (1995) J Biol.", "Chem.", "270, 11612-11618; Boggs, K. P., Rock, C. O., and Jackowski, S. (1998) Biochim.", "Biophys.", "Acta 1389, 1-12); activation of the stress activated protein kinase pathways (Gajate, C., Santos-Beneit, A., Modolell, M., and Mollinedo, F. (1998) Mol.", "Pharmacol.", "53, 602-612; Ruiter, G. A., Zerp, S. F., Bartelink, H., Van Blitterswijk, W. J., and Verheij, M. (1999) Cancer Res.", "59, 2457-2463), drug-induced increase in cellular cerarnide levels (Wieder, T., Orfanos, C. E., and Geilen, C. G. (1998) J. Biol.", "Chem.", "273, 11025-11031); nutrient starvation, inhibition of transacylase activity, enhanced lipid peroxidation and inhibition of cellular signaling pathways (reviewed in Bittman, R., and Arthur, G. (1998) in A. S. Janoff (ed.", "), Liposomes: Rational Design, pp 125-144, New York: Marcel Dekker; Arthur; G., and Bittman, R. (1998) Biochim.", "Biophys.", "Acta 1390, 85-102).", "Other studies have revealed that ALPs affect the activity of a large number of signaling molecules including protein kinase C (PKC), phosphatidylinositol 3-kinase, phosphatidylinositol-specific phospholipase C, and diacylglycerol kinase (reviewed in Arthur, G., and Bittman, R. (1998) Biochim.", "Biophys.", "Acta 1390, 85-102).", "Recently another signaling molecule, Raf-1, was added to the list with the demonstration that ET-18-OCH 3 decreased the levels of Raf-1 associating with the cell membrane in growth-factor stimulated MCF-7 cells which consequently led to decreased activation of MAP kinase (Zhou, X., Lu, X., Richard, C., Xiong, W., Litchfield, D. W., Bittman, R., and Arthur, G. (1996) J Clin.", "Invest.", "98, 937-944), a crucial enzyme required in initiating cell proliferation (Marshall, C. J.", "(1995) Cell 80, 179-185).", "It was suggested that Raf-1 is a primary target of ALPs in cells.", "The large number of molecules affected by ALPs has complicated the task of separating their primary site(s) of action from secondary events.", "In order to advance the present understanding of the mechanism(s) of action of these compounds, one has to distinguish target molecules and events that are relevant to growth inhibition from those that are irrelevant.", "Despite the progress that has been made in understanding the underlying mechanisms of antitumor ether lipids, there remains a need to develop novel compounds and compositions for treatment of disease.", "Ideally, the treatment methods would advantageously be based on anti-tumor ether lipids that are capable of acting as anti-neoplastic agents." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention is directed to the discovery of a class of anti-tumor ether lipid compounds having anti-neoplastic activity.", "Preferably, the invention provides bioactive unsaturated alkyllysophosphonocholines or pharmaceutically-acceptable salts, prodrugs or isomers thereof having the R or S configuration at the C-2 position of the glycerol backbone.", "The invention also relates to pharmaceutical compositions comprising these compounds, and methods for treating cancer.", "Neither the R enantiomer nor S enantiomer of a trans double bond (TBD) phosphonocholine etherlipid, an analogue of ET-16-OCH 3 , 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-methoxy-1-butenephosphonate [ET-16-phosphono-TDB], has previously been synthesized.", "In order to differentiate the activities of each of these enantiomers, single enantiomers must be available in sufficient chiral purity to enable a comparison of the chemical, biological and biochemical effects of the single enantiomers.", "Thus, until the present invention nothing was known about the antineoplastic effects of the single R and S enantiomers of phosphono etherlipids, in general, and ET-16-phosphono-TDB specifically.", "In one embodiment, the invention relates to compounds, or a pharmaceutically acceptable salt, prodrug or isomer thereof, having formula I below: wherein X 1 is H, R 1 , R 2 , OR 2 , NR 1 R 2 , or S(O) a R 2 , where a is an integer selected from 0, 1, 2, or 3; X 2 is H, R 3 , R 4 , OR 4 , NR 3 R 4 , or S(O) a R 4 , where a is an integer selected from 0, 1, 2, or 3; X 3 is (CH 2 ) b , where b is an integer selected from 0, 1, 2, 3, or 4; X 4 is (CH 2 ) c , where c is an integer selected from 0, 1, 2, 3, or 4; R 1 is a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R 2 is H, a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R 3 is a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R 4 is H, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R 5 is (CH 2 ) m where m is an integer selected from 0, 1, 2, 3, or 4; Z is H, X 5 is N or As; R 6 and R 7 are each independently hydrogen, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; and R 8 is hydrogen, a straight-chain alkyl-group having 1 to 3 carbon atoms or a branched or cyclic alkyl group having 3 carbon atoms.", "In another embodiment, the invention relates to compounds of formula II, or a pharmaceutically acceptable salt, prodrug or isomer thereof: wherein X 1 is H, R 1 , R 2 , OR 2 , NR 1 R 2 , or S(O) a R 2 , where a is an integer selected from 0, 1, 2, or 3; X 2 is H, R 3 , R 4 , OR 4 , NR 3 R 4 , or S(O) a R 4 , where a is an integer selected from 0, 1, 2, or 3; X 3 is (CH 2 ) b , where b is an integer selected from 0, 1, 2, 3, or 4; X 4 is (CH 2 ) c , where c is an integer selected from 0, 1, 2, 3, or 4; R 1 is a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R 2 is H, a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R 3 is a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R 4 is H, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R 5 is (CH 2 ) m where m is an integer selected from 0, 1, 2, 3, or 4; Z is H, X 5 is N or As; R 6 and R 7 are each independently hydrogen, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R 8 is hydrogen, a straight-chain alkyl group having 1 to 3 carbon atoms or a branched or cyclic alkyl group having 3 carbon atoms; and R 9 is a straight-chain alkyl group having 1 to 3 carbon atoms or a branched or cyclic alkyl group having 3 carbon atoms.", "The compounds of formula (I) or (II) above may further comprise a pharmaceutically acceptable anion.", "Preferably, R 1 and R 2 are independently a straight-chain or branched alkyl having 16 to 20 carbon atoms, or a straight-chain or a branched alkenyl group having 16 to 20 carbon atoms.", "In another preferred embodiment, R 1 and R 2 are independently a straight-chain or branched alkyl having 18 carbon atoms, or a straight-chain or a branched alkenyl group having 18 carbon atoms.", "In an embodiment of the invention, X 1 is OR 2 or NHR 2 , where R 2 is a straight-chain or branched alkyl having 16 to 20 carbon atoms, or a straight-chain or a branched alkenyl group having 16 to 20 carbon atoms.", "In a preferred embodiment, R 2 is a straight-chain or branched alkyl having 18 carbon atoms, or a straight-chain or a branched alkenyl group having 18 carbon atoms.", "In an embodiment of the invention, X 1 or X 2 is independently selected from —SR 2 , —S(═O)R 2 , —S(═O) 2 R 2 , —S(═O) 2 OR 2 , or —OS(═O) 2 R 2 .", "In an embodiment of the invention, X 1 or X 2 is —OCH 3 .", "Typical values for X 3 , X 4 , and R 5 include a direct link, a —CH 2 — group, or a —CH 2 CH 2 — group.", "Preferred values for Z include Yet another embodiment of the invention relates to compounds where X 1 is O, X 2 is O, X 4 is O and b is 0.The invention also relates to optically active compounds of formula (I) or (II), which are substantially free of the R enantiomer.", "In addition, the invention also relates to optically active compounds of formula (I) or (II), which are substantially free of the S enantiomer.", "For example, the invention relates to 2′-(trimethylanmmonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the R enantiomer, as well as 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the S enantiomer.", "Additionally, the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of formula (I) or (II).", "These pharmaceutical compositions can be used in methods for treating a mammal afflicted with a cancer, comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition.", "Typical dosages range from about 0.1 to about 1000 mg of the compound of formula (I) or (II) per kg of the body weight of the mammal per day.", "The type of cancer to be treated may be selected from the group consisting of, but not limited to: lung cancers, brain cancers, colon cancers, ovarian cancers, breast cancers, leukemias, lymphomas, sarcomas, and carcinomas.", "These treatment methods may also include administering to the mammal an additional biologically active agent.", "The additional biologically active agent may be selected from the group consisting of antineoplastic agents, antimicrobial agents, and hematopoietic cell growth stimulating agents, for example.", "In addition, the invention relates to a method for making 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the (R) enantiomer, comprising: (i) oxidizing 1-O-hexadecyl-2-O-methyl-sn-glycerol under Swern oxidation conditions sufficient to produce (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde; (ii) reacting the (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde with tetraisopropyl methylenediphosphonate under conditions sufficient to produce diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate; (iii) hydrolyzing the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate under conditions sufficient to produce the 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonic acid; and (iv) reacting the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate with choline tosylate under conditions sufficient to produce 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate.", "The invention also relates to a method for making 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the (S) enantiomer, comprising: (i) oxidizing 3-O-hexadecyl-2-O-methyl-sn-glycerol under Swern oxidation conditions sufficient to produce (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde; (ii) reacting the (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde with tetraisopropyl methylenediphosphonate under conditions sufficient to produce diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate; (iii) hydrolyzing the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate under conditions sufficient to produce the 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonic acid; and (iv) reacting the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate with choline tosylate under conditions sufficient to produce 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention provides novel unsaturated alkyllysophosphonocholines compounds and enantiomers thereof, as well as pharmaceutical compositions thereof, and methods for treating cancer.", "Synthetic methods for synthesizing enantiomers of the compounds is also provided.", "2.State of the Art Alkyllysophospholipids (ALPs) and alkylphosphocholines (APCs) represent subclasses of potential antitumor agents collectively known as antitumor ether lipids (AELs).", "They do not interact with cellular DNA and are therefore not mutagenic (Berdel, W. E. (1991) Br.", "J Cancer, 64, 208-211; Lohmeyer, M., and Bittman, R. (1994) Drugs of the Future 19, 1021-1037).", "The antitumor activities of these compounds, which are based on lysophosphatidylcholine, are now well established; the prototype of the alkyllysophospholipids (ALPs), 1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OCH3), and other ether-linked phosphocholine analogues are in clinical trials (Lohmeyer, M., and Bittman, R. (1994) Drugs of the Future 19, 1021-1037, Houlihan, W. J., Lohmeyer, M., Workman, P., and Cheon, S. H. (1995) Med Res.", "Rev.", "15, 157-223; Principe, P., and Braquet, P. (1995) Rev.", "Oncol.", "Hematol.", "18, 155-178).", "ALPs also appear to inhibit the proliferation of tumor cells without affecting the growth of normal cells (Berdel, W. E., Andreesen, R., and Munder, P. G. (1985) in Phospholipids and Cellular Regulation, Vol 2.Kuo, J (ed).", "CRC Press: Boca Raton, pp.", "41-73).", "While the mechanism of inhibition of cell proliferation has yet to be resolved, various hypotheses have been proposed.", "In some cells, ALPs and APCs appear to induce apoptosis as a consequence of inhibition of phosphatidylcholine synthesis (Boggs, K. P., Rock, C. O., and Jackowski, S. (1995) J Biol.", "Chem.", "270, 11612-11618; Boggs, K. P., Rock, C. O., and Jackowski, S. (1998) Biochim.", "Biophys.", "Acta 1389, 1-12); activation of the stress activated protein kinase pathways (Gajate, C., Santos-Beneit, A., Modolell, M., and Mollinedo, F. (1998) Mol.", "Pharmacol.", "53, 602-612; Ruiter, G. A., Zerp, S. F., Bartelink, H., Van Blitterswijk, W. J., and Verheij, M. (1999) Cancer Res.", "59, 2457-2463), drug-induced increase in cellular cerarnide levels (Wieder, T., Orfanos, C. E., and Geilen, C. G. (1998) J. Biol.", "Chem.", "273, 11025-11031); nutrient starvation, inhibition of transacylase activity, enhanced lipid peroxidation and inhibition of cellular signaling pathways (reviewed in Bittman, R., and Arthur, G. (1998) in A. S. Janoff (ed.", "), Liposomes: Rational Design, pp 125-144, New York: Marcel Dekker; Arthur; G., and Bittman, R. (1998) Biochim.", "Biophys.", "Acta 1390, 85-102).", "Other studies have revealed that ALPs affect the activity of a large number of signaling molecules including protein kinase C (PKC), phosphatidylinositol 3-kinase, phosphatidylinositol-specific phospholipase C, and diacylglycerol kinase (reviewed in Arthur, G., and Bittman, R. (1998) Biochim.", "Biophys.", "Acta 1390, 85-102).", "Recently another signaling molecule, Raf-1, was added to the list with the demonstration that ET-18-OCH3 decreased the levels of Raf-1 associating with the cell membrane in growth-factor stimulated MCF-7 cells which consequently led to decreased activation of MAP kinase (Zhou, X., Lu, X., Richard, C., Xiong, W., Litchfield, D. W., Bittman, R., and Arthur, G. (1996) J Clin.", "Invest.", "98, 937-944), a crucial enzyme required in initiating cell proliferation (Marshall, C. J.", "(1995) Cell 80, 179-185).", "It was suggested that Raf-1 is a primary target of ALPs in cells.", "The large number of molecules affected by ALPs has complicated the task of separating their primary site(s) of action from secondary events.", "In order to advance the present understanding of the mechanism(s) of action of these compounds, one has to distinguish target molecules and events that are relevant to growth inhibition from those that are irrelevant.", "Despite the progress that has been made in understanding the underlying mechanisms of antitumor ether lipids, there remains a need to develop novel compounds and compositions for treatment of disease.", "Ideally, the treatment methods would advantageously be based on anti-tumor ether lipids that are capable of acting as anti-neoplastic agents.", "SUMMARY OF THE INVENTION The invention is directed to the discovery of a class of anti-tumor ether lipid compounds having anti-neoplastic activity.", "Preferably, the invention provides bioactive unsaturated alkyllysophosphonocholines or pharmaceutically-acceptable salts, prodrugs or isomers thereof having the R or S configuration at the C-2 position of the glycerol backbone.", "The invention also relates to pharmaceutical compositions comprising these compounds, and methods for treating cancer.", "Neither the R enantiomer nor S enantiomer of a trans double bond (TBD) phosphonocholine etherlipid, an analogue of ET-16-OCH3, 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-methoxy-1-butenephosphonate [ET-16-phosphono-TDB], has previously been synthesized.", "In order to differentiate the activities of each of these enantiomers, single enantiomers must be available in sufficient chiral purity to enable a comparison of the chemical, biological and biochemical effects of the single enantiomers.", "Thus, until the present invention nothing was known about the antineoplastic effects of the single R and S enantiomers of phosphono etherlipids, in general, and ET-16-phosphono-TDB specifically.", "In one embodiment, the invention relates to compounds, or a pharmaceutically acceptable salt, prodrug or isomer thereof, having formula I below: wherein X1 is H, R1, R2, OR2, NR1R2, or S(O)aR2, where a is an integer selected from 0, 1, 2, or 3; X2 is H, R3, R4, OR4, NR3R4, or S(O)aR4, where a is an integer selected from 0, 1, 2, or 3; X3 is (CH2)b, where b is an integer selected from 0, 1, 2, 3, or 4; X4 is (CH2)c, where c is an integer selected from 0, 1, 2, 3, or 4; R1 is a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R2 is H, a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R3 is a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R4 is H, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R5 is (CH2)m where m is an integer selected from 0, 1, 2, 3, or 4; Z is H, X5 is N or As; R6 and R7 are each independently hydrogen, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; and R8 is hydrogen, a straight-chain alkyl-group having 1 to 3 carbon atoms or a branched or cyclic alkyl group having 3 carbon atoms.", "In another embodiment, the invention relates to compounds of formula II, or a pharmaceutically acceptable salt, prodrug or isomer thereof: wherein X1 is H, R1, R2, OR2, NR1R2, or S(O)aR2, where a is an integer selected from 0, 1, 2, or 3; X2 is H, R3, R4, OR4, NR3R4, or S(O)aR4, where a is an integer selected from 0, 1, 2, or 3; X3 is (CH2)b, where b is an integer selected from 0, 1, 2, 3, or 4; X4 is (CH2)c, where c is an integer selected from 0, 1, 2, 3, or 4; R1 is a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R2 is H, a straight-chain or branched alkyl having 12 to 20 carbon atoms or a straight-chain or branched alkenyl group having 12 to 20 carbon atoms; R3 is a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R4 is H, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R5 is (CH2)m where m is an integer selected from 0, 1, 2, 3, or 4; Z is H, X5 is N or As; R6 and R7 are each independently hydrogen, a straight-chain alkyl group having 1 to 3 carbon atoms, or a branched or cyclic alkyl group having 3 carbon atoms; R8 is hydrogen, a straight-chain alkyl group having 1 to 3 carbon atoms or a branched or cyclic alkyl group having 3 carbon atoms; and R9 is a straight-chain alkyl group having 1 to 3 carbon atoms or a branched or cyclic alkyl group having 3 carbon atoms.", "The compounds of formula (I) or (II) above may further comprise a pharmaceutically acceptable anion.", "Preferably, R1 and R2 are independently a straight-chain or branched alkyl having 16 to 20 carbon atoms, or a straight-chain or a branched alkenyl group having 16 to 20 carbon atoms.", "In another preferred embodiment, R1 and R2 are independently a straight-chain or branched alkyl having 18 carbon atoms, or a straight-chain or a branched alkenyl group having 18 carbon atoms.", "In an embodiment of the invention, X1 is OR2 or NHR2, where R2 is a straight-chain or branched alkyl having 16 to 20 carbon atoms, or a straight-chain or a branched alkenyl group having 16 to 20 carbon atoms.", "In a preferred embodiment, R2 is a straight-chain or branched alkyl having 18 carbon atoms, or a straight-chain or a branched alkenyl group having 18 carbon atoms.", "In an embodiment of the invention, X1 or X2 is independently selected from —SR2, —S(═O)R2, —S(═O)2R2, —S(═O)2OR2, or —OS(═O)2R2.In an embodiment of the invention, X1 or X2 is —OCH3.Typical values for X3, X4, and R5 include a direct link, a —CH2— group, or a —CH2CH2— group.", "Preferred values for Z include Yet another embodiment of the invention relates to compounds where X1 is O, X2 is O, X4 is O and b is 0.The invention also relates to optically active compounds of formula (I) or (II), which are substantially free of the R enantiomer.", "In addition, the invention also relates to optically active compounds of formula (I) or (II), which are substantially free of the S enantiomer.", "For example, the invention relates to 2′-(trimethylanmmonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the R enantiomer, as well as 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the S enantiomer.", "Additionally, the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of formula (I) or (II).", "These pharmaceutical compositions can be used in methods for treating a mammal afflicted with a cancer, comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition.", "Typical dosages range from about 0.1 to about 1000 mg of the compound of formula (I) or (II) per kg of the body weight of the mammal per day.", "The type of cancer to be treated may be selected from the group consisting of, but not limited to: lung cancers, brain cancers, colon cancers, ovarian cancers, breast cancers, leukemias, lymphomas, sarcomas, and carcinomas.", "These treatment methods may also include administering to the mammal an additional biologically active agent.", "The additional biologically active agent may be selected from the group consisting of antineoplastic agents, antimicrobial agents, and hematopoietic cell growth stimulating agents, for example.", "In addition, the invention relates to a method for making 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the (R) enantiomer, comprising: (i) oxidizing 1-O-hexadecyl-2-O-methyl-sn-glycerol under Swern oxidation conditions sufficient to produce (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde; (ii) reacting the (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde with tetraisopropyl methylenediphosphonate under conditions sufficient to produce diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate; (iii) hydrolyzing the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate under conditions sufficient to produce the 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonic acid; and (iv) reacting the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate with choline tosylate under conditions sufficient to produce 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate.", "The invention also relates to a method for making 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate substantially free of the (S) enantiomer, comprising: (i) oxidizing 3-O-hexadecyl-2-O-methyl-sn-glycerol under Swern oxidation conditions sufficient to produce (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde; (ii) reacting the (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde with tetraisopropyl methylenediphosphonate under conditions sufficient to produce diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate; (iii) hydrolyzing the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate under conditions sufficient to produce the 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonic acid; and (iv) reacting the diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate with choline tosylate under conditions sufficient to produce 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1.Graphical depiction of the effects of (R) and (S) ET-16-phosphono-TDB on the proliferation of breast epithelial cancer cell line MDA-MB-468.FIG.", "2.Graphical depiction of the effects of (R) and (S) ET-16-phosphono-TDB on the proliferation of breast epithelial cancer cell line MCF-7.FIG.", "3.Graphical depiction of the effects of (R) and (S) ET-16-phosphono-TDB on the proliferation of breast epithelial cancer cell line T47D.", "FIG.", "4.Graphical depiction of the effects of (R) and (S) ET-16-phosphono TDB on the proliferation of neuroblastoma cell line SK-N-MC.", "FIG.", "5.Graphical depiction of the effects of (R) and (S) ET-16-phosphono-TDB on the proliferation of neuroblastoma cell line SK-N-SH.", "FIG.", "6.Western blot analysis showing the effect of ET-16-phosphono-TDB preincubation on MAP kinase phosphorylation in insulin-stimulated MCF-7 cells.", "Cell lysates of quiescent MCF-7 cells, treated with (“+”) or without (“−”) (R)- or (S)-ET-16-phosphono-TDB and stimulated with insulin, were probed with anti-phospho-MAP kinase antibody.", "FIG.", "7.Western, blot analysis showing the effect of ET-16-phosphono-TDB preincubation on PKB/AKT phosphorylation in insulin-stimulated MCF-7 cells.", "Cell lysates of quiescent MCF-7 cells, treated with (“+”) or without (“−”) (R)- or (S)-ET-16-phosphono-TDB and stimulated with insulin, were probed with anti-phospho-PKB/AKT antibody.", "FIG.", "8.Western blot analysis showing the effect of ET-16-phosphono-TDB preincubation on MAP kinase phosphorylation in EGF-stimulated MCF-7 cells.", "Cell lysates of quiescent MCF-7 cells, treated with (“+”) or without (“−”) (R)- or (S)-ET-16-phosphono-TDB and stimulated with EGF, were probed with anti-phospho-MAP kinase antibody and anti MAP kinase antibody.", "DETAILED DESCRIPTION OF THE INVENTION As above, this invention relates to unsaturated alkyllysophosphonocholines or pharmaceutically-acceptable salts, prodrugs, or isomers thereof, which have utility as anti-neoplastic agents.", "In particular, the invention relates to compounds of formula (I) and (II), having the R or S configuration at the C-2 position of the glycerol backbone, substantially free of the other enantiomer.", "However, prior to describing this invention in further detail, the following terms will first be defined.", "Definitions The term “alkyl” refers to saturated aliphatic groups including straight-chain, branched-chain, cyclic groups, and combinations thereof.", "The alkyl groups preferably have between 1 to 20 carbon atoms.", "The term “alkenyl” refers to unsaturated aliphatic groups including straight-chain, branched-chain, cyclic groups, and combinations thereof, having at least one double bond and having the number of carbon atoms specified.", "The alkenyl groups preferably have between 1 to 20 carbon atoms.", "The term “cyclic alkyl” or “cycloalkyl” refers to alkyl group forming an aliphatic ring.", "Preferred cyclic alkyl groups have about 3 carbon atoms.", "The term “direct link” as used herein refers to a bond directly linking the substituents on each side of the direct link.", "“Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts that are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.", "Examples of pharmaceutically acceptable acid addition salts includes salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.", "Examples of pharmaceutically acceptable base addition salts include those salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum bases, and the like.", "Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts.", "Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like.", "Particularly preferred organic nontoxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine.", "“Prodrug” means any compound which releases an active parent drug according to formulas (I) or (II) in vivo when such prodrug is administered to a mammalian subject.", "Prodrugs of a compound may be prepared by modifying functional groups present in the compound in such a way that the modifications may be cleaved in vivo to release the parent compound.", "Prodrugs include compounds of formula (I) or (II) wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, or sulfhydryl group, respectively.", "Examples of prodrugs include, but are not limited to esters (e.g., acetate, formate, and benzoate derivatives), carbamates (e.g., N,N-dimethylamino-carbonyl), and the like.", "“Isomers” are compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space.", "Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers.” An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (−)-isomers respectively).", "A chiral compound can exist as either individual enantiomer or as a mixture thereof.", "A mixture containing equal proportions of the enantiomers is called a “racemic mixture”.", "“Treating” or “treatment” of a disease includes: (1) preventing the disease, i.e.", "causing the clinical symptoms of the disease not to develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms, or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.", "A “therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease.", "The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated.", "A “pharmaceutically acceptable carrier” means an carrier that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a pharmaceutically acceptable excipient that is acceptable for veterinary use or human pharmaceutical use.", "A “pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.", "Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose.", "The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.", "The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.", "“Cancer” refers to a group of diseases characterized by uncontrolled growth and spread of abnormal cells, often resulting in the formation of a non-structured mass or tumor.", "Illustrative tumors include carcinomas, sarcomas and melanomas, such as basal cell carcinoma, squamous cell carcinoma, melanoma, soft tissue sarcoma, solar keratoses, Kaposi's sarcoma, cutaneous malignant lymphoma, Bowen's disease, Wilm's tumor, hepatomas, colorectals cancer, brain tumors, mycosis fungoides, Hodgkin's lymphoma, polycythemia Vera, chronic granulocytic leukemia, lymphomas, oat cell sarcoma, and the like.", "Tumors may also include benign growths such as condylomata acuminata (genital warts) and moles and common warts.", "An “anti-neoplastic agent” is a pharmaceutical which inhibits or causes the death of cancer or tumor cells.", "An “antimicrobial agent” is a substance that either destroys or inhibits the growth of a microorganism at concentrations tolerated by the infected host.", "A “hematopoietic cell growth stimulating agent” is one that stimulates blood cell growth and development, i.e.", "of red blood cells, leukocytes, and , platelets.", "Such agents are well known in the art.", "For example, in order to increase infection-fighting white blood cell production, recombinant granulocyte-colony stimulating factor may be used to stimulate the growth of neutrophils.", "Another example of a hematopoietic cell growth stimulating agent is recombinant granulocyte macrophage-colony stimulating factor, which increases production of neutrophils, as well as other infection-fighting white blood cells, granulocytes and monocytes, and macrophages.", "Another hematopoietic agent is recombinant stem cell factor, which regulates and stimulates the bone marrow, specifically to produce stem cells.", "Pharmaceutical Formulations When employed as pharmaceuticals, the compounds of formulas (I) and (II) are usually administered in the form of pharmaceutical compositions.", "These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal.", "These compounds are effective as both injectable and oral compositions.", "Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.", "This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds of formula (I) or (II) above associated with pharmaceutically acceptable carriers.", "In making the compositions of this invention, the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container.", "When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.", "Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.", "In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients.", "If the active compound is-substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh.", "If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g.", "about 40 mesh.", "Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose.", "The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.", "The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.", "The compositions are preferably formulated in a unit dosage form, each dosage containing from about 5 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient.", "The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.", "Preferably, the compound of formula (I) or (II) above is employed at no more than about 20 weight percent of the pharmaceutical composition, more preferably no more than about 15 weight percent, with the balance being pharmaceutically inert carrier(s).", "The active compound is effective over a wide dosage range and is generally administered in a pharmaceutically effective amount.", "It will be understood, however, that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.", "For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.", "When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.", "This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.", "The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.", "For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.", "The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.", "A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.", "The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.", "Compositions for inhalation or insufflation include-solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.", "The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra.", "Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect.", "Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases.", "Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine.", "Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.", "The following formulation examples illustrate representative pharmaceutical compositions of the present invention.", "FORMULATION EXAMPLE 1 Hard gelatin capsules containing the following ingredients are prepared: Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0 Magnesium stearate 5.0 The above ingredients are mixed and filled into hard-gelatin capsules in 340 mg quantities.", "FORMULATION EXAMPLE 2 A tablet formula is prepared using the ingredients below: Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose, microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0 The components are blended and compressed to form tablets, each weighing 240 mg. FORMULATION EXAMPLE 3 A dry powder inhaler formulation is prepared containing the following components: Ingredient Weight % Active Ingredient 5 Lactose 95 The active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.", "FORMULATION EXAMPLE 4 Tablets, each containing 30 mg of active ingredient, are prepared as follows: Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mg Starch 45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone 4.0 mg (as 10% solution in sterile water) Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1.0 mg Total 120 mg The active ingredient, starch and cellulose are passed through a No.", "20 mesh U.S. sieve and mixed thoroughly.", "The solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve.", "The granules so produced are dried at 50° to 60° C. and passed through a 16 mesh U.S. sieve.", "The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No.", "30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 120 mg. FORMULATION EXAMPLE 5 Capsules, each containing 40 mg of medicament are made as follows: Quantity Ingredient (mg/capsule) Active Ingredient 40.0 mg Starch 109.0 mg Magnesium stearate 1.0 mg Total 150.0 mg The active ingredient, starch, and magnesium stearate are blended, passed through a No.", "20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg quantities.", "FORMULATION EXAMPLE 6 Suppositories, each containing 25 mg of active ingredient are made as follows: Ingredient Amount Active Ingredient 25 mg Saturated fatty acid glycerides 2,000 mg The active ingredient is passed through a No.", "60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary.", "The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.", "FORMULATION EXAMPLE 7 Suspensions, each containing 50 mg of medicament per 5.0 mL dose are made as follows: Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v.", "Purified water to 5.0 mL The active ingredient, sucrose and xanthan gum are blended, passed through a No.", "10 mesh U.S. sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water.", "The sodium benzoate, flavor, and color are diluted with some of the water and added with stirring.", "Sufficient water is then added to produce the required volume.", "FORMULATION EXAMPLE 8 Quantity Ingredient (mg/capsule) Active Ingredient 15.0 mg Starch 407.0 mg Magnesium stearate 3.0 mg Total 425.0 mg The active ingredient, starch, and magnesium stearate are blended, passed through a No.", "20 mesh U.S. sieve, and filled into hard gelatin capsules in 425.0 mg quantities.", "FORMULATION EXAMPLE 9 A subcutaneous formulation may be prepared as follows: Ingredient Quantity Active Ingredient 5.0 mg Corn Oil 1.0 mL FORMULATION EXAMPLE 10 A topical formulation may be prepared as follows: Ingredient Quantity Active Ingredient 1-10 g Emulsifying Wax 30 g Liquid Paraffin 20 g White Soft Paraffin to 100 g The white soft paraffin is heated until molten.", "The liquid paraffin and emulsifying wax are incorporated and stirred until dissolved.", "The active ingredient is added and stirring is continued until dispersed.", "The mixture is then cooled until solid.", "Another preferred formulation employed in the methods of the present invention employs transdermal delivery devices (“patches”).", "Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts.", "The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art.", "See, e.g., U.S. Pat.", "No.", "5,023,252, issued Jun.", "11, 1991, herein incorporated by reference in its entirety.", "Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.", "Frequently, it will be desirable or necessary to introduce the pharmaceutical composition to the brain, either directly or indirectly.", "Direct techniques usually involve placement of a drug delivery catheter into the host's ventricular system to bypass the blood-brain barrier.", "One such implantable delivery system used for the transport of biological factors to specific anatomical regions of the body is described in U.S. Pat.", "No.", "5,011,472 which is herein incorporated by reference in its entirety.", "Indirect techniques, which are generally preferred, usually involve formulating the compositions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid-soluble drugs.", "Latentiation is generally achieved through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present on the drug to render the drug more lipid soluble and amenable to transportation across the blood-brain barrier.", "Alternatively, the delivery of hydrophilic drugs may be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood-brain barrier.", "Other suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed.", "(1985).", "Utility The compounds and pharmaceutical compositions of the invention are useful as anti-neoplastic agents, and accordingly, have utility in treating cancer in mammals including humans.", "As noted above, the compounds described herein are suitable for use in a variety of drug delivery systems described above.", "Additionally, in order to enhance the in vivo serum half-life of the administered compound, the compounds may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or other conventional techniques may be employed which provide an extended serum half-life of the compounds.", "The amount of compound administered to the patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like.", "In therapeutic applications, compositions are administered to a patient already suffering from cancer in an amount sufficient to at least partially arrest further onset of the symptoms of the disease and its complications.", "An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on the judgment of the attending clinician depending upon factors such as the degree or severity of cancer in the patient, the age, weight and general condition of the patient, and the like.", "Preferably, for use as therapeutics, the compounds described herein are administered at dosages ranging from about 0.1 to about 500 mg/kg/day.", "In prophylactic applications, compositions are administered to a patient at risk of developing cancer (determined for example by genetic screening or familial trait) in an amount sufficient to inhibit the onset of symptoms of the disease.", "An amount adequate to accomplish this is defined as “prophylactically effective dose.” Amounts effective for this use will depend on the judgment of the attending clinician depending upon factors such as the age, weight and general condition of the patient, and the like.", "Preferably, for use as prophylactics, the compounds described herein are administered at dosages ranging from about 0.1 to about 500 mg/kg/day.", "As noted above, the compounds administered to a patient are in the form of pharmaceutical compositions described above.", "These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.", "When aqueous solutions are employed, these may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.", "The pH of the compound preparations typically will be between 3 and 11, more preferably from 5-9 and most preferably from 7 and 8.It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.", "Specific embodiments of the invention will now be described through examples.", "The following synthetic and biological examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention.", "Unless otherwise stated, all temperatures are in degrees Celsius.", "EXAMPLES In the examples below, the following abbreviations have the following meanings.", "If an abbreviation is not defined, it has its generally accepted meaning.", "bd=broad doublet bs=broad singlet c=concentration d=doublet dd=doublet of doublets ddd=doublet of doublets of doublets DMF=dimethylformamide DMSO=dimethyl sulfoxide g=grams hept.=heptuplet J=coupling constant m=multiplet M=molar max=maximum mg=milligram min.=minutes mL=milliliter mM=millimolar mmol=millimole N=normal ng=nanogram nm=nanometers OD=optical density q=quartet s=singlet sept=septuplet t=triplet THF=tetrahydrofuran tlc=thin layer chromatography μL=microliter The antibodies were obtained from the following vendors: Transduction Laboratories, Lexington, Ky. (Raf-1, PKB/AKT); New England Biolabs Inc, Beverly, Mass.", "(phospho-MAP kinase and phospho-PKB/AKT); Santa Cruz Inc, Santa Cruz, Calif. (ERK-1, ERK-2); fetal bovine serum (FBS) from Hyaclone (Logan, Utah).", "Additionally, the term “Aldrich” indicates that the compound or reagent used in the following procedures is commercially available from Aldrich Chemical Company, Inc., .1001 West Saint Paul Avenue, Milwaukee, Wis. 53233 USA; the term “Fluka” indicates the compound or reagent is commercially available from Fluka Chemical Corp., 980 South 2nd Street, Ronkonkoma, N.Y. 11779 USA; the term “Lancaster” indicates the compound or reagent is commercially available from Lancaster Synthesis, Inc., P.O.", "Box 100, Windham, N.H. 03087 USA; and the term “Sigma” indicates the compound or reagent is commercially available from Sigma, P.O.", "Box 14508, St. Louis, Mo.", "63178 USA.", "Unless otherwise stated, all temperatures are in degrees Celsius.", "NMR spectra were recorded on an IBM-Bruker 200-MHz or a Bruker 400-MHz Spectrometer with Me4Si as internal standard.", "Infrared spectra were recorded on a Perkin-Elmer 1600 FT spectrophotometer.", "Optical rotations were measured on a JASCO Model DIP-140 digital polarimeter-using a 1-dm cell.", "Methylene chloride and pyridine were distilled from calcium hydride and barium oxide, respectively.", "Chloroform was distilled from P2O5.All other synthetic reagents were used as received unless otherwise stated.", "In these synthetic methods, the starting materials can contain a chiral center and, when a racemic starting material is employed, the resulting product is a mixture of R,S enantiomers.", "Alternatively, a chiral isomer of the starting material can be employed and, if the reaction protocol employed does not racemize this starting material, a chiral product is obtained.", "Such reaction protocols can involve inversion of the chiral center during synthesis.", "Alternatively, chiral products can be obtained via purification techniques which separates enantiomers from a R,S mixture to provide for one or the other stereoisomer.", "Such techniques are well known in the art.", "The compounds of formula (I) or (II) may be synthesized using the methods ememplified in the examples.", "Such method may be adapted to produce analogs, derivatives and variants within the scope of formula (I) or (II).", "The compounds of formula (I) or (II) can also be prepared via several divergent synthetic routes with the particular route selected relative to the ease of compound preparation, the commercial availability of starting materials, and the like.", "Example 1 Preparation of the S and R ET-16-phosphono-TDB stereoisomers 2′-(Trimethylammonio)ethyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate [(S)-ET-16-phosphono-TDB] was synthesized in 91% enantiomeric excess from 1-O-hexadecyl-2-O-methyl-sn-glycerol (Byun, H-S., Kumar, E. R., and Bittman, R. (1994) J Org.", "Chem.", "59, 2630-2633).", "Swern oxidation of the latter compound was carried out as follows: Dimethyl sulfoxide (1.85 mL, 21.4 mmol) was added to a solution of oxalyl chloride (1.0 mL, 11.5 mmol) in 150 mL of methylene chloride at −78° C. After 10 min, 1-O-hexadecyl-2-O-methyl-sn-glycerol (3.31 g, 10.0 mmol) in 20 mL of methylene chloride was added, and the reaction mixture was stirred for 1 h at −78° C., followed by the addition of triethylamine (7.0 mL, 50 mmol).", "After 1 h at −78° C., the mixture was warmed to room temperature, diluted with diethyl ether, and washed with water, 10% aqueous sodium bisulfate solution, and brine solution.", "The organic phase was dried over sodium sulfate and concentrated to give crude (S)-1-O-hexadecyl-2-O-methoxyglyceraldehyde.", "Tetraisopropyl methylenediphosphonate (3.44 g, 10 mmol) was added to a suspension of sodium hydride (0.3 g, 10 mmol, 85% in white oil, washed with dry hexane twice) in 50 mL of dry THF at 0° C. After cessation of H2 evolution, a solution of the crude aldehyde in 10 mL of THF was added and the mixture was stirred overnight at 0° C. The mixture was concentrated under reduced pressure and the residue was dissolved in diethyl ether and washed with water and brine.", "The organic phase was dried over sodium sulfate and concentrated.", "The residue was purified by column chromatography on silica gel (elution with chlorofonn-methanol 50:1) to give 4.31 g of pure diisopropyl 4-(hexadecyloxy)-3-(S)-methoxy-1-butenephosphonate (88%) as a pale yellow oil; [α]25D −3.14° (c 50.8, CHCl3); IR (film) 2356, 1361, 1115, 1061, 1020 cm−1; 1H NMR (CDCl3) δ 6.65 (dt, 1H, J=5.1, 17.2 Hz), 5.98 (dd, 1H, J=17.2,20.1 Hz), 4.59-4.73 (m, 1H), 3.38-3.96 (m, 6H), 3.47 (s, 3H), 1.32-1.35 (m, 2H), 1.32 (s, 12H), 1.26 (s, 26H), 0.88 (t, 3H, J=5.6 Hz); 13C NMR (CDCl3) δ 153.21, 152.95; Anal.", "Calcd for C27H55O5P: C, 66.09; H, 11.30; P, 6.31.Found: C, 66.21; H, 11.22; P, 6.26.The butenephosphonate (246 mg, 0.50 mmol) was dissolved in 10 mL of methylene chloride followed by the addition of bromotrimethylsilane (1.3 mL, 2.7 mmol) and stirring for 2 h at room temperature.", "Volatile materials were removed in vacuo, the residue was dissolved in THF-water (10 mL, 9:1 by volume), and the mixture was allowed to stand for 2 h at room temperature to complete the hydrolysis of the isopropyl ester groups.", "The solvents were removed under vacuum, and the residue was dried with the aid of dry 2-propanol followed by lyophilization from benzene to give the corresponding phosphonic acid.", "A solution of the phosphonic acid, choline tosylate (0.30 g, 0.90 mmol), and trichloroacetonitrile (0.30 mL, 3.0 mmol) in 20 mL of pyridine was heated for 48 h at 50° C. After most of pyridine was removed under reduced pressure, a dark brown semi-solid residue was obtained.", "It was dissolved in THF-water (9:1 by volume) and passed through a column of Amberlite MB-3 ion exchange resin, which was previously equilibrated with THF-water (9:1).", "Pure phosphonocholine was obtained after chromatography on silica gel column two times, eluting with chloroform-methanol-water (65:35:4); 150 mg (61% yield, after filtration of a solution of the product in chloroform through Cameo filters to remove suspended silica gel); [α]25D −2.32° (c 9.1, CHCl3—CH3OH, 1:1); IR (film) 3355, 1361, 1100 cm-1; 1H NMR (400 MHz, CHCl3—CD3OD) δ 6.35 (ddd, IH, J=5.80, 17.32, 20.17 Hz), 5.98 (dd, 1H, J=17.32,17.56 Hz), 4.05 (s, 3H), 3.90-3.93 (m, 1H), 3.65-3.68 (m, 2H), 3.42-3.48 (m, 4H), 3.41 (s, 2H), 3.26 (s, 9H), 1.54-1.58 (m, 2H), 1.26 (s, 26H), 0.88 (t, 3H, J=6.64 Hz); 13C NMR (100 MHz, CHCl3—CD3OD) δ 142.82, 125.43 (d, J1H-31P=186.6 Hz), 81.12 (d, J1H-31P=20.1 Hz), 73.00, 71.71 66.63, 57.69, 57.21, 54.35, 31.81, 29.58, 29.54, 29.46, 29.41, 25.92, 22.57, 13.98.Anal.", "Calcd for C26H54O5P.2H2O: C, 59.18; H, 11.08; N, 2.65; P, 5.87.Found: C, 59.20; H, 11.11; N, 2.44; P, 5.66.2′-(Trimethylammonio)ethyl 4-(hexadecyloxy)-3-(R)-methoxy-1-butenephosphonate [(R)-ET-16-phosphono-TDB] was synthesized from 3-O-hexadecyl-2-O-methyl-sn-glycerol in 59% yield by analogous procedures to those described above; [α]25D+2.67° (c 23.1, CHCl3—CH3OH, 1:1).", "The 1H and 13C NMR spectra were identical to those of the S enantiomer.", "Stock solutions (30 mM) of (R)-ET-16-phosphono-TDB and (S)-ET-16-phosphono-TDB in absolute ethanol were kept at −20° C. The structures of (R) and (S)-ET-16-phosphono-TDB are as follows: Example 2 Cell Culture All the cell lines were grown from frozen stocks originally obtained from ATCC.", "T47D, MDA-MB-468 and MCF-7 (breast cancer cell lines) were grown in DMEM.", "SK-N-MC and SK-N-SH (neuroblastoma) were grown in minimum essential medium with non-essential amino acids and Earle's balanced salt solution.", "All media were supplemented with 10% FBS and antibiotics (Ashagbley, A., Samadder, P., Bittman, R., Erukulla, R. K., Byun, H-S., Arthur, G. (I 996) Anticancer Res.", "16, 1813-1818).", "Example 3 Effect of Ether Lipids on the Proliferation of Log-Phase Epithelial Cells The effect of the ET-16-phosphono-TDBs on cell proliferation was conducted as previously described (Ashagbley, A., Samadder, P., Bittman, R., Erukulla, R. K., Byun, H-S., Arthur, G. (1996) Anticancer Res.", "16, 1813-1818; and Lu, X., and Arthur, G. (1992) Cancer Res.", "52, 2806-2812).", "The cells were seeded in 24-well dishes at a density of 20,000 cells/well with 10% FBS-supplemented medium.", "The cell numbers were monitored daily.", "When growth was exponential, the medium was removed and replaced with 10% FBS-supplemented medium containing different concentrations (0-5 μM) of (R) or (S)-ET-16-phosphono-TDB.", "The cell numbers at the time of addition of the drugs were determined with a Coulter ZM counter.", "After 48 h incubation, the cell numbers were determined and the increase in cell number over the numbers at the time of the addition of the drugs was determined and expressed as a function of the control cells which received no drug.", "The viability of the cells was assessed by the ability to exclude trypan blue dye.", "Effect of (R)-ET-16-phosphono-TDB and (S)-ET-16-phosphono-TDB on Epithelial Cancer Cell Proliferation The antiproliferative properties of (R)- and (S)-ET-16-phosphono-TDB were assessed with breast epithelial cancer cells (FIGS.", "1, 2, and 3) and neuroblastoma cell lines (FIGS.", "4 and 5).", "The results are the means±standard deviations of quadruplicate wells from 3 different experiments.", "Differences in the susceptibility of the cell lines to each enantiomer were observed at the concentrations examined.", "The order of increasing susceptibility among the breast cell lines was MDA-MB-468<MCF-7<T47D, and among neuroblastoma cell lines, it was SK-N-MC<SK-N-SH.", "In the 3 breast epithelial cancer cell lines examined (MDA-MB-468, MCF-7 and T47D), the (S) enantiomer had a greater effect on inhibition of cell proliferation than did the R enantiomer (FIGS.", "1-3) although the magnitude of the differences varied with the cell line.", "Thus, the S enantiomer was much more active than the R enantiomer in T47D and MDA-MB-468 cells compared with MCF-7 cells.", "The IC50 values (drug concentration required to reduce cell growth by 50%) are shown in Table 1.A greater differential effect was also observed with the neuroblastoma cell line SK-N-MC than with SK-N-SH.", "Thus, for each cell line tested, the S enantiomer of ET-16-phosphono-TDB was significantly more active than the R enantiomer.", "TABLE I IC50 values (μM) Cell Line R-enantiomer S-enantiomer T47D >5 0.9 MDA-MB-468 >5 4.2 MCF-7 4.1 1.9 SK-N-MC >5 2.2 SK-N-SH 2.5 1.8 Example 4 Effect of S and R ET-16-phosphono-TDB Compounds on MAP kinase and PKB phosphorylation Quiescent MCF-7 cells were preincubated with (“+” in FIGS.", "6-8) or without (“−” in FIGS.", "6-8) 15 μM of the R or S enantiomer of ET-16-phosphono-TDB (TDB-PC in FIGS.", "7-9) in DMEM supplemented with BSA (0.5 mg/mL) for 3 h. The cells were washed twice and stimulated with epithelial growth factor (EGF) or insulin for various periods.", "At the end of the incubation, the medium was aspirated, and the cells washed with ice-cold PBS and scraped into ice cold buffer comprising 20 mM Tris-HCl (pH 7.4) 2 mM EGTA, 100 mM β-glycerophosphate, 1 mM Na3VO4, aprotinin (10 μg/mL), leupeptin (10 μg/mL), 0.2 mM aininoethylbenzylsulfonyl fluoride, 0.2 mM benzarnidine, 1 mM dithiothreitol, 1% triton-X-100, and 0.5% NP-40).", "After ultrasonication, the mixture was centrifuged at 100,000×g for 30 min.", "The cell lysates were flash frozen and stored at −70° C. until required.", "MAP kinase activity in cell lysates from EGF and insulin-stimulated MCF-7 cells were assessed by Western blot analysis with phospho-specific antibodies according to the instructions provided by the manufacturer (New England Biolabs).", "PKB/AKT activity in insulin-stimulated MCF-7 cell lysates was also assessed with phospho-specific antibodies by Western blot analysis.", "Bound antibody was visualized by chemiluminescence using reagents from Boehringer Mannheim and quantitated with a multiimager Fluor S system (BioRad).", "The results displayed in FIGS.", "6-8 indicate that there was a direct correlation between the activity of the compounds and inhibition of the MAP kinase pathway (which transduces signals from growth factors) and activation of protein kinase B pathway (which has been implicated in cell survival).", "The S enantiomer, which was more potent in inhibiting the growth of the cells, inhibited the phosphorylation of MAP kinase and PKB in insulin- and EGF-stimulated MCF-7 cells to a greater extent than the less active R enantiomer.", "The results suggest that the inhibitory effects of the compounds are related to their ability to inhibit MAP kinase, which is implicated in proliferation (Marshall (1995) Cell 80: 179-185), and the PKB pathway, which is implicated in cell survival (Coffer et al.", "(1998) Biochem.", "J.", "335: 1-13).", "Example 5 Growth Inhibition (GI50s) For growth inhibitory evaluation, five human tumor cell lines (U937; HT29; A549; MCF7; MCF7/ADR) and two normal fibroblast cell lines (NIH-3T3, murine; WI-38, human) were used.", "For comparison, the activity of free L-ET18OCH3 and D-ET18OCH3 was examined.", "As shown in Table 3, both L and D isomers of ET18OCH3 gave essentially identical results with the order of sensitivity for the cells lines being U937>HT29>A549>MCF7>MCF7/ADR, NIH-3T3 (normal cell line).", "WI-38 cells were moderately sensitive to both ether lipids with GI50 values of 10-13 μM, which was 3-4 times lower than that for the NIH-3T3 cells at 41-47 μM.", "Compounds (R)- and (S)-ET-16-phosphono-TDB show remarkable selectivity with significantly higher GI50 values for the normal cell lines as compared to the tumor cell lines.", "TABLE 2 Growth Inhibition of Tumor/Normal Cells GI50 (μM) Tumor Cell Lines Normal Cell MCF7/ Lines* Compound U937 HT29 A549 MCF7 ADR NIH-3T3 WI38 L-ET18OCH3 1.0-1.5 5.5, 6.0 6.5-9.1 9.7-18.6 25.7->40 46.6 10-12.8 D-ET18OCH3 1.4 5.1 8.0 14.6 25.1 41.4 10-13.5 (R)-ET-16- 2.2 5.9 15.3 35.1 >40 77.3 57.5 phosphono- TDB (S)-ET-16- 1.1 2.8 9.0 32.4 >40 66.3 14.4 phosphono- TDB The invention has been described with reference to specific embodiments.", "Substitutions, omissions, additions and deletions may be made without departing from the spirit and scope of the invention defined in the appended claims.", "From the foregoing description, various modifications and changes in the composition and method will occur to those skilled in the art.", "All such modifications coming within the scope of the appended claims are intended to be included therein.", "All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety." ] ]	Patent_10468766
[ [ "Rear cover assembly for washing machine and dryer and washing system using the same", "Rear cover assemblies (100, 200) for washing machines and dryers including front panels(110, 210) each mounted on rear of a top cover fitted to an upper part of each of the washing machines (1) and the dryers (2), the front panels each having a seat (111, 211) of identical shape, and back panels (120, 210) each being formed to be fitted to the seat (111, 211) of the front panel (110, 210), the back panel being configured to allow accessories required for each of the washing machines (1) or each of the dryers (2) to be fitted thereto.", "These rear cover assemblies form a unified particular outer appearance in one set consisting of any selected two of the washing machines (1) and the dryers (2), and they are exchangeable such that the particular outer appearance can be maintained even if relative positions of the washing machines and the dryers within the one set are changed." ], [ "1.Rear cover assemblies for washing machines and dryers comprising: front panels each mounted on rear of a top cover fitted to an upper part of each of the washing machines and the dryers, the front panels each having a seat of identical shape; and back panels each being formed to be fitted to the seat of the front panel, the back panels be configured to allow accessories required for each of the washing machines or each of the dryers to be fitted thereto, wherein the rear cover assemblies form a unified particular outer appearance in one set consisting of any selected two of the washing machines and the dryers, and the rear cover assemblies are exchangeable in the one set such that the particular outer appearance can be maintained for the one set even if relative positions of the washing machines and the dryers within the one set are changed.", "2.The rear cover assemblies as claimed in claim 1, wherein the particular outer appearance is formed by the front panels.", "3.The rear cover assemblies as claimed in claim 2, wherein the particular outer appearance is formed by upper part outlines of the front panels.", "4.The rear cover assemblies as claimed in claim 2, wherein the front panels are symmetric within the one set.", "5.The rear cover assemblies as claimed in claim 1, wherein the front panel includes a control panel enabling a user to operate the washing machine or the dryer.", "6.The rear cover assemblies as claimed in claim 5, wherein the control panel includes various buttons for giving operational orders and a display window for displaying an operation state.", "7.The rear cover assemblies as claimed in claim 5, wherein the control panel is unified with the front panel, or detachable from the front panel.", "8.The rear cover assemblies as claimed in claim 7, wherein the windows and buttons have identical positions in the different control panels in the one set.", "9.The rear cover assemblies as claimed in claim 1, wherein the seat is formed by cutting off a rear surface of the front panel.", "10.The rear cover assemblies as claimed in claim 1, wherein the accessories for the washing machine include a water supply tube and wires.", "11.The rear cover assemblies as claimed in claim 1, wherein the accessories for the dryer include wire.", "12.The rear cover assemblies as claimed in claim 1, wherein the back panel of the dryer is formed of a metal.", "13.The rear cover assemblies as claimed in claim 1, wherein the back panel is formed unified with the top cover.", "14.A washing machine comprising: a cabinet; a washing tub, or drum mounted in the cabinet rotatable by power means for washing laundry; a top cover for covering an upper part of the cabinet; and a rear cover including; a front panel mounted in a rear part of the top cover and having a seat of a predetermined shape, and a back panel configured to allow various accessories to be fitted thereto, the back panel being formed to be fitted to the seat in the front panel, wherein, when the washing machine is combined with another washing machine or a dryer having identical rear cover, the rear covers form a unified particular outer appearance, and the rear covers are exchangeable such that the particular outer appearance is maintained even if positions of the washing machine and the another washing machine or the dryer are interchanged.", "15.The washing machine as claimed in claim 14, wherein the particular outer appearance is formed by the front panels.", "16.The washing machine as claimed in claim 15, wherein the particular outer appearance is formed by upper part outlines of the front panels.", "17.The washing machine as claimed in claim 15, wherein the front panels are symmetric.", "18.The washing machine as claimed in claim 14, wherein the front panel includes a control panel enabling a user to operate the washing machine.", "19.The washing machine as claimed in claim 18, wherein the control panel includes various buttons for giving operational orders and a display window for displaying an operation state.", "20.The washing machine as claimed in claim 18, wherein the control panel is unified with the front panel, or detachable from the front panel.", "21.The washing machine as claimed in claim 20, wherein the windows and buttons have identical positions in the different control panels.", "22.The washing machine as claimed in claim 14, wherein the seat is formed by cutting off a rear surface of the front panel.", "23.The washing machine as claimed in claim 14, wherein the accessories include a water supply tube and wires.", "24.The washing machine as claimed in claim 14, wherein the back panel is formed unified with the top cover.", "25.A dryer comprising: a cabinet; a drum mounted in the cabinet rotatable by power means for drying laundry; a top cover for covering an upper part of the cabinet; and a rear cover including; a front panel mounted in a rear part of the top cover and having a seat of a predetermined shape, and a back panel be configured to allow various accessories to be fitted thereto, the back panel being formed to be fitted to the seat in the front panel, wherein when the dryer is combined with another dryer or a washing having identical rear cover, the rear covers form a unified particular outer appearance, and the rear covers are exchangeable such that the particular outer appearance is maintained even if positions of the dryer and the another dryer or the washing machine are interchanged.", "26.The dryer as claimed in claim 25, wherein the particular outer appearance is formed by the front panels.", "27.The dryer as claimed in claim 26, wherein the particular outer appearance is formed by upper part outlines of the front panels.", "28.The dryer as claimed in claim 26, wherein the front panels are symmetric.", "29.The dryer as claimed in claim 25, wherein the front panel includes a control panel enabling a user to operate the dryer.", "30.The dryer as claimed in claim 29, wherein the control panel includes various buttons for giving operational orders and a display window for displaying an operation state.", "31.The dryer as claimed in claim 29, wherein the control panel is unified with the front panel, or detachable from the front panel.", "32.The dryer as claimed in claim 31, wherein the windows and buttons have identical positions in the different control panels.", "33.The dryer as claimed in claim 25, wherein the seat is formed by cutting off a rear surface of the front panel.", "34.The dryer as claimed in claim 25, wherein the accessories include wires.", "35.The dryer as claimed in claim 25, wherein the back panel is formed of a metal.", "36.The dryer as claimed in claim 25, wherein the back panel is formed unified with the top cover.", "37.A washing system comprising: a washing machine including a cabinet, a washing tub or drum mounted in the cabinet rotatable by power means for washing laundry, a top cover for covering an upper part of the cabinet, and a rear cover having a front panel mounted in a rear part of the top cover and having a seat of a predetermined shape and a back panel configured to allow various accessories to fitted thereto and formed to be fitted to the seat in the front panel; and a dryer including a cabinet, a drum mounted in the cabinet rotatable by power means for drying laundry, a top cover for covering an upper part of the cabinet, and a rear cover having a front panel mounted in a rear part of the top cover and having a seat identical with the seat of the washing machine and a back panel configured to allow fitting various accessories to be fitted thereto and formed to be fitted to the seat in the front panel, wherein the rear covers of the washing machine and dryer form a unified particular outer appearance on the whole, and are exchangeable to maintain the outer appearance.", "38.The washing system as claimed in claim 37, wherein the particular outer appearance is formed by the front panels of the washing machine and the dryer.", "39.The washing system as claimed in claim 3S, wherein the particular outer appearance is formed by upper part outlines of the front panels of the washing machine and the dryer.", "40.The washing system as claimed in claim 2, wherein the front panels of the washing machine and the dryer are symmetric.", "41.The washing system as claimed in claim 37, wherein the front panels include control panels each enabling a user to operate the washing machine or the dryer.", "42.The washing system as claimed in claim 41, wherein the control panel includes various buttons for giving operational orders and a display window for displaying an operation state.", "43.The washing system as claimed in claim 41, wherein the control panel is unified with the front panel, or detachable from the front panel.", "44.The washing system as claimed in claim 43, wherein the windows and buttons have identical positions in the control panels of the washing machine and the dryer.", "45.The washing system as claimed in claim 37, wherein the seats are formed by cutting off rear surfaces of the front panels of the washing machine and the dryer.", "46.The washing system as claimed in claim 37, wherein the accessories for the washing machine include a water supply tube and wires.", "47.The washing system as claimed in claim 37, wherein the accessories for the dryer include wires.", "48.The washing system as claimed in claim 37, wherein the back panel of the dryer is formed of a metal.", "49.The washing system as claimed in claim 37, wherein the back panel is unified with the top cover." ], [ "<SOH> BACKGROUND ART <EOH>As known, since the washing machine and the dryer provide function supplementary to each other, the washing machine and the dryer are installed even in parallel when required.", "According to this, a washing system having independent washing machine and dryer combined as one set therein is put into practical use, recently.", "The washing system has the washing machine and the dryer coupled in view of functions, though independent from each other in view of structure.", "Moreover, for giving a sense of one system to a user, the washing system has a unified design on the whole.", "Depending on specification, the washing system may be provided with a control panel, and a rear cover assembly projected from rear of the washing system.", "Accordingly, in the washing system, particularly, the rear cover assemblies on the washing machine and dryer respectively are designed to have one specific appearance.", "In the meantime, an air discharge structure for the dryer, and water supply/drain structures for the washing machine may vary with a structure of a house.", "Therefore, it is required that the washing machine and the dryer in the washing system are installed in the vicinity of the water supply/drain structures and the air discharge structure.", "However, even if the washing system is installed suitable to the house structure, the washing system may not be harmonious in view of the outer appearance.", "For an example, in the washing system, the dryer may be disposed on a right side of the washing machine, and under this arrangement, the washing system may be designed to have a particular outer appearance on the whole.", "However, if it is required that the dryer is disposed on a left side of the washing machine suitable to a structure of a house, arrangement of the washing machine and the dryer may not be harmonious." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>The accompanying drawings, which are included to provide a further understanding of the invention, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention: In the drawings: FIG.", "1 illustrates a perspective view of a washing system in accordance with a preferred embodiment of the present invention; FIG.", "2 illustrates a back side perspective view of a washing system of the present invention showing a rear cover assembly in detail; and FIGS.", "3 A˜ 3 D illustrate back side perspective views each showing the steps of a process for interchanging the rear cover assemblies in the washing system of the present invention.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to a washing system for washing and drying laundry, and more particularly, to a rear cover assembly for a washing machine or a dryer in the washing system.", "BACKGROUND ART As known, since the washing machine and the dryer provide function supplementary to each other, the washing machine and the dryer are installed even in parallel when required.", "According to this, a washing system having independent washing machine and dryer combined as one set therein is put into practical use, recently.", "The washing system has the washing machine and the dryer coupled in view of functions, though independent from each other in view of structure.", "Moreover, for giving a sense of one system to a user, the washing system has a unified design on the whole.", "Depending on specification, the washing system may be provided with a control panel, and a rear cover assembly projected from rear of the washing system.", "Accordingly, in the washing system, particularly, the rear cover assemblies on the washing machine and dryer respectively are designed to have one specific appearance.", "In the meantime, an air discharge structure for the dryer, and water supply/drain structures for the washing machine may vary with a structure of a house.", "Therefore, it is required that the washing machine and the dryer in the washing system are installed in the vicinity of the water supply/drain structures and the air discharge structure.", "However, even if the washing system is installed suitable to the house structure, the washing system may not be harmonious in view of the outer appearance.", "For an example, in the washing system, the dryer may be disposed on a right side of the washing machine, and under this arrangement, the washing system may be designed to have a particular outer appearance on the whole.", "However, if it is required that the dryer is disposed on a left side of the washing machine suitable to a structure of a house, arrangement of the washing machine and the dryer may not be harmonious.", "DISCLOSURE OF INVENTION An object of the present invention for solving the foregoing problem is to provide a washing system which can maintain an initial outer appearance even if an arrangement of independent appliances in the washing system is changed.", "The object of the present invention can be achieved by providing rear- cover assemblies for washing machines and dryers including front panels each mounted on rear of a top cover fitted to an upper part of each of the washing machines and the dryers, the front panel having a seat, and back panels each fitted to the seat of the front panel, the back panel having a system to fit accessories required for each of the washing machines or each of the dryers thereto, wherein a unified particular outer appearance is formed for one set consisting of any selected two of the washing machines and the dryers, such that the particular outer appearance can be maintained for the one set even if relative positions of the washing machines and the dryers within the one set are changed.", "The particular outer appearance is formed by the front panels, and more precisely formed by upper part outlines of the front panels.", "For forming the particular outer appearance, it is preferable that the front panels are symmetric within the one set.", "The front panel includes a control panel having a system enabling a user to operate the washing machine or the dryer, and the control panel includes various buttons for giving operational orders and a display window for displaying an operation state.", "The control panel is unified with the front panel, or detachable from the front panel, and the windows and buttons have identical positions in the different control panels in the one set.", "The seat is formed by cutting off a rear surface of the front panel.", "In the back panel, the accessories for the washing machine include a water supply tube and wires, and the accessories for the dryer include wire.", "It is preferable that the back panel of the dryer is formed of a metal.", "More preferably, the back panel is formed unified with the top cover.", "In another aspect of the present invention, there is provided a washing machine including a cabinet, a washing tub, or drum mounted in the cabinet rotatable by power means for washing laundry, a top cover for covering an upper part of the cabinet, and a rear cover having a front panel mounted in a rear part of the top cover having a form of seat and a back panel having a system for fitting various accessories thereto and a shape to be fitted to the seat in the front panel, wherein, when the washing machine is combined with another washing machine or a dryer having identical rear cover, the rear covers form a unified particular outer appearance such that the particular outer appearance is maintained even if positions of the washing machine and the another washing machine or the dryer are interchanged.", "In further aspect of the present invention, there is provided a dryer including a cabinet, a drum mounted in the cabinet rotatable by power means for drying laundry, a top cover for covering an upper part of the cabinet, and a rear cover having a front panel mounted in a rear part of the top cover having a form of seat and a back panel having a system for fitting various accessories thereto and a shape to be fitted to the seat in the front panel, wherein, when the dryer is combined with another dryer or a washing having identical rear cover, the rear covers form a unified particular outer appearance such that the particular outer appearance is maintained even if positions of the dryer and the another dryer or the washing machine are interchanged.", "In still further aspect of the present invention, there is provided a washing system including a washing machine having a cabinet, a washing tub, or drum mounted in the cabinet rotatable by power means for washing laundry, a top cover for covering an upper part of the cabinet, and a rear cover having a front panel mounted in a rear part of the top cover having a form of seat and a back panel having a system for fitting various accessories thereto and a shape to be fitted to the seat in the front panel, and a dryer having a cabinet, a drum mounted in the cabinet rotatable by power means for drying laundry, a top cover for covering an upper part of the cabinet, and a rear cover having a front panel mounted in a rear part of the top cover having a form of seat and a back panel having a system for fitting various accessories thereto and a shape to be fitted to the seat in the front panel, wherein the rear covers of the washing machine and dryer form a unified particular outer appearance on the whole, and are interchangeable to maintain the outer appearance.", "BRIEF DESCRIPTION OF DRAWINGS The accompanying drawings, which are included to provide a further understanding of the invention, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention: In the drawings: FIG.", "1 illustrates a perspective view of a washing system in accordance with a preferred embodiment of the present invention; FIG.", "2 illustrates a back side perspective view of a washing system of the present invention showing a rear cover assembly in detail; and FIGS.", "3A˜3D illustrate back side perspective views each showing the steps of a process for interchanging the rear cover assemblies in the washing system of the present invention.", "BEST MODE FOR CARRYING OUT THE INVENTION Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings.", "In description of the embodiments, the same parts will be given the same names and reference numerals, and iterative description of which will be omitted.", "FIG.", "1 illustrates a perspective view of a washing system in accordance with a preferred embodiment of the present invention, and FIG.", "2 illustrates a back side perspective view of a washing system of the present invention showing a rear cover assembly in detail, referring to which the washing system of the present invention will be described.", "The washing system of the present invention includes one set having a washing machine 1 and a dryer 2 combined therein.", "As shown, the washing machine 1 and the dryer 2 have independent and separated structures, but are combined to form the washing system.", "The washing machine 1 includes a housing and various components fitted in the housing for washing, and the dray 2 also has a similar structure.", "Each of the housings includes a cabinet 11 or 21 in a lower part, a top cover 12 or 22 on the cabinet 11 or 21, and a rear cover 100 or 200 in rear of the top cover 12 or 22.The lower cabinet 11 or 21 holds and mounts key components of the washing machine 1 or the dryer 2 therein.", "The top cover 12 or 22 covers an upper part of the cabinet 11 or 21, for protection of the components.", "The rear cover 100 or 200 includes a control panel 112 or 212 for operation of the washing machine 1 or the dryer 2, and covers a controller operably connected to the panel 113 or 212.Meanwhile, the door 13 or 23 may be fitted to the top cover 12 or the cabinet 21 depending on an arrangement of the components.", "In the case of the washing machine 1, there are a washing tub and- driving means for driving the washing tub inside of the housing.", "Laundry is introduced into the washing tub through the door 13, and washed and extracted of water as the washing tub rotated.", "Moreover, in the case of the dryer 2, there are a drying drum, driving means for rotating the drying drum, and devices for heating air inside of the housing.", "Laundry washed in the washing machine is introduced into the drying drum through the door 23, and dried by using the heating devices.", "For better drying of the laundry, the drying drum is rotated by the driving means.", "Therefore, a general washing process started from the washing machine 1 is finished at the dryer 1.In the washing system of the present invention, the washing machine 1 and the dryer 2 are arranged in parallel such that functions of the washing machine 1 and the dryer 2 are connected, closely.", "According to this, the user can process an entire washing process in a lot, more conveniently.", "That is, after washing the laundry at the washing machine 1, the user takes out the laundry from the washing machine and dries the laundry at an adjacent dryer 2 at once.", "Moreover, as shown, not only the functions, but also the designs are preferably harmonized, the washing machine 1 and the dryer 2 in the washing system are designed to have the same heights and widths.", "In the meantime, in general the rear covers 100 and 200 are projected from the top covers 12 and 22 for easy handling by the user.", "In view of overall design of the washing system, it is important to unify the designs of the rear covers 100 and 200 into one.", "Furthermore, it is preferable that a combination of rear covers 100 and 200 has a particular outer appearance.", "For an example, FIG.", "1 illustrates the rear covers 100 and 200 which form a smooth semi-elliptic form.", "Such rear covers 100 and 200 of the present invention will be explained in more detail.", "Referring to FIG.", "2, the rear cover 100 or 200 includes the front panel 110 or 210 on the top cover 12 or 22 and a back panel 120 or 220 in rear of the front panel 110 or 210.The front panel 110 or 210 is detachably fitted to rear of the top cover 12 or 22.As described before, the front panel 110 or 210 is projected from the top cover 12 or 22 to a height so that the user can handle the control panel 112 or 212 in the front panel 110 or 210 with easy.", "Meanwhile, referring to FIGS.", "1 and 2, since the front panels 110 and 210 are exposed to a front face of the washing system, and represents a whole shape of the rear covers 100 and 200, it can be actually known that the front panels 110 and 210 form a unified outer appearance, i.e., a smooth semi-elliptic form, at the time the rear covers 100 and 200 are combined.", "Particularly, since the projected front panels 110 and 210 form a top part outline of entire washing system, the top part outline of the front panels 110 and 210 form the semi-elliptic form, more significantly.", "Moreover, for forming a particular outer appearance, the front panels 110 and 210 may be asymmetrical.", "However, for easy design and fabrication, it is preferable that the front panels 110 and 210 are symmetrical.", "Referring to FIG.", "2, in the rear part of the front panel 110 or 210, a seat 111 or 211 for a back cover 120 is provided.", "The seat 111 or 211 is formed by cutting a part of the rear surface of the panel 110 or 210 away so that the cut away part can receive the back cover 120, actually.", "In more detail, the seat 111 or 211 has a form in the front panel 110 or 210.That is, the seat 111 of the washing machine and the seat 211 of the dryer have identical sizes and forms.", "The control panel 112 or 212 is fitted to a front part of the front panel 110 or 210.The control panel 112 or 212 includes a display window 112a or 212a for displaying an operation state and a plurality of buttons 112b and 212b for giving operational orders.", "The user presses the buttons 112b or 212b for generating an electrical signal to control operation, in response to which signal the controller makes the washing machine or the dryer to carry out a particular function.", "Information on the operation is provided to the user through the window 112a or 212a.", "In more detail, the control panel 112 or 212 may be unified with, or detachably fitted to the front panel 110 or 210.As shown in FIG.", "1, in the case of the unified control panels 112 and 212, the windows 112a and 212a are formed at identical positions in different control panels 112 and 212, and the buttons 112b and 212b are also formed in the same styles.", "That is, as shown, the window 112a of the washing machine and the window 212a of the dryer are positioned in right parts of the control panels 112 and 212 in the drawing, and buttons 112b of the washing machine and buttons 212b of the dryer are positioned left parts of the control panels 112 and 212.Therefore, even if the control panel 112 of the washing machine is fitted in place of the control panel 212 of the dryer, the control panel 112 can carry out the same functions for the dryer described before.", "Moreover, under the same reason, it is preferable that other characteristics except the positions, i.e., shapes colors of the windows and buttons are formed identical to each other.", "On the other hand, in the case of the detachable control panel 112 or 212, the detachable control panel 112 or 212 may be detached from a prior panel, and attached to a new panel again, when the front panel 110 or 210 is interchanged with the new panel.", "Therefore, since the detachable control panels 112 and 212 can be remained at original positions, windows and buttons thereof may be identical or have different positions and forms, selectively.", "The back panel 120 or 220 is required to have accessories required for the washing machine 1 or the dryer 2 fitted thereto.", "As known, at least the washing machine 1 and the dryer require the drain structure and the air discharge structure positioned at outsides thereof.", "The accessories not only connect the drain and the air discharge structure and the washing machine 1 and the dryer 2 for making normal operation, but also have various supplementary functions.", "As shown in FIG.", "2, in a case of the washing machine 1, the accessories include a water supply tube 121a and various wires 122a.", "The water supply tube 121a connects an external water supply source (city water tap) and a washing tub inside of the washing machine, for supplying washing water from the water supply source to an inside of the washing machine.", "The wires 122a electrically connect an external power source to inside devices.", "Therefore, for extending the wires 122a and the water supply tube 121a, the back panel 120 actually has openings 121b and 122b suitable for the wires 122a and the water supply tube 121a.", "In the case of the dryer 2, the accessories may be various wires 221a, for connecting the dryer to the external power source.", "When,the dryer 2 has a burner, the dryer 2 is required to have an exhaust pipe 222a and a ground structure 223a.", "The exhaust pipe 222a is provided for discharging hot air which is produced by the burner and dries the laundry through an exhaust structure of the house.", "The ground structure 223a is provided for fire protection since the dryer 2 has a combustion device, such as the burner.", "When the dryer 2 uses a gas, a gas tube is provided additionally.", "Therefore, alike the back panel of the washing machine, the back panel 220 of the dryer has openings 221b, 222b, and 223b for the accessories.", "Owing to the hot air, the exhaust tube 222a is heated to a substantial temperature.", "Therefore, it is preferable that the back panel 220 of the dryer is formed of a metal of good heat resistance.", "Opposite to this, the back panel 120 of the washing machine, which requires no heat resistance, may be formed of plastic.", "The back panel 120 or 220 is formed to fit to the seat 111 or 211 of the front panel.", "That is, a shape and a size of the back panel 120 or 220 is defined by the seat 111 or 211 for seating thereon.", "Therefore, as explained, since the seats 111 and 211 are identical, the back panels 120 and 220 are also identical in view of shapes and sizes.", "The back panel 126 or 220 is seated on the seat 111 or 211, to form a part of a rear surface of the front panel 110 or 210, in another words, to cover a part of rear surface.", "Since the back panel 120 or 220 has a system suitable to particular accessories, the back panel 120 or 220 may be unified with the top cover 12 or 22, unable to detach therefrom.", "Such unified back panel 120 or 220 is more favorable for stable support of the accessories, too.", "As described, seats 111 and 112 of the washing machine and the dryer are formed identical to each other.", "Alikely, the back panels 120 and 220 have the same shapes, such that the back panels 120 and 220 can be seated on any of the seats 111 and 112.Eventually, the front panels 110 and 220 are interchangeable.", "Furthermore, even if an overall particular outer appearance of the washing system is not harmonious owing to a relative positional change of the washing machine 1 and the dryer 2, the front panels 110 and 210 may be interchanged for restoration of the particular outer appearance.", "That is, in overall, the interchange of the front panels 110 and 210 brings about a result in which the rear covers 100 and 120 are interchanged, entirely.", "A process for interchanging such rear covers will be described in detail with reference to related drawings.", "FIGS.", "3A˜3D illustrate back side perspective views each showing the steps of a process for interchanging the rear cover assemblies in the washing system of the present invention.", "Alike FIG.", "2, for clarity and simplicity of the drawings, only key parts of the entire washing system are shown.", "FIG.", "3A illustrates a washing system of which initial particular outer appearance is changed.", "In general, the washing system is designed such that one of arrangements of the washing machine 1 and the dryer 2 (for an example, the washing machine 1 is positioned on a left side of the dryer 2) exhibits a unified outer appearance.", "Therefore, it can be known that the outer appearance in FIG.", "3A shows positions of the washing machine 1 and the dryer 2 are interchanged.", "On the other hand, as described, since widths and heights of the washing machine 1 and the dryer 2 in the unified washing system are identical basically, even if the positions are interchanged, the positional interchange gives no influence to an outer appearance unification of the entire washing system.", "However, since the rear covers 100 and 200 form a particular unified outer appearance in a state the rear covers 100 and 200 are projected, and combined to each other, when the positions of the washing machine I and the dryer 2 are interchanged, despite of the identical heights and widths, an overall outer appearance of the washing system is not harmonious.", "In FIG.", "3A, though the washing machine 1 is positioned on a right side of the dryer 1 initially when seen from rear of the washing machine 1, the washing machine 1 is shifted to a left side of the dryer 2 due to various reasons, such as a structure of the house, and the like.", "Referring to FIG.", "3B, in a case like this, at first, the front panel 110 or 210 is detached from the top cover 12 or 22.When the control panel 112 or 212 is detachable from the front panel 110 or 210, only the front panel 110 or 210 is detached except the control panel 112 or 212.If the control panel 112 or 212 and the front panel 110 or 210 are unified, the control panel 112 or 212 is detached together with the front panel 110 or 210.As shown, since the back panels 120 and 220 have systems to receive accessories of the washing machine 1 and the dryer 2 respectively, the back panels 120 and 220 are not interchangeable.", "Then, the front panels 110 and 210 are interchanged.", "As shown in FIG.", "3C, according to this, the front panel 110 of the washing machine is moved from a left side to the right side on an upper side 6f the dryer 2, and the front panel 210 of the dryer is moved from a right side to a left side on an upper side of the washing machine.", "Referring to FIG.", "3D, finally, the interchanged front panels 110 and 220 are fastened to the washing machine and the dryer 2, respectively.", "That is, as shown, the front panel 110 of the washing machine is fitted to the top cover 22 of the dryer 2, and the front panel 210 of the dryer is fitted to the top cover 12 of the washing machine.", "At a same time, the back panel 120 of the washing machine is fitted to the seat 211 of the dryer, and the back panel 220 of the dryer is fitted to the seat 111 of the washing machine.", "As described before, this interchange of the front panels 110 and 210 is made possible by the identical seats 111 and 112 and the back covers 120 and 220.The control panels 112 and 212 detachable from the front panels are left at originally fitted washing machine 1 and the dryer 2, and fitted to the interchanged front panels.", "On the other hand, the unified control panels 112 and 212 are interchanged together with the front panels 110 and 210.However, the windows 112a and 212a and the buttons 112b and 212b are formed at the same positions, the windows 112a and 212a and the buttons 112b and 212b can carry out the same functions even after the front panels 110 and 210 are interchanged.", "Moreover, it is preferable that fitting structures for the front covers are formed identical on the top covers 12 and 22 for interchange of the front panels 110 and 210.For an example, the fitting structures are inclusive of the seats and fastening parts for the front panels 110 and 210.However, in the washing system, sizes of the washing machine 1 and the dryer 2 and sizes of the rear covers 100 and 200 dependent thereto are basically identical, the fitting structures are of course identical from the beginning.", "Even if there is a little difference of the fitting strictures, under the same reason, the front panels 110 and 210 can be made interchangeable by using adapters.", "The interchange of the front panels 110 and 210 restores the unharmonious outer appearance of the rear covers 100 and 200 into the initial unified particular outer appearance again, which brings about a result in which the outer appearance of the washing system is harmonious, actually.", "Moreover, the back panels 120 and 220 also permit the washing machine 1 and the dryer 2 to have continuous rear surfaces together with the seats 111 and 211, which also permit an overall outer appearance of the washing system more harmonious.", "As a result, in the present invention, the interchange of the front panels 110 and 210 has an effect of an interchange of the rear covers 100 and 210 actually, and moreover makes the user unable to aware of a change of the outer appearance.", "In the meantime, though the foregoing embodiment discloses a washing system including one washing machine and one dryer, the present invention is not limited to this.", "Rather, the washing system of the present invention may include two washing machines as one set, or two dryers as one set, as the case demands.", "As described before, in those embodiments too, the rear covers may have interchangeable system, to permit the washing system to maintain one unified particular outer appearance even if positions of the appliances in the washing system are changed.", "Thus, since those embodiments are identical to the embodiment described in detail actually, detailed description of those embodiments will be omitted.", "It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention.", "Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.", "Industrial Applicability In the washing system of the present invention, owing to seats of front panels formed identical to each other and the back panel fitted to the seats and also formed identical to each other, the front panels are made mountable to any of the washing machine and the dryer.", "Therefore, even if the washing system becomes to have an unharmonious outer appearance due to change of positions of the washing machine and the dryer, the front panels may be interchanged so that a particular outer appearance of the washing system is restored.", "As a result, the positions of the washing machine and the dryer in the washing system can be changed as desired without change of the outer appearance, thereby permitting the user convenient." ] ]	Patent_10468778
[ [ "Equipment for producing high-pressure saturated steam", "This invention relates to an equipment for producing high-pressure saturated steam.", "The equipment includes a water tank, a pump, a check valve, an atomizing nozzle, a heat chamber and a steam outlet.", "The pump is connected between water tank and the check valve.", "The check valve is connected with the heat chamber via the atomizing nozzle.", "The heat chamber having a steam outlet is hollow into which the atomizing nozzle extends.", "The hollow chamber is divided into many small chambers which are connected with each other successively.", "The equipment can produce high temperature steam rapidly and save energy." ], [ "1.Device for generating high-pressure saturated steam, comprising water tank (1), water pump (2), one-way valve (3), atomising nozzle (4), heating chamber (5) and steam outlet (6), in which the water pump (2) links the water tank (1) and one-way valve (3), which connects to atomising nozzle (4), atomising nozzle (4) is connected to heating chamber (5) which has a steam outlet (6) on one side, characterised in that the heating chamber (5) is hollow, installed in which are heating plates (8) that subdivide the chamber into multiple interconnected smaller heating cavities, the atomising nozzle (4) is connected to the first heating cavity (1) in the hollow chamber (5), whilst the last heating cavity in the hollow chamber (5) is connected to steam outlet (6).", "2.Device for generating high-pressure saturated steam according to claim 1, characterised in that the hollow chamber (5) is covered externally with a thermal insulating layer (7).", "3.Device for generating high-pressure saturated steam according to claim 1, characterised in that the said chamber is a spherical, hollow chamber.", "4.Device for generating high-pressure saturated steam according to claim 1, characterised in that the heating plate (8) has a circular main body, in which a heating plate positioning hole (11) is provided to retain the heating bar." ], [ "THE TECHNICAL SECTOR THIS DEVICE IS RELATED TO This invention relates to a device for generating high-pressure, saturated steam.", "THE PRIOR ART The application of steam is very broad, ranging from industrial, agricultural, domestic to medical applications.", "The traditional method to generate steam is to heat water to boiling point.", "Industrial boilers utilizing waste heat, domestic boilers or thermo-electric boilers may vary in their method of generating steam; the principle, however, remains the same, that is, to transmit heat via a heat conductor into water, which, through natural convection, reaches the boiling point and steam is generated.", "Such a method is time-consuming and uses expensive natural resources; when steam is not required, the boiling water cools off and energy is wasted.", "The Chinese patent 00228251.8 describes a device for generating high-pressure saturated steam.", "It comprises a motor, a water tank, a pressure gauge, a steam outlet, a steam chamber, a pressure pump or a gear pump, an atomising nozzle and a heating chamber, which are all interconnected.", "The water in the water tank is pumped to an atomising nozzle via a pressure pump or gear pump driven by a motor.", "The head of the nozzle then generates atomised mist, which turns into high-pressure, saturated steam in the heat chamber immediately after being heated.", "The steam is then transferred to the steam chamber, which is linked to the heat chamber.", "Due to the combined effect of heat and pressure from a pressure pump or gear pump, the steam in the steam chamber generated through continuous atomisation becomes high-pressure, saturated steam that meets the pressure requirements so that it can be delivered through a steam outlet with a shut-off valve to the device using the steam.", "Although the said device has already addressed the shortcomings and problems of long production time, high consumption of resources, waste of energy and high cost, the steam generated is at a relatively low pressure and temperature; when the steam is compressed to achieve high pressure, it tends to liquefy due to low temperature, which is unhelpful when utilising the steam.", "OBJECT OF THE INVENTION The object of this invention is to provide a device for generating high-pressure, saturated steam with the advantages of saving time and energy, lower running cost and being able to provide higher steam temperature and pressure.", "THE TECHNICAL PROPOSAL OF THIS INVENTION In order to achieve the above object, the technical proposal adopted by this invention is as follows.", "The said device for generating high-pressure, saturated steam is comprised of water tank, water pump, one-way valve, atomising nozzle, heating chamber and steam outlet.", "The pump links the tank and one-way valve, which connects to the atomising nozzle.", "Following the atomising nozzle, the heating chamber, which has a steam outlet, is hollow and has heating plates installed that subdivide the chamber into multiple interconnected, smaller heating cavities.", "The atomising nozzle is connected to the first heating cavity in the hollow chamber, whilst the last heating cavity in the chamber is connected to the steam outlet.", "Compared to the state of the art, this device has the following advantages and benefits: (1) After being atomised by the atomising nozzle and heated in the heating chamber, the water from the water tank turns into steam immediately, which avoids heating up a large amount of water for steam production.", "This saves time and energy.", "(2) Whenever the device or the equipment to which steam is supplied does not require steam, the power supply can be switched off and atomisation will be stopped immediately.", "Thus the heating chamber will also stop the heating and steam generating process, thereby saving a large amount of energy.", "(3) The heating plates subdivide the heating chamber into many interconnected, smaller heating cavities.", "As steam flows through the multiple heating cavities a curved steam current is formed.", "Thus the steam is heated adequately and is generated at high temperature, which prevents the steam from liquefaction.", "DESCRIPTION OF ATTACHED DRAWINGS FIG.", "1: Depicts the interconnection of the device according to the invention, FIG.", "2: Shows the construction of the device according to the invention FIG.", "3: A cross-section along C-C of FIG.", "2 FIG.", "4: A cross-section along D-D of FIG.", "2 FIG.", "5: The main body structure of a heating plate FIG.", "6: A cross-section along L-L of FIG.", "5 FIG.", "7: A cross-section along A-A of FIG.", "5 FIG.", "8: An alternative construction of a heating chamber of this device APPLICATION EXAMPLES Example 1 As shown in FIGS.", "1, 2, 3 and 4, the said device for generating high-pressure, saturated steam is comprised of water tank 1, water pump 2, one-way valve 3, atomising nozzle 4, heating chamber 5, and steam outlet 6.They are interconnected as follows: The water pump 2 links the water tank 1 and one-way valve 3, which connects to atomising nozzle 4.Joining the atomising nozzle 4, the heat chamber 5 with a steam outlet 6, is a spherical, hollow chamber 5 covered with an external thermal insulating layer 7.Installed in the spherical, hollow chamber 5 are heating plates, which subdivide the chamber into five interconnected smaller heating cavities.", "The atomising nozzle 4 is connected to the first heating cavity I in the spherical, hollow chamber 5 and becomes an integral part of heating plate 8, whilst the last heating cavity V in the chamber is connected to steam outlet 6.Thermal sensors and temperature gauges are installed in the last heating chamber, i.e.", "the fifth heating cavity V. The construction of the heating plate is shown in FIG.", "2, whilst the construction of the main body of the heating plate is shown in FIGS.", "5, 6, and 7.The heating plate's main body 9 is a circular plate, in which a heating plate positioning hole 11 is provided to retain the heating bar.", "Threaded holes 10 are evenly distributed at the circumference of the plate to connect and fasten the top and bottom semi-spheres.", "The working process of the above device is as follows: The arrows in FIGS.", "1 and 2 show the steam flow direction in the heating chamber; the water from water tank 1 is delivered by water pump 2 via a one-way valve 3 to atomising nozzle 4 and then injected into the first heating cavity I of the spherical, hollow chamber 5 after being atomised by the atomising nozzle 4.Since the atomising nozzle 4 is heated by heating plate 8, the water mist from the atomising nozzle reaches a relatively high temperature.", "It is therefore easier for the water mist to turn into saturated steam at a super-high temperature after being further heated in the first heating cavity I.", "The arrows in FIG.", "2 show the flow direction of the steam in the heating chamber.", "The steam flows then to the second heating cavity II via the cylindrical holes 12 located at the edge of heating cavity I.", "The steam, after being further heated in heating cavity II, flows to the third heating cavity III via a tube 13 between the second and third heating cavities.", "After being further heated in the third heating cavity III the steam flows to the fourth heating cavity IV.", "Following further heating in cavity IV, it enters the fifth heating cavity V and exits at outlet 6.A pressure gauge and an automatic switch can be attached to the outlet 16.Example 2 FIG.", "8 is an alternative structural diagram for subdivided heating cavities in the spherical chamber.", "All other details are the same as in Example 1." ] ]	Patent_10468808
[ [ "Recombinant narbonolide polyketide synthase", "Recombinant DNA compounds that encode all or a portion of the narbonolide polyketide synthase are used to express recombinant polyketide synthase genes in host cells for the production of narbonolide, narbonolide derivatives, and polyketides that are useful as antibiotics and as intermediates in the synthesis of compounds with pharmaceutical value." ], [ "1.An isolated recombinant DNA compound that comprises a coding sequence for a domain of a narbonolide PKS.", "2.The isolated recombinant DNA compound of claim 1, wherein said domain is selected from the group consisting of a thioesterase domain, a KSQ domain, an AT domain, a KS domain, an ACP domain, a KR domain, a DH domain, and an ER domain.", "3.The isolated recombinant DNA compound of claim 2 that comprises the coding sequence for a loading module, thioesterase domain, and all six extender modules of the narbonolide PKS.", "4.An isolated recombinant DNA compound that comprises a coding sequence for a desosamine biosynthetic gene or a desosaminyl transferase gene or a beta-glucosidase gene of Streptomyces venezuelae.", "5.An isolated recombinant DNA compound that comprises a coding sequence for a picK hydroxylase gene of Streptomyces venezuelae.", "6.An isolated DNA compound of any of claim 1 that further comprises a promoter operably linked to said coding sequence.", "7.The isolated recombinant DNA compound of claim 6, wherein said promoter is a promoter derived from a cell other than a Streptomyces venezuelae cell.", "8.The isolated recombinant DNA compound of claim 7 that is a recombinant DNA expression vector.", "9.The recombinant DNA expression vector of claim 8 that expresses a PKS in Streptomyces host cells.", "10.The recombinant DNA expression vector of claim 9 that encodes a hybrid PKS comprising at least a portion of a narbonolide PKS gene and at least a portion of a second PKS gene for a macrolide aglycone other than narbonolide.", "11.The recombinant DNA compound of claim 10, wherein said second PKS gene is a DEBS gene.", "12.The recombinant DNA compound of claim 11, wherein said hybrid PKS is composed of a loading module and extender modules 1 through 6 of DEBS excluding a KR domain of extender module 6 of DEBS and an ACP of extender module 6 and a thioesterase domain of the narbonolide PKS.", "13.A recombinant host cell, which in its untransformed state does not produce 10-deoxymethynolide or narbonolide, that comprises a recombinant DNA expression vector of claim 9 that encodes a narbonolide PKS and said cell produces 10-deoxymethynolide or narbonolide.", "14.The recombinant host cell of claim 13 that further comprises a picB gene.", "15.The recombinant host cell of claim 13 that further comprises desosamine biosynthetic genes and a gene for desosaminyl transferase and produces YC17 or narbomycin.", "16.The recombinant host cell of claim 15 that further comprises a picK gene and produces methymycin, neomethymycin, or picromycin.", "17.The recombinant host cell of any of claim 16 that is Streptomyces coelicolor or Streptomyces lividans.", "18.A recombinant host cell other than a Streptomyces venezuelae cell that expresses a picK hydroxylase gene of S. venezuelae encoded by the DNA compound of claim 5.19.A recombinant host cell other than a Streptomyces venezuelae host cell that expresses a desosamine biosynthetic gene or desosaminyl transferase gene of S. venezuelae encoded by the DNA compound of claim 4.20.A method for increasing the yield of a desosaminylated polyketide in a cell, which method comprises transforming the cell with a recombinant expression vector that encodes a functional beta-glucosidase gene." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications.", "Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes.", "There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities.", "Tetracycline, erythromycin, FK506, FK520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin, are examples of such compounds.", "Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds.", "See PCT publication Nos.", "WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat.", "Nos.", "4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew.", "Chem.", "Int.", "Ed.", "Engl.", "34(8): 881-888, each of which is incorporated herein by reference.", "Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes.", "These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids.", "PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs).", "Each ORF typically comprises two or more “modules” of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three or more enzymatic activities or “domains.” Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis.", "These two major types of PKS enzymes are commonly referred to as Type I or “modular” and Type II “iterative” PKS enzymes.", "Modular PKSs are responsible for producing a large number of 12, 14, and 16-membered macrolide antibiotics including methymycin, erythromycin, narbomycin, picromycin, and tylosin.", "These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying β-carbon processing activities (see O'Hagan, D. The polyketide metabolites ; E. Horwood: New York, 1991, incorporated herein by reference).", "During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Pat.", "Nos.", "5,672,491 and 5,712,146, each of which is incorporated herein by reference).", "The advantages to this plasmid-based genetic system for DEBS were that it overcame the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythaea , allowed more facile construction of recombinant PKSs, and reduced the complexity of PKS analysis by providing a “clean” host background.", "This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No.", "WO 98/49315, incorporated herein by reference).", "The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of β-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, Curr.", "Opin.", "Microbiol.", "1: 319-329; Carreras and Santi, 1998, Curr.", "Opin.", "Biotech.", "9: 403-411; and U.S. Pat.", "Nos.", "5,712,146 and 5,672,491, each of which is incorporated herein by reference).", "This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes.", "The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide.", "The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.", "The present invention provides methods and reagents relating to the PKS gene cluster for the polyketide antibiotics known as narbomycin and picromycin.", "Narbomycin is produced in Streptomyces narbonensis , and both narbomycin and picromycin are produced in S. venezuelae .", "These species are unique among macrolide producing organisms in that they produce, in addition to the 14-membered macrolides narbomycin and picromycin (picromycin is shown in FIG.", "1 , compound 1), the 12-membered macrolides neomethymycin and methymycin (methymycin is shown in FIG.", "1 , compound 2).", "Based on the structural similarities between picromycin and methymycin, it was speculated that methymycin would result from premature cyclization of a hexaketide intermediate in the picromycin pathway.", "Glycosylation of the C5 hydroxyl group of the polyketide precursor, narbonolide, is achieved through an endogenous desosaminyl transferase to produce narbomycin.", "In Streptomyces venezuelae , narbomycin is then converted to picromycin by the endogenously produced narbomycin hydroxylase.", "Thus, as in the case of other macrolide antibiotics, the macrolide product of the narbonolide PKS is further modified by hydroxylation and glycosylation.", "Picromycin ( FIG.", "1 , compound 1) is of particular interest because of its close structural relationship to ketolide compounds (e.g.", "HMR 3004, FIG.", "1 , compound 3).", "The ketolides are a new class of semi-synthetic macrolides with activity against pathogens resistant to erythromycin (see Agouridas et al., 1998, J. Med.", "Chem.", "41: 4080-4100, incorporated herein by reference).", "Thus, genetic systems that allow rapid engineering of the narbonolide PKS would be valuable for creating novel ketolide analogs for pharmaceutical applications.", "Furthermore, the production of picromycin as well as novel compounds with useful activity could be accomplished if the heterologous expression of the narbonolide PKS in Streptomyces lividans and other host cells were possible.", "The present invention meets these and other needs." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides recombinant methods and materials for expressing PKSs derived in whole and in part from the narbonolide PKS and other genes involved in narbomycin and picromycin biosynthesis in recombinant host cells.", "The invention also provides the polyketides derived from the narbonolide PKS.", "The invention provides the complete PKS gene cluster that ultimately results, in Streptomyces venezuelae , in the production of picromycin.", "The ketolide product of this PKS is narbonolide.", "Narbonolide is glycosylated to obtain narbomycin and then hydroxylated at C12 to obtain picromycin.", "The enzymes responsible for the glycosylation and hydroxylation are also provided in recombinant form by the invention.", "Thus, in one embodiment, the invention is directed to recombinant materials that contain nucleotide sequences encoding at least one domain, module, or protein encoded by a narbonolide PKS gene.", "The invention also provides recombinant materials useful for conversion of ketolides to antibiotics.", "These materials include recombinant DNA compounds that encode the C12 hydroxylase (the picK gene), the desosamine biosynthesis and desosaminyl transferase enzymes, and the beta-glucosidase enzyme involved in picromycin biosynthesis in S. venezuelae and the recombinant proteins that can be produced from these nucleic acids in the recombinant host cells of the invention.", "In one embodiment, the invention provides a recombinant expression vector that comprises a heterologous promoter positioned to drive expression of the narbonolide PKS.", "In a preferred embodiment, the promoter is derived from a PKS gene.", "In a related embodiment, the invention provides recombinant host cells comprising the vector that produces narbonolide.", "In a preferred embodiment, the host cell is Streptomyces lividans or S. coelicolor.", "In another embodiment, the invention provides a recombinant expression vector that comprises the desosamine biosynthetic genes as well as the desosaminyl transferase gene.", "In a related embodiment, the invention provides recombinant host cells comprising the vector that produces the desosamine biosynthetic gene products and desosaminyl transferase gene product.", "In a preferred embodiment, the host cell is Streptomyces lividans or S. coelicolor.", "In another embodiment, the invention provides a method for desosaminylating polyketide compounds in recombinant host cells, which method comprises expressing the PKS for the polyketide and the desosaminyl transferase and desosamine biosynthetic genes in a host cell.", "In a preferred embodiment, the host cell expresses a beta-glucosidase gene as well.", "This preferred method is especially advantageous when producing desosaminylated polyketides in Streptomyces host cells, because such host cells typically glucosylate desosamine residues of polyketides, which can decrease desired activity, such as antibiotic activity.", "By coexpression of beta-glucosidase, the glucose residue is removed from the polyketide.", "In another embodiment, the invention provides the picK hydroxylase gene in recombinant form and methods for hydroxylating polyketides with the recombinant gene product.", "The invention also provides polyketides thus produced and the antibiotics or other useful compounds derived therefrom.", "In another embodiment, the invention provides a recombinant expression vector that comprises a promoter positioned to drive expression of a hybrid PKS comprising all or part of the narbonolide PKS and at least a part of a second PKS.", "In a related embodiment, the invention provides recombinant host cells comprising the vector that produces the hybrid PKS and its corresponding polyketide.", "In a preferred embodiment, the host cell is Streptomyces lividans or S. coelicolor.", "In a related embodiment, the invention provides recombinant materials for the production of libraries of polyketides wherein the polyketide members of the library are synthesized by hybrid PKS enzymes of the invention.", "The resulting polyketides can be further modified to convert them to other useful compounds, such as antibiotics, typically through hydroxylation and/or glycosylation.", "Modified macrolides provided by the invention that are useful intermediates in the preparation of antibiotics are of particular benefit.", "In another related embodiment, the invention provides a method to prepare a nucleic acid that encodes a modified PKS, which method comprises using the narbonolide PKS encoding sequence as a scaffold and modifying the portions of the nucleotide sequence that encode enzymatic activities, either by mutagenesis, inactivation, insertion, or replacement.", "The thus modified narbonolide PKS encoding nucleotide sequence can then be expressed in a suitable host cell and the cell employed to produce a polyketide different from that produced by the narbonolide PKS.", "In addition, portions of the narbonolide PKS coding sequence can be inserted into other PKS coding sequences to modify the products thereof.", "The narbonolide PKS can itself be manipulated, for example, by fusing two or more of its open reading frames, particularly those for extender modules 5 and 6, to make more efficient the production of 14-membered as opposed to 12-membered macrolides.", "In another related embodiment, the invention is directed to a multiplicity of cell colonies, constituting a library of colonies, wherein each colony of the library contains an expression vector for the production of a modular PKS derived in whole or in part from the narbonolide PKS.", "Thus, at least a portion of the modular PKS is identical to that found in the PKS that produces narbonolide and is identifiable as such.", "The derived portion can be prepared synthetically or directly from DNA derived from organisms that produce narbonolide.", "In addition, the invention provides methods to screen the resulting polyketide and antibiotic libraries.", "The invention also provides novel polyketides and antibiotics or other useful compounds derived therefrom.", "The compounds of the invention can be used in the manufacture of another compound.", "In a preferred embodiment, the antibiotic compounds of the invention are formulated in a mixture or solution for administration to an animal or human.", "These and other embodiments of the invention are described in more detail in the following description, the examples, and claims set forth below." ], [ "REFERENCE TO GOVERNMENT FUNDING This invention was supported in part by SBIR grant 1R43-CA75792-01.The U.S. government has certain rights in this invention.", "FIELD OF THE INVENTION The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology.", "The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology.", "BACKGROUND OF THE INVENTION Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications.", "Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes.", "There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities.", "Tetracycline, erythromycin, FK506, FK520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin, are examples of such compounds.", "Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds.", "See PCT publication Nos.", "WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat.", "Nos.", "4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew.", "Chem.", "Int.", "Ed.", "Engl.", "34(8): 881-888, each of which is incorporated herein by reference.", "Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes.", "These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids.", "PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs).", "Each ORF typically comprises two or more “modules” of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three or more enzymatic activities or “domains.” Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis.", "These two major types of PKS enzymes are commonly referred to as Type I or “modular” and Type II “iterative” PKS enzymes.", "Modular PKSs are responsible for producing a large number of 12, 14, and 16-membered macrolide antibiotics including methymycin, erythromycin, narbomycin, picromycin, and tylosin.", "These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying β-carbon processing activities (see O'Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).", "During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Pat.", "Nos.", "5,672,491 and 5,712,146, each of which is incorporated herein by reference).", "The advantages to this plasmid-based genetic system for DEBS were that it overcame the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythaea, allowed more facile construction of recombinant PKSs, and reduced the complexity of PKS analysis by providing a “clean” host background.", "This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No.", "WO 98/49315, incorporated herein by reference).", "The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of β-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, Curr.", "Opin.", "Microbiol.", "1: 319-329; Carreras and Santi, 1998, Curr.", "Opin.", "Biotech.", "9: 403-411; and U.S. Pat.", "Nos.", "5,712,146 and 5,672,491, each of which is incorporated herein by reference).", "This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes.", "The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide.", "The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.", "The present invention provides methods and reagents relating to the PKS gene cluster for the polyketide antibiotics known as narbomycin and picromycin.", "Narbomycin is produced in Streptomyces narbonensis, and both narbomycin and picromycin are produced in S. venezuelae.", "These species are unique among macrolide producing organisms in that they produce, in addition to the 14-membered macrolides narbomycin and picromycin (picromycin is shown in FIG.", "1, compound 1), the 12-membered macrolides neomethymycin and methymycin (methymycin is shown in FIG.", "1, compound 2).", "Based on the structural similarities between picromycin and methymycin, it was speculated that methymycin would result from premature cyclization of a hexaketide intermediate in the picromycin pathway.", "Glycosylation of the C5 hydroxyl group of the polyketide precursor, narbonolide, is achieved through an endogenous desosaminyl transferase to produce narbomycin.", "In Streptomyces venezuelae, narbomycin is then converted to picromycin by the endogenously produced narbomycin hydroxylase.", "Thus, as in the case of other macrolide antibiotics, the macrolide product of the narbonolide PKS is further modified by hydroxylation and glycosylation.", "Picromycin (FIG.", "1, compound 1) is of particular interest because of its close structural relationship to ketolide compounds (e.g.", "HMR 3004, FIG.", "1, compound 3).", "The ketolides are a new class of semi-synthetic macrolides with activity against pathogens resistant to erythromycin (see Agouridas et al., 1998, J. Med.", "Chem.", "41: 4080-4100, incorporated herein by reference).", "Thus, genetic systems that allow rapid engineering of the narbonolide PKS would be valuable for creating novel ketolide analogs for pharmaceutical applications.", "Furthermore, the production of picromycin as well as novel compounds with useful activity could be accomplished if the heterologous expression of the narbonolide PKS in Streptomyces lividans and other host cells were possible.", "The present invention meets these and other needs.", "SUMMARY OF THE INVENTION The present invention provides recombinant methods and materials for expressing PKSs derived in whole and in part from the narbonolide PKS and other genes involved in narbomycin and picromycin biosynthesis in recombinant host cells.", "The invention also provides the polyketides derived from the narbonolide PKS.", "The invention provides the complete PKS gene cluster that ultimately results, in Streptomyces venezuelae, in the production of picromycin.", "The ketolide product of this PKS is narbonolide.", "Narbonolide is glycosylated to obtain narbomycin and then hydroxylated at C12 to obtain picromycin.", "The enzymes responsible for the glycosylation and hydroxylation are also provided in recombinant form by the invention.", "Thus, in one embodiment, the invention is directed to recombinant materials that contain nucleotide sequences encoding at least one domain, module, or protein encoded by a narbonolide PKS gene.", "The invention also provides recombinant materials useful for conversion of ketolides to antibiotics.", "These materials include recombinant DNA compounds that encode the C12 hydroxylase (the picK gene), the desosamine biosynthesis and desosaminyl transferase enzymes, and the beta-glucosidase enzyme involved in picromycin biosynthesis in S. venezuelae and the recombinant proteins that can be produced from these nucleic acids in the recombinant host cells of the invention.", "In one embodiment, the invention provides a recombinant expression vector that comprises a heterologous promoter positioned to drive expression of the narbonolide PKS.", "In a preferred embodiment, the promoter is derived from a PKS gene.", "In a related embodiment, the invention provides recombinant host cells comprising the vector that produces narbonolide.", "In a preferred embodiment, the host cell is Streptomyces lividans or S. coelicolor.", "In another embodiment, the invention provides a recombinant expression vector that comprises the desosamine biosynthetic genes as well as the desosaminyl transferase gene.", "In a related embodiment, the invention provides recombinant host cells comprising the vector that produces the desosamine biosynthetic gene products and desosaminyl transferase gene product.", "In a preferred embodiment, the host cell is Streptomyces lividans or S. coelicolor.", "In another embodiment, the invention provides a method for desosaminylating polyketide compounds in recombinant host cells, which method comprises expressing the PKS for the polyketide and the desosaminyl transferase and desosamine biosynthetic genes in a host cell.", "In a preferred embodiment, the host cell expresses a beta-glucosidase gene as well.", "This preferred method is especially advantageous when producing desosaminylated polyketides in Streptomyces host cells, because such host cells typically glucosylate desosamine residues of polyketides, which can decrease desired activity, such as antibiotic activity.", "By coexpression of beta-glucosidase, the glucose residue is removed from the polyketide.", "In another embodiment, the invention provides the picK hydroxylase gene in recombinant form and methods for hydroxylating polyketides with the recombinant gene product.", "The invention also provides polyketides thus produced and the antibiotics or other useful compounds derived therefrom.", "In another embodiment, the invention provides a recombinant expression vector that comprises a promoter positioned to drive expression of a hybrid PKS comprising all or part of the narbonolide PKS and at least a part of a second PKS.", "In a related embodiment, the invention provides recombinant host cells comprising the vector that produces the hybrid PKS and its corresponding polyketide.", "In a preferred embodiment, the host cell is Streptomyces lividans or S. coelicolor.", "In a related embodiment, the invention provides recombinant materials for the production of libraries of polyketides wherein the polyketide members of the library are synthesized by hybrid PKS enzymes of the invention.", "The resulting polyketides can be further modified to convert them to other useful compounds, such as antibiotics, typically through hydroxylation and/or glycosylation.", "Modified macrolides provided by the invention that are useful intermediates in the preparation of antibiotics are of particular benefit.", "In another related embodiment, the invention provides a method to prepare a nucleic acid that encodes a modified PKS, which method comprises using the narbonolide PKS encoding sequence as a scaffold and modifying the portions of the nucleotide sequence that encode enzymatic activities, either by mutagenesis, inactivation, insertion, or replacement.", "The thus modified narbonolide PKS encoding nucleotide sequence can then be expressed in a suitable host cell and the cell employed to produce a polyketide different from that produced by the narbonolide PKS.", "In addition, portions of the narbonolide PKS coding sequence can be inserted into other PKS coding sequences to modify the products thereof.", "The narbonolide PKS can itself be manipulated, for example, by fusing two or more of its open reading frames, particularly those for extender modules 5 and 6, to make more efficient the production of 14-membered as opposed to 12-membered macrolides.", "In another related embodiment, the invention is directed to a multiplicity of cell colonies, constituting a library of colonies, wherein each colony of the library contains an expression vector for the production of a modular PKS derived in whole or in part from the narbonolide PKS.", "Thus, at least a portion of the modular PKS is identical to that found in the PKS that produces narbonolide and is identifiable as such.", "The derived portion can be prepared synthetically or directly from DNA derived from organisms that produce narbonolide.", "In addition, the invention provides methods to screen the resulting polyketide and antibiotic libraries.", "The invention also provides novel polyketides and antibiotics or other useful compounds derived therefrom.", "The compounds of the invention can be used in the manufacture of another compound.", "In a preferred embodiment, the antibiotic compounds of the invention are formulated in a mixture or solution for administration to an animal or human.", "These and other embodiments of the invention are described in more detail in the following description, the examples, and claims set forth below.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 shows the structures of picromycin (compound 1), methymycin (compound 2), and the ketolide HMR 3004 (compound 3).", "FIG.", "2 shows a restriction site and function map of cosmid pKOS023-27.FIG.", "3 shows a restriction site and function map of cosmid pKOS023-26.FIG.", "4 has three parts.", "In Part A, the structures of picromycin (A(a)) and methymycin (A(b)) are shown, as well as the related structures of narbomycin, narbonolide, and methynolide.", "In the structures, the bolded lines indicate the two or three carbon chains produced by each module (loading and extender) of the narbonolide PKS.", "Part B shows the organization of the narbonolide PKS genes on the chromosome of Streptomyces venezuelae, including the location of the various module encoding sequences (the loading module domains are identified as sKS, sAT, and sACP), as well as the picB thioesterase gene and two desosamine biosynthesis genes (picCII and picCIII).", "Part C shows the engineering of the S. venezuelae host of the invention in which the picAI gene has been deleted.", "In the Figure, ACP is acyl carrier protein; AT is acyltransferase; DH is dehydratase; ER is enoylreductase; KR is ketoreductase; KS is ketosynthase; and TE is thioesterase.", "FIG.", "5 shows the narbonolide PKS genes encoded by plasmid pKOS039-86, the compounds synthesized by each module of that PKS and the narbonolide (compound 4) and 10-deoxymethynolide (compound 5) products produced in heterologous host cells transformed with the plasmid.", "The Figure also shows a hybrid PKS of the invention produced by plasmid pKOS038-18, which encodes a hybrid of DEBS and the narbonolide PKS.", "The Figure also shows the compound, 3,6-dideoxy-3-oxo-erythronolide B (compound 6), produced in heterologous host cells comprising the plasmid.", "FIG.", "6 shows a restriction site and function map of plasmid pKOS039-104, which contains the desosamine biosynthetic, beta-glucosidase, and desosaminyl transferase genes under transcriptional control of actII-4.DETAILED DESCRIPTION OF THE INVENTION The present invention provides useful compounds and methods for producing polyketides in recombinant host cells.", "As used herein, the term recombinant refers to a compound or composition produced by human intervention.", "The invention provides recombinant DNA compounds encoding all or a portion of the narbonolide PKS.", "The invention also provides recombinant DNA compounds encoding the enzymes that catalyze the further modification of the ketolides produced by the narbonolide PKS.", "The invention provides recombinant expression vectors useful in producing the narbonolide PKS and hybrid PKSs composed of a portion of the narbonolide PKS in recombinant host cells.", "Thus, the invention also provides the narbonolide PKS, hybrid PKSs, and polyketide modification enzymes in recombinant form.", "The invention provides the polyketides produced by the recombinant PKS and polyketide modification enzymes.", "In particular, the invention provides methods for producing the polyketides 10-deoxymethynolide, narbonolide, YC17, narbomycin, methymycin, neomethymycin, and picromycin in recombinant host cells.", "To appreciate the many and diverse benefits and applications of the invention, the description of the invention below is organized as follows.", "First, a general description of polyketide biosynthesis and an overview of the synthesis of narbonolide and compounds derived therefrom in Streptomyces venezuelae are provided.", "This general description and overview are followed by a detailed description of the invention in six sections.", "In Section I, the recombinant narbonolide PKS provided by the invention is described.", "In Section II, the recombinant desosamine biosynthesis genes, the desosaminyl transferase gene, and the beta-glucosidase gene provided by the invention are described.", "In Section III, the recombinant picK hydroxylase gene provided by the invention is described.", "In Section IV, methods for heterologous expression of the narbonolide PKS and narbonolide modification enzymes provided by the invention are described.", "In Section V, the hybrid PKS genes provided by the invention and the polyketides produced thereby are described.", "In Section VI, the polyketide compounds provided by use invention and pharmaceutical compositions of those compounds are described.", "The detailed description is followed by a variety of working examples illustrating the invention.", "The narbonolide synthase gene, like other PKS genes, is composed of coding sequences organized in a loading module, a number of extender modules, and a thioesterase domain.", "As described more fully below, each of these domains and modules is a polypeptide with one or more specific functions.", "Generally, the loading module is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first extender module.", "The building blocks used to form complex polyketides are typically acylthioesters, most commonly acetyl, propionyl, malonyl, methylmalonyl, and ethylmalonyl CoA.", "Other building blocks include amino acid like acylthioesters.", "PKSs catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between the acylthioester building blocks.", "Each module is responsible for binding a building block, performing one or more functions on that building block, and transferring the resulting compound to the next module.", "The next module, in turn, is responsible for attaching the next building block and transferring the growing compound to the next module until synthesis is complete.", "At that point, an enzymatic thioesterase activity cleaves the polyketide from the PKS.", "Such modular organization is characteristic of the class of PKS enzymes that synthesize complex polyketides and is well known in the art.", "The polyketide known as 6-deoxyerythronolide B is a classic example of this type of complex polyketide.", "The genes, known as eryAI, eryAII, and eryAIII (also referred to herein as the DEBS genes, for the proteins, known as DEBS1, DEBS2, and DEBS3, that comprise the 6-dEB synthase), that code for the multi-subunit protein known as DEBS that synthesizes 6-dEB are described in U.S. Pat.", "No.", "5,824,513, incorporated herein by reference.", "Recombinant methods for manipulating modular PKS genes are described in U.S. Pat.", "Nos.", "5,672,491; 5,843,718; 5,830,750; and 5,712,146; and in PCT publication Nos.", "98/49315 and 97/02358, each of which is incorporated herein by reference.", "The loading module of DEBS consists of two domains, an acyl-transferase (AT) domain and an acyl carrier protein (ACP) domain.", "Each extender module of DEBS, like those of other modular PKS enzymes, contains a ketosynthase (KS), AT, and ACP domains, and zero, one, two, or three domains for enzymatic activities that modify the beta-carbon of the growing polyketide chain.", "A module can also contain domains for other enzymatic activities, such as, for example, a methyltransferase or dimethyltransferase activity.", "Finally, the releasing domain contains a thioesterase and, often, a cyclase activity.", "The AT domain of the loading module recognizes a particular acyl-CoA (usually acetyl or propionyl but sometimes butyryl) and transfers it as a thiol ester to the ACP of the loading module.", "Concurrently, the AT on each of the extender modules recognizes a particular extender-CoA (malonyl or alpha-substituted malonyl, i.e., methylmalonyl, ethylmalonyl, and carboxylglycolyl) and transfers it to the ACP of that module to form a thioester.", "Once the PKS is primed with acyl- and malonyl-ACPs, the acyl group of the loading module migrates to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, extender module 1 possesses an acyl-KS adjacent to a malonyl (or substituted malonyl) ACP.", "The acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading unit (elongation or extension).", "The growing polyketide chain is transferred from the ACP to the KS of the next module, and the process continues.", "The polyketide chain, growing by two carbons each module, is sequentially passed as covalently bound thiol esters from module to module, in an assembly line-like process.", "The carbon chain produced by this process alone would possess a ketone at every other carbon atom, producing a polyketone, from which the name polyketide arises.", "Most commonly, however, additional enzymatic activities modify the beta keto group of each two-carbon unit just after it has been added to the growing polyketide chain but before it is transferred to the next module.", "Thus, in addition to the minimal module containing KS, AT, and ACP domains necessary to form the carbon-carbon bond, modules may contain a ketoreductase (ICR) that reduces the keto group to an alcohol.", "Modules may also contain a KR plus a dehydratase (DH) that dehydrates the alcohol to a double bond.", "Modules may also contain a KR, a DH, and an enoylreductase (ER) that converts the double bond to a saturated single bond using the beta carbon as a methylene function.", "As noted above, modules may contain additional enzymatic activities as well.", "Once a polyketide chain traverses the final extender module of a PKS, it encounters the releasing domain or thioesterase found at the carboxyl end of most PKSs.", "Here, the polyketide is cleaved from the enzyme and cyclyzed.", "The resulting polyketide can be modified further by tailoring enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule.", "While the above description applies generally to modular PKS enzymes, there are a number of variations that exist in nature.", "For example, some polyketides, such as epothilone, incorporate a building block that is derived from an amino acid.", "PKS enzymes for such polyketides include an activity that functions as an amino acid ligase or as a non-ribosomal peptide synthetase (NRPS).", "Another example of a variation, which is actually found more often than the two domain loading module construct found in DEBS, occurs when the loading module of the PKS is not composed of an AT and an ACP but instead utilizes an inactivated KS, an AT, and an ACP.", "This inactivated KS is in most instances called KSQ, where the superscript letter is the abbreviation for the amino acid, glutamine, that is present instead of the active site cysteine required for activity.", "For example, the narbonolide PKS loading module contains aKSQ.", "Yet another example of a variation has been mentioned above in the context of modules that include a methyltransferase or dimethyltransferase activity; modules can also include an epimerase activity.", "These variations will be described further below in specific reference to the narbonolide PKS and the various recombinant and hybrid PKSs provided by the invention.", "With this general description of polyketide biosynthesis, one can better appreciate the biosynthesis of narbonolide related polyketides in Streptomyces venezuelae and S. narbonensis.", "The narbonolide PKS produces two polyketide products, narbonolide and 10-deoxymethynolide.", "Narbonolide is the polyketide product of all six extender modules of the narbonolide PKS.", "10-deoxymethynolide is the polyketide product of only the first five extender modules of the narbonolide PKS.", "These two polyketides are desosaminylated to yield narbomycin and YC17, respectively.", "These two glycosylated polyketides are the final products produced in S. narbonensis.", "In S. venezuelae, these products are hydroxylated by the picK gene product to yield picromycin and either methymycin (hydroxylation at the C10 position of YC7) or neomethymycin (hydroxylation at the C12 position of YC17).", "The present invention provides the genes required for the biosynthesis of all of these polyketides in recombinant form.", "Section 1: The Narbonolide PKS The narbonolide PKS is composed of a loading module, six extender modules, and a thioesterase domain.", "FIG.", "4, part B, shows the organization of the narbonolide PKS genes on the Streptomyces venezuelae chromosome, as well as the location of the module encoding sequences in those genes, and the various domains within those modules.", "In the Figure, the loading module is not numbered, and its domains are indicated as sKS, sAT, and ACP.", "Also shown in the Figure, part A, are the structures of picromycin and methymycin.", "The loading and six extender modules and the thioesterase domain of the narbonolide PKS reside on four proteins, designated PICAI, PICAII, PICAII, and PICAIV.", "PICAI includes the loading module and extender modules 1 and 2 of the PKS.", "PICAII includes extender modules 3 and 4.PICAIII includes extender module 5.PICAIV includes extender module 6 and a thioesterase domain.", "There is a second thioesterase domain (TEII) on a separate protein, designated PICB.", "The amino acid sequences of these proteins are shown below.", "Amino acid sequence of narbonolide synthase subunit 1, PICAI 1 MSTVSKSESE EFVSVSNDAG SAHGTAEPVA VVGISCRVPG ARDPPEFWEL LAAGGQAVTD 61 VPADRWNAGD FYDPDRSAPG RSNSRWGGFI EDVDRFDAAF FGISPREAAE MDPQQRLALE 121 LGWEALERAG IDPSSLTGTR TGVFAGAIWD DYATLKHRQG GAAITPHTVT GLHRGIIANR 181 LSYTLGLRGP SMVVDSGQSS SLVAVHLACE SLRRGESELA LAGGVSLNLV PDSIIGASKF 241 GGLSPDGRAY TFDARANGYV RGEGGGFVVL KRLSRAVADG DPVLAVIRGS AVNNGGAAQG 301 MTTPDAQAQE AVLREAHERA GTAPADVRYV ELHGTGTPVG DPIEAAALGA ALGTGRPAGQ 361 PLLVGSVKTN IGHLEGAAGI AGLIKAVLAV RGRALPASLN YETPNPAIPF EELNLRVNTE 421 YLPWEPEHDG QRMVVGVSSF GMGGTNAHVV LEEAPGVVEG ASVVESTVGG SAVGGGVVPW 481 VVSAKSAAAL DAQIERLAAF ASRDRTDGVD AGAVDAGAVD AGAVARVLAG GRAQFEHRAV 541 VVGSGPDDLA AALAAPEGLV RGVASGVGRV AFVFPGQGTQ WAGMGAELLD SSAVFAAAMA 601 ECEAALSPYV DWSLEAVVRQ APGAPTLERV DVVQPVTFAV MVSLARVWQH HGVTPQAVVG 661 HSQGEIAAAY VAGALSLDDA ARVVTLRSKS IAAHLAGKGG MLSLALSEDA VLERLAGFDG 721 LSVAAVNGPT ATVVSGDPVQ IEELARACEA DGVRARVIPV DYASHSRQVE IIESELAEVL 781 AGLSPQAPRV PFFSTLEGAW ITEPVLDGGY WYRNLRHRVG FAPAVETLAT DEGFTHFVEV 841 SAHPVLTMAL PGTVTGLATL RRDNGGQDRL VASLAEAWAN GLAVDWSPLL PSATGHHSDL 901 PTYAFQTERH WLGEIEALAP AGEPAVQPAV LRTEAAEPAE LDRDEQLRVI LDKVRAQTAQ 961 VLGYATGGQI EVDRTFREAG CTSLTGVDLR NRINAAFGVR MAPSMIFDFP TPEALAEQLL 1021 LVVHGEAAAN PAGAEPAPVA AAGAVDEPVA IVGMACRLPG GVASPEDLWR LVAGGGDAIS 1081 EFPQDRGWDV EGLYHPDPEH PGTSYVRQGG FIENVAGFDA AFFGISPREA LAMDPQQRLL 1141 LETSWEAVED AGIDPTSLRG RQVGVFTGAM THEYGPSLRD GGEGLDGYLL TGNTASVMSG 1201 RVSYTLGLEG PALTVDTACS SSLVALHLAV QALRKGEVDM ALAGGVAVMP TPGMFVEFSR 1261 QRGLAGDGRS KAFAASADGT SWSEGVGVLL VERLSDARRN GHQVLAVVRG SAVNQDGASN 1321 GLTAPNGPSQ ORVIRRALAD ARLTTSDVDV VEAHGTGTRL GDPIEAQALI ATYGQGRDDE 1381 QPLRLGSLKS NIGHTQAAAG VSGVIKMVQA MRHGLLPKTL HVDEPSDQID WSAGAVELLT 1441 EAVDWPEKQD GGLRRAAVSS FGISGTNAHV VLEEAPVVVE GASVVEPSVG GSAVGGGVTP 1501 WVVSAKSAAA LDAQIERLAA FASRDRTDDA DAGAVDAGAV AHVLADGRAQ FEHRAVALGA 1561 GADDLVQALA DPDGLIRGTA SGVGRVAFVF PGQGTQWAGM GAELLDSSAV FAAAMAECEA 1621 ALSPYVDWSL EAVVRQAPGA PTLERVDVVQ PVTFAVMVSL ARVWQHHGVT PQAVVGHSQG 1681 EIAAAYVAGA LPLDDAARVV TLRSKSIAAH LAGKGGMLSL ALNEDAVLER LSDFDGLSVA 1741 AVNGPTATVV SGDPVQIEEL AQACKADGFR ARIIPVDYAS HSRQVEIIES ELAQVLAGLS 1801 PQAPRVPFFS TLEGTWITEP VLDGTYWYRN LRHRVGFAPA IETLAVDEGF THFVEVSAHP 1861 VLTMTLPETV TGLGTLRREQ GGQERLVTSL AEAWVNGLPV AWTSLLPATA SRPGLPTYAF 1921 QAERYWLENT PAALATGDDW RYRIDWKRLP AAEGSERTGL SGRWLAVTPE DHSAQAAAVL 1981 TALVDAGAKV EVLTAGADDD REALAARLTA LTTGDGFTGV VSLLDGLVPQ VAWVQALGDA 2041 GIKAPLWSVT QGAVSVGRLD TPADPDRAML WGLGRVVALE HPERWAGLVD LPAQPDAAAL 2101 AHLVTALSGA TGEDQIAIRT TGLHARRLAR APLHGRRPTR DWQPHGTVLI TGGTGALGSH 2161 AARWMAHHGA EHLLLVSRSG EQAPGATQLT AELTASGARV TIAACDVADP HAMRTLLDAI 2221 PAETPLTAVV HTAGALDDGI VDTLTAEQVR RAHRAKAVGA SVLDELTRDL DLDAFVLFSS 2281 VSSTLGIPGQ GNYAPHNAYL DALAARRRAT GRSAVSVAWG PWDGGGMAAG DGVAERLRNH 2341 GVPGMDPELA LAALESALGR DETAITVADI DWDRFYLAYS SGRPQPLVEE LPEVRRIIDA 2401 RDSATSGQGG SSAQGANPLA RRLAAAAPGE RTEILLGLVR AQAAAVLRMR SPEDVAADRA 2461 FKDIGFDSLA GVELRNRLTR ATGLQLPATL VFDHPTPLAL VSLLRSEFLG DEETADARRS 2521 AALPATVGAG AGAGAGTDAD DDPIAIVAMS CRYPGDIRSP EDLWRMLSEG GEGITPFPTD 2581 RGWDLDGLYD ADPDALGRAY VREGGFLHDA AEFDAEFFGV SPREALAMDP QQRMLLTTSW 2641 EAFERAGIEP ASLRGSSTGV FIGLSYQDYA ARVPNAPRGV EGYLLTGSTP SVASGRIAYT 2701 FGLEGPATTV DTACSSSLTA LHLAVRALRS GECTMALAGG VAMMATPHMF VEFSRQRALA 2761 PDGRSKAFSA DADGFGAAEG VGLLLVERLS DARRNGHPVL AVVRGTAVNQ DGASNGLTAP 2821 NGPSQQRVIR QALADARLAP GDIDAVETHG TGTSLGDPIE AQGLQATYGK ERPAERPLAI 2881 GSVKSNIGHT QAAAGAAGII KMVLAMRHGT LPKTLHADEP SPHVDWANSG LALVTEPIDW 2941 PAGTGPRRAA VSSFGISGTN AHVVLEQAPD AAGEVLGADE VPEVSETVAM AGTAGTSEVA 3001 EGSEASEAPA APGSREASLP GHLPWVLSAK DEQSLRGQAA ALHAWLSEPA ADLSDADGPA 3061 RLRDVGYTLA TSRTAFAHRA AVTAADRDGF LDGLATLAQG GTSAHVHLDT ARDGTTAFLF 3121 TGQGSQRPGA GRELYDRHPV FARALDEICA HLDGHLELPL LDVMFAAEGS AEAALLDETR 3181 YTQCALFALE VALFRLVESW GMRPAALLGH SVGEIAAAHV AGVFSLADAA RLVAARGRLM 3241 QELPAGGAML AVQAAEDEIR VWLETEERYA GRLDVAAVNG PEAAVLSGDA DAAREAEAYW 3301 SGLGRRTRAL RVSHAFHSAH MDGMLDGFRA VLETVEFRRP SLTVVSNVTG LAAGPDDLCD 3361 PEYWVRHVRG TVRFLDGVRV LRDLGVRTCL ELGPDGVLTA MAADGLADTP ADSAAGSPVG 3421 SPAGSPADSA AGALRPRPLL VALLRRKRSE TETVADALGR AHAHGTGPDW HAWFAGSGAH 3481 RVDLPTYSFR RDRYWLDAPA ADTAVDTAGL GLGTADHPLL GAVVSLPDRD GLLLTGRLSL 3541 RTHPWLADHA VLGSVLLPGA AMVELAAHAA ESAGLRDVRE LTLLEPLVLP EHGGVELRVT 3601 VGAPAGEPGG ESAGDGARPV SLHSRLADAP AGTAWSCHAT GLLATDRPEL PVAPDRAAMW 3661 PPQGAEEVPL DGLYERLDGN GLAFGPLFQG LNAVWRYEGE VFADIALPAT TNATAPATAN 3721 GGGSAAAAPY GIHPALLDAS LHAIAVGGLV DEPELVRVPF HWSGVTVHAA GAAAARVRLA 3781 SAGTDAVSLS LTDGEGRPLV SVERLTLRPV TADQAAASRV GGLMHRVAWR PYALASSGEQ 3841 DPHATSYGPT AVLGKDELKV AAALESAGVE VGLYPDLAAL SQDVAAGAPA PRTVLAPLPA 3901 GPADGGAEGV RGTVARTLEL LQAWLADEHL AGTRLLLVTR GAVRDPEGSG ADDGGEDLSH 3961 AAAWGLVRTA QTENPGRFGL LDLADDASSY RTLPSVLSDA GLRDEPQLAL HDGTIRLARL 4021 ASVRPETGTA APALAPEGTV LLTGGTGGLG GLVARHVVGE WGVRRLLLVS RRGTDAPGAD 4081 ELVHELEALG ADVSVAACDV ADREALTAVL DAIPAEHPLT AVVHTAGVLS DGTLPSMTTE 4141 DVEHVLRPKV DAAFLLDELT STPAYDLAAF VMFSSAAAVF GGAGQGAYAA ANATLDALAW 4201 RRRAAGLPAL SLGWGLWAET SGMTGELGQA DLRRMSRAGI GGISDAEGIA LLDAALRDDR 4261 HPVLLPLRLD AAGLRDAAGN DPAGIPALFR DVVGARTVRA RPSAASASTT AGTAGTPGTA 4321 DGAAETAAVT LADRAATVDG PARQRLLLEF VVGEVAEVLG HARGHRIDAE RGFLDLGFDS 4381 LTAVELRNRL NSAGGLALPA TLVFDHPSPA ALASHLDAEL PRGASDQDGA GNRNGNENGT 4441 TASRSTAETD ALLAQLTRLE GALVLTGLSD APGSEEVLEH LRSLRSMVTG ETGTGTASGA 4501 PDGAGSGAED RPWAAGDGAG GGSEDGAGVP DFMNASAEEL FGLLDQDPST D Amino acid sequence of narbonolide synthase subunit 2, PICAII 1 VSTVNEEKYL DYLRRATADL HEARGRLREL EAKAGEPVAI VGMACRLPGG VASPEDLWRL 61 VAGGEDAISE FPQDRGWDVE GLYDPNPEAT GKSYAREAGF LYEAGEFDAD FFGISPREAL 121 AMDPQQRLLL EASWEAFEHA GIPAATARGT SVGVFTGVMY HDYATRLTDV PEGIEGYLGT 181 GNSGSVASGR VAYTLGLEGP AVTVDTACSS SLVALHLAVQ ALRKGEVDMA LAGGVTVMST 241 PSTFVEFSRQ RGLAPDGRSK SFSSTADGTS WSEGVGVLLV ERLSDARRKG HRILAVVRGT 301 AVNQDGASSG LTAPNGPSQQ RVIRRALADA RLTTSDVDVV EAHGTGTRLG DPIEAQAVIA 361 TYGQGRDGEQ PLRLGSLKSN IGHTQAAAGV SGVIKMVQAM RHGVLPKTLH VEKPTDQVDW 421 SAGAVELLTE AMDWPDKGDG GLRRAAVSSF GVSGTNAHVV LEEAPAAEET PASEATPAVE 481 PSVGAGLVPW LVSAKTPAAL DAQIGRLAAF ASQGRTDAAD PGAVARVLAG GRAEFEHRAV 541 VLGTGQDDFA QALTAPEGLI RGTPSDVGRV AFVFPGQGTQ WAGMGAELLD VSKEFAAAMA 601 ECESALSRYV DWSLEAVVRQ APGAPTLERV DVVQPVTFAV MVSLAKVWQH HGVTPQAVVG 661 HSQGEIAAAY VAGALTLDDA ARVVTLRSKS IAAHLAGKGG MISLALSEEA TRQRIENLHG 721 LSIAAVNGPT ATVVSGDPTQ IQELAQACEA DGVRARIIPV DYASHSAHVE TIESELAEVL 781 AGLSPRTPEV PFFSTLEGAW ITEPVLDGTY WYRNLRHRVG FAPAVETLAT DEGFTHFIEV 841 SAHPVLTMTL PETVTGLGTL RREQGGQERL VTSLAEAWTN GLTIDWAPVL PTATGHHPEL 901 PTYAFQRRHY WLHDSPAVQG SVQDSWRYRI DWKRLAVADA SERAGLSGRW LVVVPEDRSA 961 EAAPVLAALS GAGADPVQLD VSPLGDRQRL AATLGEALAA AGGAVDGVLS LLAWDESAHP 1021 GHPAPFTRGT GATLTLVQAL EDAGVAAPLW CVTHGAVSVG RADHVTSPAQ AMVWGMGRVA 1081 ALEHPERWGG LIDLPSDADR AALDRMTTVL AGGTGEDQVA VRASGLLARR LVRASLPAHG 1141 TASPWWQADG TVLVTGAEEP AAAEAARRLA RDGAGHLLLH TTPSGSEGAE GTSGAAEDSG 1201 LAGLVAELAD LGATATVVTC DLTDAEAAAR LLAGVSDAHP LSAVLHLPPT VDSEPLAATD 1261 ADALARVVTA KATAALHLDR LLREAAAAGG RPPVLVLFSS VAAIWGGAGQ GAYAAGTAFL 1321 DALAGQHRAD GPTVTSVAWS PWEGSRVTEG ATGERLRRLG LRPLAPATAL TALDTALGHG 1381 DTAVTIADVD WSSFAPGFTT ARPGTLLADL PEARRALDEQ QSTTAADDTV LSRELGALTG 1441 AEQQRRMQEL VREHLAVVLN HPSPEAVDTG RAFRDLGFDS LTAVELRNRL KNATGLALPA 1501 TLVFDYPTPR TLAEFLLAEI LGEQAGAGEQ LPVDGGVDDE PVAIVGMACR LPGGVASPED 1561 LWRLVAGGED AISGFPQDRG WDVEGLYDPD PDASGRTYCR AGGFLDEAGE FDADFFGISP 1621 REALAMDPQQ RLLLETSWEA VEDAGIDPTS LQGQQVGVFA GTNGPHYEPL LRNTAEDLEG 1681 YVGTGNAASI MSGRVSYTLG LEGPAVTVDT ACSSSLVALH LAVQALRKGE CGLALAGGVT 1741 VMSTPTTFVE FSRQRGLAED GRSKAFAASA DGFGPAEGVG MLLVERLSDA RRNGHRVLAV 1801 VRGSAVNQDG ASNGLTAPNG PSQQRVIRRA LADARLTTAD VDVVEAHGTG TRLGDPIEAQ 1861 ALIATYGQGR DTEQPLRLGS LKSNIGHTQA AAGVSGIIKM VQAMRHGVLP KTLHVDRPSD 1921 QIDWSAGTVE LLTEAMDWPR KQEGGLRRAA VSSFGISGTN AHIVLEEAPV DEDAPADEPS 1981 VGGVVPWLVS AKTPAALDAQ IGRLAAFASQ GRTDAADPGA VARVLAGGRA QFEHRAVALG 2041 TGQDDLAAAL AAPEGLVRGV ASGVGRVAFV FPGQGTQWAG MGAELLDVSK EFAAAMAECE 2101 AALAPYVDWS LEAVVRQAPG APTLERVDVV QPVTFAVMVS LAKVWQHHGV TPQAVVGHSQ 2161 GEIAAAYVAG ALSLDDAARV VTLRSKSIGA HLAGQGGMLS LALSEAAVVE RLAGFDGLSV 2221 AAVNGPTATV VSGDPTQIQE LAQACEADGV RARIIPVDYA SHSAHVETIE SELADVLAGL 2281 SPQTPQVPFF STLEGAWITE PALDGGYWYR NLRHRVGFAP AVETLATDEG FTHFVEVSAH 2341 PVLTMALPET VTGLGTLRRD NGGQHRLTTS LAEAWANGLT VDWASLLPTT TTHPDLPTYA 2401 FQTERYWPQP DLSAAGDITS AGLGAAEHPL LGAAVALADS DGCLLTGSLS LRTHPWLADH 2461 AVAGTVLLPG TAFVELAFRA GDQVGCDLVE ELTLDAPLVL PRRGAVRVQL SVGASDESGR 2521 RTFGLYAHPE DAPGEAEWTR HATGVLAARA DRTAPVADPE AWPPPGAEPV DVDGLYERFA 2581 ANGYGYGPLF QGVRGVWRRG DEVFADVALP AEVAGAEGAR FGLHPALLDA AVQAAGAGGA 2641 FGAGTRLPFA WSGISLYAVG ATALRVRLAP AGPDTVSVSA ADSSGQPVFA ADSLTVLPVD 2701 PAQLAAFSDP TLDALHLLEW TAWDGAAQAL PGAVVLGGDA DGLAAALRAG GTEVLSFPDL 2761 TDLVEAVDRG ETPAPATVLV ACPAAGPGGP EHVREALHGS LALMQAWLAD ERFTDGRLVL 2821 VTRDAVAARS GDGLRSTGQA AVWGLGRSAQ TESPGRFVLL DLAGEARTAG DATAGDGLTT 2881 GDATVGGTSG DAALGSALAT ALGSGEPQLA LRDGALLVPR LARAAAPAAA DGLAAADGLA 2941 ALPLPAAPAL WRLEPGTDGS LESLTAAPGD AETLAPEPLG PGQVRIAIRA TGLNFRDVLI 3001 ALGMYPDPAL MGTEGAGVVT ATGPGVTHLA PGDRVMGLLS GAYAPVVVAD ARTVARMPEG 3061 WTFAQGASVP VVFLTAVYAL RDLADVKPGE RLLVHSAAGG VGMAAVQLAR HWGVEVHGTA 3121 SHGKWDALRA LGLDDAHIAS SRTLDFESAF RAASGGAGMD VVLNSLAREF VDASLRLLGP 3181 GGRFVEMGKT DVRDAERVAA DHPGVGYRAF DLGEAGPERI GEMLAEVIAL FEDGVLRHLP 3241 VTTWDVRRAR DAFRHVSQAR HTGKVVLTMP SGLDPEGTVL LTGGTGALGG IVARHVVGEW 3301 GVRRLLLVSR RGTDAPGAGE LVHELEALGA DVSVAACDVA DREALTAVLD SIPAEHPLTA 3361 VVHTAGVLSD GTLPSMTAED VEHVLRPKVD AAFLLDELTS TPGYDLAAFV MFSSAAAVFG 3421 GAGQGAYAAA NATLDALAWR RRTAGLPALS LGWGLWAETS GMTGGLSDTD RSRLARSGAT 3481 PMDSELTLSL LDAAMRRDDP ALVPIALDVA ALRAQQRDGM LAPLLSGLTR GSRVGGAPVN 3541 QRRAAAGGAG EADTDLGGRL AAMTPDDRVA HLRDLVRTHV ATVLGHGTPS RVDLERAFRD 3601 TGFDSLTAVE LRNRLNAATG LRLPATLVFD HPTPGELAGH LLDELATAAG GSWAEGTGSG 3661 DTASATDRQT TAALAELDRL EGVLASLAPA AGGRPELAAR LRALAAALGD DGDDATDLDE 3721 ASDDDLFSFI DKELGDSDF Amino acid sequence of narbonolide synthase subunit 3, PICAIII 1 MANNEDKLRD YLKRVTAELQ QNTRRLREIE GRTHEPVAIV GMACRLPGGV ASPEDLWQLV 61 AGDGDAISEF PQDRGWDVEG LYDPDPDASG RTYCRSGGFL HDAGEFDADF FGISPREALA 121 MDPQQRLSLT TAWEAIESAG IDPTALKGSG LGVFVGGWHT GYTSGQTTAV QSPELEGHLV 181 SGAALGFLSG RIAYVLGTDG PALTVDTACS SSLVALHLAV QALRKGECDM ALAGGVTVMP 241 NADLFVQFSR QRGLAADGRS KAFATSADGF GPAEGAGVLL VERLSDARRN GHRILAVVRG 301 SAVNQDGASN GLTAPHGPSQ QRVIRRALAD ARLAPGDVDV VEAHGTGTRL GDPIEAQALI 361 ATYGQEKSSE QPLRLGALKS NIGHTQAAAG VAGVIKMVQA MRHGLLPKTL HVDEPSDQID 421 WSAGTVELLT EAVDWPEKQD GGLRRAAVSS FGISGTNAHV VLEEAPAVED SPAVEPPAGG 481 GVVPWPVSAK TPAALDAQIG QLAAYADGRT DVDPAVAARA LVDSRTAMEH RAVAVGDSRE 541 ALRDALRMPE GLVRGTSSDV GRVAFVFPGQ GTQWAGMGAE LLDSSPEFAA SMAECETALS 601 RYVDWSLEAV VRQEPGAPTL DRVDVVQPVT FAVMVSLAKV WQHHGITPQA VVGHSQGEIA 661 AAYVAGALTL DDAARVVTLR SKSIAAHLAG KGGMISLALD EAAVLKRLSD FDGLSVAAVN 721 GPTATVVSGD PTQIEELART CEADGVRARI IPVDYASHSR QVEIIEKELA EVLAGLAPQA 781 PHVPFFSTLE GTWITEPVLD GTYWYRNLRH RVGFAPAVET LAVDGFTHFI EVSAHPVLTM 841 TLPETVTGLG TLRREQGGQE RLVTSLAEAW ANGLTIDWAP ILPTATGHHP ELPTYAFQTE 901 RFWLQSSAPT SAADDWRYRV EWKPLTASGQ ADLSGRWIVA VGSEPEAELL GALKAAGAEV 961 DVLEAGADDD REALAARLTA LTTGDGFTGV VSLLDDLVPQ VAWVQALGDA GIKAPLWSVT 1021 QGAVSVGRLD TPADPDRAML WGLGRVVALE HPERWAGLVD LPAQPDAAAL AHLVTALSGA 1081 TGEDQIAIRT TGLHARRLAR APLHGRRPTR DWQPHGTVLI TGGTGALGSH AARWMAHHGA 1141 EHLLLVSRSG EQAPGATQLT AELTASGARV TIAACDVADP HAMRTLLDAI PAETPLTAVV 1201 HTAGAPGGDP LDVTGPEDIA RILGAKTSGA EVLDDLLRGT PLDAFVLYSS NAGVWGSGSQ 1261 GVYAAANAHL DALAARRRAR GETATSVAWG LWAGDGMGRG ADDAYWQRRG IRPMSPDRAL 1321 DELAKALSHD ETFVAVADVD WERFAPAFTV SRPSLLLDGV PEARQALAAP VGAPAPGDAA 1381 VAPTGQSSAL AAITALPEPE RRPALLTLVR THAAAVLGHS SPDRVAPGRA FTELGFDSLT 1441 AVQLRNQLST VVGNRLPATT VFDHPTPAAL AAHLHEAYLA PAEPAPTDWE GRVRRALAEL 1501 PLDRLRDAGV LDTVLRLTGI EPEPGSGGSD GGAADPGAEP EASIDDLDAE ALIRMALGPR 1561 NT Amino acid sequence of narbonolide synthase subunit 4, PICAIV 1 MTSSNEQLVD ALRASLKENE ELRKESRRRA DRRQEPMAIV GMSCRFAGGI RSPEDLWDAV 61 AAGKDLVSEV PEERGWDIDS LYDPVPGRKG TTYVRNAAFL DDAAGFDAAF FGISPREALA 121 MDPQQRQLLE ASWEVFERAG IDPASVRGTD VGVYVGCGYQ DYAPDIRVAP EGTGGYVVTG 181 NSSAVASGRI AYSLGLEGPA VTVDTACSSS LVALHLALKG LRNGDCSTAL VGGVAVLATP 241 GAFIEFSSQQ AMAADGRTKG FASAADGLAW GEGVAVLLLE RLSDARRKGH RVLAVVRGSA 301 INQDGASNGL TAPHGPSQQR LIRQALADAR LTSSDVDVVE GHGTGTRLGD PIEAQALLAT 361 YGQGRAPGQP LRLGTLKSNI GHTQAASGVA GVIKMVQALR HGVLPKTLHV DEPTDQVDWS 421 AGSVELLTEA VDWPERPGRL RRAGVSAFGV GGTNAHVVLE EAPAVEESPA VEPPAGGGVV 481 PWPVSAKTSA ALDAQIGQLA AYAEDRTDVD PAVAARALVD SRTAMEHRAV AVGDSREALR 541 DALRMPEGLV RGTVTDPGRV AFVFPGQGTQ WAGMGAELLD SSPEFAAAMA ECETALSPYV 601 DWSLEAVVRQ APSAPTLDRV DVVQPVTFAV MVSLAKVWQH HGITPEAVIG HSQGEIAAAY 661 VAGALTLDDA ARVVTLRSKS IAAHLAGKGG MISLALSEEA TRQRIENLHG LSIAAVNGPT 721 ATVVSGDPTQ IQELAQACEA DGIRARIIPV DYASHSAHVE TIENELADVL AGLSPQTPQV 781 PFFSTLEGTW ITEPALDGGY WYRNLRHRVG FAPAVETLAT DEGFTHFIEV SAHPVLTMTL 841 PDKVTGLATL RREDGGQHRL TTSLAEAWAN GLALDWASLL PATGALSPAV PDLPTYAFQH 901 RSYWISPAGP GEAPAHTASG REAVAETGLA WGPGAEDLDE EGRRSAVLAM VMRQAASVLR 961 CDSPEEVPVD RPLREIGFDS LTAVDFRNRV NRLTGLQLPP TVVFEHPTPV ALAERISDEL 1021 AERNWAVAEP SDHEQAEEEK AAAPAGARSG ADTGAGAGMF RALFRQAVED DRYGEFLDVL 1081 AEASAFRPQF ASPEACSERL DPVLLAGGPT DRAEGRAVLV GCTGTAANGG PHEFLRLSTS 1141 FQEERDFLAV PLPGYGTGTG TGTALLPADL DTALDAQARA ILRAAGDAPV VLLGHSGGAL 1201 LAHELAFRLE RAHGAPPAGI VLVDPYPPGH QEPIEVWSRQ LGEGLFAGEL EPMSDARLLA 1261 MGRYARFLAG PRPGRSSAPV LLVRASEPLG DWQEERGDWR AHWDLPHTVA DVPGDHFTMM 1321 RDHAPAVAEA VLSWLDAIEG IEGAGK Amino acid sequence of typeII thioesterase, PICB 1 VTDRPLNVDS GLWIRRFHPA PNSAVRLVCL PHAGGSASYF FRFSEELHPS VEALSVQYPG 61 RQDRRAEPCL ESVEELAEHV VAATEPWWQE GRLAFFGHSL GASVAFETAR ILEQRHGVRP 121 EGLYVSGRRA PSLAPDRLVH QLDDRAFLAE IRRLSGTDER FLQDDELLRL VLPALRSDYK 181 AAETYLHRPS AKLTCPVMAL AGDRDPKAPL NEVAEWRRHT SGPFCLRAYS GGHFYLNDQW 241 HEICNDISDH LLVTRGAPDA RVVQPPTSLI EGAAKRWQNP R The DNA encoding the above proteins can be isolated in recombinant form from the recombinant cosmid pKOS023-27 of the invention, which was deposited with the American Type Culture Collection under the terms of the Budapest Treaty on 20 Aug. 1998 and is available under accession number ATCC 203141.Cosmid pKOS023-27 contains an insert of Streptomyces venezuelae DNA of −38506 nucleotides.", "The complete sequence of the insert from cosmid pKOS023-27 is shown below.", "The location of the various ORFs in the insert, as well as the boundaries of the sequences that encode the various domains of the multiple modules of the PKS, are summarized in the Table below.", "FIG.", "2 shows a restriction site and function map of pKOS023-27, which contains the complete coding sequence for the four proteins that constitute narbonolide PKS and four additional ORFs.", "One of these additional ORFs encodes the picB gene product, the type II thioesterase mentioned above.", "PICB shows a high degree of similarity to other type II thioesterases, with an identity of 51%, 49%, 45% and 40% as compared to those of Amycolatopsis mediterranae, S. griseus, S. fradiae and Saccharopolyspora erythraea, respectively.", "The three additional ORFs in the cosmid pKOS023-27 insert DNA sequence, from the picCII, picCIII, and picCVI, genes, are involved in desosamine biosynthesis and transfer and described in the following section.", "From Nucleotide To Nucleotide Description 70 13725 picAI 70 13725 narbonolide synthase 1 (PICAI) 148 3141 loading module 148 1434 KS loading module 1780 2802 AT loading module 2869 3141 ACP loading module 3208 7593 extender module 1 3208 4497 KS1 4828 5847 AT1 6499 7257 KR1 7336 7593 ACP1 7693 13332 extender module 2 7693 8974 KS2 9418 10554 AT2 10594 11160 DH2 12175 12960 KR2 13063 13332 ACP2 13830 25049 picAII 13830 25049 narbonolide synthase 2 (PICAII) 13935 18392 extender module 3 13935 15224 KS3 15540 16562 AT3 17271 18071 KR3 (inactive) 18123 18392 ACP3 18447 24767 extender module 4 18447 19736 KS4 20031 21050 AT4 21093 21626 DH4 22620 23588 ER4 23652 24423 KR4 24498 24765 ACP4 25133 29821 picAIII 25133 29821 narbonolide synthase 3 (PICAIII) 25235 29567 extender module 5 25235 26530 KS5 26822 27841 AT5 28474 29227 KR5 29302 29569 ACP5 29924 33964 picAIV 29924 33964 narbonolide synthase 4 (PICAIV) 30026 32986 extender module 6 30026 31312 KS6 31604 32635 AT6 32708 32986 ACP6 33068 33961 PKS thioesterase domain 33961 34806 picB 33961 34806 type II thioesterase homolog 34863 36011 picCII 34863 36011 4-keto-6-deoxyglucose isomerase 36159 37439 picCIII 36159 37439 desosaminyl transferase 37529 38242 picCVI 37529 38242 3-amino dimethyltransferase Sequence of the Insert DNA in Cosmid pKOS023-27 1 GATCATGCGG AGCACTCCTT CTCTCGTGCT CCTACCGGTG ATGTGCGCGC CGAATTGATT 61 CGTGGAGAGA TGTCGACAGT GTCCAAGAGT GAGTCCGAGG AATTCGTGTC CGTGTCGAAC 121 GACGCCGGTT CCGCGCACGG CACAGCGGAA CCCGTCGCCG TCGTCGGCAT CTCCTGCCGG 181 GTGCCCGGCG CCCGGGACCC GAGAGAGTTC TGGGAACTCC TGGCGGCAGG CGGCCAGGCC 241 GTCACCGACG TCCCCGCGGA CCGCTGGAAC GCCGGCGACT TCTACGACCC GGACCGCTCC 301 GCCCCCGGCC GCTCGAACAG CCGGTGGGGC GGGTTCATCG AGGACGTCGA CCGGTTCGAC 361 GCCGCCTTCT TCGGCATCTC GCCCCGCGAG GCCGCGGAGA TGGACCCGCA GCAGCGGCTC 421 GCCCTGGAGC TGGGCTGGGA GGCCCTGGAG CGCGCCGGGA TCGACCCGTC CTCGCTCACC 481 GGCACCCGCA CCGGCGTCTT CGCCGGCGCC ATCTGGGACG ACTACGCCAC CCTGAAGCAC 541 CGCCAGGGCG GCGCCGCGAT CACCCCGCAC ACCGTCACCG GCCTCCACCG CGGCATCATC 601 GCGAACCGAC TCTCGTACAC GCTCGGGCTC CGCGGCCCCA GCATGGTCGT CGACTCCGGC 661 CAGTCCTCGT CGCTCGTCGC CGTCCACCTC GCGTGCGAGA GCCTGCGGCG CGGCGAGTCC 721 GAGCTCGCCC TCGCCGGCGG CGTCTCGCTC AACCTGGTGC CGGACAGCAT CATCGGGGCG 781 AGCAAGTTCG GCGGCCTCTC CCCCGACGGC CGCGCCTACA CCTTCGACGC GCGCGCCAAC 841 GGCTACGTAC GCGGCGAGGG CGGCGGTTTC GTCGTCCTGA AGCGCCTCTC CCGGGCCGTC 901 GCCGACGGCG ACCCGGTGCT CGCCGTGATC CGGGGCAGCG CCGTCAACAA CGGCGGCGCC 961 GCCCAGGGCA TGACGACCCC CGACGCGCAG GCGCAGGAGG CCGTGCTCCG CGAGGCCCAC 1021 GAGCGGGCCG GGACCGCGCC GGCCGACGTG CGGTACGTCG AGCTGCACGG CACCGGCACC 1081 CCCGTGGGCG ACCCGATCGA GGCCGCTGCG CTCGGCGCCG CCCTCGGCAC CGGCCGCCCG 1141 GCCGGACAGC CGCTCCTGGT CGGCTCGGTC AAGACGAACA TCGGCCACCT GGAGGGCGCG 1201 GCCGGCATCG CCGGCCTCAT CAAGGCCGTC CTGGCGGTCC GCGGTCGCGC GCTGCCCGCC 1261 AGCCTGAACT ACGAGACCCC GAACCCGGCG ATCCCGTTCG AGGAACTGAA CCTCCGGGTG 1321 AACACGGAGT ACCTGCCGTG GGAGCCGGAG CACGACGGGC AGCGGATGGT CGTCGGCGTG 1381 TCCTCGTTCG GCATGGGCGG CACGAACGCG CATGTCGTGC TCGAAGAGGC CCCGGGGGTT 1441 GTCGAGGGTG CTTCGGTCGT GGAGTCGACG GTCGGCGGGT CGGCGGTCGG CGGCGGTGTG 1501 GTGCCGTGGG TGGTGTCGGC GAAGTCCGCT GCCGCGCTGG ACGCGCAGAT CGAGCGGCTT 1561 GCCGCGTTCG CCTCGCGGGA TCGTACGGAT GGTGTCGACG CGGGCGCTGT CGATGCGGGT 1621 GCTGTCGATG CGGGTGCTGT CGCTCGCGTA CTGGCCGGCG GGCGTGCTCA GTTCGAGCAC 1681 CGGGCCGTCG TCGTCGGCAG CGGGCCGGAC GATCTGGCGG CAGCGCTGGC CGCGCCTGAG 1741 GGTCTGGTCC GGGGCGTGGC TTCCGGTGTC GGGCGAGTGG CGTTCGTGTT CCCCGGGCAG 1801 GGCACGCAGT GGGCCGGCAT GGGTGCCGAA CTGCTGGACT CTTCCGCGGT GTTCGCGGCG 1861 GCCATGGCCG AATGCGAGGC CGCACTCTCC CCGTACGTCG ACTGGTCGCT GGAGGCCGTC 1921 GTACGGCAGG CCCCCGGTGC GCCCACGCTG GAGCGGGTCG ATGTCGTGCA GCCTGTGACG 1981 TTCGCCGTCA TGGTCTCGCT GGCTCGCGTG TGGCAGCACC ACGGGGTGAC GCCCCAGGCG 2041 GTCGTCGGCC ACTCGCAGGG CGAGATCGCC GCCGCGTACG TCGCCGGTGC CCTGAGCCTG 2101 GACGACGCCG CTCGTGTCGT GACCCTGCGC AGCAAGTCCA TCGCCGCCCA CCTCGCCGGC 2161 AAGGGCGGCA TGCTGTCCCT CGCGCTGAGC GAGGACGCCG TCCTGGAGCG ACTGGCCGGG 2221 TTCGACGGGC TGTCCGTCGC CGCTGTGAAC GGGCCCACCG CCACCGTGGT CTCCGGTGAC 2281 CCCGTACAGA TCGAAGAGCT TGCTCGGGCG TGTGAGGCCG ATGGGGTCCG TGCGCGGGTC 2341 ATTCCCGTCG ACTACGCGTC CCACAGCCGG CAGGTCGAGA TCATCGAGAG CGAGCTCGCC 2401 GAGGTCCTCG CCGGGCTCAG CCCGCAGGCT CCGCGCGTGC CGTTCTTCTC GACACTCGAA 2461 GGCGCCTGGA TCACCGAGCC CGTGCTCGAC GGCGGCTACT GGTACCGCAA CCTGCGCCAT 2521 CGTGTGGGCT TCGCCCCGGC CGTCGAGACC CTGGCCACCG ACGAGGGCTT CACCCACTTC 2581 GTCGAGGTCA CCGCCCACCC CGTCCTCACC ATGGCCCTCC CCGGGACCGT CACCGGTCTG 2641 GCGACCCTGC GTCGCGACAA CGGCGGTCAG GACCGCCTCG TCGCCTCCCT CGCCGAAGCA 2701 TGGGCCAACG GACTCGCGGT CGACTGGAGC CCGCTCCTCC CCTCCGCGAC CGGCCACCAC 2761 TCCGACCTCC CCACCTACGC GTTCCAGACC GAGCGCCACT GGCTGGGCGA GATCGAGGCG 2821 CTCGCCCCGG CGGGCGAGCC GGCGGTGCAG CCCGCCGTCC TCCGCACGGA GGCGGCCGAG 2881 CCGGCGGAGC TCGACCGGGA CGAGCAGCTG CGCGTGATCC TGGACAAGGT CCGGGCGCAG 2941 ACGGCCCAGG TGCTGGGGTA CGCGACAGGC GGGCAGATCG AGGTCGACCG GACCTTCCGT 3001 GAGGCCGGTT GCACCTCCCT GACCGGCGTG GACCTGCGCA ACCGGATCAA CGCCGCCTTC 3061 GGCGTACGGA TGGCGCCGTC CATGATCTTC GACTTCCCCA CCCCCGAGGC TCTCGCGGAG 3121 CAGCTGCTCC TCGTCGTGCA CGGGGAGGCG GCGGCGAACC CGGCCGGTGC GGAGCCGGCT 3181 CCGGTGGCGG CGGCCGGTGC CGTCGACGAG CCGGTGGCGA TCGTCGGCAT GGCCTGCCGC 3241 CTGCCCGGTG GGGTCGCCTC GCCGGAGGAC CTGTGGCGGC TGGTGGCCGG CGGCGGGGAC 3301 GCGATCTCGG AGTTCCCGCA GGACCGCGGC TGGGACGTGG AGGGGCTGTA CCACCCGGAT 3361 CCCGAGCACC CCGGCACGTC GTACGTCCGC CAGGGCGGTT TCATCGAGAA CGTCGCCGGC 3421 TTCGACGCGG CCTTCTTCGG GATCTCGCCG CGCGAGGCCC TCGCCATGGA CCCGCAGCAG 3481 CGGCTCCTCC TCGAAACCTC CTGGGAGGCC GTCGAGGACG CCGGGATCGA CCCGACCTCC 3541 CTGCGGGGAC GGCAGGTCGG CGTCTTCACT GGGGCGATGA CCCACGAGTA CGGGCCGAGC 3601 CTGCGGGACG GCGGGGAAGG CCTCGACGGC TACCTGCTGA CCGGCAACAC GGCCAGCGTG 3661 ATGTCGGGCC GCGTCTCGTA CACACTCGGC CTTGAGGGCC CCGCCCTGAC GGTGGACACG 3721 GCCTGCTCGT CGTCGCTGGT CGCCCTGCAC CTCGCCGTGC AGGCCCTGCG CAAGGGCGAG 3781 GTCGACATGG CGCTCGCCGG CGGCGTGGCC GTGATGCCCA CGCCCGGGAT GTTCGTCGAG 3841 TTCAGCCGGC AGCGCGGGCT GGCCGGGGAC GGCCGGTCGA AGGCGTTCGC CGCGTCGGCG 3901 GACCGGACCA GCTGGTCCGA GGGCGTCGGC GTCCTCCTCG TCGAGCGCCT GTCGGACGCC 3961 CGCCGCAACG GACACCAGGT CCTCGCGGTC GTCCGCGGCA GCGCCGTGAA CCAGGACGGC 4021 GCGAGCAACG GCCTCACGGC TCCGAACGGG CCCTCGCAGC AGCGCGTCAT CCGGCGCGCG 4081 CTGGCGGACG CCCGGCTGAC GACCTCCGAC GTGGACGTCG TCGAGGCACA CGGCACGGGC 4141 ACGCGACTCG GCGACCCGAT CGAGGCGCAG GCCCTGATCG CCACCTACGG CCAGGGCCGT 4201 GACGACGAAC AGCCGCTGCG CCTCGGGTCG TTGAAGTCCA ACATCGGGCA CACCCAGGCC 4261 GCGGCCGGCG TCTCCGGTGT CATCAAGATG GTCCAGGCGA TGCGCCACGG ACTGCTGCCG 4321 AAGACGCTGC ACGTCGACGA GCCCTCGGAC CAGATCGACT GGTCGGCTGG CGCCGTGGAA 4381 CTCCTCACCG AGGCCGTCGA CTGGCCGGAG AAGCAGGACG GCGGGCTGCG CCGGGCCGCC 4441 GTCTCCTCCT TCGGGATCAG CGGCACCAAT GCGCATGTGG TGCTCGAAGA GGCCCCGGTG 4501 GTTGTCGAGG GTGCTTCGGT CGTCGAGCCG TCGGTTGGCG GGTCGGCGGT CGGCGGCGGT 4561 GTGACGCCTT GGGTGGTGTC GGCGAAGTCC GCTGCCGCGC TCGACGCGCA GATCGAGCGG 4621 CTTGCCGCAT TCGCCTCGCG GGATCGTACG GATGACGCCG ACGCCGGTGC TGTCGACGCG 4681 GGCGCTGTCG CTCACGTACT GGCTGACGGG CGTGCTCAGT TCGAGCACCG GGCCGTCGCG 4741 CTCGGCGCCG GGGCGGACGA CCTCGTACAG GCGCTGGCCG ATCCGGACGG GCTGATACGC 4801 GGAACGGCTT CCGGTGTCGG GCGAGTGGCG TTCGTGTTCC CCGGTCAGGG CACGCAGTGG 4861 GCTGGCATGG GTGCCGAACT GCTGGACTCT TCCGCGGTGT TCGCGGCGGC CATGGCCGAG 4921 TGTGAGGCCG CGCTGTCCCC GTACGTCGAC TGGTCGCTGG AGGCCGTCGT ACGGCAGGCC 4981 CCCGGTGCGC CCACGCTGGA GCGGGTCGAT GTCGTGCAGC CTGTGACGTT CGCCGTCATG 5041 GTCTCGCTGG CTCGCGTGTG GCAGCACCAC GGTGTGACGC CCCAGGCGGT CGTCGGCCAC 5101 TCGCAGGGCG AGATCGCCGC CGCGTACGTC GCCGGAGCCC TGCCCCTGGA CGACGCCGCC 5161 CGCGTCGTCA CCCTGCGCAG CAAGTCCATC GCCGCCCACC TCGCCGGCAA GGGCGGCATG 5221 CTGTCCCTCG CGCTGAACGA GGACGCCGTC CTGGAGCGAC TGAGTGACTT CGACGGGCTG 5281 TCCGTCGCCG CCGTCAACGG GCCCACCGCC ACTGTCGTGT CGGGTGACCC CGTACAGATC 5341 GAAGAGCTTG CTCAGGCGTG CAAGGCGGAC GGATTCCGCG CGCGGATCAT TCCCGTCGAC 5401 TACGCGTCCC ACAGCCGGCA GGTCGAGATC ATCGAGAGCG AGCTCGCCCA GGTCCTCGCC 5461 GGTCTCAGCC CGCAGGCCCC GCGCGTGCCG TTCTTCTCGA CGCTCGAAGG CACCTGGATC 5521 ACCGAGCCCG TCCTCGACGG CACCTACTGG TACCGCAACC TCCGTCACCG CGTCGGCTTC 5581 GCCCCCGCCA TCGAGACCCT GGCCGTCGAC GAGGGCTTCA CGCACTTCGT CGAGGTCAGC 5641 GCCCACCCCG TCCTCACCAT GACCCTCCCC GAGACCGTCA CCGGCCTCGG CACCCTCCGT 5701 CGCGAACAGG GAGGCCAAGA GCGTCTGGTC ACCTCGCTCG CCGAGGCGTG GGTCAACGGG 5761 CTTCCCGTGG CATGGACTTC GCTCCTGCCC GCCACGGCCT CCCGCCCCGG TCTGCCCACC 5821 TACGCCTTCC AGGCCGAGCG CTACTGGCTC GAGAACACTC CCGCCGCCCT GGCCACCGGC 5881 GACGACTGGC GCTACCGCAT CGACTGGAAG CGCCTCCCGG CCGCCGAGGG GTCCGAGCGC 5941 ACCGGCCTGT CCGGCCGCTG GCTCGCCGTC ACGCCGGAGG ACCACTCCGC GCAGGCCGCC 6001 GCCGTGCTCA CCGCGCTGGT CGACGCCGGG GCGAAGGTCG AGGTGCTGAC GGCCGGGGCG 6061 GACGACGACC GTGAGGCCCT CGCCGCCCGG CTCACCGCAC TGACGACCGG TGACGGCTTC 6121 ACCGGCGTGG TCTCGCTCCT CGACGGACTC GTACCGCAGG TCGCCTGGGT CCAGGCGCTC 6181 GGCGACGCCG GAATCAAGGC GCCCCTGTGG TCCGTCACCC AGGGCGCGGT CTCCGTCGGA 6241 CGTCTCGACA CCCCCGCCGA CCCCGACCGG GCCATGCTCT GGGGCCTCGG CCGCGTCGTC 6301 GCCCTTGAGC ACCCCGAACG CTGGGCCGGC CTCGTCGACC TCCCCGCCCA GCCCGATGCC 6361 GCCGCCCTCG CCCACCTCGT CACCGCACTC TCCGGCGCCA CCGGCGAGGA CCAGATCGCC 6421 ATCCGCACCA CCGGACTCCA CGCCCGCCGC CTCGCCCGCG CACCCCTCCA CGGACGTCGG 6481 CCCACCCGCG ACTGGCAGCC CCACGGCACC GTCCTCATCA CCGGCGGCAC CGGAGCCCTC 6541 GGCAGCCACG CCGCACGCTG GATGGCCCAC CACGGAGCCG AACACCTCCT CCTCGTCAGC 6601 CGCAGCGGCG AACAAGCCCC CGGAGCCACC CAACTCACCG CCGAACTCAC CGCATCGGGC 6661 GCCCGCGTCA CCATCGCCGC CTGCGACGTC GCCGACCCCC ACGCCATGCG CACCCTCCTC 6721 GACGCCATCC CCGCCGAGAC GCCCGTCACC GCCGTCGTCC ACACCGCCGG CGCGCTCGAC 6781 GACGGCATCG TGGACACGCT GACCGCCGAG CAGGTCCGGC GGGCCCACCG TGCGAAGGCC 6841 GTCGGCGCCT CGGTGCTCGA CGAGCTGACC CGGGACCTCG ACCTCGACGC GTTCGTGCTC 6901 TTCTCGTCCG TGTCGAGCAC TCTGGGCATC CCCGGTCAGG GCAACTACGC CCCGCACAAC 6961 GCCTACCTCG ACGCCCTCGC GGCTCGCCGC CGGGCCACCG GCCGGTCCGC CGTCTCGGTG 7021 GCCTGGGGAC CGTGGGACGG TGGCGGCATG GCCGCCGGTG ACGGCGTGGC CGAGCGGCTG 7081 CGCAACCACG GCGTGCCCGG CATGGACCCG GAACTCGCCC TGGCCGCACT GGAGTCCGCG 7141 CTCGGCCGGG ACGAGACCGC GATCACCGTC GCGGACATCG ACTGGGACCG CTTCTACCTC 7201 GCGTACTCCT CCGGTCGCCC GCAGCCCCTC GTCGAGGAGC TGCCCGAGGT GCGGCGCATC 7261 ATCGACGCAC GGGACAGCGC CACGTCCGGA CAGGGCGGGA GCTCCGCCCA GGGCGCCAAC 7321 CCCCTGGCCG AGCGGCTGGC CGCCGCGGCT CCCGGCGAGC GTACGGAGAT CCTCCTCGGT 7381 CTCGTACGGG CGCAGGCCGC CGCCGTGCTC CGGATGCGTT CGCCGGAGGA CGTCGCCGCC 7441 GACCGCGCCT TCAAGGACAT CGGCTTCGAC TCGCTCGCCG GTGTCGAGCT GCGCAACAGG 7501 CTGACCCGGG CGACCGGGCT CCAGCTGCCC GCGACGCTCG TCTTCGACCA CCCGACGCCG 7561 CTGGCCCTCG TGTCGCTGCT CCGCAGCGAG TTCCTCGGTG ACGAGGAGAC GGCGGACGCC 7621 CGGCGGTCCG CGGCGCTGCC CGCGACTGTC GGTGCCGGTG CCGGCGCCGG CGCCGGCACC 7681 GATGCCGACG ACGATCCGAT CGCGATCGTC GCGATGAGCT GCCGCTACCC CGGTGACATC 7741 CGCAGCCCGG AGGACCTGTG GCGGATGCTG TCCGAGGGCG GCGAGGGCAT CACGCCGTTC 7801 CCCACCGACC GCGGCTGGGA CCTCGACGGC CTGTACGACG CCGACCCGGA CGCGCTCGGC 7861 AGGGCGTACG TCCGCGAGGG CGGGTTCCTG CACGACGCGG CCGAGTTCGA CGCGGAGTTC 7921 TTCGGCGTCT CGCCGCGCGA GGCGCTGGCC ATGGACCCGC AGCAGCGGAT GCTCCTGACG 7981 ACGTCCTGGG AGGCCTTCGA GCGGGCCGGC ATCGAGCCGG CATCGCTGCG CGGCAGCAGC 8041 ACCGGTGTCT TCATCGGCCT CTCCTACCAG GACTACGCGG CCCGCGTCCC GAACGCCCCG 8101 CGTGGCGTGG AGGGTTACCT GCTGACCGGC AGCACGCCGA GCGTCGCGTC GGGCCGTATC 8161 GCGTACACCT TCGGTCTCGA AGGGCCCGCG ACGACCGTCG ACACCGCCTG CTCGTCGTCG 8221 CTGACCGCCC TGCACCTGGC GGTGCGGGCG CTGCGCAGCG GCGAGTGCAC GATGGCGCTC 8281 GCCGGTGGCG TGGCGATGAT GGCGACCCCG CACATGTTCG TGGAGTTCAG CCGTCAGCGG 8341 GCGCTCGCCC CGGACGGCCG CAGCAAGGCC TTCTCGGCGG ACGCCGACGG GTTCGGCGCC 8401 GCGGAGGGCG TCGGCCTGCT GCTCGTGGAG CGGCTCTCGG ACGCGCGGCG CAACGGTCAC 8461 CCGGTGCTCG CCGTGGTCCG CGGTACCGCC GTCAACCAGG ACGGCGCCAG CAACGGGCTG 8521 ACCGCGCCCA ACGGACCCTC GCAGCAGCGG GTGATCCGGC AGGCGCTCGC CGACGCCCGG 8581 CTGGCACCCG GCGACATCGA CGCCGTCGAG ACGCACGGCA CGGGAACCTC GCTGGGCGAC 8641 CCCATCGAGG CCCAGGGCCT CCAGGCCACG TACGGCAAGG AGCGGCCCGC GGAACGGCCG 8701 CTCGCCATCG GCTCCGTGAA GTCCAACATC GGACACACCC AGGCCGCGGC CGGTGCGGCG 8761 GGCATCATCA AGATGGTCCT CGCGATGCGC CACGGCACCC TGCCGAAGAC CCTCCACGCC 8821 GACGAGCCGA GCCCGCACGT CGACTGGGCG AACAGCGGCC TGGCCCTCGT CACCGAGCCG 8881 ATCGACTGGC CGGCCGGCAC CGGTCCGCGC CGCGCCGCCG TCTCCTCCTT CGGCATCAGC 8941 GGGACGAACG CGCACGTCGT GCTGGAGCAG GCGCCGGATG CTGCTGGTGA GGTGCTTGGG 9001 GCCGATGAGG TGCCTGAGGT GTCTGAGACG GTAGCGATGG CTGGGACGGC TGGGACCTCC 9061 GAGGTCGCTG AGGGCTCTGA GGCCTCCGAG GCCCCCGCGG CCCCCGGCAG CCGTGAGGCG 9121 TCCCTCCCCG GGCACCTGCC CTGGGTGCTG TCCGCCAAGG ACGAGCAGTC GCTGCGCGGC 9181 CAGGCCGCCG CCCTGCACGC GTGGCTGTCC GAGCCCGCCG CCGACCTGTC GGACGCGGAC 9241 GGACCGGCCC GCCTGCGGGA CGTCGGGTAC ACGCTCGCCA CGAGCCGTAC CGCCTTCGCG 9301 CACCGCGCCG CCGTGACCGC CGCCGACCGG GACGGGTTCC TGGACGGGCT GGCCACGCTG 9361 GCCCAGGGCG GCACCTCGGC CCACGTCCAC CTGGACACCG CCCGGGACGG CACCACCGCG 9421 TTCCTCTTCA CCGGCCAGGG CAGTCAGCGC CCCGGCGCCG GCCGTGAGCT GTACGACCGG 9481 CACCCCGTCT TCGCCCGGGC GCTCGACGAG ATCTGCGCCC ACCTCGACGG TCACCTCGAA 9541 CTGCCCCTGC TCGACGTGAT GTTCGCGGCC GAGGGCAGCG CGGAGGCCGC GCTGCTCGAC 9601 GAGACGCGGT ACACGCAGTG CGCGCTGTTC GCCCTGGAGG TCGCGCTCTT CCGGCTCGTC 9661 GAGAGCTGGG GCATGCGGCC GGCCGCACTG CTCGGTCACT CGGTCGGCGA GATCGCCGCC 9721 GCGCACGTCG CCGGTGTGTT CTCGCTCGCC GACGCCGCCC GCCTGGTCGC CGCGCGCGGC 9781 CGGCTCATGC AGGAGCTGCC CGCCGGTGGC GCGATGCTCG CCGTCCAGGC CGCGGAGGAC 9841 GAGATCCGCG TGTGGCTGGA GACGGAGGAG CGGTACGCGG GACGTCTGGA CGTCGCCGCC 9901 GTCAACGGCC CCGAGGCCGC CGTCCTGTCC GGCGACGCGG ACGCGGCGCG GGAGGCGGAG 9961 GCGTACTGGT CCGGGCTCGG CCGCAGGACC CGCGCGCTGC GGGTCAGCCA CGCCTTCCAC 10021 TCCGCGCACA TGGACGGCAT GCTCGACGGG TTCCGCGCCG TCCTGGAGAC GGTGGAGTTC 10081 CGGCGCCCCT CCCTGACCGT GGTCTCGAAC GTCACCGGCC TGGCCGCCGG CCCGGACGAC 10141 CTGTGCGACC CCGAGTACTG GGTCCGGCAC GTCCGCGGCA CCGTCCGCTT CCTCGACGGC 10201 GTCCGTGTCC TGCGCGACCT CGGCGTGCGG ACCTGCCTGG AGCTGGGCCC CGACGGGGTC 10261 CTCACCGCCA TGGCGGCCGA CGGCCTCGCG GACACCCCCG CGGATTCCGC TGCCGGCTCC 10321 CCCGTCGGCT CTCCCGCCGG CTCTCCCGCC GACTCCGCCG CCGGCGCGCT CCGGCCCCGG 10381 CCGCTGCTCG TGGCGCTGCT GCGCCGCAAG CGGTCGGAGA CCGAGACCGT CGCGGACGCC 10441 CTCGGCAGGG CGCACGCCCA CGGCACCGGA CCCGACTGGC ACGCCTGGTT CGCCGGCTCC 10501 GGGGCGCACC GCGTGGACCT GCCCACGTAC TCCTTCCGGC GCGACCGCTA CTGGCTGGAC 10561 GCCCCGGCGG CCGACACCGC GGTGGACACC GCCGGCCTCG GTCTCGGCAC CGCCGACCAC 10621 CCGCTGCTCG GCGCCGTGGT CAGCCTTCCG GACCGGGACG GCCTGCTGCT CACCGGCCGC 10681 CTCTCCCTGC GCACCCACCC GTGGCTCGCG GACCACGCCG TCCTGGGGAG CGTCCTGCTC 10741 CCCGGCGCCG CGATGGTCGA ACTCGCCGCG CACGCTGCGG AGTCCGCCGG TCTGCGTGAC 10801 GTGCGGGAGC TGACCCTCCT TGAACCGCTG GTACTGCCCG AGCACGGTGG CGTCGAGCTG 10861 CGCGTGACGG TCGGGGCGCC GGCCGGAGAG CCCGGTGGCG AGTCGGCCGG GGACGGCGCA 10921 CGGCCCGTCT CCCTCCACTC GCGGCTCGCC GACGCGCCCG CCGGTACCGC CTGGTCCTGC 10981 CACGCGACCG GTCTGCTGGC CACCGACCGG CCCGAGCTTC CCGTCGCGCC CGACCGTGCG 11041 GCCATGTGGC CGCCGCAGGG CGCCGAGGAG GTGCCGCTCG ACGGTCTCTA CGAGCGGCTC 11101 GACGGGAACG GCCTCGCCTT CGGTCCGCTG TTCCAGGGGC TGAACGCGGT GTGGCGGTAC 11161 GAGGGTGAGG TCTTCGCCGA CATCGCGCTC CCCGCCACCA CGAATGCGAC CGCGCCCGCG 11221 ACCGCGAACG GCGGCGGGAG TGCGGCGGCG GCCCCCTACG GCATCCACCC CGCCCTGCTC 11281 GACGCTTCGC TGCACGCCAT CGCGGTCGGC GGTCTCGTCG ACGAGCCCGA GCTCGTCCGC 11341 GTCCCCTTCC ACTGGAGCGG TGTCACCGTG CACGCGGCCG GTGCCGCGGC GGCCCGGGTC 11401 CGTCTCGCCT CCGCGGGGAC GGACGCCGTC TCGCTGTCCC TGACGGACGG CGAGGGACGC 11461 CCGCTGGTCT CCGTGGAACG GCTCACGCTG CGCCCGGTCA CCGCCGATCA GGCGGCGGCG 11521 AGCCGCGTCG GCGGGCTGAT GCACCGGGTG GCCTGGCGTC CGTACGCCCT CGCCTCGTCC 11581 GGCGAACAGG ACCCGCACGC CACTTCGTAC GGGCCGACCG CCGTCCTCGG CAAGGACGAG 11641 CTGAAGGTCG CCGCCGCCCT GGAGTCCGCG GGCGTCGAAG TCGGGCTCTA CCCCGACCTG 11701 GCCGCGCTGT CCCAGGACGT GGCGGCCGGC GCCCCGGCGC CCCGTACCGT CCTTGCGCCG 11761 CTGCCCGCGG GTCCCGCCGA CGGCGGCGCG GAGGGTGTAC GGGGCACGGT GGCCCGGACG 11821 CTGGAGCTGC TCCAGGCCTG GCTGGCCGAC GAGCACCTCG CGGGCACCCG CCTGCTCCTG 11881 GTCACCCGCG GTGCGGTGCG GGACCCCGAG GGGTCCGGCG CCGACGATGG CGGCGAGGAC 11941 CTGTCGCACG CGGCCGCCTG GGGTCTCGTA CGGACCGCGC AGACCGAGAA CCCCGGCCGC 12001 TTCGGCCTTC TCGACCTGGC CGACGACGCC TCGTCGTACC GGACCCTGCC GTCGGTGCTC 12061 TCCGACGCGG GCCTGCGCGA CGAACCGCAG CTCGCCCTGC ACGACGGCAC CATCAGGCTG 12121 GCCCGCCTGG CCTCCGTCCG GCCCGAGACC GGCACCGCCG CACCGGCGCT CGCCCCGGAG 12181 GGCACGGTCC TGCTGACCGG CGGCACCGGC GGCCTGGGCG GACTGGTCGC CCGGCACGTG 12241 GTGGGCGAGT GGGGCGTACG ACGCCTGCTG CTGGTGAGCC GGCGGGGCAC GGACGCCCCG 12301 GGCGCCGACG AGCTCGTGCA CGAGCTGGAG GCCCTGGGAG CCGACGTCTC GGTGGCCGCG 12361 TGCGACGTCG CCGACCGCGA AGCCCTCACC GCCGTACTCG ACGCCATCCC CGCCGAACAC 12421 CCGCTCACCG CGGTCGTCCA CACGGCAGGC GTCCTCTCCG ACGGCACCCT CCCGTCCATG 12481 ACGACGGAGG ACGTGGAACA CGTACTGCGG CCCAAGGTCG ACGCCGCGTT CCTCCTCGAC 12541 GAACTCACCT CGACGCCCGC ATACGACCTG GCAGCGTTCG TCATGTTCTC CTCCGCCGCC 12601 GCCGTCTTCG GTGGCGCGGG GCAGGGCGCC TACGCCGCCG CCAACGCCAC CCTCGACGCC 12661 CTCGCCTGGC GCCGCCGGGC AGCCGGACTC CCCGCCCTCT CCCTCGGCTG GGGCCTCTGG 12721 GCCGAGACCA GCGGCATGAC CGGCGAGCTC GGCCAGGCGG ACCTGCGCCG GATGAGCCGC 12781 GCGGGCATCG GCGGGATCAG CGACGCCGAG GGCATCGCGC TCCTCGACGC CGCCCTCCGC 12841 GACGACCGCC ACCCGGTCCT GCTGCCCCTG CGGCTCGACG CCGCCGGGCT GCGGGACGCG 12901 GCCGGGAACG ACCCGGCCGG AATCCCGGCG CTCTTCCGGG ACGTCGTCGG CGCCAGGACC 12961 GTCCGGGCCC GGCCGTCCGC GGCCTCCGCC TCGACGACAG CCGGGACGGC CGGCACGCCG 13021 GGGACGGCGG ACGGCGCGGC GGAAACGGCG GCGGTCACGC TCGCCGACCG GGCCGCCACC 13081 GTGGACGGGC CCGCACGGCA GCGCCTGCTG CTCGAGTTCG TCGTCGGCGA GGTCGCCGAA 13141 GTACTCGGCC ACGCCCGCGG TCACCGGATC GACGCCGAAC GGGGCTTCCT CGACCTCGGC 13201 TTCGACTCCC TGACCGCCGT CGAACTCCGC AACCGGCTCA ACTCCGCCGG TGGCCTCGCC 13261 CTCCCGGCGA CCCTGGTCTT CGACCACCCA AGCCCGGCGG CACTCGCCTC CCACCTGGAC 13321 GCCGAGCTGC CGCGCGGCGC CTCGGACCAG GACGGAGCCG GGAACCGGAA CGGGAACGAG 13381 AACGGGACGA CGGCGTCCCG GAGCACCGCC GAGACGGACG CGCTGCTGGC ACAACTGACC 13441 CGCCTGGAAG GCGCCTTGGT GCTGACGGGC CTCTCGGACG CCCCCGGGAG CGAAGAAGTC 13501 CTGGAGCACC TGCGGTCCCT GCGCTCGATG GTCACGGGCG AGACCGGGAC CGGGACCGCG 13561 TCCGGAGCCC CGGACGGCGC CGGGTCCGGC GCCGAGGACC GGCCCTGGGC GGCCGGGGAC 13621 GGAGCCGGGG GCGGGAGTGA GGACGGCGCG GGAGTGCCGG ACTTCATGAA CGCCTCGGCC 13681 GAGGAACTCT TCGGCCTCCT CGACCAGGAC CCCAGCACGG ACTGATCCCT GCCGCACGGT 13741 CGCCTCCCGC CCCGGACCCC GTCCCGGGCA CCTCGACTCG AATCACTTCA TGCGCGCCTC 13801 GGGCGCCTCC AGGAACTCAA GGGGACAGCG TGTCCACGGT GAACGAAGAG AAGTACCTCG 13861 ACTACCTGCG TCGTGCCACG GCGGACCTCC ACGAGGCCCG TGGCCGCCTC CGCGAGCTGG 13921 AGGCGAAGGC GGGCGAGCCG GTGGCGATCG TCGGCATGGC CTGCCGCCTG CCCGGCGGCG 13981 TCGCCTCGCC CGAGGACCTG TGGCGGCTGG TGGCCGGCGG CGAGGACGCG ATCTCGGAGT 14041 TCCCCCAGGA CCGCGGCTGG GACGTGGAGG GCCTGTACGA CCCGAACCCG GAGGCCACGG 14101 GCAAGAGTTA CGCCCGCGAG GCCGGATTCC TGTACGAGGC GGGCGAGTTC GACGCCGACT 14161 TCTTCGGGAT CTCGCCGCGC GAGGCCCTCG CCATGGACCC GCAGCAGCGT CTCCTCCTGG 14221 AGGCCTCCTG GGAGGCGTTC GAGCACGCCG GGATCCCGGC GGCCACCGCG CGCGGCACCT 14281 CGGTCGGCGT CTTCACCGGC GTGATGTACC ACGACTACGC CACCCGTCTC ACCGATGTCC 14341 CGGAGGGCAT CGAGGGCTAC CTGGGCACCG GCAACTCCGG CAGTGTCGCC TCGGGCCGCG 14401 TCGCGTACAC GCTTGGCCTG GAGGGGCCGG CCGTCACGGT CGACACCGCC TGCTCGTCCT 14461 CGCTGGTCGC CCTGCACCTC GCCGTGCAGG CCCTGCGCAA GGGCGAGGTC GACATGGCGC 14521 TCGCCGGCGG CGTGACGGTC ATGTCGACGC CCAGCACCTT CGTCGAGTTC AGCCGTCAGC 14581 GCGGGCTGGC GCCGGACGGC CGGTCGAAGT CCTTCTCGTC GACGGCCGAC GGCACCAGCT 14641 GGTCCGAGGG CGTCGGCGTC CTCCTCGTCG AGCGCCTGTC CGACGCGCGT CGCAAGGGCC 14701 ATCGGATCCT CGCCGTGGTC CGGGGCACCG CCGTCAACCA GGACGGCGCC AGCAGCGGCC 14761 TCACGGCTCC GAACGGGCCG TCGCAGCAGC GCGTCATCCG ACGTGCCCTG GCGGACGCCC 14821 GGCTCACGAC CTCCGACGTG GACGTCGTCG AGGCCCACGG CACGGGTACG CGACTCGGCG 14881 ACCCGATCGA GGCGCAGGCC GTCATCGCCA CGTACGGGCA GGGCCGTGAC GGCGAACAGC 14941 CGCTGCGCCT CGGGTCGTTG AAGTCCAACA TCGGACACAC CCAGGCCGCC GCCGGTGTCT 15001 CCGGCGTGAT CAAGATGGTC CAGGCGATGC GCCACGGCGT CCTGCCGAAG ACGCTCCACG 15061 TGGAGAAGCC GACGGACCAG GTGGACTGGT CCGCGGGCGC GGTCGAGCTG CTCACCGAGG 15121 CCATGGACTG GCCGGACAAG GGCGACGGCG GACTGCGCAG GGCCGCGGTC TCCTCCTTCG 15181 GCGTCAGCGG GACGAACGCG CACGTCGTGC TCGAAGAGGC CCCGGCGGCC GAGGAGACCC 15241 CTGCCTCCGA GGCGACCCCG GCCGTCGAGC CGTCGGTCGG CGCCGGCCTG GTGCCGTGGC 15301 TGGTGTCGGC GAAGACTCCG GCCGCGCTGG ACGCCCAGAT CGGACGCCTC GCCGCGTTCG 15361 CCTCGCAGGG CCGTACGGAC GCCGCCGATC CGGGCGCGGT CGCTCGCGTA CTGGCCGGCG 15421 GGCGCGCCGA GTTCGAGCAC CGGGCCGTCG TGCTCGGCAC CGGACAGGAC GATTTCGCGC 15481 AGGCGCTGAC CGCTCCGGAA GGACTGATAC GCGGCACGCC CTCGGACGTG GGCCGGGTGG 15541 CGTTCGTGTT CCCCGGTCAG GGCACGCAGT GGGCCGGGAT GGGCGCCGAA CTCCTCGACG 15601 TGTCGAAGGA GTTCGCGGCG GCCATGGCCG AGTGCGAGAG CGCGCTCTCC CGCTATGTCG 15661 ACTGGTCGCT GGAGGCCGTC GTCCGGCAGG CGCCGGGCGC GCCCACGCTG GAGCGGGTCG 15721 ACGTCGTCCA GCCCGTGACC TTCGCTGTCA TGGTTTCGCT GGCGAAGGTC TGGCAGCACC 15781 ACGGCGTGAC GCCGCAGGCC GTCGTCGGCC ACTCGCAGGG CGAGATCGCC GCCGCGTACG 15841 TCGCCGGTGC CCTCACCCTC GACGACGCCG CCCGCGTCGT CACCCTGCGC AGCAAGTCCA 15901 TCGCCGCCCA CCTCGCCGGC AAGGGCGGCA TGATCTCCCT CGCCCTCAGC GAGGAAGCCA 15961 CCCGGCAGCG CATCGAGAAC CTCCACGGAC TGTCGATCGC CGCCGTCAAC GGCCCCACCG 16021 CCACCGTGGT TTCGGGCGAC CCCACCCAGA TCCAAGAGCT CGCTCAGGCG TGTGAGGCCG 16081 ACGGGGTCCG CGCACGGATC ATCCCCGTCG ACTACGCCTC CCACAGCGCC CACGTCGAGA 16141 CCATCGAGAG CGAACTCGCC GAGGTCCTCG CCGGGCTCAG CCCGCGGACA CCTGAGGTGC 16201 CGTTCTTCTC GACACTCGAA GGCGCCTGGA TCACCGAGCC GGTGCTCGAC GGCACCTACT 16261 GGTACCGCAA CCTCCGCCAC CGCGTCGGCT TCGCCCCCGC CGTCGAGACC CTCGCCACCG 16321 ACGAAGGCTT CACCCACTTC ATCGAGGTCA GCGCCCACCC CGTCCTCACC ATGACCCTCC 16381 CCGAGACCGT CACCGGCCTC GGCACCCTCC GCCGCGAACA GGGAGGCCAG GAGCGTCTGG 16441 TCACCTCACT CGCCGAAGCC TGGACCAACG GCCTCACCAT CGACTCGGCG CCCGTCCTCC 16501 CCACCGCAAC CGGCCACCAC CCCGAGCTCC CCACCTACGC CTTCCAGCGC CGTCACTACT 16561 GGCTCCACGA CTCCCCCGCC GTCCAGGGCT CCGTGCAGGA CTCCTGGCGC TACCGCATCG 16621 ACTGGAAGCG CCTCGCGGTC GCCGACGCGT CCGAGCGCGC CGGGCTGTCC GGGCGCTGGC 16681 TCGTCGTCGT CCCCGAGGAC CGTTCCGCCG AGGCCGCCCC GGTGCTCGCC GCGCTGTCCG 16741 GCGCCGGCGC CGACCCCGTA CAGCTGGACG TGTCCCCGCT GGGCGACCGG CAGCGGCTCG 16801 CCGCGACGCT GGGCGAGGCC CTGGCGGCGG CCGGTGGAGC CGTCGACGGC GTCCTCTCGC 16861 TGCTCGCGTG GGACGAGAGC GCGCACCCCG GCCACCCCGC CCCCTTCACC CGGGGCACCG 16921 GCGCCACCCT CACCCTGGTG CAGGCGCTGG AGGACGCCGG CGTCGCCGCC CCGCTGTGGT 16981 GCGTGACCCA CGGCGCGGTG TCCGTCGGCC GGGCCGACCA CGTCACCTCC CCCGCCCAGG 17041 CCATGGTGTG GGGCATGGGC CGGGTCGCCG CCCTGGAGCA CCCCGAGCGG TGGGGCGGCC 17101 TGATCGACCT GCCCTCGGAC GCCGACCGGG CGGCCCTGGA CCGCATGACC ACGGTCCTCG 17161 CCGGCGGTAC GGGTGAGGAC CAGGTCGCGG TACGCGCCTC CGGGCTGCTC GCCCGCCGCC 17221 TCGTCCGCGC CTCCCTCCCG GCGCACGGCA CGGCTTCGCC GTGGTGGCAG GCCGACGGCA 17281 CGGTGCTCGT CACCGGTGCC GAGGAGCCTG CGGCCGCCGA GGCCGCACGC CGGCTGGCCC 17341 GCGACGGCGC CGGACACCTC CTCCTCCACA CCACCCCCTC CGGCAGCGAA GGCGCCGAAG 17401 GCACCTCCGG TGCCGCCGAG GACTCCGGCC TCGCCGGGCT CGTCGCCGAA CTCGCGGACC 17461 TGGGCGCGAC GGCCACCGTC GTGACCTGCG ACCTCACGGA CGCGGAGGCG GCCGCCCGGC 17521 TGCTCGCCGG CGTCTCCGAC GCGCACCCGC TCAGCGCCGT CCTCCACCTG CCGCCCACCG 17581 TCGACTCCGA GCCGCTCGCC GCGACCGACG CGGACGCGCT CGCCCGTGTC GTGACCGCGA 17641 AGGCCACCGC CGCGCTCCAC CTGGACCGCC TCCTGCGGGA GGCCGCGGCT GCCGGAGGCC 17701 GTCCGCCCGT CCTGGTCCTC TTCTCCTCGG TCGCCGCGAT CTGGGGCGGC GCCGGTCAGG 17761 GCGCGTACGC CGCCGGTACG GCCTTCCTCG ACGCCCTCGC CGGTCAGCAC CGGGCCGACG 17821 GCCCCACCGT GACCTCGGTG GCCTGGAGCC CCTGGGAGGG CAGCCGCGTC ACCGAGGGTG 17881 CGACCGGGGA GCGGCTGCGC CGCCTCGGCC TGCGCCCCCT CGCCCCCGCG ACGGCGCTCA 17941 CCGCCCTGGA CACCGCGCTC GGCCACGGCG ACACCGCCGT CACGATCGCC GACGTCGACT 18001 GGTCGAGCTT CGCCCCCGGC TTCACCACGG CCCGGCCGGG CACCCTCCTC GCCGATCTGC 18061 CCGAGGCGCG CCGCGCGCTC GACGAGCAGC AGTCGACGAC GGCCGCCGAC GACACCGTCC 18121 TGAGCCGCGA GCTCGGTGCG CTCACCGGCG CCGAACAGCA GCGCCGTATG CAGGAGTTGG 18181 TCCGCGAGCA CCTCGCCGTG GTCCTCAACC ACCCCTCCCC CGAGGCCGTC GACACGGGGC 18241 GGGCCTTCCG TGACCTCGGA TTCGACTCGC TGACGGCGGT CGAGCTCCGC AACCGCCTCA 18301 AGAACGCCAC CGGCCTGGCC CTCCCGGCCA CTCTGGTCTT CGACTACCCG ACCCCCCGGA 18361 CGCTGGCGGA GTTCCTCCTC GCGGAGATCC TGGGCGAGCA GGCCGGTGCC GGCGAGCAGC 18421 TTCCGGTGGA CGGCGGGGTC GACGACGAGC CCGTCGCGAT CGTCGGCATG GCGTGCCGCC 18481 TGCCGGGCGG TGTCGCCTCG CCGGAGGACC TGTGGCGGCT GGTGGCCGGC GGCGAGGACG 18541 CGATCTCCGG CTTCCCGCAG GACCGCGGCT GGGACGTGGA GGGGCTGTAC GACCCGGACC 18601 CGGACGCGTC CGGGCGGACG TACTGCCGTG CCGGTGGCTT CCTCGACGAG GCGGGCGAGT 18661 TCGACGCCGA CTTCTTCGGG ATCTCGCCGC GCGAGGCCCT CGCCATGGAC CCGCAGCAGC 18721 GGCTCCTCCT GGAGACCTCC TGGGAGGCCG TCGAGGACGC CGGGATCGAC CCGACCTCCC 18781 TTCAGGGGCA GCAGGTCGGC GTGTTCGCGG GCACCAACGG CCCCCACTAC GAGCCGCTGC 18841 TCCGCAACAC CGCCGAGGAT CTTGAGGGTT ACGTCGGGAC GGGCAACGCC GCCAGCATCA 18901 TGTCGGGCCG TGTCTCGTAC ACCCTCGGCC TGGAGGGCCC GGCCGTCACG GTCGACACCG 18961 CCTGCTCCTC CTCGCTGGTC GCCCTGCACC TCGCCGTGCA GGCCCTGCGC AAGGGCGAAT 19021 GCGGACTGGC GCTCGCGGGC GGTGTGACGG TCATGTCGAC GCCCACGACG TTCGTGGAGT 19081 TCAGCCGGCA GCGCGGGCTC GCGGAGGACG GCCGGTCGAA GGCGTTCGCC GCGTCGGCGG 19141 ACGGCTTCGG CCCGGCGGAG GGCGTCGGCA TGCTCCTCGT CGAGCGCCTG TCGGACGCCC 19201 GCCGCAACGG ACACCGTGTG CTGGCGGTCG TGCGCGGCAG CGCGGTCAAC CAGGACGGCG 19261 CGAGCAACGG CCTGACCGCC CCGAACGGGC CCTCGCAGCA GCGCGTCATC CGGCGCGCGC 19321 TCGCGGACGC CCGACTGACG ACCGCCGACG TGGACGTCGT CGAGGCCCAC GGCACGGGCA 19381 CGCGACTCGG CGACCCGATC GAGGCACAGG CCCTCATCGC CACCTACGGC CAGGGGCGCG 19441 ACACCGAACA GCCGCTGCGC CTGGGGTCGT TGAAGTCCAA CATCGGACAC ACCCAGGCCG 19501 CCGCCGGTGT CTCCGGCATC ATCAAGATGG TCCAGGCGAT GCGCCACGGC GTCCTGCCGA 19561 AGACGCTCCA CGTGGACCGG CCGTCGGACC AGATCGACTG GTCGCCGGGC ACGGTCGAGC 19621 TGCTCACCGA GGCCATGGAC TGGCCGAGGA AGCAGGAGGG CGGGCTGCGC CGCGCGGCCG 19681 TCTCCTCCTT CGGCATCAGC GGCACGAACG CGCACATCGT GCTCGAAGAA GCCCCGGTCG 19741 ACGAGGACGC CCCGGCGGAC GAGCCGTCGG TCGGCGGTGT GGTGCCGTGG CTCGTGTCCG 19801 CGAAGACTCC GGCCGCGCTG GACGCCCAGA TCGGACGCCT CGCCGCGTTC GCCTCGCAGG 19861 GCCGTACGGA CGCCGCCGAT CCGGGCGCGG TCGCTCGCGT ACTGGCCGGC GGGCGTGCGC 19921 AGTTCGAGCA CCGGGCCGTC GCGCTCGGCA CCGGACAGGA CGACCTGGCG GCCGCACTGG 19981 CCGCGCCTGA GGGTCTGGTC CGGGGTGTGG CCTCCGGTGT GGGTCGAGTG GCGTTCGTGT 20041 TCCCGGGACA GGGCACGCAG TGGGCCGGGA TGGGTGCCGA ACTCCTCGAC GTGTCGAAGG 20101 AGTTCGCGGC GGCCATGGCC GAGTGCGAGG CCGCGCTCGC TCCGTACGTG GACTGGTCGC 20161 TGGAGGCCGT CGTCCGACAG GCCCCCGGCG CGCCCACGCT GGAGCGGGTC GATGTCGTCC 20221 AGCCCGTGAC GTTCGCCGTC ATGGTCTCGC TGGCGAAGGT CTGGCAGCAC CACGGGGTGA 20281 CCCCGCAAGC CGTCGTCGGC CACTCGCAGG GCGAGATCGC CGCCGCGTAC GTCGCCGGTG 20341 CCCTGAGCCT GGACGACGCC GCTCGTGTCG TGACCCTGCG CAGCAAGTCC ATCGGCGCCC 20401 ACCTCGCGGG CCAGGGCGGC ATGCTGTCCC TCGCGCTGAG CGAGGCGGCC GTTGTGGAGC 20461 GACTGGCCGG GTTCGACGGG CTGTCCGTCG CCGCCGTCAA CGGGCCTACC GCCACCGTGG 20521 TTTCGGGCGA CCCGACCCAG ATCCAAGAGC TCGCTCAGGC GTGTGAGGCC GACGGGGTCC 20581 GCGCACGGAT CATCCCCGTC GACTACGCCT CCCACAGCGC CCACGTCGAG ACCATCGAGA 20641 GCGAACTCGC CGACGTCCTG GCGGGGTTGT CCCCCCAGAC ACCCCAGGTC CCCTTCTTCT 20701 CCACCCTCGA AGGCGCCTGG ATCACCGAAC CCGCCCTCGA CGGCGGCTAC TGGTACCGCA 20761 ACCTCCGCCA TCGTGTGGGC TTCGCCCCGG CCGTCGAAAC CCTGGCCACC GACGAAGGCT 20821 TCACCCACTT CGTCGAGGTC AGCGCCCACC CCGTCCTCAC CATGGCCCTG CCCGAGACCG 20881 TCACCGGCCT CGGCACCCTC CGCCGTGACA ACGGCGGACA GCACCGCCTC ACCACCTCCC 20941 TCGCCGAGGC CTGGGCCAAC GGCCTCACCG TCGACTGGGC CTCTCTCCTC CCCACCACGA 21001 CCACCCACCC CGATCTGCCC ACCTACGCCT TCCAGACCGA GCGCTACTGG CCGCAGCCCG 21061 ACCTCTCCGC CGCCGGTGAC ATCACCTCCG CCGGTCTCGG GGCGGCCGAG CACCCGCTGC 21121 TCGGCGCGGC CGTGGCGCTC GCGGACTCCG ACGGCTGCCT GCTCACGGGG AGCCTCTCCC 21181 TCCGTACGCA CCCCTGGCTG GCGGACCACG CGGTGGCCGG CACCGTGCTG CTGCCGGGAA 21241 CGGCGTTCGT GGAGCTGGCG TTCCGAGCCG GGGACCAGGT CGGTTGCGAT CTGGTCGAGG 21301 AGCTCACCCT CGACGCGCCG CTCGTGCTGC CCCGTCGTGG CGCGGTCCGT GTGCAGCTGT 21361 CCGTCGGCGC GAGCGACGAG TCCGGGCGTC GTACCTTCGG GCTCTACGCG CACCCGGAGG 21421 ACGCGCCGGG CGAGGCGGAG TGGACGCGGC ACGCCACCGG TGTGCTGGCC GCCCGTGCGG 21481 ACCGCACCGC CCCCGTCGCC GACCCGGAGG CCTGGCCGCC GCCGGGCGCC GAGCCGGTGG 21541 ACGTGGACGG TCTGTACGAG CGCTTCGCGG CGAACGGCTA CGGCTACGGC CCCCTCTTCC 21601 AGGGCGTCCG TGGTGTCTGG CGGCGTGGCG ACGAGGTGTT CGCCGACGTG GCCCTGCCGG 21661 CCGAGGTCGC CGGTGCCGAG GGCGCGCGGT TCGGCCTTCA CCCGGCGCTG CTCGACGCCG 21721 CCGTGCAGGC GGCCGGTGCG GGCGGGGCGT TCGGCGCGGG CACGCGGCTG CCGTTCGCCT 21781 GGAGCGGGAT CTCCCTGTAC GCGGTCGGCG CCACCGCCCT CCGCGTGCGG CTGGCCCCCG 21841 CCGGCCCGGA CACGGTGTCC GTGAGCGCCG CCGACTCCTC CGGGCAGCCG GTGTTCGCCG 21901 CGGACTCCCT CACGGTGCTG CCCGTCGACC CCGCGCAGCT GGCGGCCTTC AGCGACCCGA 21961 CTCTGGACGC GCTGCACCTG CTGGAGTGGA CCGCCTGGGA CGGTGCCGCG CACGCCCTGC 22021 CCGGCGCGGT CGTGCTGGGC GGCGACGCCG ACGGTCTCGC CGCGGCGCTG CGCGCCGGTG 22081 GCACCGAGGT CCTGTCCTTC CCGGACCTTA CGGACCTGGT GGAGGCCGTC GACCGGGGCG 22141 AGACCCCGGC CCCGGCGACC GTCCTGGTGG CCTGCCCCGC CGCCGGCCCC GGTGGGCCGG 22201 AGCATGTCCG CGAGGCCCTG CACGGGTCGC TCGCGCTGAT GCAGGCCTGG CTGGCCGACG 22261 AGCGGTTCAC CGATGGGCGC CTGGTGCTCG TGACCCGCGA CGCGGTCGCC GCCCGTTCCG 22321 GCGACGGCCT GCGGTCCACG GGACAGGCCG CCGTCTGGGG CCTCGGCCGG TCCGCGCAGA 22381 CGGAGAGCCC GGGCCGGTTC GTCCTGCTCG ACCTCGCCGG GGAAGCCCGG ACGGCCGGGG 22441 ACGCCACCGC CGGGGACGGC CTGACGACCG GGGACGCCAC CGTCGGCGGC ACCTCTGGAG 22501 ACGCCGCCCT CGGCAGCGCC CTCGCGACCG CCCTCGGCTC GGGCGAGCCG CAGCTCGCCC 22561 TCCGGGACGG GGCGCTCCTC GTACCCCGCC TGGCGCGGGC CGCCGCGCCC GCCGCGGCCG 22621 ACGGCCTCGC CGCGGCCGAC GGCCTCGCCG CTCTGCCGCT GCCCGCCGCT CCGGCCCTCT 22681 GGCGTCTGGA GCCCGGTACG GACGGCAGCC TGGAGAGCCT CACGGCGGCG CCCGGCGACG 22741 CCGAGACCCT CGCCCCGGAG CCGCTCGGCC CGGGACAGGT CCGCATCGCG ATCCGGGCCA 22801 CCGGTCTCAA CTTCCGCGAC GTCCTGATCG CCCTCGGCAT GTACCCCGAT CCGGCGCTGA 22861 TGGGCACCGA GGGAGCCGGC GTGGTCACCG CGACCGGCCC CGGCGTCACG CACCTCGCCC 22921 CCGGCGACCG GGTCATGGGC CTGCTCTCCG GCGCGTACGC CCCGGTCGTC GTGGCGGACG 22981 CGCGGACCGT CGCGCGGATG CCCGAGGGGT GGACGTTCGC CCAGGGCGCC TCCGTGCCGG 23041 TGGTGTTCCT GACGGCCGTC TACGCCCTGC GCGACCTGGC GGACGTCAAG CCCGGCGAGC 23101 GCCTCCTGGT CCACTCCGCC GCCGGTGGCG TGGGCATGGC CGCCGTGCAG CTCGCCCGGC 23161 ACTGGGGCGT GGAGGTCCAC GGCACGGCGA GTCACGGGAA GTGGGACGCC CTGCGCGCGC 23221 TCGGCCTGGA CGACGCGCAC ATCGCCTCCT CCCGCACCCT GGACTTCGAG TCCGCGTTCC 23281 GTGCCGCTTC CGGCGGGGCG GGCATGGACG TCGTACTGAA CTCGCTCGCC CGCGAGTTCG 23341 TCGACGCCTC GCTGCGCCTG CTCGGGCCGG GCGGCCGGTT CGTGGAGATG GGGAAGACCG 23401 ACGTCCGCGA CGCGGAGCGG GTCGCCGCCG ACCACCCCGG TGTCGGCTAC CGCGCCTTCG 23461 ACCTGGGCGA GGCCGGGCCG GAGCGGATCG GCGAGATGCT CGCCGAGGTC ATCGCCCTCT 23521 TCGAGGACGG GGTGCTCCGG CACCTGCCCG TCACGACCTG GGACGTGCGC CGGGCCCGCG 23581 ACGCCTTCCG GCACGTCAGC CAGGCCCGCC ACACGGGCAA GGTCGTCCTC ACGATGCCGT 23641 CGGGCCTCGA CCCGGAGGGT ACGGTCCTGC TGACCGGCGG CACCGGTGCG CTGGGGGGCA 23701 TCGTGGCCCG GCACGTGGTG GGCGAGTGGG GCGTACGACG CCTGCTGCTC GTGAGCCGGC 23761 GGGGCACGGA CGCCCCGGGC GCCGGCGAGC TCGTGCACGA GCTGGAGGCC CTGGGAGCCG 23821 ACGTCTCGGT GGCCGCGTGC GACGTCGCCG ACCGCGAAGC CCTCACCGCC GTACTCGACT 23881 CGATCCCCGC CGAACACCCG CTCACCGCGG TCGTCCACAC GGCAGGCGTC CTCTCCGACG 23941 GCACCCTCCC CTCGATGACA GCGGAGGATG TGGAACACGT ACTGCGTCCC AAGGTCGACG 24001 CCGCGTTCCT CCTCGACGAA CTCACCTCGA CGCCCGGCTA CGACCTGGCA GCGTTCGTCA 24061 TGTTCTCCTC CGCCGCCGCC GTCTTCGGTG GCGCGGGGCA GGGCGCCTAC GCCGCCGCCA 24121 ACGCCACCCT CGACGCCCTC GCCTGGCGCC GCCGGACAGC CGGACTCCCC GCCCTCTCCC 24181 TCGGCTGGGG CCTCTGGGCC GAGACCAGCG GCATGACCGG CGGACTCAGC GACACCGACC 24241 GCTCGCGGCT GGCCCGTTCC GGGGCGACGC CCATGGACAG CGAGCTGACC CTGTCCCTCC 24301 TGGACGCGGC CATGCGCCGC GACGACCCGG CGCTCGTCCC GATCGCCCTG GACGTCGCCG 24361 CGCTCCGCGC CCAGCAGCGC GACGGCATGC TGGCGCCGCT GCTCAGCGGG CTCACCCGCG 24421 GATCGCGGGT CGGCGGCGCG CCGGTCAACC AGCGCAGGGC AGCCGCCGGA GGCGCGGGCG 24481 AGGCGGACAC GGACCTCGGC GGGCGGCTCG CCGCGATGAC ACCGGACGAC CGGGTCGCGC 24541 ACCTGCGGGA CCTCGTCCGT ACGCACGTGG CGACCGTCCT GGGACACGGC ACCCCGAGCC 24601 GGGTGGACCT GGAGCGGGCC TTCCGCGACA CCGGTTTCGA CTCGCTCACC GCCGTCGAAC 24661 TCCGCAACCG TCTCAACGCC GCGACCGGGC TGCGGCTGCC GGCCACGCTG GTCTTCGACC 24721 ACCCCACCCC GGGGGAGCTC GCCGGGCACC TGCTCGACGA ACTCGCCACG GCCGCGGGCG 24781 GGTCCTGGGC GGAAGGCACC GGGTCCGGAG ACACGGCCTC GGCGACCGAT CGGCAGACCA 24841 CGGCGGCCCT CGCCGAACTC GACCGGCTGG AAGGCGTGCT CGCCTCCCTC GCGCCCGCCG 24901 CCGGCGGCCG TCCGGAGCTC GCCGCCCGGC TCAGGGCGCT GGCCGCGGCC CTGGGGGACG 24961 ACGGCGACGA CGCCACCGAC CTGGACGAGG CGTCCGACGA CGACCTCTTC TCCTTCATCG 25021 ACAAGGAGCT GGGCGACTCC GACTTCTGAC CTGCCCGACA CCACCGGCAC CACCGGCACC 25081 ACCAGCCCCC CTCACACACG GAACACGGAA CGGACAGGCG AGAACGGGAG CCATGGCGAA 25141 CAACGAAGAC AAGCTCCGCG ACTACCTCAA GCGCGTCACC GCCGAGCTGC AGCAGAACAC 25201 CAGGCGTCTG CGCGAGATCG AGGGACGCAC GCACGAGCCG GTGGCGATCG TGGGCATGGC 25261 CTGCCGCCTG CCGGGCGGTG TCGCCTCGCC CGAGGACCTG TGGCAGCTGG TGGCCGGGGA 25321 CGGGGACGCG ATCTCGGAGT TCCCGCAGGA CCGCGGCTGG GACGTGGAGG GGCTGTACGA 25381 CCCCGACCCG GACGCGTCCG GCAGGACGTA CTGCCGGTCC GGCGGATTCC TGCACGACGC 25441 CGGCGAGTTC GACGCCGACT TCTTCGGGAT CTCGCCGCGC GAGGCCCTCG CCATGGACCC 25501 GCAGCAGCGA CTGTCCCTCA CCACCGCGTG GGAGGCGATC GAGAGCGCGG GCATCGACCC 25561 GACGGCCCTG AAGGGCAGCG GCCTCGGCGT CTTCGTCGGC GGCTGGCACA CCGGCTACAC 25621 CTCGGGGCAG ACCACCGCCG TGCAGTCGCC CGAGCTGGAG GGCCACCTGG TCAGCGGCGC 25681 GGCGCTGGGC TTCCTGTCCG GCCGTATCGC GTACGTCCTC GGTACGGACG GACCGGCCCT 25741 GACCGTGGAC ACGGCCTGCT CGTCCTCGCT GGTCGCCCTG CACCTCGCCG TGCAGGCCCT 25801 CCGCAAGGGC GAGTGCGACA TGGCCCTCGC CGGTGGTGTC ACGGTCATGC CCAACGCGGA 25861 CCTGTTCGTG CAGTTCAGCC GGCAGCGCGG GCTGGCCGCG GACGGCCGGT CGAAGGCGTT 25921 CGCCACCTCG GCGGACGGCT TCGGCCCCGC GGAGGGCGCC GGAGTCCTGC TGGTGGAGCG 25981 CCTGTCGGAC GCCCGCCGCA ACGGACACCG GATCCTCGCG GTCGTCCGCG GCAGCGCGGT 26041 CAACCAGGAC GGCGCCAGCA ACGGCCTCAC GGCTCCGCAC GGGCCCTCCC AGCAGCGCGT 26101 CATCCGACGG GCCCTGGCGG ACGCCCGGCT CGCGCCGGGT GACGTGGACG TCGTCGAGGC 26161 GCACGGCACG GGCACGCGGC TCGGCGACCC GATCGAGGCG CAGGCCCTCA TCGCCACCTA 26221 CGGCCAGGAG AAGAGCAGCG AACAGCCGCT GAGGCTGGGC GCGTTGAAGT CGAACATCGG 26281 GCACACGCAG GCCGCGGCCG GTGTCGCAGG TGTCATCAAG ATGGTCCAGG CGATGCGCCA 26341 CGGACTGCTG CCGAAGACGC TGCACGTCGA CGAGCCCTCG GACCAGATCG ACTGGTCGGC 26401 GGGCACGGTG GAACTCCTCA CCGAGGCCGT CGACTGGCCG GAGAAGCAGG ACGGCGGGCT 26461 GCGCCGCGCG GCTGTCTCCT CCTTCGGCAT CAGCGGGACG AACGCGCACG TCGTCCTGGA 26521 GGAGGCCCCG GCGGTCGAGG ACTCCCCGGC CGTCGAGCCG CCGGCCGGTG GCGGTGTGGT 26581 GCCGTGGCCG GTGTCCGCGA AGACTCCGGC CGCGCTGGAC GCCCAGATCG GGCAGCTCGC 26641 CGCGTACGCG GACGGTCGTA CGGACGTGGA TCCGGCGGTG GCCGCCCGCG CCCTGGTCGA 26701 CAGCCGTACG GCGATGGAGC ACCGCGCGGT CGCGGTCGGC GACAGCCGGG AGGCACTGCG 26761 GGACGCCCTG CGGATGCCGG AAGGACTGGT ACGCGGCACG TCCTCGGACG TGGGCCGGGT 26821 GGCGTTCGTC TTCCCCGGCC AGGGCACGCA GTGGGCCGGC ATGGGCGCCG AACTCCTTGA 26881 CAGCTCACCG GAGTTCGCTG CCTCGATGGC CGAATGCGAG ACCGCGCTCT CCCGCTACGT 26941 CGACTGGTCT CTTGAAGCCG TCGTCCGACA GGAACCCGGC GCACCCACGC TCGACCGCGT 27001 CGACGTCGTC CAGCCCGTGA CCTTCGCTGT CATGGTCTCG CTGGCGAAGG TCTGGCAGCA 27061 CCACGGCATC ACCCCCCAGG CCGTCGTCGG CCACTCGCAG GGCGAGATCG CCGCCGCGTA 27121 CGTCGCCGGT GCACTCACCC TCGACGACGC CGCCCGCGTC GTCACCCTGC GCAGCAAGTC 27181 CATCGCCGCC CACCTCGCCG GCAAGGGCGG CATGATCTCC CTCGCCCTCG ACGAGGCGGC 27241 CGTCCTGAAG CGACTGAGCG ACTTCGACGG ACTCTCCGTC GCCGCCGTCA ACGGCCCCAC 27301 CGCCACCGTC GTCTCCGGCG ACCCGACCCA GATCGAGGAA CTCGCCCGCA CCTGCGAGGC 27361 CGACGGCGTC CGTGCGCGGA TCATCCCGGT CGACTACGCC TCCCACAGCC GGCAGGTCGA 27421 GATCATCGAG AAGGAGCTGG CCGAGGTCCT CGCCGGACTC GCCCCGCAGG CTCCGCACGT 27481 GCCGTTCTTC TCCACCCTCG AAGGCACCTG GATCACCGAG CCGGTGCTCG ACGGCACCTA 27541 CTGGTACCGC AACCTGCGCC ATCGCGTGGG CTTCGCCCCC GCCGTGGAGA CCTTGGCGGT 27601 TGACGGCTTC ACCCACTTCA TCGAGGTCAG CGCCCACCCC GTCCTCACCA TGACCCTCCC 27661 CGAGACCGTC ACCGGCCTCG GCACCCTCCG CCGCGAACAG GGAGGCCAGG AGCGTCTGGT 27721 CACCTCACTC GCGGAAGCCT GGGCCAACGG CCTCACCATC GACTGGGCGC CCATCCTCCC 27781 CACCGCAACC GGCCACCACC CCGAGCTCCC CACCTACGCC TTCCAGACCG AGCGCTTCTG 27841 GCTGCAGAGC TCCGCGCCCA CCAGCGCCGC CGACGACTGG CGTTACCGCG TCGAGTGGAA 27901 GCCGCTGACG GCCTCCGGCC AGGCGGACCT GTCCGGGCGG TGGATCGTCG CCGTCGGGAG 27961 CGAGCCAGAA GCCGAGCTGC TGGGCGCGCT GAAGGCCGCG GGAGCGGAGG TCGACGTACT 28021 GGAAGCCGGG GCGGACGACG ACCGTGAGGC CCTCGCCGCC CGGCTCACCG CACTGACGAC 28081 CGGCGACGGC TTCACCGGCG TGGTCTCGCT CCTCGACGAC CTCGTGCCAC AGGTCGCCTG 28141 GGTGCAGGCA CTCGGCGACG CCGGAATCAA GGCGCCCCTG TGGTCCGTCA CCCAGGGCGC 28201 GGTCTCCGTC GGACGTCTCG ACACCCCCGC CGACCCCGAC CGGGCCATGC TCTGGGGCCT 28261 CGGCCGCGTC GTCGCCCTTG AGCACCCCGA ACGCTGGGCC GGCCTCGTCG ACCTCCCCGC 28321 CCAGCCCGAT GCCGCCGCCC TCGCCCACCT CGTCACCGCA CTCTCCGGCG CCACCGGCGA 28381 GGACCAGATC GCCATCCGCA CCACCGGACT CCACGCCCGC CGCCTCGCCC GCGCACCCCT 28441 CCACGGACGT CGGCCCACCC GCGACTGGCA GCCCCACGGC ACCGTCCTCA TCACCGGCGG 28501 CACCGGAGCC CTCGGCAGCC ACGCCGCACG CTGGATGGCC CACCACGGAG CCGAACACCT 28561 CCTCCTCGTC AGCCGCAGCG GCGAACAAGC CCCCGGAGCC ACCCAACTCA CCGCCGAACT 28621 CACCGCATCG GGCGCCCGCG TCACCATCGC CGCCTGCGAC GTCGCCGACC CCCACGCCAT 28681 GCGCACCCTC CTCGACGCCA TCCCCGCCGA GACGCCCCTC ACCGCCGTCG TCCACACCGC 28741 CGGCGCACCG GGCGGCGATC CGCTGGACGT CACCGGCCCG GAGGACATCG CCCGCATCCT 28801 GGGCGCGAAG ACGAGCGGCG CCGAGGTCCT CGACGACCTG CTCCGCGGCA CTCCGCTGGA 28861 CGCCTTCGTC CTCTACTCCT CGAACGCCGG GGTCTGGGGC AGCGGCAGCC AGGGCGTCTA 28921 CGCGGCGGCC AACGCCCACC TCGACGCGCT CGCCGCCCGG CGCCGCGCCC GGGGCGAGAC 28981 GGCGACCTCG GTCGCCTGGG GCCTCTGGGC CGGCGACGGC ATGGGCCGGG GCGCCGACGA 29041 CGCGTACTGG CAGCGTCGCG GCATCCGTCC GATGAGCCCC GACCGCGCCC TGGACGAACT 29101 GGCCAAGGCC CTGAGCCACG ACGAGACCTT CGTCGCCGTG GCCGATGTCG ACTGGGAGCG 29161 GTTCGCGCCC GCGTTCACGG TGTCCCGTCC CAGCCTTCTG CTCGACGGCG TCCCGGAGGC 29221 CCGGCAGGCG CTCGCCGCAC CCGTCGGTGC CCCGGCTCCC GGCGACGCCG CCGTGGCGCC 29281 GACCGGGCAG TCGTCGGCGC TGGCCGCGAT CACCGCGCTC CCCGAGCCCG AGCGCCGGCC 29341 GGCGCTCCTC ACCCTCGTCC GTACCCACGC GGCGGCCGTA CTCGGCCATT CCTCCCCCGA 29401 CCGGGTGGCC CCCGGCCGTG CCTTCACCGA GCTCGGCTTC GACTCGCTGA CGGCCGTGCA 29461 GCTCCGCAAC CAGCTCTCCA CGGTGGTCGG CAACAGGCTC CCCGCCACCA CGGTCTTCGA 29521 CCACCCGACG CCCGCCGCAC TCGCCGCGCA CCTCCACGAG GCGTACCTCG CACCGGCCGA 29581 GCCGGCCCCG ACGGACTGGG AGGGGCGGGT GCGCCGGGCC CTGGCCGAAC TGCCCCTCGA 29641 CCGGCTGCGG GACGCGGGGG TCCTCGACAC CGTCCTGCGC CTCACCGGCA TCGAGCCCGA 29701 GCCGGGTTCC GGCGGTTCGG ACGGCGGCGC CGCCGACCCT GGTGCGGAGC CGGAGGCGTC 29761 GATCGACGAC CTGGACGCCG AGGCCCTGAT CCGGATGGCT CTCGGCCCCC GTAACACCTQ 29821 ACCCGACCGC GGTCCTGCCC CACGCGCCGC ACCCCGCGCA TCCCGCGCAC CACCCGCCCC 29881 CACACGCCCA CAACCCCATC CACGAGCGGA AGACCACACC CAGATGACGA GTTCCAACGA 29941 ACAGTTGGTG GACGCTCTGC GCGCCTCTCT CAAGGAGAAC GAAGAACTCC GGAAAGAGAG 30001 CCGTCGCCGG GCCGACCGTC GGCAGGAGCC CATGGCGATC GTCGGCATGA GCTGCCGGTT 30061 CGCGGGCGGA ATCCGGTCCC CCGAGGACCT CTGGGACGCC GTCGCCGCGG GCAAGGACCT 30121 GGTCTCCGAG GTACCGGAGG AGCGCGGCTG GGACATCGAC TCCCTCTACG ACCCGGTGCC 30181 CGGGCGCAAG GGCACGACGT ACGTCCGCAA CGCCGCGTTC CTCGACGACG CCGCCGGATT 30241 CGACGCGGCC TTCTTCGGGA TCTCGCCGCG CGAGGCCCTC GCCATGGACC CGCAGCAGCG 30301 GCAGCTCCTC GAAGCCTCCT GGGAGGTCTT CGAGCGGGCC GGCATCGACC CCGCGTCGGT 30361 CCGCGGCACC GACGTCGGCG TGTACGTGGG CTGTGGCTAC CAGGACTACG CGCCGGACAT 30421 CCGGGTCGCC CCCGAAGGCA CCGGCGGTTA CGTCGTCACC GGCAACTCCT CCGCCGTGGC 30481 CTCCGGGCGC ATCGCGTACT CCCTCGGCCT GGAGGGACCC GCCGTGACCG TGGACACGGC 30541 GTGCTCCTCT TCGCTCGTCG CCCTGCACCT CGCCCTGAAG GGCCTGCGGA ACGGCGACTG 30601 CTCGACGGCA CTCGTGGGCG GCGTGGCCGT CCTCGCGACG CCGGGCGCGT TCATCGAGTT 30661 CAGCAGCCAG CAGGCCATGG CCGCCGACGG CCGGACCAAG GGCTTCGCCT CGGCGGCGGA 30721 CGGCCTCGCC TGGGGCGAGG GCGTCGCCGT ACTCCTCCTC GAACGGCTCT CCGACGCGCG 30781 GCGCAAGGGC CACCGGGTCC TGGCCGTCGT GCGCGGCAGC GCCATCAACC AGGACGGCGC 30841 GAGCAACGGC CTCACGGCTC CGCACGGGCC CTCCCAGCAG CGCCTGATCC GCCAGGCCCT 30901 GGCCGACGCG CGGCTCACGT CGAGCGACGT GGACGTCGTG GAGGGCCACG GCACGGGGAC 30961 CCGTCTCGGC GACCCGATCG AGGCGCAGGC GCTGCTCGCC ACGTACGGGC AGGGGCGCGC 31021 CCCGGGGCAG CCGCTGCGGC TGGGGACGCT GAAGTCGAAC ATCGGGCACA CGCAGGCCGC 31081 TTCGGGTGTC GCCGGTGTCA TCAAGATGGT GCAGGCGCTG CGCCACGGGG TGCTGCCGAA 31141 GACCCTGCAC GTGGACGAGC CGACGGACCA GGTCGACTGG TCGGCCGGTT CGGTCGAGCT 31201 GCTCACCGAG GCCGTGGACT GGCCGGAGCG GCCGGGCCGG CTCCGCCGGG CGGGCGTCTC 31261 CGCGTTCGGC GTGGGCGGGA CGAACGCGCA CGTCGTCCTG GAGGAGGCCC CGGCGGTCGA 31321 GGAGTCCCCT GCCGTCGAGC CGCCGGCCGG TGGCGGCGTG GTGCCGTGGC CGGTGTCCGC 31381 GAAGACCTCG GCCGCACTGG ACGCCCAGAT CGGGCAGCTC GCCGCATACG CGGAAGACCG 31441 CACGGACGTG GATCCGGCGG TGGCCGCCCG CGCCCTGGTC GACAGCCGTA CGGCGATGGA 31501 GCACCGCGCG GTCGCGGTCG GCGACAGCCG GGAGGCACTG CGGGACGCCC TGCGGATGCC 31561 GGAAGGACTG GTACGGGGCA CGGTCACCGA TCCGGGCCGG GTGGCGTTCG TCTTCCCCGG 31621 CCAGGGCACG CAGTGGGCCG GCATGGGCGC CGAACTCCTC GACAGCTCAC CCGAATTCGC 31681 CGCCGCCATG GCCGAATGCG AGACCGCACT CTCCCCGTAC GTCGACTGGT CTCTCGAAGC 31741 CGTCGTCCGA CAGGCTCCCA GCGCACCGAC ACTCGACCGC GTCGACGTCG TCCAGCCCGT 31801 CACCTTCGCC GTCATGGTCT CCCTCGCCAA GGTCTGGCAG CACCACGGCA TCACCCCCGA 31861 GGCCGTCATC GGCCACTCCC AGGGCGAGAT CGCCGCCGCG TACGTCGCCG GTGCCCTCAC 31921 CCTCGACGAC GCCGCTCGTG TCGTGACCCT CCGCAGCAAG TCCATCGCCG CCCACCTCGC 31981 CGGCAAGGGC GGCATGATCT CCCTCGCCCT CAGCGAGGAA GCCACCCGGC AGCGCATCGA 32041 GAACCTCCAC GGACTGTCGA TCGCCGCCGT CAACGGGCCT ACCGCCACCG TGGTTTCGGG 32101 CGACCCCACC CAGATCCAAG AACTTGCTCA GGCGTGTGAG GCCGACGGCA TCCGCGCACG 32161 GATCATCCCC GTCGACTACG CCTCCCACAG CGCCCACGTC GAGACCATCG AGAACGAACT 32221 CGCCGACGTC CTGGCGGGGT TGTCCCCCCA GACACCCCAG GTCCCCTTCT TCTCCACCCT 32281 CGAAGGCACC TGGATCACCG AACCCGCCCT CGACGGCGGC TACTGGTACC GCAACCTCCG 32341 CCATCGTGTG GGCTTCGCCC CGGCCGTCGA GACCCTCGCC ACCGACGAAG GCTTCACCCA 32401 CTTCATCGAG GTCAGCGCCC ACCCCGTCCT CACCATGACC CTCCCCGACA AGGTCACCGG 32461 CCTGGCCACC CTCCGACGCG AGGACGGCGG ACAGCACCGC CTCACCACCT CCCTTGCCGA 32521 GGCCTGGGCC AACGGCCTCG CCCTCGACTG GGCCTCCCTC CTGCCCGCCA CGGGCGCCCT 32581 CAGCCCCGCC GTCCCCGACC TCCCGACGTA CGCCTTCCAG CACCGCTCGT ACTGGATCAG 32641 CCCCGCGGGT CCCGGCGAGG CGCCCGCGCA CACCGCTTCC GGGCGCGAGG CCGTCGCCGA 32701 GACGGGGCTC GCGTGGGGCC CGGCTGCCGA GGACCTCGAC GAGGAGGGCC GGCGCAGCGC 32761 CGTACTCGCG ATGGTGATGC GGCAGGCGGC CTCCGTGCTC CGGTGCGACT CGCCCGAAGA 32821 GGTCCCCGTC GACCGCCCGC TGCGGGAGAT CGGCTTCGAC TCGCTGACCG CCGTCGACTT 32881 CCGCAACCGC GTCAACCGGC TGACCGGTCT CCAGCTGCCG CCCACCGTCG TGTTCGAGCA 32941 CCCGACGCCC GTCGCGCTCG CCGAGCGCAT CAGCGACGAG CTGGCCGAGC GGAACTGGGC 33001 CGTCGCCGAG CCGTCGGATC ACGAGCAGGC GGAGGAGGAG AAGGCCGCCG CTCCGGCGGG 33061 GGCCCGCTCC GGGGCCGACA CCGGCGCCGG CGCCGGGATG TTCCGCGCCC TGTTCCGGCA 33121 GGCCGTGGAG GACGACCGGT ACGGCGAGTT CCTCGACGTC CTCGCCGAAG CCTCCGCGTT 33181 CCGCCCGCAG TTCGCCTCGC CCGAGGCCTG CTCGGAGCGG CTCGACCCGG TGCTGCTCGC 33241 CGGCGGTCCG ACGGACCGGG CGGAAGGCCG TGCCGTTCTC GTCGGCTGCA CCGGCACCGC 33301 GGCGAACGGC GGCCCGCACG AGTTCCTGCG GCTCAGCACC TCCTTCCAGG AGGAGCGGGA 33361 CTTCCTCGCC GTACCTCTCC CCGGCTACGG CACGGGTACG GGCACCGGCA CGGCCCTCCT 33421 CCCGGCCGAT CTCGACACCG CGCTCGACGC CCAGGCCCGG GCGATCCTCC GGGCCGCCGG 33481 GGACGCCCCG GTCGTCCTGC TCGGGCACTC CGGCGGCGCC CTGCTCGCGC ACGAGCTGGC 33541 CTTCCGCCTG GAGCGGGCGC ACGGCGCGCC GCCGGCCGGG ATCGTCCTGG TCGACCCCTA 33601 TCCGCCGGGC CATCAGCAGC CCATCGAGGT GTGGAGCAGG CAGCTGGGCG AGGGCCTGTT 33661 CGCGGGCGAG CTGGAGCCGA TGTCCGATGC GCGGCTGCTG GCCATGGGCC GGTACGCGCG 33721 GTTCCTCGCC GGCCCGCGGC CGGGCCGCAG CAGCGCGCCC GTGCTTCTGG TCCGTGCCTC 33781 CGAACCGCTG GGCGACTGGC AGGAGGAGCG GGGCGACTGG CGTGCCCACT GGGACCTTCC 33841 GCACACCGTC GCGGACGTGC CGGGCGACCA CTTCACGATG ATGCGGGACC ACGCGCCGGC 33901 CGTCGCCGAG GCCGTCCTCT CCTGGCTCGA CGCCATCGAG GGCATCGAGG GGGCGGGCAA 33961 GTGACCGACA GACCTCTGAA CGTGGACAGC GGACTGTGGA TCCGGCGCTT CCACCCCGCG 34021 CCGAACAGCG CGGTGCGGCT GGTCTGCCTG CCGCACGCCG GCGGCTCCGC CAGCTACTTC 34081 TTCCGCTTCT CGGAGGAGCT GCACCCCTCC GTCGAGGCCC TGTCGGTGCA GTATCCGGGC 34141 CGCCAGGACC GGCGTGCCGA GCCGTGTCTG GAGAGCGTCG AGGAGCTCGC CGAGCATGTG 34201 GTCGCGGCCA CCGAACCCTG GTGGCAGGAG GGCCGGCTGG CCTTCTTCGG GCACAGCCTC 34261 GGCGCCTCCG TCGCCTTCGA GACGGCCCGC ATCCTGGAAC AGCGGCACGG GGTACGGCCC 34321 GAGGGCCTGT ACGTCTCCGG TCGGCGCGCC CCGTCGCTGG CGCCGGACCG GCTCGTCCAC 34381 CAGCTGGACG ACCGGGCGTT CCTGGCCGAG ATCCGGCGGC TCAGCGGCAC CGACGAGCGG 34441 TTCCTCCAGG ACGACGAGCT GCTGCGGCTG GTGCTGCCCG CGCTGCGCAG CGACTACAAG 34501 GCGGCCGAGA CGTACCTGCA CCGGCCGTCC GCCAAGCTCA CCTGCCCGGT GATGGCCCTG 34561 GCCGGCCACC GTGACCCGAA GGCGCCGCTG AACGAGGTGG CCGAGTGGCG TCGGCACACC 34621 AGCGGGCCGT TCTGCCTCCG GGCGTACTCC GGCGGCCACT TCTACCTCAA CGACCAGTGG 34681 CACGAGATCT GCAACGACAT CTCCGACCAC CTGCTCGTCA CCCGCGGCGC GCCCGATGCC 34741 CGCGTCGTGC AGCCCCCGAC CAGCCTTATC GAAGGAGCGG CGAAGAGATG GCAGAACCCA 34801 CGGTGACCGA CGACCTGACG GGGGCCCTCA CGCAGCCCCC GCTGGGCCGC ACCGTCCGCG 34861 CGGTGGCCGA CCGTGAACTC GGCACCCACC TCCTGGAGAC CCGCGGCATC CACTGGATCC 34921 ACGCCGCGAA CGGCGACCCG TACGCCACCG TGCTGCGCGG CCAGGCGGAC GACCCGTATC 34981 CCGCGTACGA GCGGGTGCGT GCCCGCGGCG CGCTCTCCTT CAGCCCGACG GGCAGCTGGG 35041 TCACCGCCGA TCACGCCCTG GCGGCGAGCA TCCTCTGCTC GACGGACTTC GGGGTCTCCG 35101 GCGCCGACGG CGTCCCGGTG CCGCAGCAGG TCCTCTCGTA CGGGGAGGGC TGTCCGCTGG 35161 AGCGCGAGCA GGTGCTGCCG GCGGCCGGTG ACGTGCCGGA GGGCGGGCAG CGTGCCGTGG 35221 TCGAGGGGAT CCACCGGGAG ACGCTGGAGG GTCTCGCGCC GGACCCGTCG GCGTCGTACG 35281 CCTTCGAGCT GCTGGGCGGT TTCGTCCGCC CGGCGGTGAC GGCCGCTGCC GCCGCCGTGC 35341 TGGGTGTTCC CGCGGACCGG CGCGCGGACT TCGCGGATCT GCTGGAGCGG CTCCGGCCGC 35401 TGTCCGACAG CCTGCTGGCC CCGCAGTCCC TGCGGACGGT ACGGGCGGCG GACGGCGCGC 35461 TGGCCGAGCT CACGGCGCTG CTCGCCGATT CGGACGACTC CCCCGGGGCC CTGCTGTCGG 35521 CGCTCGGGGT CACCGCAGCC GTCCAGCTCA CCGGGAACGC GGTGCTCGCG CTCCTCGCGC 35581 ATCCCGAGCA GTGGCGGGAG CTGTGCGACC GGCCCGGGCT CGCGGCGGCC GCGGTGGAGG 35641 AGACCCTCCG CTACGACCCG CCGGTGCAGC TCGACGCCCG GGTGGTCCGC GGGGAGACGG 35701 AGCTGGCGGG CCGGCGGCTG CCGGCCGGGG CGCATGTCGT CGTCCTGACC GCCGCGACCG 35761 GCCGGGACCC GGAGGTCTTC ACGGACCCGG AGCGCTTCGA CCTCGCGCGC CCCGACGCCG 35821 CCGCGCACCT CGCGCTGCAC CCCGCCGGTC CGTACGGCCC GGTGGCGTCC CTGGTCCGGC 35881 TTCAGGCGGA GGTCGCGCTG CGGACCCTGG CCGGGCGTTT CCCCGGGCTG CGGCAGGCGG 35941 GGGACGTGCT CCGCCCCCGC CGCGCGCCTG TCGGCCGCGG GCCGCTGAGC GTCCCGGTCA 36001 GCAGCTCCTG AGACACCGGG GCCCCGGTCC GCCCGGCCCC CCTTCGGACG GACCGGACGG 36061 CTCGGACCAC GGGGACGGCT CAGACCGTCC CGTGTGTCCC CGTCCGGCTC CCGTCCGCCC 36121 CATCCCGCCC CTCCACCGGC AAGGAAGGAC ACGACGCCAT GCGCGTCCTG CTGACCTCGT 36181 TCGCACATCA CACGCACTAC TACGGCCTGG TGCCCCTGGC CTGGGCGCTG CTCGCCGCCG 36241 GGCACGAGGT GCGGGTCGCC AGCCAGCCCG CGCTCACGGA CACCATCACC GGGTCCGGGC 36301 TCGCCGCGGT GCCGGTCGGC ACCGACCACC TCATCCACGA GTACCGGGTG CGGATGGCGG 36361 GCGAGCCGCG CCCGAACCAT CCGGCGATCG CCTTCGACGA GGCCCGTCCC GAGCCGCTGG 36421 ACTGGGACCA CGCCCTCGGC ATCGAGGCGA TCCTCGCCCC GTACTTCTAT CTGCTCGCCA 36481 ACAACGACTC GATGGTCGAC GACCTCGTCG ACTTCGCCCG GTCCTGGCAG CCGGACCTGG 36541 TGCTGTGGGA GCCGACCACC TACGCGGGCG CCGTCGCCGC CCAGGTCACC GGTGCCGCGC 36601 ACGCCCGGGT CCTGTGGGGG CCCGACGTGA TGGGCAGCGC CCGCCGCAAG TTCGTCGCGC 36661 TGCGGGACCG GCAGCCGCCC GAGCACCGCG AGGACCCCAC CGCGGAGTGG CTGACGTGGA 36721 CGCTCGACCG GTACGGCGCC TCCTTCGAAG AGGAGCTGCT CACCGGCCAG TTCACGATCG 36781 ACCCGACCCC GCCGAGCCTG CGCCTCGACA CGGGCCTGCC GACCGTCGGG ATGCGTTATG 36841 TTCCGTACAA CGGCACGTCG GTCGTGCCGG ACTGGCTGAG TGAGCCGCCC GCGCGGCCCC 36901 GGGTCTGCCT GACCCTCGGC GTCTCCGCGC GTGAGGTCCT CGGCGGCGAC GGCGTCTCGC 36961 AGGGCGACAT CCTGGAGGCG CTCGCCGACC TCGACATCGA GCTCGTCGCC ACGCTCGACG 37021 CGAGTCAGCG CGCCGAGATC CGCAACTACC CGAAGCACAC CCGGTTCACG GACTTCGTGC 37081 CGATGCACGC GCTCCTGGCCG AGCTGCTCGG CGATCATCCA CCACGGCGGG GCGGGCACCT 37141 ACGCGACCGC CGTGATCAAC GCGGTGCCGC AGGTCATGCT CGCCGAGCTG TGGGACGCGC 37201 CGGTCAAGGC GCGGGCCGTC GCCGAGCAGG GGGCGGGGTT CTTCCTGCCG CCGGCCGAGC 37261 TCACGCCGCA GGCCGTGCGG GACGCCGTCG TCCGCATCCT CGACGACCCC TCGGTCGCCA 37321 CCGCCGCGCA CCGGCTGCGC GAGGAGACCT TCGGCGACCC CACCCCGGCC GGGATCGTCC 37381 CCGAGCTGGA GCGGCTCGCC GCGCAGCACC GCCGCCCGCC GGCCGACGCC CGGCACTGAG 37441 CCGCACCCCT CGCCCCAGGC CTCACCCCTG TATCTGCGCC GGGGGACGCC CCCGGCCCAC 37501 CCTCCGAAAG ACCGAAAGCA GGAGCACCGT GTACGAAGTC GACCACGCCG ACGTCTACGA 37561 CCTCTTCTAC CTGGGTCGCG GCAAGGACTA CGCCGCCGAG GCCTCCGACA TCGCCGACCT 37621 GGTGCGCTCC CGTACCCCCG AGGCCTCCTC GCTCCTGGAC GTGGCCTGCG GTACGGGCAC 37681 GCATCTGGAG CACTTCACCA AGGAGTTCGG CGACACCGCC GGCCTGGAGC TGTCCGAGGA 37741 CATGCTCACC CACGCCCGCA AGCGGCTGCC CGACGCCACG CTCCACCAGG GCGACATGCG 37801 GGACTTCCGG CTCGGCCGGA AGTTCTCCGC CGTGGTCAGC ATGTTCAGCT CCGTCGGCTA 37861 CCTGAAGACG ACCGAGGAAC TCGGCGCGGC CGTCGCCTCG TTCGCGGAGC ACCTGGAGCC 37921 CGGTGGCGTC GTCGTCGTCG AGCCGTGGTG GTTCCCGGAG ACCTTCGCCG ACGGCTGGGT 37981 CAGCGCCGAC GTCGTCCGCC GTGACGGGCG CACCGTGGCC CGTGTCTCGC ACTCGGTGCG 38041 GGAGGGGAAC GCGACGCGCA TGGAGGTCCA CTTCACCGTG GCCGACCCGG GCAAGGGCGT 38101 GCGGCACTTC TCCGACGTCC ATCTCATCAC CCTGTTCCAC CAGGCCGAGT ACGAGGCCGC 38161 GTTCACGGCC GCCGGGCTGC GCGTCGAGTA CCTGGAGGGC GGCCCGTCGG GCCGTGGCCT 38221 CTTCGTCGGC GTCCCCGCCT GAGCACCGCC CAAGACCCCC CGGGGCGGGA CGTCCCGGGT 38281 GCACCAAGCA AAGAGAGAGA AACGAACCGT GACAGGTAAG ACCCGAATAC CGCGTGTCCG 38341 CCGCGGCCGC ACCACGCCCA GGGCCTTCAC CCTGGCCGTC GTCGGCACCC TGCTGGCGGG 38401 CACCACCGTG GCGGCCGCCG CTCCCGGCGC CGCCGACACG GCCAATGTTC AGTACACGAG 38461 CCGGGCGGCG GAGCTCGTCG CCCAGATGAC GCTCGACGAG AAGATC Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given amino acid sequence of the invention.", "The native DNA sequence encoding the narbonolide PKS of Streptomyces venezuelae is shown herein merely to illustrate a preferred embodiment of the invention, and the invention includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the invention.", "In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity.", "The present invention includes such polypeptides with alternate amino acid sequences, and the amino acid sequences shown merely illustrate preferred embodiments of the invention.", "The recombinant nucleic acids, proteins, and peptides of the invention are many and diverse.", "To facilitate an understanding of the invention and the diverse compounds and methods provided thereby, the following description of the various regions of the narbonolide PKS and corresponding coding sequences is provided.", "The loading module of the narbonolide PKS contains an inactivated KS domain, an AT domain, and an ACP domain.", "The AT domain of the loading module binds propionyl CoA.", "Sequence analysis of the DNA encoding the KS domain indicates that this domain is enzymatically inactivated, as a critical cysteine residue in the motif TVDACSSSL, which is highly conserved among KS domains, is replaced by a glutamine and so is referred to as a KSQ domain.", "Such inactivated KS domains are also found in the PKS enzymes that synthesize the 16-membered macrolides carbomycin, spiromycin, tylosin, and niddamycin.", "While the KS domain is inactive for its usual function in extender modules, it is believed to serve as a decarboxylase in the loading module.", "The present invention provides recombinant DNA compounds that encode the loading module of the narbonolide PKS and useful portions thereof.", "These recombinant DNA compounds are useful in the construction of PKS coding sequences that encode all or a portion of the narbonolide PKS and in the construction of hybrid PKS encoding DNA compounds of the invention, as described in the section concerning hybrid PKSs below.", "To facilitate description of the invention, reference to a PKS, protein, module, or domain herein can also refer to DNA compounds comprising coding sequences therefor and vice versa.", "Also, reference to a heterologous PKS refers to a PKS or DNA compounds comprising coding sequences therefor from an organism other than Streptomyces venezuelae.", "In addition, reference to a PKS or its coding sequence includes reference to any portion thereof.", "The present invention provides recombinant DNA compounds that encode one or more of the domains of each of the six extender modules (modules 1-6, inclusive) of the narbonolide PKS.", "Modules 1 and 5 of the narbonolide PKS are functionally similar.", "Each of these extender modules contains a KS domain, an AT domain specific for methylmalonyl CoA, a KR domain, and an ACP domain.", "Module 2 of the narbonolide PKS contains a KS domain, an AT domain specific for malonyl CoA, a KR domain, a DH domain, and an ACP domain.", "Module 3 differs from extender modules f and 5 only in that it contains an inactive ketoreductase domain.", "Module 4 of the narbonolide PKS contains a KS domain, an AT domain specific for methylmalonyl CoA, a IR domain, a DH domain, an ER domain, and an ACP domain.", "Module 6 of the narbonolide PKS contains a KS domain, an AT domain specific for methylmalonyl CoA, and an ACP domain.", "In one important embodiment, the invention provides a recombinant narbonolide PKS that can be used to express only narbonolide (as opposed to the mixture of narbonolide and 10-deoxymethynolide that would otherwise be produced) in recombinant host cells.", "This recombinant narbonolide PKS results from a fusion of the coding sequences of the picAIII and picAIV genes so that extender modules 5 and 6 are present on a single protein.", "This recombinant PKS can be constructed on the Streptomyces venezuelae or S. narbonensis chromosome by homologous recombination.", "Alternatively, the recombinant PKS can be constructed on an expression vector and introduced into a heterologous host cell.", "This recombinant PKS is preferred for the expression of narbonolide and its glycosylated and/or hydroxylated derivatives, because a lesser amount or no 10-deoxymethynolide is produced from the recombinant PKS as compared to the native PKS.", "In a related embodiment, the invention provides a recombinant narbonolide PKS in which the picAIV gene has been rendered inactive by an insertion, deletion, or replacement.", "This recombinant PKS of the invention is useful in the production of 10-deoxymethynolide and its derivatives without production of narbonolide.", "In similar fashion, the invention provides recombinant narbonolide PKS in which any of the domains of the native PKS have been deleted or rendered inactive to make the corresponding narbonolide or 10-deoxymethynolide derivative.", "Thus, the invention also provides recombinant narbonolide PKS genes that differ from the narbonolide PKS gene by one or more deletions.", "The deletions can encompass one or more modules and/or can be limited to a partial deletion within one or more modules.", "When a deletion encompasses an entire module, the resulting narbonolide derivative is at least two carbons shorter than the polyketide produced from the PKS encoded by the gene from which deleted PKS gene and corresponding polyketide were derived.", "When a deletion is within a module, the deletion typically encompasses a KR, DH, or ER domain, or both DH and ER domains, or both KR and DH domains, or all three KR, DH, and ER domains.", "This aspect of the invention is illustrated in FIG.", "4, parts B and C, which shows how a vector of the invention, plasmid pKOS039-16 (not shown), was used to delete or “knock out” the picAI gene from the Streptomyces venezuelae chromosome.", "Plasmid pKOS039-16 comprises two segments (shown as cross-hatched boxes in FIG.", "4, part B) of DNA flanking the picAI gene and isolated from cosmid pKOS023-27 (shown as a linear segment in the Figure) of the invention.", "When plasmid pKOS039-16 was used to transform S. venezuelae and a double crossover homologous recombination event occurred, the picAI gene was deleted.", "The resulting host cell, designated K039-03 in the Figure, does not produce picromycin unless a functional picAI gene is introduced.", "This Streptomyces venezuelae K039-03 host cell and corresponding host cells of the invention are especially useful for the production of polyketides produced from hybrid PKS or narbonolide PKS derivatives.", "Especially preferred for production in this host cell are narbonolide derivatives produced by PKS enzymes that differ from the narbonolide PKS only in the loading module and/or extender modules 1 and/or 2.These are especially preferred, because one need only introduce into the host cell the modified picAI gene or other corresponding gene to produce the desired PKS and corresponding polyketide.", "These host cells are also preferred for desosaminylating polyketides in accordance with the method of the invention in which a polyketide is provided to an S. venezuelae cell and desosaminylated by the endogenous desosamine biosynthesis and desosaminyl transferase gene products.", "The recombinant DNA compounds of the invention that encode each of the domains of each of the modules of the narbonolide PKS are also useful in the construction of expression vectors for the heterologous expression of the narbonolide PKS and for the construction of hybrid PKS expression vectors, as described further below.", "Section II: The Genes for Desosamine Biosynthesis and Transfer and for Beta-glucosidase Narbonolide and 10-deoxymethynolide are desosaminylated in Streptomyces venezuelae and S. narbonensis to yield narbomycin and YC-17, respectively.", "This conversion requires the biosynthesis of desosamine and the transfer of the desosamine to the substrate polyketides by the enzyme desosaminyl transferase.", "Like other Streptomyces, S. venezuelae and S. narbonensis produce glucose and a glucosyl transferase enzyme that glucosylates desosamine at the 2′ position.", "However, S. venezuelae and S. narbonensis also produce an enzyme called beta-glucosidase, which removes the glucose residue from the desosamine.", "The present invention provides recombinant DNA compounds and expression vectors for each of the desosamine biosynthesis enzymes, desosaminyl transferase, and beta-glucosidase.", "As noted above, cosmid pKOS023-27 contains three ORFs that encode proteins involved in desosamine biosynthesis and transfer.", "The first ORF is from the picCII gene, also known as des VIII, a homologue of enyCII, believed to encode a 4-keto-6-deoxyglucose isomerase.", "The second ORF is from the picCIII gene, also known as des VII, a homologue of eryCIII, which encodes a desosaminyl transferase.", "The third ORF is from the picCVI gene, also known as desVI, a homologue of eryCVI, which encodes a 3-amino dimethyltransferase.", "The three genes above and the remaining desosamine biosynthetic genes can be isolated from cosmid pKOS023-26, which was deposited with the American Type Culture Collection on 20 Aug. 1998 under the Budapest Treaty and is available under the accession number ATCC 203141.FIG.", "3 shows a restriction site and function map of cosmid pKOS023-26.This cosmid contains a region of overlap with cosmid pKOS02327 representing nucleotides 14252 to nucleotides 38506 of pKOS023-27.The remaining desosamine biosynthesis genes on cosmid pKOS023-26 include the following genes.", "ORF11, also known as desR, encodes beta-glucosidase and has no ery gene homologue.", "The picCI gene, also known as desV, is a homologue of eryCI.", "ORF14, also known as desIV, has no known ery gene homologue and encodes an NDP glucose 4,6-dehydratase.", "ORF13, also known as desIII, has no known ery gene homologue and encodes an NDP glucose synthase.", "The picCV gene, also known as desII, a homologue of eryCV is required for desosamine biosynthesis.", "The picCIV gene also known as desI, is a homologue of eryCIV, and its product is believed to be a 3,4-dehydratase.", "Other ORFs on cosmid pKOS02326 include ORF12, believed to be a regulatory gene; ORF15, which encodes an S-adenosyl methionine synthase; and ORF16, which is a homolog of the M. tuberculosis cbhK gene.", "Cosmid pKOS023-26 also encodes the picK gene, which encodes the cytochrome P450 hydroxylase that hydroxylates the C12 of narbomycin and the C10 and C12 positions of YC-17.This gene is described in more detail in the following section.", "Below, the amino acid sequences or partial amino acid sequences of the gene products of the desosamine biosynthesis and transfer and beta-glucosidase genes are shown.", "These amino acid sequences are followed by the DNA sequences that encode them.", "Amino acid sequence of PICCI 1 VSSRAETPRV PFLDLKAAYE ELRAETDAAI ARVLDSGRYL LGPELEGFEA EFAAYCETDH 61 AVGVNSGMDA LQLALRGLGI GPGDEVIVPS HTYIASWLAV SATGATPVPV EPHEDHPTLD 121 PLLVEKAITP RTRALLPVHL YGHPADMDAL RELADRHGLH IVEDAAQAHG ARYRGRRIGA 181 GSSVAAFSFY PGKNLGCFGD GGAVVTGDPE LAERLRMLRN YGSRQKYSHE TKGTNSRLDE 241 MQAAVLRIRL XHLDSWNGRR SALAAEYLSG LAGLPGIGLP VTAPDTDPVW HLFTVRTERR 301 DELRSHLDAR GIDTLTHYPV PVHLSPAYAG EAPPEGSLPR AESFARQVLS LPIGPHLERP 361 QALRVIDAVR EWAERVDQA Amino acid sequence of 3-keto-6-deoxyglucose isomerase, PICCII 1 VADRELGTHL LETRGIHWIH AANGDPYATV LRGQADDPYP AYERVRARGA LSFSPTGSWV 61 TADHALAASI LCSTDFGVSG ADGVPVPQQV LSYGEGCPLE REQVLPAAGD VPEGGQRAVV 121 EGIHRETLEG LAPDPSASYA FELLGGFVRP AVTAAAAAVL GVPADRRADF ADLLERLRPL 181 SDSLLAPQSL RTVRAADGAL AELTALLADS DDSPGALLSA LGVTAAVQLT GNAVLALLAH 241 PEQWRELCDR PGLAAAAVEE TLRYDPPVQL DARVVRGETE LAGRRLPAGA HVVVLTAATG 301 RDPEVFTDPE RFDLARPDAA AHLALHPAGP YGPVASLVRL QAEVALRTLA GRFPGLRQAG 361 DVLRPRRAPV GRGPLSVPVS SS Amino acid sequence of desosaminyl transferase, PICCIII 1 MRVLLTSFAH HTHYYGLVPL AWALLAAGHE VRVASQPALT DTITGSGLAA VPVGTDHLIH 61 EYRVRMAGEP RPNHPAIAFD EARPEPLDWD HALGIEAILA PYFYLLANND SMVDDLVDFA 121 RSWQPDLVLW EPITYAGAVA AQVTGAAHAR VLWGPDVMGS ARRKFVALRD RQPPEHREDP 181 TAEWLTWTLD RYGASFEEEL LTGQFTIDPT PPSLRLDTGL PTVGMRYVPY NGTSVVPDWL 241 SEPPARPRVC LTLGVSAREV LGGDGVSQGD ILEALADLDI ELVATLDASQ RAEIRNYPKH 301 TRFTDFVPMH ALLPSCSAII HHGGAGTYAT AVINAVPQVM LAELWDAPVK ARAVAEQGAG 361 FFLPPAELTP QAVRDAVVRI LDDPSVATAA HRLREETFGD PTPAGIVPEL ERLAAQHRRP 421 PADARH Partial amino acid sequence of aminotransferase-dehydrase, PICCIV 1 VKSALSDLAF FGGPAAFDQP LLVGRPNRID RARLYERLDR ALDSQWLSNG GPLVREFEER 61 VAGLAGVRHA VATCNATAGL QLLAHAAGLT GEVIMPSMTF AATPHALRWI GLTPVFADID 121 PDTGNLDPDQ VAAAVTPRTS AVVGVHLWGR PCAADQLRKV ADEHGLRLYF DAAHALGCAV 181 DGRPAGSLGD AEVFSFHATK AVNAFEGGAV VTDDADLAAR IRALHNFGFD LPGGSPAGGT 241 NAKMSEAAAA MGLTSLDAFP EVIDRNRRNH AXYREHLADL PGVLVADHDR HGLNNHQYVI 301 VEIDEATTGI HRDLVMEVLK AEGVHTRAYF S Amino acid sequence of PICCV 1 MTAPALSATA PAERCAHPGA DLGAAVHAVG QTLAAGGLVP PDEAGTTARH LVRLAVRYGN 61 SPFTPLEEAR HDLGVDRDAF RRLLALFGQV PELRTAVETG PAGAYWKNTL LPLEQRGVFD 121 AALARKPVFP YSVGLYPGPT CMFRCHFCVR VTGARYDPSA LDAGNAMFRS VIDEIPAGNP 181 SAMYFSGGLE PLTNPGLGSL AAHATDHGLR PTVYTNSFAL TERTLERQPG LWGLHAIRTS 241 LYGLNDEEYE QTTGKKAAFR RVRENLRRFQ QLRAERESPI NLGFAYIVLP GRASRLLDLV 301 DFIADLNDAG QGRTIDFVNI REDYSGRDDG KLPQEERAEL QEALNAFEER VRERTPGLHI 361 DYGYALNSLR TGADAELLRI KPATMRPTAH PQVAVQVDLL GDVYLYREAG FPDLDGATRY 421 IAGRVTPDTS LTEVVRDFVE RGGEVAAVDG DEYFMDGFDQ VVTARLNQLE RDAADGWEEA 481 RGFLR Amino acid sequence of 3-amino dimethyl transferase, PICCVI 1 VYEVDHADVY DLFYLGRGKD YAAEASDIAD LVRSRTPEAS SLLDVACGTG THLEHFTKEF 61 GDTAGLELSE DMLTHARKRL PDATLHQGDM RDFRLGRKFS AVVSMFSSVG YLKTTEELGA 121 AVASFAEHLE PGGVVVVEPW WFPETFADGW VSADVVRRDG RTVARVSHSV REGNATRMEV 181 HFTVADPGKG VRHFSDVHLI TLFHQAEYEA AFTAAGLRVE YLEGGPSGRG LFVGVPA Partial amino acid sequence of beta-glucosidase, ORF11 1 MTLDEKISFV HWALDPDRQN VGYLPGVPRL GIPELRAADG PNGIRLVGQT ATALPAPVAL 61 ASTFDDTMAD SYGKVMGRDG RALNQDMVLG PMMNNIRVPH GGRNYETFSE DPLVSSRTAV 121 AQIKGIQGAG LMTTAKHFAA NNQENNRFSV NANVDEQTLR EIEFPAFEAS SKAGAGSFMC 181 AYNGLNGKPS CGNDELLNNV LRTQWGFQGW VMSDWLATPG TDAITKGLDQ EMGVELPGDV 241 PKGEPSPPAK FFGEALKTAV LNGTVPEAAV TRSAERIVGQ MEKFGLLLAT PAPRPERDKA 301 GAQAVSRKVA ENGAVLLRNE GQALPLAGDA GKSIAVIGPT AVDPKVTGLG SAHVVPDSAA 361 APLDTIKARA GAGATVTYET GEETFGTQIP AGNLSPAFNQ GHQLEPGKAG ALYDGTLTVP 421 ADGEYRIAVR ATGGYATVQL GSHTIEAGQV YGKVSSPLLK LTKGTHKLTI SGFAMSATPL 481 SLELGWVTPA AADATIAKAV ESARKARTAV VFAYDDGTEG VDRPNLSLPG TQDKLISAVA 541 DANPNTIVVL NTGSSVLMPW LSKTRAVLDM WYPGQAGAEA TAALLYGDVN PSGKLTQSFP 601 AAENQHAVAG DPTSYPGVDN QQTYREGIHV GYRWFDKENV KPLFPFGHGL SYTSFTQSAP 661 TVVRTSTGGL KVTVTVRNSG KRAGQEVVQA YLGASPNVTA PQAKKKLVGY TKVSLAAGEA 721 KTVTVNVDRR QLQFWDAATD NWKTGTGNRL LQTGSSSADL RGSATVNVW Amino acid sequence of transcriptional activator, ORF12 1 MNLVERDGEI AHLRAVLDAS AAGDGTLLLV SGPAGSGKTE LLRSLRRLAA ERETPVWSVR 61 ALPGDRDIPL GVLCQLLRSA EQHGADTSAV RDLLDAASRR AGTSPPPPTR RSASTRHTAC 121 TTGCSPSPAG TPFLVAVDDL THADTASLRF LLYCAAHHDQ GGIGFVMTER ASQRAGYRVF 181 RAELLRQPHC RNMWLSGLPP SGVRQLLAHY YGPEAAERRA PAYHATTGGN PLLLRALTQD 241 RQASHTTLGA AGGDEPVHGD AFAQAVLDCL HRSAEGTLET ARWLAVLEQS DPLLVERLTG 301 TTAAAVERHI QELAAIGLLD EDGTLGQPAI REAALQDLPA GERTELHRRA AEQLHRDGAD 361 EDTVARHLLV GGAPDAPWAL PLLERGAQQA LFDDRLDDAF RILEFAVRSS TDNTQLARLA 421 PHLVAASWRM NPHMTTRALA LFDRLLSGEL PPSHPVMALI RCLVWYGRLP EAADALSRLR 481 PSSDNDALEL SLTRMWLAAL CPPLLESLPA TPEPERGPVP VRLAPRTTAL QAQAGVFQRG 541 PDNASVAQAE QILQGCRLSE ETYEALETAL LVLVHADRLD RALFWSDALL AEAVERRSLG 601 WEAVFAATRA MIAIRCGDLP TARERAELAL SHAAPESWGL AVGMPLSALL LACTEAGEYE 661 QAERVLRQPV PDAMFDSRHG MEYMHARGRY WLAXGRLHAA LGEFMLCGEI LGSWNLDQPS 721 IVPWRTSAAE VYLRLGNRQK ARALAEAQLA LVRPGRSRTR GLTLRVLAAA VDGQQAERLH 781 AEAVDMLHDS GDRLEHARAL AGMSRHQQAQ GDNYRARMTA RLAGDMAWAC GAYPLAEEIV 841 PGRGGRRAKA VSTELELPGG PDVGLLSEAE RRVAALAARG LTNRQIARRL CVTASTVEQH 901 LTRVYRKLNV TRRADLPISL AQDKSVTA Amino acid sequence of dNDP-glucose synthase (glucose-1-phosphate thymidyl transferase), ORF13 1 MKGIVLAGGS GTRLHPATSV ISKQILPVYN KPMIYYPLSV LMLGGIREIQ IISTPQHIEL 61 FQSLLGNGRH LGIELDYAVQ KEPAGIADAL LVGAEHIGDD TCALILGDNI FHGPGLYTLL 121 RDSIARLDGC VLFGYPVKDP ERYGVAEVDA TGRLTDLVEK PVKPRSNLAV TGLYLYDNDV 181 VDIAKNIRPS PRGELEITDV NRVYLERGRA ELVNLGRGFA WLDTGTHDSL LRAAQYVQVL 241 EERQGVWIAG LEEIAFRMGF IDAEACHGLG EGLSRTEYGS YLMEIAGREG AP Amino acid sequence of dNDP-glucose 4,6-dehydratase, ORF14 1 VRLLVTGGAG FIGSHFVRQL LAGAYPDVPA DEVIVLDSLT YAGNRANLAP VDADPRLRFV 61 HGDIRDAGLL ARELRGVDAI VHFAAESHVD RSIAGASVFT ETNVQGTQTL LQCAVDAGVG 121 RVVHVSTDEV YGSIDSGSWT ESSPLEPNSP YAASKAGSDL VARAYHRTYG LDVRITRCCN 181 NYGPYQHPEK LIPLFVTNLL DGGTLPLYGD GANVREWVHT DDHCRGIALV LAGGRAGEIY 241 HIGGGLELTN RELTGILLDS LGADWSSVRK VADRKGHDLR YSLDGGKIER ELGYRPQVSF 301 ADGLARTVRW YRENRGWWEP LKATAPQLPA TAVEVSA Partial amino acid sequence of S-adenosylmethionine synthase, ORF15 1 IGYDSSKKGF DGASCGVSVS IGSQSPDIAQ GVDTAYEKRV EGASQRDEGD ELDKQGAGDQ 61 GLMFGYASDE TPELMPLPIH LAHRLSRRLT EVRKNGTIPY LRPDGKTQVT IEYDGDRAVR 121 LDTVVVSSQH ASDIDLESLL APDVRKFVVE HVLAQLVEDG IKLDTDGYRL LVNPTGRFEI 181 GGPMGDAGLT GRKIIIDTYG GMARHGGGAF SGKDPSKVDR SAAYAMRWVA KNVVAAGLAS 241 RCEVQVAYAI GKAEPVGLFV ETFGTHKIET EKIENAIGEV FDLRPAAIIR DLDLLRPIYS 301 QTAAYGHFGR ELPDFTWERT DRVDALKKAA GL Partial amino acid sequence of ORF16 (homologous to M. tuberculosis cbhK) 1 MRIAVTGSIA TDHLMTFPGR FAEQILPDQL AHVSLSFLVD TLDIRHGGVA ANIAYGLGLL 61 GRRPVLVGAV GKDFDGYGQL LRAAGVDTDS VRVSDRQHTA RFMCTTDEDG NQLASFYAGA 121 MAEARDIDLG ETAGRPGGID LVLVGADDPE AMVRHTRVCR ELGLRPAADP SQQLARLEGD 181 SVRELVDGAE LLFTNAYERA LLLSKTGWTE QEVLARVGTW ITTLGAKGCR While not all of the insert DNA of cosmid pKOS02326 has been sequenced, five large contigs shown of FIG.", "3 have been assembled and provide sufficient sequence information to manipulate the genes therein in accordance with the methods of the invention.", "The sequences of each of these five contigs are shown below.", "Contig 001 from cosmid pKOS023-26 contains 2401 nucleotides, the first 100 bases of which correspond to 100 bases of the insert sequence of cosmid pKOS023-27.Nucleotides 80-2389 constitute ORF11, which encodes 1 beta glucosidase.", "1 CGTGGCGGCC GCCGCTCCCG GCGCCGCCGA CACGGCCAAT GTTCAGTACA CGAGCCGGGC 61 GGCGGAGCTC GTCGCCCAGA TGACGCTCGA CGAGAAGATC AGCTTCGTCC ACTGGGCGCT 121 GGACCCCGAC CGGCAGAACG TCGGCTACCT TCCCGGCGTG CCGCGTCTGG GCATCCCGGA 181 GCTGCGTGCC GCCGACGGCC CGAACGGCAT CCGCCTGGTG GGGCAGACCG CCACCGCGCT 241 GCCCGCGCCG GTCGCCCTGG CCAGCACCTT CGACGACACC ATGGCCGACA GCTACGGCAA 301 GGTCATGGGC CGCGACGGTC GCGCGCTCAA CCAGGACATG GTCCTGGGCC CGATGATGAA 361 CAACATCCGG GTGCCGCACG GCGGCCGGAA CTACGAGACC TTCAGCGAGG ACCCCCTGGT 421 CTCCTCGCGC ACCGCGGTCG CCCAGATCAA GGGCATCCAG GGTGCGGGTC TGATGACCAC 481 GGCCAAGCAC TTCGCGGCCA ACAACCAGGA GAACAACCGC TTCTCCGTGA ACGCCAATGT 541 CGACGAGCAG ACGCTCCGCG AGATCGAGTT CCCGGCGTTC GAGGCGTCCT CCAAGGCCGG 601 CGCGGGCTCC TTCATGTGTG CCTACAACGG CCTCAACGGG AAGCCGTCCT GCGGCAACGA 661 CGAGCTCCTC AACAACGTGC TGCGCACGCA GTGGGGCTTC CAGGGCTGGG TGATGTCCGA 721 CTGGCTCGCC ACCCCGGGCA CCGACGCCAT CACCAAGGGC CTCGACCAGG AGATGGGCGT 781 CGAGCTCCCC GGCGACGTCC CGAAGGGCGA GCCCTCGCCG CCGGCCAAGT TCTTCGGCGA 841 GGCGCTGAAG ACGGCCGTCC TGAACGGCAC GGTCCCCGAG GCGGCCGTGA CGCGGTCGGC 901 GGAGCGGATC GTCGGCCAGA TGGAGAAGTT CGGTCTGCTC CTCGCCACTC CGGCCCCGCG 961 GCCCGAGCGC GACAAGGCGG GTGCCCAGGC GGTGTCCCGC AAGGTCGCCG AGAACGGCGC 1021 GGTGCTCCTG CGCAACGAGG GCCAGGCCCT GCCGCTCGCC GGTGACGCCG GCAAGAGCAT 1081 CGCGGTCATC GGCCCGACGG CCGTCGACCC CAAGGTCACC GGCCTGGGCA GCGCCCACGT 1141 CGTCCCGGAC TCGGCGGCGG CGCCACTCGA CACCATCAAG GCCCGCGCGG GTGCGGGTGC 1201 GACGGTGACG TACGAGACGG GTGAGGAGAC CTTCGGGACG CAGATCCCGG CGGGGAACCT 1261 CAGCCCGGCG TTCAACCAGG GCCACCAGCT CGAGCCGGGC AAGGCGGGGG CGCTGTACGA 1321 CGGCACGCTG ACCGTGCCCG CCGACGGCGA GTACCGCATC GCGGTCCGTG CCACCGGTGG 1381 TTACGCCACG GTGCAGCTCG GCAGCCACAC CATCGAGGCC GGTCAGGTCT ACGGCAAGGT 1441 GAGCAGCCCG CTCCTCAAGC TGACCAAGGG CACGCACAAG CTCACGATCT CGGGCTTCGC 1501 GATGAGTGCC ACCCCGCTCT CCCTGGAGCT GGGCTGGGTN ACGCCGGCGG CGGCCGACGC 1561 GACGATCGCG AAGGCCGTGG AGTCGGCGCG GAAGGCCCGT ACGGCGGTCG TCTTCGCCTA 1621 CGACGACGGC ACCGAGGGCG TCGACCGTCC GAACCTGTCG CTGCCGGGTA CGCAGGACAA 1681 GCTGATCTCG GCTGTCGCGG ACGCCAACCC GAACACGATC GTGGTCCTCA ACACCGGTTC 1741 GTCGGTGCTG ATGCCGTGGC TGTCCAAGAC CCGCGCGGTC CTGGACATGT GGTACCCGGG 1801 CCAGGCGGGC GCCGAGGCCA CCGCCGCGCT GCTCTACGGT GACGTCAACC CGAGCGGCAA 1861 GCTCACGCAG AGCTTCCCGG CCGCCGAGAA CCAGCACGCG GTCGCCGGCG ACCCGACCAG 1921 CTACCCGGGC GTCGACAACC AGCAGACGTA CCGCGAGGGC ATCCACGTCG GGTACCGCTG 1981 GTTCGACAAG GAGAACGTCA AGCCGCTGTT CCCGTTCGGG CACGGCCTGT CGTACACCTC 2041 GTTCACGCAG AGCGCCCCGA CCGTCGTGCG TACGTCCACG GGTGGTCTGA AGGTCACGGT 2101 CACGGTCCGC AACAGCGGGA AGCGCGCCGG CCAGGAGGTC GTCCAGGCGT ACCTCGGTGC 2161 CAGCCCGAAC GTGACGGCTC CGCAGGCGAA GAAGAAGCTC GTGGGCTACA CGAAGGTCTC 2221 GCTCGCCGCG GGCGAGGCGA AGACGGTGAC GGTGAACGTC GACCGCCGTC AGCTGCAGTT 2281 CTGGGATGCC GCCACGGACA ACTGGAAGAC GGGAACGGGC AACCGCCTCC TGCAGACCGG 2341 TTCGTCCTCC GCCGACCTGC GGGGCAGCGC CACGGTCAAC GTCTGGTGAC GTGACGCCGT 2401 G Contig 002 from cosmid pKOS023-26 contains 5970 nucleotides and the following ORFs: from nucleotide 995 to 1 is an ORF of picCIV that encodes a partial sequence of an amino transferase-dehydrase; from nucleotides 1356 to 2606 is an ORF of picK that encodes a cytochrome P450 hydroxylase; and from nucleotides 2739 to 5525 is ORF12, which encodes a transcriptional activator.", "1 GGCGAGAAGT AGGCGCGGGT GTGCACGCCT TCGGCCTTCA GGACCTCCAT GACGAGGTCG 61 CGGTGGATGC CGGTGGTGGC CTCGTCGATC TCGACGATCA CGTACTGGTG GTTGTTGAGG 121 CCGTGGCGGT CGTGGTCGGC GACGAGGACG CCGGGGAGGT CCGCGAGGTG CTCGCGGTAG 181 SCGGCGTGGT TGCGCCGGTT CCGGTCGATG ACCTCGGGAA ACGCGTCGAG GGAGGTGAGG 241 CCCATGGCGG CGGCGGCCTC GCTCATCTTG GCGTTGGTCC CGCCGGCGGG GCTGCCGCCG 301 GGCAGGTCGA AGCCGAAGTT GTGGAGGGCG CGGATCCGGG CGGCGAGGTC GGCGTCGTCG 361 GTGACGACGG CGCCGCCCTC GAAGGCGTTG ACGGCCTTGG TGGCGTGGAA GCTGAAGACC 421 TCGGCGTCGC CGAGGCTGCC GGCGGGCCGG CCGTCGACCG CGCAGCCGAG GGCGTGCGCG 481 GCGTCGAAGT ACAGCCGCAG GCCGTGCTCG TCGGCGACCT TCCGCAGCTG GTCGGCGGCG 541 CAGGGGCGGC CCCAGAGGTG GACGCCGACG ACGGCCGAGG TGCGGGGTGT GACCGCGGCG 601 GCCACCTGGT CCGGGTCGAG GTTGCCGGTG TCCGGGTCGA TGTCGGCGAA GACCGGGGTG 661 AGGCCGATCC AGCGCAGTGC GTGCGGGGTG GCGGCGAACG TCATCGACGG CATGATCACT 721 TCGCCGGTGA GGCCGGCGGC GTGCGCGAGG AGCTGGAGCC CGGCCGTGGC GTTGCAGGTG 781 GCCACGGCAT GCCGGACCCC GGCGAGCCCG GCGACGCGCT CCTCGAACTC GCGGACGAGC 841 GGGCCGCCGT TGGACAGCCA CTGGCTGTCG AGGGCCCCGT CGAGCCGCTC GTACAGCCTG 901 GCGCGGTCGA TGCGGTTGGG CCGCCCCACG AGGAGCGGCT GGTCGAAAGC GGCGGGGCCG 961 CCGAAGAATG CGAGGTCGGA TAAGGCGCTT TTCACGGATG TTCCCTCCGG GCCACCGTCA 1021 CGAAATGATT CGCCGATCCG GGAATCCCGA ACGAGGTCGC CGCGCTCCAC CGTGACGTAC 1081 GACGAGATGG TCGATTGTGG TGGTCGATTT CGGGGGGACT CTAATCCGCG CGGAACGGGA 1141 CCGACAAGAG CACGCTATGC GCTCTCGATG TGCTTCGGAT CACATCCGCC TCCGGGGTAT 1201 TCCATCGGCG GCCCGAATGT GATGATCCTT GACAGGATCC GGGAATCAGC CGAGCCGCCG 1261 GGAGGGCCGG GGCGCGCTCC GCGGAAGAGT ACGTGTGAGA AGTCCCGTTC CTCTTCCCGT 1321 TTCCGTTCCG CTTCCGGCCC GGTCTGGAGT TCTCCGTGCG CCGTACCCAG CAGGGAACGA 1381 CCGCTCCTCC CCCGGTACTC GACCTCGGGG CCCTGGGGCA GGATTTCGCG GCCGATCCGT 1441 ATCCGACGTA CGCGAGACTG CGTGCCGAGG GTCCGGCCCA CCGGGTGCGC ACCCCCGAGG 1501 GGGACGAGGT GTGGCTGGTC GTCGGCTACG ACCGGGCGCG GGCGGTCCTC GCCGATCCCC 1561 GGTTCAGCAA GGACTGGCGC AACTCCACGA CTCCCCTGAC CGAGGCCGAG GCCGCGCTCA 1621 ACCACAACAT GCTGGAGTCC GACCCGCCGC GGCACACCCG GCTGCGCAAG CTGGTGGCCC 1681 GTGAGTTCAC CATGCGCCGG GTCGAGTTGC TGCGGCCCCG GGTCCAGGAG ATCGTCGACG 1741 GGCTCGTGGA CGCCATGCTG GCGGCGCCCG ACGGCCGCGC CGATCTGATG GAGTCCCTGG 1801 CCTGGCCGCT GCCGATCACC GTGATCTCCG AACTCCTCGG CGTGCCCGAG CCGGACCGCG 1861 CCGCCTTCCG CGTCTGGACC GACGCCTTCG TCTTCCCGGA CGATCCCGCC CAGGCCCAGA 1921 CCGCCATGGC CGAGATGAGC GGCTATCTCT CCCGGCTCAT CGACTCCAAG CGCGGGCAGG 1981 ACGGCGAGGA CCTGCTCAGC GCGCTCGTGC GGACCAGCGA CGAGGACGGC TCCCGGCTGA 2041 CCTCCGAGGA GCTGCTCGGT ATGGCCCACA TCCTGCTCGT CGCGGGGCAC GAGACCACGG 2101 TCAATCTGAT CGCCAACGGC ATGTACGCGC TGCTCTCGCA CCCCGACCAG CTGGCCGCCC 2161 TGCGGGCCGA CATGACGCTC TTGGACGGCG CGGTGGAGGA GATGTTGCGC TACGAGGGCC 2221 CGGTGGAATC CGCGACCTAC CGCTTCCCGG TCGAGCCCGT CGACCTGGAC GGCACGGTCA 2281 TCCCGGCCGG TGACACGGTC CTCGTCGTCC TGGCCGACGC CCACCGCACC CCCGAGCGCT 2341 TCCCGGACCC GCACCGCTTC GACATCCGCC GGGACACCGC CGGCCATCTC GCCTTCGGCC 2401 ACGGCATCCA CTTCTGCATC GGCGCCCCCT TGGCCCGGTT GGAGGCCCGG ATCGCCGTCC 2461 GCGCCCTTCT CGAACGCTGC CCGGACCTCG CCCTGGACGT CTCCCCCGGC GAACTCGTGT 2521 GGTATCCGAA CCCGATGATC CGCGGGCTCA AGGCCCTGCC GATCCGGTGG CGGCGAGGAC 2581 GGGAGGCGGG CCGCCGTACC GGTTGAACCC GCACGTCACC CATTACGACT CCTTGTCACG 2641 GAAGCCCCGG ATCGGTCCCC CCTCGCCGTA ACAAGACCTG GTTAGAGTGA TGGAGGACGA 2701 CGAAGGGTTC GGCGCCCGGA CGAGGGGGGA CTTCCGCGAT GAATCTGGTG GAACGCGACG 2761 GGGAGATAGC CCATCTCAGG GCCGTTCTTG ACGCATCCGC CGCAGGTGAC GGGACGCTCT 2821 TACTCGTCTC CGGACCGGCC GGCAGCGGGA AGACGGAGCT GCTGCGGTCG CTCCGCCGGC 2881 TGGCCGCCGA GCGGGAGACC CCCGTCTGGT CGGTCCGGGC GCTGCCGGGT GACCGCGACA 2941 TCCCCCTGGG CGTCCTCTGC CAGTTACTCC GCAGCGCCGA ACAACACGGT GCCGACACCT 3001 CCGCCGTCCG CGACCTGCTG GACGCCGCCT CGCGGCGGGC CGGAACCTCA CCTCCCCCGC 3061 CGACGCGCCG CTCCGCGTCG ACGAGACACA CCGCCTGCAC GACTGGCTGC TCTCCGTCTC 3121 CCGCCGGCAC CCCGTTCCTC GTCGCCGTCG ACGACCTGAC CCACGCCGAC ACCGCGTCCC 3181 TGAGGTTCCT CCTGTACTGC GCCGCCCACC ACGACCAGGG CGGCATCGGC TTCGTCATGA 3241 CCGAGCGGGC CTCGCAGCGC GCCGGATACC GGGTGTTCCG CGCCGAGCTC CTCCGCCAGC 3301 CGCACTGCCG CAACATGTGG CTCTCCGGGC TTCCCCCCAG CGGGGTACGC CAGTTACTCG 3361 CCCACTACTA CGGCCCCGAG GCCGCCGAGC GGCGGGCCCC CGCGTACCAC GCGACGACCG 3421 GCGGGAACCC GCTGCTCCTG CGGGCGCTGA CCCAGGACCG GCAGGCCTCC CACACCACCC 3481 TCGGCGCGGC CGGCGGCGAC GAGCCCGTCC ACGGCGACGC CTTCGCCCAG GCCGTCCTCG 3541 ACTGCCTGCA CCGCAGCGCC GAGGGCACAC TGGAGACCGC CCGCTGGCTC GCGGTCCTCG 3601 AACAGTCCGA CCCGCTCCTG GTGGAGCGGC TCACGGGAAC GACCGCCGCC GCCGTCGAGC 3661 GCCACATCCA GGAGCTCGCC GCCATCGGCC TCCTGGACGA GGACGGCACC CTGGGACAGC 3721 CCGCGATCCG CGAGGCCGCC CTCCAGGACC TGCCGGCCGG CGAGCGCACC GAACTGCACC 3781 GGCGCGCCGC GGAGCAGCTG CACCGGGACG GCGCCGACGA GGACACCGTG GCCCGCCACC 3841 TGCTGGTCGG CGGCGCCCCC GACGCTCCCT GGGCGCTGCC CCTGCTCGAA CGGGGCGCGC 3901 AGCAGGCCCT GTTCGACGAC CGACTCGACG ACGCCTTCCG GATCCTCGAG TTCGCCGTGC 3961 GGTCGAGCAC CGACAACACC CAGCTGGCCC GCCTCGCCCC ACACCTGGTC GCGGCCTCCT 4021 GGCGGATGAA CCCGCACATG ACGACCCGGG CCCTCGCACT CTTCGACCGG CTCCTGAGCG 4081 GTGAACTGCC GCCCAGCCAC CCGGTCATGG CCCTGATCCG CTGCCTCGTC TGGTACGGNC 4141 GGCTGCCCGA GGCCGCCGAC GCGCTGTCCC GGCTGCGGCC CAGCTCCGAC AACGATGCCT 4201 TGGAGCTGTC GCTCACCCGG ATGTGGCTCG CGGCGCTGTG CCCGCCGCTC CTGGAGTCCC 4261 TGCCGGCCAC GCCGGAGCCG GAGCGGGGTC CCGTCCCCGT ACGGCTCGCG CCGCGGACGA 4321 CCGCGCTCCA GGCCCAGGCC GGCGTCTTCC AGCGGGGCCC GGACAACGCC TCGGTCGCGC 4381 AGGCCGAACA GATCCTGCAG GGCTGCCGGC TGTCGGAGGA GACGTACGAG GCCCTGGAGA 4441 CGGCCCTCTT GCTCCTCGTC CACGCCGACC GGCTCGACCG GGCGCTGTTC TGGTCGGACG 4501 CCCTGCTCGC CGAGGCCGTG GAGCGGCGGT CGCTCGGCTG GGAGGCGGTC TTCGCCGCGA 4561 CCCGGGCGAT GATCGCGATC CGCTGCGGCG ACCTCCCGAC GGCGCGGGAG CGGGCCGAGC 4621 TGGCGCTCTC CCACGCGGCG CCGGAGAGCT GGGGCCTCGC CGTGGGCATG CCCCTCTCCG 4681 CGCTGCTGCT CGCCTGCACG GAGGCCGGCG AGTACGAACA GGCGGAGCGG GTCCTGCGGC 4741 AGCCGGTGCC GGACGCGATG TTCGACTCGC GGCACGGCAT GGAGTACATG CACGCCCGGG 4801 GCCGCTACTG GCTGGCGANC GGCCGGCTGC ACGCGGCGCT GGGCGAGTTC ATGCTCTGCG 4861 GGGAGATCCT GGGCAGCTGG AACCTCGACC AGCCCTCGAT CGTGCCCTGG CGGACCTCCG 4921 CCGCCGAGGT GTACCTGCGG CTCGGCAACC GCCAGAAGGC CAGGGCGCTG GCCGAGGCCC 4981 AGCTCGCCCT GGTGCGGCCC GGGCGCTCCC GCACCCGGGG TCTCACCCTG CGGGTCCTGG 5041 CGGCGGCGGT GGACGGCCAG CAGGCGGAGC GGCTGCACGC CGAGGCGGTC GACATGCTGC 5101 ACGACAGCGG CGACCGGCTC GAACACGCCC GCGCGCTCGC CGGGATGAGC CGCCACCAGC 5161 AGGCCCAGGG GGACAACTAC CGGGCGAGGA TGACGGCGCG GCTCGCCGGC GACATGGCGT 5221 GGGCCTGCGG CGCGTACCCG CTGGCCGAGG AGATCGTGCC GGGCCGCGGC GGCCGCCGGG 5281 CGAAGGCGGT GAGCACGGAG CTGGAACTGC CGGGCGGCCC GGACGTCGGC CTGCTCTCGG 5341 AGGCCGAACG CCGGGTGGCG GCCCTGGCAG CCCGAGGATT GACGAACCGC CAGATAGCGC 5401 GCCGGCTCTG CGTCACCGCG AGCACGGTCG AACAGCACCT GACGCGCGTC TACCGCAAAC 5461 TGAACGTGAC CCGCCGAGCA GACCTCCCGA TCAGCCTCGC CCAGGACAAG TCCGTCACGG 5521 CCTGAGCCAC CCCCGGTGTC CCCGTGCGAC GACCCGCCGC ACGGGCCACC GGGCCCGCCG 5581 GGACACGCCG GTGCGACACG GGGGCGCGCC AGGTGCCATG GGGACCTCCG TGACCGCCCG 5641 AGGCGCCCGA GGCGCCCGGT GCGGCACCCG GAGACGCCAG GACCGCCGGG ACCACCGGAG 5701 ACGCCAGGGA CCGCTGGGGA CACCGGGACC TCAGGGACCG CCGGGACCGC CCGAGTTGCA 5761 CCCGGTGCGC CCGGGGACAC CAGACCGCCG GGACCACCCG AGGGTGCCCG GTGTGGCCCC 5821 GGCGGCCGGG GTGTCCTTCA TCGGTGGGCC TTCATCGGCA GGAGGAAGCG ACCGTGAGAC 5881 CCGTCGTGCC GTCGGCGATC AGCCGCCTGT ACGGGCGTCG GACTCCCTGG CGGTCCCGGA 5941 CCCGTCGTAC GGGCTCGCGG GACCCGGTGC Contig 003 from cosmid pKOS023-26 contains 3292 nucleotides and the following ORFs: from nucleotide 104 to 982 is ORF13, which encodes dNDP glucose synthase (glucose-1-phosphate thymidyl transferase); from nucleotide 1114 to 2127 is ORF14, which encodes dNDP-glucose 4,6-dehydratase; and from nucleotide 2124 to 3263 is the picCI ORF.", "1 ACCCCCCAAA GGGGTGGTGA CACTCCCCCT GCGCAGCCCC TAGCGCCCCC CTAACTCGCC 61 ACGCCGACCG TTATCACCGG CGCCCTGCTG CTAGTTTCCG ACAATGAAGG GAATAGTCCT 121 GGCCGGCGGG AGCGGAACTC GGCTGCATCC GGCGACCTCG GTCATTTCGA AGCAGATTCT 181 TCCGGTCTAC AACAAACCGA TGATCTACTA TCCGCTGTCG GTTCTCATGC TCGGCGGTAT 241 TCGCGAGATT CAAATCATCT CGACCCCCCA GCACATCGAA CTCTTCCAGT CGCTTCTCGG 301 AAACGGCAGG CACCTGGGAA TAGAACTCGA CTATGCGGTC CAGAAAGAGC CCGCAGGAAT 361 CGCGGACGCA CTTCTCGTCG GAGCCGAGCA CATCGGCGAC GACACCTGCG CCCTGATCCT 421 GGGCGACAAC ATCTTCCACG GGCCCGGCCT CTACACGCTC CTGCGGGACA GCATCGCGCG 481 CCTCGACGGC TGCGTGCTCT TCGGCTACCC GGTCAAGGAC CCCGAGCGGT ACGGCGTCGC 541 CGAGGTGGAC GCGACGGGCC GGCTGACCGA CCTCGTCGAG AAGCCCGTCA AGCCGCGCTC 601 CAACCTCGCC GTCACCGGCC TCTACCTCTA CGACAACGAC GTCGTCGACA TCGCCAAGAA 661 CATCCGGCCC TCGCCGCGCG GCGAGCTGGA GATCACCGAC GTCAACCGCG TCTACCTGGA 721 GCGGGGCCGG GCCGAACTCG TCAACCTGGG CCGCGGCTTC GCCTGGCTGG ACACCGGCAC 781 CCACGACTCG CTCCTGCGGG CCGCCCAGTA CGTCCAGGTC CTGGAGGAGC GGCAGGGCGT 841 CTGGATCGCG GGCCTTGAGG AGATCGCCTT CCGCATGGGC TTCATCGACG CCGAGGCCTG 901 TCACGGCCTG GGAGAAGGCC TCTCCCGCAC CGAGTACGGC AGCTATCTGA TGGAGATCGC 961 CGGCCGCGAG GGAGCCCCGT GAGGGCACCT CGCGGCCGAC GCGTTCCCAC GACCGACAGC 1021 GCCACCGACA GTGCGACCCA CACCGCGACC CGCACCGCCA CCGACAGTGC GACCCACACC 1081 GCGACCTACA GCGCGACCGA AAGGAAGACG GCAGTGCGGC TTCTGGTGAC CGGAGGTGCG 1141 GGCTTCATCG GCTCGCACTT CGTGCGGCAG CTCCTCGCCG GGGCGTACCC CGACGTGCCC 1201 GCCGATGAGG TGATCGTCCT GGACAGCCTC ACCTACGCGG GCAACCGCGC CAACCTCGCC 1261 CCGGTGGACG CGGACCCGCG ACTGCGCTTC GTCCACGGCG ACATCCGCGA CGCCGGCCTC 1321 CTCGCCCGGG AACTGCGCGG CGTGGACGCC ATCGTCCACT TCGCGGCCGA GAGCCACGTG 1381 GACCGCTCCA TCGCGGGCGC GTCCGTGTTC ACCGAGACCA ACGTGCAGGG CACGCAGACG 1441 CTGCTCCAGT GCGCCGTCGA CGCCGGCGTC GGCCGGGTCG TGCACGTCTC CACCGACGAG 1501 GTGTACGGGT CGATCGACTC CGGCTCCTGG ACCGAGAGCA GCCCGCTGGA GCCCAACTCG 1561 CCCTACGCGG CGTCCAAGGC CGGCTCCGAC CTCGTTGCCC GCGCCTACCA CCGGACGTAC 1621 GGCCTCGACG TACGGATCAC CCGCTGCTGC AACAACTACG GGCCGTACCA GCACCCCGAG 1681 AAGCTCATCC CCCTCTTCGT GACGAACCTC CTCGACGGCG GGACGCTCCC GCTGTACGGC 1741 GACGGCGCGA ACGTCCGCGA GTGGGTGCAC ACCGACGACC ACTGCCGGGG CATCGCGCTC 1801 GTCCTCGCGG GCGGCCGGGC CGGCGAGATC TACCACATCG GCGGCGGCCT GGAGCTGACC 1861 AACCGCGAAC TCACCGGCAT CCTCCTGGAC TCGCTCGGCG CCGACTGGTC CTCGGTCCGG 1921 AAGGTCGCCG ACCGCAAGGG CCACGACCTG CGCTACTCCC TCGACGGCGG CAAGATCGAG 1981 CGCGAGCTCG GCTACCGCCC GCAGGTCTCC TTCGCGGACG GCCTCGCGCG GACCGTCCGC 2041 TGGTACCGGG AGAACCGCGG CTGGTGGGAG CCGCTCAAGG CGACCGCCCC GCAGCTGCCC 2101 GCCACCGCCG TGGAGGTGTC CGCGTGAGCA GCCGCGCCGA GACCCCCCGC GTCCCCTTCC 2161 TCGACCTCAA GGCCGCCTAC GACGAGCTCC GCGCGGAGAC CGACGCCGCG ATCGCCCGCG 2221 TCCTCGACTC GGGGCGCTAC CTCCTCGGAC CCGAACTCGA AGGATTCGAG GCGGAGTTCG 2281 CCGCGTACTG CGAGACGGAC CACGCCGTCG GCGTGAACAG CGGGATGGAC GCCCTCCAGC 2341 TCGCCCTCCG CGGCCTCGGC ATCGGACCCG GGGACGAGGT GATCGTCCCC TCGCACACGT 2401 ACATCGCCAG CTGGCTCGCG GTGTCCGCCA CCGGCGCGAC CCCCGTGCCC GTCGAGCCGC 2461 ACGAGGACCA CCCCACCCTG GACCCGCTGC TCGTCGAGAA GGCGATCACC CCCCGCACCC 2521 GGGCGCTCCT CCCCGTCCAC CTCTACGGGC ACCCCGCCGA CATGGACGCC CTCCGCGAGC 2581 TCGCGGACCG GCACGGCCTG CACATCGTCG AGGACGCCGC GCAGGCCCAC GGCGCCCGCT 2641 ACCGGGGCCG GCGGATCGGC GCCGGGTCGT CGGTGGCCGC GTTCAGCTTC TACCCGGGCA 2701 AGAACCTCGG CTGCTTCGGC GACGGCGGCG CCGTCGTCAC CGGCGACCCC GAGCTCGCCG 2761 AACGGCTCCG GATGCTCCGC AACTACGGCT CGCGGCAGAA GTACAGCCAC GAGACGAAGG 2821 GCACCAACTC CCGCCTGGAC GAGATGCAGG CCGCCGTGCT GCGGATCCGG CTCGNCCACC 2881 TGGACAGCTG GAACGGCCGC AGGTCGGCGC TGGCCGCGGA GTACCTCTCC GGGCTCGCCG 2941 GACTGCCCGG CATCGGCCTG CCGGTGACCG CGCCCGACAC CGACCCGGTC TGGCACCTCT 3001 TCACCGTGCG CACCGAGCGC CGCGACGAGC TGCGCAGCCA CCTCGACGCC CGCGGCATCG 3061 ACACCCTCAC GCACTACCCG GTACCCGTGC ACCTCTCGCC CGCCTACGCG GGCGAGGCAC 3121 CGCCGGAAGG CTCGCTCCCG CGGGCCGAGA GCTTCGCGCG GCAGGTCCTC AGCCTGCCGA 3181 TCGGCCCGCA CCTGGAGCGC CCGCAGGCGC TGCGGGTGAT CGACGCCGTG CGCGAATGGG 3241 CCGAGCGGGT CGACCAGGCC TAGTCAGGTG GTCCGGTAGA CCCAGCAGGC CG Contig 004 from cosmid pKOS023-26 contains 1693 nucleotides and the following ORFs: from nucleotide 1692 to 694 is ORF15, which encodes a part of S-adenosylmethionine synthetase; and from nucleotide 692 to 1 is ORF16, which encodes a part of a protein homologous to the M. tuberculosis cbhK gene.", "1 ATGCGGCACC CCTTGGCGCC GAGCGTGGTG ATCCAGGTGC CGACCCGGGC GAGCACCTCC 61 TGCTCGGTCC AGCCCGTCTT GCTGAGCAGC AGCGCCCGCT CGTAGGCGTT CGTGAACAGC 121 AGCTCGGCTC CGTCGACGAG CTCCCGGACG CTGTCGCCCT CCAGCCGGGC GAGCTGCTGC 181 GAGGGGTCCG CGGCCCGGCG GAGGCCCAGC TCGCGGCAGA CCCGCGTGTG CCGCACCATC 241 GCCTCGGGGT CGTCCGCGCC GACGAGGACG AGGTCGATCC CGCCGGGCCG GCCGGCCGTC 301 TCGCCCAGGT CGATGTCGCG CGCCTCGGCC ATCGCGCCCG CGTAGAACGA GGCGAGCTGA 361 TTGCCGTCCT CGTCGGTGGT GCACATGAAG CGGGCGGTGT GCTGACGGTC CGACACCCGC 421 ACGGAGTCGG TGTCGACGCC CGCGGCGCGG AGCAGCTGCC CGTACCCGTC GAAGTCCTTG 481 CCGACGGCGC CGACGAGGAC GGGGCGGCGA CCGAGCAGGC CGAGGCCGTA CGCGATGTTG 541 GCGGCGACGC CGCCGTGCCG GATGTCCAGG GTGTCGACGA GGAACGACAG GGACACGTGG 601 GCGAGCTGGT CCGGCAGGAT CTGCTCGGCG AAGCGGCCCG GGAAGGTCAT CAGGTGGTCG 661 GTGGCGATCG ACCCGGTGAC GGCTATACGC ATGTCAGAGC CCCGCGGCCT TCTTCAGGGC 721 GTCCACGCGG TCGGTGCGCT CCCAGGTGAA GTCCGGCAGC TCGCGGCCGA AGTGGCCGTA 781 GGCGGCGGTC TGGGAGTAGA TCGGGCGGAG CAGGTCGAGG TCGCGGATGA TCGCGGCCGG 841 GCGGAGGTCG AAGACCTCGC CGATGGCGTT CTCGATCTTC TCGGTCTCGA TCTTGTGGGT 901 GCCGAAGGTC TCGACGAAGA GGCCGACGGG CTCGGCCTTG CCGATCGCGT ACGCGACCTG 961 GACCTCGCAG CGCGAGGCGA GACCGGCGGC GACGACGTTC TTCGCCACCC AGCGCATCGC 1021 GTACGCGGCG GAGCGGTCGA CCTTCGACGG GTCCTTGCCG GAGAAGGCGC CGCCACCGTG 1081 GCGGGCCATG CCGCCGTAGG TGTCGATGAT GATCTTGCGG CCGGTGAGGC CGGCGTCGCC 1141 CATCGGGCCG CCGATCTCGA AGCGACCGGT CGGGTTCACG AGCAGGCGGT AGCCGTCGGT 1201 GTCGAGCTTG ATGCCGTCCT CGACGAGCTG CGCAAGCACG TGCTCGACGA CGAACTTCCG 1261 CACGTCGGGG GCGAGCAGCG ACTCCAGGTC GATGTCCGAG GCGTGCTGCG AGGAGACGAC 1321 GACCGTGTCG AGACGGACCG CCCTGTCGCC GTCGTACTCG ATGGTGACCT GGGTCTTGCC 1381 GTCGGGACGC AGGTACGGGA TGGTCCCGTT CTTGCGGACC TCGGTCAGGC GGCGCGAGAG 1441 ACGGTGCGCG AGGTGGATCG GCAGCGGCAT CAGCTCGGGC GTCTCGTCCG AGGCATAGCC 1501 GAACATCAGG CCCTGGTCAC CGGCGCCCTG CTTGTCGAGC TCGTCCCCCT CGTCCCGCTG 1561 GGAGGCACCC TCGACCCGCT TCTCGTACGC GGTGTCGACA CCCTGGGCGA TGTCCGGGGA 1621 CTGCGACCCG ATGGACACCG ACACGCCGCA GGAGGCGCCG TCGAAGCCCT TCTTCGAGGA 1681 GTCGTACCCG ATC Contig 005 from cosmid pKOS023-26 contains 1565 nucleotides and contains the ORF of the picCV gene that encodes PICCV, involved in desosamine biosynthesis.", "1 CCCCGCTCGC GGCCCCCCAG ACATCCACGC CCACGATTGG ACGCTCCCGA TGACCGCCCC 61 CGCCCTCTCC GCCACCGCCC CGGCCGAACG CTGCGCGCAC CCCGGAGCCG ATCTGGGGGC 121 GGCGGTCCAC GCCGTCGGCC AGACCCTCGC CGCCGGCGGC CTCGTGCCGC CCGACGAGGC 181 CGGAACGACC GCCCGCCACC TCGTCCGGCT CGCCGTGCGC TACGGCAACA GCCCCTTCAC 241 CCCGCTGGAG GAGGCCCGCC ACGACCTGGG CGTCGACCGG GACGCCTTCC GGCGCCTCCT 301 CGCCCTGTTC GGGCAGGTCC CCGAGCTCCG CACCGCGGTC GAGACCGGCC CCGCCGGGGC 361 GTACTGGAAC AACACCCTGC TCCCGCTCGA ACAGCGCGGC GTCTTCGACG CGGCGCTCGC 421 CAGGAAGCCC GTCTTCCCGT ACAGCGTCGG CCTCTACCCC GGCCCGACCT GCATGTTCCG 481 CTGCCACTTC TGCGTCCGTG TGACCGGCGC CCGCTACGAC CCGTCCGCCC TCGACGCCGG 541 CAACGCCATG TTCCGGTCGG TCATCCACGA GATACCCGCG GGCAACCCCT CGGCGATGTA 601 CTTCTCCGGC GGCCTGGAGC CGCTCACCAA CCCCGGCCTC GGGAGCCTGG CCGCGCACGC 661 CACCGACCAC GGCCTGCGGC CCACCGTCTA CACGAACTCC TTCGCGCTCA CCGAGCGCAC 721 CCTGGAGCGC CAGCCCGGCC TCTGGGGCCT GCACGCCATC CCCACCTCGC TCTACGGCCT 781 CAACGACGAG GAGTACGAGC AGACCACCGG CAAGAAGGCC GCCTTCCGCC GCGTCCGCGA 841 GAACCTGCGC CGCTTCCAGC AGCTGCGCGC CGAGCGCGAG TCGCCGATCA ACCTCGGCTT 901 CGCCTACATC GTGCTCCCGG GCCGTGCCTC CCGCCTGCTC GACCTGGTCG ACTTCATCGC 961 CGACCTCAAC GACGCCGGGC AGGGCAGGAC GATCGACTTC GTCAACATTC GCGAGGACTA 1021 CAGCGGCCGT GACGACGGCA AGCTGCCGCA GGAGGAGCGG GCCGAGCTCC AGGAGGCCCT 1081 CAACGCCTTC GAGGAGCGGG TCCGCGAGCG CACCCCCGGA CTCCACATCG ACTACGGCTA 1141 CGCCCTGAAC AGCCTGCGCA CCGGGGCCGA CGCCGAACTG CTGCCGATCA AGCCCGCCAC 1201 CATGCGGCCC ACCGCGCACC CGCAGGTCGC GGTGCAGGTC GATCTCCTCG GCGACGTGTA 1261 CCTGTACCGC GAGGCCGGCT TCCCCGACCT GGACGGCGCG ACCCGCTACA TCGCGGGCCG 1321 CGTGACCCCC GACACCTCCC TCACCGAGGT CGTCAGGGAC TTCGTCGAGC GCGGCGGCGA 1381 GGTGGCGGCC GTCGACGGCG ACGAGTACTT CATGGACGGC TTCGATCAGG TCGTCACCGC 1441 CCGCCTGAAC CAGCTGGAGC GCGACGCCGC GGACGGCTGG GAGGAGGCCC GCGGCTTCCT 1501 GCGCTGACCC GCACCCGCCC CGATCCCCC GATCCCCCCC CCACGATCCC CCCACCTGAG 1561 GGCCC The recombinant desosamine biosynthesis and transfer and beta-glucosidase genes and proteins provided by the invention are useful in the production of glycosylated polyketides in a variety of host cells, as described in Section IV below.", "Section III.", "The picK Hydroxylase Gene The present invention provides the picK gene in recombinant form as well as recombinant PicK protein.", "The availability of the hydroxylase encoded by the picK gene in recombinant form is of significant benefit in that the enzyme can convert narbomycin into picromycin and accepts in addition a variety of polyketide substrates, particularly those related to narbomycin in structure.", "The present invention also provides methods of hydroxylating polyketides, which method comprises contacting the polyketide with the recombinant PicK enzyme under conditions such that hydroxylation occurs.", "This methodology is applicable to large numbers of polyketides.", "DNA encoding the picK gene can be isolated from cosmid pKOS023-26 of the invention.", "The DNA sequence of the picK gene is shown in the preceding section.", "This DNA sequence encodes one of the recombinant forms of the enzyme provided by the invention.", "The amino acid sequence of this form of the picK gene is shown below.", "The present invention also provides a recombinant picK gene that encodes a picK gene product in which the PicK protein is fused to a number of consecutive histidine residues, which facilitates purification from recombinant host cells.", "Amino acid sequence of picromycin/methymycin cytochrome P450 hydroxylase, PicK 1 VRRTQQGTTA SPPVLDLGAL GQDFAADPYP TYARLRAEGP AHRVRTPEGD EVWLVVGYDR 61 ARAVLADPRF SKDWRNSTTP LTEAEAALNH NMLESDPPRH TRLRKLVARE FTMRRVELLR 121 PRVQEIVDGL VDAMLAAPDG RADLMESLAW PLPITVISEL LGVPEPDRAA FRVWTDAFVF 181 PDDPAQAQTA MAEMSGYLSR LIDSKRGQDG EDLLSALVRT SDEDGSRLTS EELLGMAHIL 241 LVAGHETTVN LIANGMYALL SHPDQLAALR ADMTLLDGAV EEMLRYEGPV ESATYRFPVE 301 PVDLDGTVIP AGDTVLVVLA DAHRTPERFP DPHRFDIRRD TAGHLAFGHG IHFCIGAPLA 361 RLEARIAVRA LLERCPDLAL DVSPGELVWY PNPMIRGLKA LPIRWRRGRE AGRRTG The recombinant PicK enzyme of the invention hydroxylates narbomycin at the C12 position and YC-17 at either the C10 or C12 position.", "Hydroxylation of these compounds at the respective positions increases the antibiotic activity of the compound relative to the unhydroxylated compound.", "Hydroxylation can be achieved by a number of methods.", "First, the hydroxylation may be performed in vitro using purified hydroxylase, or the relevant hydroxylase can be produced recombinantly and utilized directly in the cell that produces it.", "Thus, hydroxylation may be effected by supplying the nonhydroxylated precursor to a cell that expresses the hydroxylase.", "These and other details of this embodiment of the invention are described in additional detail below in Section IV and the examples.", "Section IV: Heterologous Expression of the Narbonolide PKS; the Desosamine Biosynthetic and transferase Genes; the Beta-Glucosidase Gene; and the picK Hydroxylase Gene In one important embodiment, the invention provides methods for the heterologous expression of one or more of the genes involved in picromycin biosynthesis and recombinant DNA expression vectors useful in the method.", "Thus, included within the scope of the invention in addition to isolated nucleic acids encoding domains, modules, or proteins of the narbonolide PKS, glycosylation, and/or hydroxylation enzymes, are recombinant expression systems.", "These systems contain the coding sequences operably linked to promoters, enhancers, and/or termination sequences that operate to effect expression of the coding sequence in compatible host cells.", "The host cells are modified by transformation with the recombinant DNA expression vectors of the invention to contain these sequences either as extrachromosomal elements or integrated into the chromosome.", "The invention also provides methods to produce PKS and post-PKS tailoring enzymes as well as polyketides and antibiotics using these modified host cells.", "As used herein, the term expression vector refers to a nucleic acid that can be introduced into a host cell or cell-free transcription and translation medium.", "An expression vector can be maintained stably or transiently in a cell, whether as part of the chromosomal or other DNA in the cell or in any cellular compartment, such as a replicating vector in the cytoplasm.", "An expression vector also comprises a gene that serves to produce RNA, which typically is translated into a polypeptide in the cell or cell extract.", "To drive production of the RNA, the expression vector typically comprises one or more promoter elements.", "Furthermore, expression vectors typically contain additional functional elements, such as, for example, a resistance-conferring gene that acts as a selectable marker.", "The various components of an expression vector can vary widely, depending on the intended use of the vector.", "In particular, the components depend on the host cell(s) in which the vector will be introduced or in which it is intended to function.", "Components for expression and maintenance of vectors in E. coli are widely known and commercially available, as are components for other commonly used organisms, such as yeast cells and Streptomyces cells.", "One important component is the promoter, which can be referred to as, or can be included within, a control sequence or control element, which drives expression of the desired gene product in the heterologous host cell.", "Suitable promoters include those that function in eucaryotic or procaryotic host cells.", "In addition to a promoter, a control element can include, optionally, operator sequences, and other elements, such as ribosome binding sites, depending on the nature of the host.", "Regulatory sequences that allow for regulation of expression of the heterologous gene relative to the growth of the host cell may also be included.", "Examples of such regulatory sequences known to those of skill in the art are those that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus.", "Preferred host cells for purposes of selecting vector components include fungal host cells such as yeast and procaryotic, especially E. coli and Streptomyces, host cells, but single cell cultures of, for example, mammalian cells can also be used.", "In hosts such as yeasts, plants, or mammalian cells that ordinarily do not produce polyketides, it may be necessary to provide, also typically by recombinant means, suitable holo-ACP synthases to convert the recombinantly produced PKS to functionality.", "Provision of such enzymes is described, for example, in PCI publication Nos.", "WO 97/13845 and 98/27203, each of which is incorporated herein by reference.", "Control systems for expression in yeast, including controls that effect secretion are widely available and can be routinely used.", "For E. coli or other bacterial host cells, promoters such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac), and maltose, can be used.", "Additional examples include promoters derived from genes encoding biosynthetic enzymes, and the tryptophan (trp), the beta-lactamase (bla), bacteriophage lambda PL, and T5 promoters.", "In addition, synthetic promoters, such as the tac promoter (U.S. Pat.", "No.", "4,551,433), can also be used.", "Particularly preferred are control sequences compatible with Streptomyces spp.", "Particularly useful promoters for Streptomyces host cells include those from PKS gene clusters that result in the production of polyketides as secondary metabolites, including promoters from aromatic (Type II) PKS gene clusters.", "Examples of Type II PKS gene cluster promoters are act gene promoters and tcm gene promoters; an example of a Type I PKS gene cluster promoter is the spiramycin PKS gene promoter.", "If a Streptomyces or other host ordinarily produces polyketides, it may be desirable to modify the host so as to prevent the production of endogenous polyketides prior to its use to express a recombinant PKS of the invention.", "Such hosts have been described, for example, in U.S. Pat.", "No.", "5,672,491, incorporated herein by reference.", "In such hosts, it may not be necessary to provide enzymatic activities for all of the desired post-translational modifications of the enzymes that make up the recombinantly produced PKS, because the host naturally expresses such enzymes.", "In particular, these hosts generally contain holo-ACP synthases that provide the pantotheinyl residue needed for functionality of the PKS.", "Thus, in one important embodiment, the vectors of the invention are used to transform Streptomyces host cells to provide the recombinant Streptomyces host cells of the invention.", "Streptomyces is a convenient host for expressing narbonolide or 10-deoxymethynolide or derivatives of those compounds, because narbonolide and 10-deoxymethynolide are naturally produced in certain Streptomyces species, and Streptomyces generally produce the precursors needed to form the desired polyketide.", "The present invention also provides the narbonolide PKS gene promoter in recombinant form, located upstream of the picAI gene on cosmid pKOS023-27.This promoter can be used to drive expression of the narbonolide PKS or any other coding sequence of interest in host cells in which the promoter functions, particularly S. venezuelae and generally any Streptomyces species.", "As described below, however, promoters other than the promoter of the narbonolide PKS genes will typically be used for heterologous expression.", "For purposes of the invention, any host cell other than Streptomyces venezuelae is a heterologous host cell.", "Thus, S. narbonensis, which produces narbomycin but not picromycin is a heterologous host cell of the invention, although other host cells are generally preferred for purposes of heterologous expression.", "Those of skill in the art will recognize that, if a Streptomyces host that produces a picromycin or methymycin precursor is used as the host cell, the recombinant vector need drive expression of only a portion of the genes constituting the picromycin gene cluster.", "As used herein, the picromycin gene cluster includes the narbonolide PKS, the desosamine biosynthetic and transferase genes, the beta-glucosidase gene, and the picK hydroxylase gene.", "Thus, such a vector may comprise only a single ORF, with the desired remainder of the polypeptides encoded by the picromycin gene cluster provided by the genes, on the host cell chromosomal DNA.", "The present invention also provides compounds and recombinant DNA vectors useful for disrupting any gene in the picromycin gene cluster (as described above and illustrated in the examples below).", "Thus, the invention provides a variety of modified host cells (particularly, S. narbonensis and S. venezuelae) in which one or more of the genes in the picromycin gene cluster have been disrupted.", "These cells are especially useful when it is desired to replace the disrupted function with a gene product expressed by a recombinant DNA vector.", "Thus, the invention provides such Streptomyces host cells, which are preferred host cells for expressing narbonolide derivatives of the invention.", "Particularly preferred host cells of this type include those in which the coding sequence for the loading module has been disrupted, those in which one or more of any of the PKS gene ORFs has been disrupted, and/or those in which the picK gene has been disrupted.", "In a preferred embodiment, the expression vectors of the invention are used to construct a heterologous recombinant Streptomyces host cell that expresses a recombinant PKS of the invention.", "As noted above, a heterologous host cell for purposes of the present invention is any host cell other than S. venezuelae, and in most cases other than S. narbonensis as well.", "Particularly preferred heterologous host cells are those which lack endogenous functional PKS genes.", "Illustrative host cells of this type include the modified Streptomyces coelicolor CH999 and similarly modified S. lividans described in PCT publication No.", "WO 96/40968.The invention provides a wide variety of expression vectors for use in Streptomyces.", "For replicating vectors, the origin of replication can be, for example and without limitation, a low copy number vector, such as SCP2* (see Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, U.K., 1985); Lydiate et al., 1985, Gene 35: 223-235; and Kieser and Melton, 1988, Gene 65: 83-91, each of which is incorporated herein by reference), SLP1.2 (Thompson et al., 1982, Gene 20: 51-62, incorporated herein by reference), and pSG5(ts) (Muth et al., 1989, Mol.", "Gen. Genet.", "219: 341-348, and Bierman et al., 1992, Gene 116: 43-49, each of which is incorporated herein by reference), or a high copy number vector, such as pIJ101 and pJV1 (see Katz et al., 1983, J. Gen. Microbiol.", "129: 2703-2714; Vara et al., 1989, J. Bacteriol.", "171: 5782-5781; and Servin-Gonzalez, 1993, Plasmid 30: 131-140, each of which is incorporated herein by reference).", "High copy number vectors are generally, however, not preferred for expression of large genes or multiple genes.", "For non-replicating and integrating vectors and generally for any vector, it is useful to include at least an E. coli origin of replication, such as from pUC, p1P, p1I, and pBR.", "For phage based vectors, the phage phiC31 and its derivative KC515 can be employed (see Hopwood et al., supra).", "Also, plasmid pSET152, plasmid pSAM, plasmids pSE101 and pSE211, all of which integrate site-specifically in the chromosomal DNA of S. lividans, can be employed.", "Preferred Streptomyces host cell/vector combinations of the invention include S. coelicolor CH999 and, S. lividans K4-114 host cells, which do not produce actinorhodin, and expression vectors derived from the pRM1 and pRM5 vectors, as described in U.S. Pat.", "No.", "5,830,750 and U.S. patent application Ser.", "No.", "08/828,898, filed 31 Mar.", "1997, and Ser.", "No.", "09/181,833, filed 28 Oct. 1998, each of which is incorporated herein by reference.", "As described above, particularly useful control sequences are those that alone or together with suitable regulatory systems activate expression during transition from growth to stationary phase in the vegetative mycelium.", "The system contained in the illustrative plasmid pRM5, i.e., the actI/actIII promoter pair and the actII-ORF4 activator gene, is particularly preferred.", "Other useful Streptomyces promoters include without limitation those from the ermE gene and the melC1 gene, which act constitutively, and the tipA gene and the merA gene, which can be induced at any growth stage.", "In addition, the T7 RNA polymerase system has been transferred to Streptomyces and can be employed in the vectors and host cells of the invention.", "In this system, the coding sequence for the T7 RNA polymerase is inserted into a neutral site of the chromosome or in a vector under the control of the inducible merA promoter, and the gene of interest is placed under the control of the T7 promoter.", "As noted above, one or more activator genes can also be employed to enhance the activity of a promoter.", "Activator genes in addition to the actII-ORF4 gene described above include dnrI, redD, and ptpA genes (see U.S. patent application Ser.", "No.", "09/181,833, supra).", "Typically, the expression vector will comprise one or more marker genes by which host cells containing the vector can be identified and/or selected.", "Selectable markers are often preferred for recombinant expression vectors.", "A variety of markers are known that are useful in selecting for transformed cell lines and generally comprise a gene that confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.", "Such markers include, for example, genes that confer antibiotic resistance or sensitivity to the plasmid.", "Alternatively, several polyketides are naturally colored, and this characteristic can provide a built-in marker for identifying cells.", "Preferred selectable markers include antibiotic resistance conferring genes.", "Preferred for use in Streptomyces host cells are the ermE (confers resistance to erythromycin and lincomycin), tsr (confers resistance to thiostrepton), aadA (confers resistance to spectinomycin and streptomycin), aacC4 (confers resistance to apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin), hyg (confers resistance to hygromycin), and vph (confers resistance to viomycin) resistance conferring genes.", "To provide a preferred host cell and vector for purposes of the invention, the narbonolide PKS genes were placed on a recombinant expression vector that was transferred to the non-macrolide producing host Streptomyces lividans K4-114, as described in Example 3.Transformation of S. lividans K4-114 with this expression vector resulted in a strain which produced two compounds in similar yield (5-10 mg/L each).", "Analysis of extracts by LC/MS followed by 1H-NMR spectroscopy of the purified compounds established their identity as narbonolide (FIG.", "5, compound 4) and 10-deoxymethynolide (FIG.", "5, compound 5), the respective 14 and 12-membered polyketide precursors of narbomycin and YC17.To provide a host cell of the invention that produces the narbonolide PKS as well as an additional narbonolide biosynthetic gene and to investigate the possible role of the Pik TEII in picromycin biosynthesis, the picB gene was integrated into the chromosome to provide the host cell of the invention Streptomyces lividans K39-18.The picB gene was cloned into the Streptomyces genome integrating vector pSET152 (see Bierman et al., 1992, Gene 116:43, incorporated herein by reference) under control of the same promoter (PactI) as the PKS on plasmid pKOS039-86.A comparison of strains Streptomyces lividans K39-18/pKOS039-86 and KS 114/pKOS039-86 grown under identical conditions indicated that the strain containing TEII produced 47 times more total polyketide.", "This increased production indicates that the enzyme is functional in this strain and is consistent with the observation that yields fall to below 5% for both picromycin and methymycin when picB is disrupted in S. venezuelae.", "Because the production levels of compound 4 and 5 from K39-18/pKOS03986 increased by the same relative amounts, TEII does not appear to influence the ratio of 12 and 14-membered lactone ring formation.", "Thus, the invention provides methods of coexpressing the picB gene product or any other type II thioesterase with the narbonolide PKS or any other PKS in heterologous host cells to increase polyketide production.", "In accordance with the methods of the invention, picromycin biosynthetic genes in addition to the genes encoding the PKS and Pik TEII can be introduced into heterologous host cells.", "In particular, the picK gene, desosamine biosynthetic genes, and the desosaminyl transferase gene can be expressed in the recombinant host cells of the invention to produce any and all of the polyketides in the picromycin biosynthetic pathway (or derivatives thereof).", "Those of skill will recognize that the present invention enables one to select whether only the 12-membered polyketides, or only the 14-membered polyketides, or both 12- and 14-membered polyketides will be produced.", "To produce only the 12-membered polyketides, the invention provides expression vectors in which the last module is deleted or the KS domain of that module is deleted or rendered inactive.", "To produce only the 14-membered polyketides, the invention provides expression vectors in which the coding sequences of extender modules 5 and 6 are fused to provide only a single polypeptide.", "In one important embodiment, the invention provides methods for desosaminylating polyketides or other compounds.", "In this method, a host cell other than Streptomyces venezuelae is transformed with one or more recombinant vectors of the invention comprising the desosamine biosynthetic and desosaminyl transferase genes and control sequences positioned to express those genes.", "The host cells so transformed can either produce the polyketide to be desosaminylated naturally or can be transformed with expression vectors encoding the PKS that produces the desired polyketide.", "Alternatively, the polyketide can be supplied to the host cell containing those genes.", "Upon production of the polyketide and expression of the desosamine biosynthetic and desosaminyl transferase genes, the desired desosaminylated polyketide is produced.", "This method is especially useful in the production of polyketides to be used as antibiotics, because the presence of the desosamine residue is known to increase, relative to their undesosaminylated counterparts, the antibiotic activity of many polyketides significantly.", "The present invention also provides a method for desosaminylating a polyketide by transforming an S. venezuelae or S. narbonensis host cell with a recombinant vector that encodes a PKS that produces the polyketide and culturing the transformed cell under conditions such that said polyketide is produced and desosaminylated.", "In this method, use of an S. venezuelae or S. narbonensis host cell of the invention that does not produce a functional endogenous narbonolide PKS is preferred.", "In a related aspect, the invention provides a method for improving the yield of a desired desosaminylated polyketide in a host cell, which method comprises transforming the host cell with a beta-glucosidase gene.", "This method is not limited to host cells that have been transformed with expression vectors of the invention encoding the desosamine biosynthetic and desosaminyl transferase genes of the invention but instead can be applied to any host cell that desosaminylates polyketides or other compounds.", "Moreover, while the beta-glucosidase gene from Streptomyces venezuelae provided by the invention is preferred for use in the method, any beta-glucosidase gene may be employed.", "In another embodiment, the beta-glucosidase treatment is conducted in a cell free extract.", "Thus, the invention provides methods not only for producing narbonolide and 10-deoxymethynolide in heterologous host cells but also for producing narbomycin and YC-17 in heterologous host cells.", "In addition, the invention provides methods for expressing the picK gene product in heterologous host cells, thus providing a means to produce picromycin, methymycin, and neomethymycin in heterologous host cells.", "Moreover, because the recombinant expression vectors provided by the invention enable the artisan to provide for desosamine biosynthesis and transfer and/or C10 or C12 hydroxylation in any host cell, the invention provides methods and reagents for producing a very wide variety of glycosylated and/or hydroxylated polyketides.", "This variety of polyketides provided by the invention can be better appreciated upon consideration of the following section relating to the production of polyketides from heterologous or hybrid PKS enzymes provided by the invention.", "Section V: Hybrid PKS Genes The present invention provides recombinant DNA compounds encoding each of the domains of each of the modules of the narbonolide PKS, the proteins involved in desosamine biosynthesis and transfer to narbonolide, and the PicK protein.", "The availability of these compounds permits their use in recombinant procedures for production of desired portions of the narbonolide PKS fused to or expressed in conjunction with all or a portion of a heterologous PKS.", "The resulting hybrid PKS can then be expressed in a host cell, optionally with the desosamine biosynthesis and transfer genes and/or the picK hydroxylase gene to produce a desired polyketide.", "Thus, in accordance with the methods of the invention, a portion of the narbonolide PKS coding sequence that encodes a particular activity can be isolated and manipulated, for example, to replace the corresponding region in a different modular PKS.", "In addition, coding sequences for individual modules of the PKS can be ligated into suitable expression systems and used to produce the portion of the protein encoded.", "The resulting protein can be isolated and purified or can may be employed in situ to effect polyketide synthesis.", "Depending on the host for the recombinant production of the domain, module, protein, or combination of proteins, suitable control sequences such as promoters, termination sequences, enhancers, and the like are ligated to the nucleotide sequence encoding the desired protein in the construction of the expression vector.", "In one important embodiment, the invention thus provides a hybrid PKS and the corresponding recombinant DNA compounds that encode those hybrid PKS enzymes.", "For purposes of the invention, a hybrid PKS is a recombinant PKS that comprises all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a first PKS and all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a second PKS.", "In one preferred embodiment, the first PKS is most but not all of the narbonolide PKS, and the second PKS is only a portion or all of a non-narbonolide PKS.", "An illustrative example of such a hybrid PKS includes a narbonolide PKS in which the natural loading module has been replaced with a loading module of another PKS.", "Another example of such a hybrid PKS is a narbonolide PKS in which the AT domain of extender module 3 is replaced with an AT domain that binds only malonyl CoA.", "In another preferred embodiment, the first PKS is most but not all of a non-narbonolide PKS, and the second PKS is only a portion or all of the narbonolide PKS.", "An illustrative example of such a hybrid PKS includes a DEBS PKS in which an AT specific for methylmalonyl CoA is replaced with the AT from the narbonolide PKS specific for malonyl CoA.", "Those of skill in the art will recognize that all or part of either the first or second PKS in a hybrid PKS of the invention need not be isolated from a naturally occurring source.", "For example, only a small portion of an AT domain determines its specificity.", "See U.S. provisional patent application Ser.", "No.", "60/091,526, and Lau et al., infra, incorporated herein by reference.", "The state of the art in DNA synthesis allows the artisan to construct de novo DNA compounds of size sufficient to construct a useful portion of a PKS module or domain.", "Thus, the desired derivative coding sequences can be synthesized using standard solid phase synthesis methods such as those described by Jaye et al., 1984, J. Biol.", "Chem.", "259: 6331, and instruments for automated synthesis are available commercially from, for example, Applied Biosystems, Inc. For purposes of the invention, such synthetic DNA compounds are deemed to be a portion of a PKS.", "With this general background regarding hybrid PKSs of the invention, one can better appreciate the benefit provided by the DNA compounds of the invention that encode the individual domains, modules, and proteins that comprise the narbonolide PKS.", "As described above, the narbonolide PKS is comprised of a loading module, six extender modules composed of a KS, AT, ACP, and zero, one, two, or three KR, DH, and ER domains, and a thioesterase domain.", "The DNA compounds of the invention that encode these domains individually or in combination are useful in the construction of the hybrid PKS encoding DNA compounds of the invention.", "The recombinant DNA compounds of the invention that encode the loading module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.", "In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.", "The resulting construct, in which the coding sequence for the loading module of the heterologous PKS is replaced by that for the coding sequence of the narbonolide PKS loading module provides a novel PKS.", "Examples include the 6-deoxyerythronolide B, rapamycin, FK506, FK520, rifamycin, and avermectin PKS coding sequences.", "In another embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.", "In another embodiment, a portion of the loading module coding sequence is utilized in conjunction with a heterologous coding sequence.", "In this embodiment, the invention provides, for example, replacing the propionyl CoA specific AT with an acetyl CoA, butyryl CoA, or other CoA specific AT.", "In addition, the KSQ and/or ACP can be replaced by another inactivated KS and/or another ACP.", "Alternatively, the KSQ, AT, and ACP of the loading module can be replaced by an AT and ACP of a loading module such as that of DEBS.", "The resulting heterologous loading module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.", "The recombinant DNA compounds of the invention that encode the first extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.", "In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS first extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.", "The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the first extender module of the narbonolide PKS or the latter is merely added to coding sequences for modules of the heterologous PKS, provides a novel PKS coding sequence.", "In another embodiment, a DNA compound comprising a sequence that encodes the first extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.", "In another embodiment, a portion or all of the first extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.", "In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (which includes inactivating) the KR; inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, or a DH, KR, and ER.", "In addition, the KS and/or ACP can be replaced with another KS and/or ACP.", "In each of these replacements or insertions, the heterologous MS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a gene for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.", "The resulting heterologous first extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.", "In an illustrative embodiment of this aspect of the invention, the invention provides recombinant PKSs and recombinant DNA compounds and vectors that encode such PKSs in which the KS domain of the first extender module has been inactivated.", "Such constructs are especially useful when placed in translational reading frame with the remaining modules and domains of a narbonolide PKS or narbonolide derivative PKS.", "The utility of these constructs is that host cells expressing, or cell free extracts containing, the PKS encoded thereby can be fed or supplied with N-acetylcysteamine thioesters of novel precursor molecules to prepare narbonolide derivatives.", "See U.S. patent application Ser.", "No.", "60/117,384, filed 27 Jan. 1999, and PCT publication Nos.", "WO 99/03986 and 97/02358, each of which is incorporated herein by reference.", "The recombinant DNA compounds of the invention that encode the second extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.", "In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS second extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.", "The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the second extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS.", "In another embodiment, a DNA compound comprising a sequence that encodes the second extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.", "In another embodiment, a portion or all of the second extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.", "In this embodiment, the invention provides, for example, replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (or inactivating) the KR, the DH, or both the DH and KR; replacing the KR or the KR and DH with a KR, a KR and a DH, or a KR, DH, and ER; and/or inserting an ER.", "In addition, the KS and/or ACP can be replaced with another KS and/or ACP.", "In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.", "The resulting heterologous second extender module coding sequence can be utilized in conjunction with a coding sequence from a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.", "The recombinant DNA compounds of the invention that encode the third extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.", "In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS third extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.", "The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the third extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS.", "In another embodiment, a DNA compound comprising a sequence that encodes the third extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.", "In another embodiment, a portion or all of the third extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.", "In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting the inactive KR; and/or inserting a KR, or a KR and DH, or a KR, DH, and ER.", "In addition, the KS and/or ACP can be replaced with another KS and/or ACP.", "In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a gene for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.", "The resulting heterologous third extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.", "The recombinant DNA compounds of the invention that encode the fourth extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.", "In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS fourth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.", "The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fourth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS.", "In another embodiment, a DNA compound comprising a sequence that encodes the fourth extender module of the narbonolide.", "PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.", "In another embodiment, a portion of the fourth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.", "In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting any one, two, or all three of the ER, DH, and KR; and/or replacing any one, two, or all three of the ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER.", "In addition, the KS and/or ACP can be replaced with another KS and/or ACP.", "In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.", "The resulting heterologous fourth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.", "The recombinant DNA compounds of the invention that encode the fifth extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.", "In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS fifth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.", "The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fifth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS.", "In another embodiment, a DNA compound comprising a sequence that encodes the fifth extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequence for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.", "In another embodiment, a portion or all of the fifth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.", "In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (or inactivating) the KR; inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, or a DH, KR, and ER.", "In addition, the KS and/or ACP can be replaced with another KS and/or ACP.", "In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.", "The restyling heterologous fifth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.", "The recombinant DNA compounds of the invention that encode the sixth extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications.", "In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS sixth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.", "The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the sixth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS.", "In another embodiment, a DNA compound comprising a sequence that encodes the sixth extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.", "In another embodiment, a portion or all of the sixth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.", "In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; and/or inserting a KR, a KR and DH, or a KR, DH, and an ER.", "In addition, the KS and/or ACP can be replaced with another KS and/or ACP.", "In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis.", "The resulting heterologous sixth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.", "The sixth extender module of the narbonolide PKS is followed by a thioesterase domain.", "This domain is important in the cyclization of the polyketide and its cleavage from the PKS.", "The present invention provides recombinant DNA compounds that encode hybrid PKS enzymes in which the narbonolide PKS is fused to a heterologous thioesterase or a heterologous PKS is fused to the narbonolide synthase thioesterase.", "Thus, for example, a thioesterase domain coding sequence from another PKS gene can be inserted at the end of the sixth extender module coding sequence in recombinant DNA compounds of the invention.", "Recombinant DNA compounds encoding this thioesterase domain are therefore useful in constructing DNA compounds that encode the narbonolide PKS, a PKS that produces a narbonolide derivative, and a PKS that produces a polyketide other than narbonolide or a narbonolide derivative.", "The following Table lists references describing illustrative PKS genes and corresponding enzymes that can be utilized in the construction of the recombinant hybrid PKSs and the corresponding DNA compounds that encode them of the invention.", "Also presented are various references describing tailoring enzymes and corresponding genes that can be employed in accordance with the methods of the invention.", "Avermectin U.S. Pat.", "No.", "5,252,474 to Merck.", "MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds.", "(ASM), pp.", "245-256, A Comparison of the Genes Encoding the Polyketide Synthases for Avermectin, Erythromycin, and Nemadectin.", "MacNeil et al., 1992, Gene 115: 119-125, Complex Organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.", "Candicidin (FR008) Hu et al., 1994, Mol.", "Microbiol.", "14 163-172.Epothilone U.S. patent application Ser.", "No.", "60/130,560, filed 22 Apr.", "1999, and Ser.", "No.", "60/122,620, filed 3 Mar.", "1999.Erythromycin PCT Pub.", "No.", "93/13663 to Abbott.", "U.S. Pat.", "No.", "5,824,513 to Abbott.", "Donadio et al., 1991, Science 252:675-9.Cortes et al., 8 Nov. 1990, Nature 348:176-8, An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea.", "Glycosylation Enzymes PCT Pat.", "App.", "Pub.", "No.", "97/23630 to Abbott.", "FK506 Motamedi et al., 1998, The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506, Eur.", "J. biochem.", "256: 528-534.Motamedi et al., 1997, Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506, Eur.", "J. Biochem.", "244: 74-80.Methyltransferase U.S. Pat.", "No.", "5,264,355, issued 23 Nov. 1993, Methylating enzyme from Streptomyces MA6858.31-O-desmethyl-FK506 methyltransferase.", "Motamedi et al., 1996, Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520, J. Bacteriol.", "178: 5243-5248.FK520 U.S. patent application Ser.", "No.", "60/123,810, filed 11 Mar.", "1999.Nielsen et al., 1991, Biochem.", "30:5789-96.Lovastatin U.S. Pat.", "No.", "5,744,350 to Merck.", "Nemadectin MacNeil et al., 1993, supra.", "Niddamycin Kakavas et al., 1997, Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis, J. Bacteriol.", "179: 7515-7522.Oleandomycin Swan et al., 1994, Characterisation of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence, Mol.", "Gen. Genet.", "242: 358-362.Olano et al., 1998, Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the riacrolactone ring, Mol.", "Gen. Genet.", "259(3): 299-308.U.S.", "patent application Ser.", "No.", "60/120,254, filed 16 Feb. 1999, and Ser.", "No.", "60/106,000, filed 29 Oct. 1998.Platenolide EP Pat.", "App.", "Pub.", "No.", "791,656 to Lilly.", "Pradimicin PCI Pat.", "Pub.", "No.", "WO 98/11230 to Bristol-Myers Squibb.", "Rapamycin Schwecke et al., August 1995, The biosynthetic gene cluster for the polyketide rapamycin, Proc.", "Natl.", "Acad.", "Sci.", "USA 92:7839-7843.Aparicio et al., 1996, Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase, Gene 169: 9-16.Rifamycin August et al., 13 Feb. 1998, Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S669, Chemistry & Biology, 5(2): 69-79.Soraphen U.S. Pat.", "No.", "5,716,849 to Novartis.", "Schupp et al., 1995, J. Bacteriology 177: 3673-3679.A Sorangium cellulosum (Myxobacterium) Gene Cluster for the Biosynthesis of the Macrolide Antibiotic Soraphen A: Cloning, Characterization, and Homology to Polyketide Synthase Genes from Actinomycetes.", "Spiramycin U.S. Pat.", "No.", "5,098,837 to Lilly.", "Activator Gene U.S. Pat.", "No.", "5,514,544 to Lilly.", "Tylosin EP Pub.", "No.", "791,655 to Lilly.", "Kuhstoss et al., 1996, Gene 183:231-6., Production of a novel polyketide through the construction of a hybrid polyketide synthase.", "25.U.S.", "Pat.", "No.", "5,876,991 to Lilly.", "Tailoring Enzymes Merson-Davies and Cundliffe, 1994, Mol.", "Microbiol.", "13: 349-355.Analysis of five tylosin biosynthetic genes from the tylBA region of the Streptomyces fradiae genome.", "As the above Table illustrates, there are a wide variety of PKS genes that serve as readily available sources of DNA and sequence information for use in constructing the hybrid PKS-encoding DNA compounds of the invention.", "Methods for constructing hybrid PKS-encoding DNA compounds are described without reference to the narbonolide PKS in U.S. Pat.", "Nos.", "5,672,491 and 5,712,146 and PCT publication No.", "98/49315, each of which is incorporated herein by reference.", "In constructing hybrid PKSs of the invention, certain general methods may be helpful.", "For example, it is often beneficial to retain the framework of the module to be altered to make the hybrid PKS.", "Thus, if one desires to add DH and ER functionalities to a module, it is often preferred to replace the KR domain of the original module with a KR, DH, and ER domain-containing segment from another module, instead of merely inserting DH and ER domains.", "One can alter the stereochemical specificity of a module by replacement of the KS domain with a KS domain from a module that specifies a different stereochemistry.", "See Lau et al., 1999, Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units” Biochemistry 38(5):1643-1651, incorporated herein by reference.", "One can alter the specificity of an AT domain by changing only a small segment of the domain.", "See Lau et al., supra.", "One can also take advantage of known linker regions in PKS proteins to link modules from two different PKSs to create a hybrid PKS.", "See Gokhale et al., 16 Apr.", "1999, Dissecting and Exploiting Intermodular Communication in Polyketide Synthases”, Science 284: 482-485, incorporated herein by reference.", "The hybrid PKS-encoding DNA compounds of the invention can be and often are hybrids of more than two PKS genes.", "Even where only two genes are used, there are often two or more modules in the hybrid gene in which all or part of the module is derived from a second (or third) PKS gene.", "Thus, as one illustrative example, the invention provides a hybrid narbonolide PKS that contains the naturally occurring loading module and thioesterase domain as well as extender modules one, two, four, and six of the narbonolide PKS and further contains hybrid or heterologous extender modules three and five.", "Hybrid or heterologous extender modules three and five contain AT domains specific for malonyl CoA and derived from, for example, the rapamycin PKS genes.", "To construct a hybrid PKS or narbonolide derivative PKS of the invention, one can employ a technique, described in PCT Pub.", "No.", "98/27203, which is incorporated herein by reference, in which the large PKS gene cluster is divided into two or more, typically three, segments, and each segment is placed on a separate expression vector.", "In this manner, each of the segments of the gene can be altered, and various altered segments can be combined in a single host cell to provide a recombinant PKS gene of the invention.", "This technique makes more efficient the construction of large libraries of recombinant PKS genes, vectors for expressing those genes, and host cells comprising those vectors.", "The invention also provides libraries of PKS genes, PKS proteins, and ultimately, of polyketides, that are constructed by generating modifications in the narbonolide PKS so that the protein complexes produced have altered activities in one or more respects and thus produce polyketides other than the natural product of the PKS.", "Novel polyketides may thus be prepared, or polyketides in general prepared more readily, using this method.", "By providing a large number of different genes or gene clusters derived from a naturally occurring PKS gene cluster, each of which has been modified in a different way from the native cluster, an effectively combinatorial library of polyketides can be produced as a result of the multiple variations in these activities.", "As will be further described below, the metes and bounds of this embodiment of the invention can be described on both the protein level and the encoding nucleotide sequence level.", "As described above, a modular PKS “derived from” the narbonolide or other naturally occurring PKS includes a modular PKS (or its corresponding encoding gene(s)) that retains the scaffolding of the utilized portion of the naturally occurring gene.", "Not all modules need be included in the constructs.", "On the constant scaffold, at least one enzymatic activity is mutated, deleted, replaced, or inserted so as to alter the activity of the resulting PKS relative to the original PKS.", "Alteration results when these activities are deleted or are replaced by a different version of the activity, or simply mutated in such a way that a polyketide other then the natural product results from these collective activities.", "This occurs because there has been a resulting alteration of the starter unit and/or extender unit, and/or stereochemistry, and/or chain length or cyclization, and/or reductive or dehydration cycle outcome at a corresponding position in the product polyketide.", "Where a deleted activity is replaced, the origin of the replacement activity may come from a corresponding activity in a different naturally occurring PKS or from a different region of the narbonolide PKS.", "Any or all of the narbonolide PKS genes may be included in the derivative or portions of any of these may be included, but the scaffolding of the PKS protein is retained in whatever derivative is constructed.", "The derivative preferably contains a thioesterase activity from the narbonolide or another PKS.", "In summary, a PKS derived from the narbonolide PKS includes a PKS that contains the scaffolding of all or a portion of the narbonolide PKS.", "The derived PKS also contains at least two extender modules that are functional, preferably three extender modules, and more preferably four or more extender modules, and most preferably six extender modules.", "The derived PKS also contains mutations, deletions, insertions, or replacements of one or more of the activities of the functional modules of the narbonolide PKS so that the nature of the resulting polyketide is altered.", "This definition applies both at the protein and DNA sequence levels.", "Particular preferred embodiments include those wherein a KS, AT, KR, DH, or ER has been deleted or replaced by a version of the activity from a different PKS or from another location within the same PKS.", "Also preferred are derivatives where at least one non-condensation cycle enzymatic activity (KR, DH, or ER) has been deleted or added or wherein any of these activities has been mutated so as to change the structure of the polyketide synthesized by the PKS.", "Conversely, also included within the definition of a PKS derived from the narbonolide PKS are functional PKS modules or their encoding genes wherein at least one portion, preferably two portions, of the narbonolide PKS activities have bean inserted.", "Exemplary is the use of the narbonolide AT for extender module 2 which accepts a malonyl CoA extender unit rather than methylmalonyl CoA to replace a methylmalonyl specific AT in a PKS.", "Other examples include insertion of portions of non-condensation cycle enzymatic activities or other regions of narbonolide synthase activity into a heterologous PKS.", "Again, the derived from definition applies to the PKS at both the genetic and protein levels.", "Thus, there are at least five degrees of freedom for constructing a hybrid PKS in terms of the polyketide that will be produced.", "First, the polyketide chain length is determined by the number of modules in the PKS.", "Second, the nature of the carbon skeleton of the PKS is determined by the specificities of the acyl transferases that determine the nature of the extender units at each position, e.g., malonyl, methylmalonyl, ethylmalonyl, or other substituted malonyl.", "Third, the loading module specificity also has an effect on the resulting carbon skeleton of the polyketide.", "The loading module may use a different starter unit, such as acetyl, butyryl, and the like.", "As noted above and in the examples below, another method for varying loading module specificity involves inactivating the KS activity in extender module 1 (KS1) and providing alternative substrates, called diketides that are chemically synthesized analogs of extender module 1 diketide products, for extender module 2.This approach was illustrated in PCT publication Nos.", "97/02358 and 99/03986, incorporated herein by reference, wherein the KS1 activity was inactivated through mutation.", "Fourth, the oxidation state at various positions of the polyketide will be determined by the dehydratase and reductase portions of the modules.", "This will determine the presence and location of ketone and alcohol moieties and C—C double bonds or C—C single bonds in the polyketide.", "Finally, the stereochemistry of the resulting polyketide is a function of three aspects of the synthase.", "The first aspect is related to the AT/KS specificity associated with substituted malonyls as extender units, which affects stereochemistry only when the reductive cycle is missing or when it contains only a ketoreductase, as the dehydratase would abolish chirality.", "Second, the specificity of the ketoreductace may determine the chirality of any beta-OH.", "Finally, the enoylreductase specificity for substituted malonyls as extender units may influence the result when there is a complete KR/DH/ER available.", "Thus, the modular PKS systems, and in particular, the narbonolide PKS system, permit a wide range of polyketides to be synthesized.", "As compared to the aromatic PKS systems, a wider range of starter units including aliphatic monomers (acetyl, propionyl, butyryl, isovaleryl, etc.", "), aromatics (aminohydroxybenzoyl), alicyclics (cyclohexanoyl), and heterocyclics (thiazolyl) are found in various macrocyclic polyketides.", "Recent studies have shown that modular PKSs have relaxed specificity for their starter units (Kao et al., 1994, Science, supra).", "Modular PKSs also exhibit considerable variety with regard to the choice of extender units in each condensation cycle.", "The degree of beta-ketoreduction following a condensation reaction has also been shown to be altered by genetic manipulation (Donadio et al., 1991, Science, supra; Donadio et al., 1993, Proc.", "Natl.", "Acad.", "Sci.", "USA 90: 7119-7123).", "Likewise, the size of the polyketide product can be varied by designing mutants with the appropriate number of modules (Kao et al., 1994, J.", "Am.", "Chem.", "Soc.", "116:1612-11613).", "Lastly, these enzymes are particularly well known for generating an impressive range of asymmetric centers in their products in a highly controlled manner.", "The polyketides and antibiotics produced by the methods of the invention are typically single stereoisomeric forms.", "Although the compounds of the invention can occur as mixtures of stereoisomers, it may be beneficial in some instances to generate individual stereoisomers.", "Thus, the combinatorial potential within modular PKS pathways based on any naturally occurring modular, such as the narbonolide, PKS scaffold is virtually unlimited.", "The combinatorial potential is increased even further when one considers that mutations in DNA encoding a polypeptide can be used to introduce, alter, or delete an activity in the encoded polypeptide.", "Mutations can be made to the native sequences using conventional techniques.", "The substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes.", "Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion.", "See, e.g., Kunkel, 1985, Proc.", "Natl.", "Acad.", "Sci.", "USA 82: 448; Geisselsoder et al., 1987, BioTechniques 5:786.Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence, at a temperature below the melting temperature of the mismatched duplex.", "The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located, See Zoller and Smith, 1983, Methods Enzymol.", "100:468.Primer extension is effected using DNA polymerase, the product cloned, and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected.", "Identification can be accomplished using the mutant primer as a hybridization probe.", "The technique is also applicable for generating multiple point mutations.", "See, e.g., Dalbie-McFarland et al., 1982, Proc.", "Natl.", "Acad.", "Sci.", "USA 79: 6409.PCR mutagenesis can also be used to effect the desired mutations.", "Random mutagenesis of selected portions of the nucleotide sequences encoding enzymatic activities can also be accomplished by several different techniques known in the art, e.g., by inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing synthetic mutants, or by damaging plasmid DNA in vitro with chemicals.", "Chemical mutagens include, for example, sodium bisulfite, nitrous acid, nitrosoguanidine, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as 5-bromouracil, 2-aminopurine, or acrdine intercaculating agents such as proflavine, acriflavine, quinacrine, and the like.", "Generally, plasmid DNA or DNA fragments are treated with chemicals, transformed into E. coli and propagated as a pool or library of mutant plasmids.", "In constructing a hybrid PKS of the invention, regions encoding enzymatic activity, i.e., regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS, can be recovered, for example, using PCR techniques with appropriate primers.", "By “corresponding” activity encoding regions is meant those regions encoding the same general type of activity.", "For example, a KR activity encoded at one location of a gene cluster “corresponds” to a KR encoding activity in another location in the gene cluster or in a different gene cluster.", "Similarly, a complete reductase cycle could be considered corresponding.", "For example, KR/DH/ER corresponds to KR alone.", "If replacement of a particular target region in a host PKS is to be made, this replacement can be conducted in vitro using suitable restriction enzymes.", "The replacement can also be effected in vivo using recombinant techniques involving homologous sequences framing the replacement gene in a donor plasmid and a receptor region in a recipient plasmid.", "Such systems, advantageously involving plasmids of differing temperature sensitivities are described, for example, in PCT publication No.", "WO 96/40968, incorporated herein by reference.", "The vectors used to perform the various operations to replace the enzymatic activity in the host PKS genes or to support mutations in these regions of the host PKS genes can be chosen to contain control sequences operably linked to the resulting coding sequences in a manner such that expression of the coding sequences can be effected in an appropriate host.", "However, simple cloning vectors may be used as well.", "If the cloning vectors employed to obtain PKS genes encoding derived PKS lack control sequences for expression operably linked to the encoding nucleotide sequences, the nucleotide sequences are inserted into appropriate expression vectors.", "This need not be done individually, but a pool of isolated encoding nucleotide sequences can be inserted into expression vectors, the resulting vectors transformed or transfected into host cells, and the resulting cells plated out into individual colonies.", "The various PKS nucleotide sequences can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter.", "The PKS subunit encoding regions can include flanking restriction sites to allow for the easy deletion and insertion of other PKS subunit encoding sequences so that hybrid PKSs can be generated.", "The design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site-directed mutagenesis and PCR.", "The expression vectors containing nucleotide sequences encoding a variety of PKS enzymes for the production of different polyketides are then transformed into the appropriate host cells to construct the library.", "In one straightforward approach, a mixture of such vectors is transformed into the selected host cells and the resulting cells plated into individual colonies and selected to identify successful transformants.", "Each individual colony has the ability to produce a particular PKS synthase and ultimately a particular polyketide.", "Typically, there will be duplications in some, most, or all of the colonies; the subset of the transformed colonies that contains a different PKS in each member colony can be considered the library.", "Alternatively, the expression vectors can be used individually to transform hosts, which transformed hosts are then assembled into a library.", "A variety of strategies are available to obtain a multiplicity of colonies each containing a PKS gene cluster derived from the naturally occurring host gene cluster so that each colony in the library produces a different PKS and ultimately a different polyketide.", "The number of different polyketides that are produced by the library is typically at least four, more typically at least ten, and preferably at least 20, and more preferably at least 50, reflecting similar numbers of different altered PKS gene clusters and PKS gene products.", "The number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length is quite large.", "Methods for introducing the recombinant vectors of the invention into suitable hosts are known to those of skill in the art and typically include the use of CaCl2 or agents such as other divalent cations, lipofection, DMSO, protoplast transformation, infection, transfection, and electroporation.", "The polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized.", "The polyketides produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity.", "The libraries of the invention can thus be considered at four levels: (1) a multiplicity of colonies each with a different PKS encoding sequence; (2) colonies that contain the proteins that are members of the PKS library produced by the coding sequences; (3) the polyketides produced; and (4) antibiotics or compounds with other desired activities derived from the polyketides.", "Of course, combination libraries can also be constructed wherein-members of a library derived, for example, from the narbonolide PKS can be considered as a part of the same library as those derived from, for example, the rapamycin PKS or DEBS.", "Colonies in the library are induced to produce the relevant synthases and thus to produce the relevant polyketides to obtain a library of polyketides.", "The polyketides secreted into the media can be screened for binding to desired targets, such as receptors, signaling proteins, and the like.", "The supernatants per se can be used for screening, or partial or complete purification of the polyketides can first be effected.", "Typically, such screening methods involve detecting the binding of each member of the library to receptor or other target ligand.", "Binding can be detected either directly or through a competiton assay.", "Means to screen such libraries for binding are well known in the art.", "Alternatively, individual polyketide members of the library can be tested against a desired target.", "In this event, screens wherein the biological response of the target is measured can more readily be included.", "Antibiotic activity can be verified using typical screening assays such as those set forth in Lehrer et al., 1991, J. Immunol.", "Meth.", "137:167-173, incorporated herein by reference, and in the examples below.", "The invention provides methods for the preparation of a large number of polyketides.", "These polyketides are useful intermediates in formation of compounds with antibiotic or other activity through hydroxylation and glycosylation reactions as described above.", "In general, the polyketide products of the PKS must be further modified, typically by hydroxylation and glycosylation, to exhibit antibiotic activity.", "Hydroxylation results in the novel polyketides of the invention that contain hydroxyl groups at C6, which can be accomplished using the hydroxylase encoded by the erF gene, and/or C12, which can be accomplished using the hydroxylase encoded by the picK or eryK gene.", "The presence of hydroxyl groups at these positions can enhance the antibiotic activity of the resulting compound relative to its unhydroxylated counterpart.", "Gycosylation is important in conferring antibiotic activity to a polyketide as well.", "Methods for glycosylating the polyketides are generally known in the art; the glycosylation may be effected intracellularly by providing the appropriate glycosylation enzymes or may be effected in vitro using chemical synthetic means as described herein and in PCT publication No.", "WO 98/49315, incorporated herein by reference.", "Preferably, glycosylation with desosamine is effected in accordance with the methods of the invention in recombinant host cells provided by the invention.", "In general, the approaches to effecting glycosylation mirror those described above with respect to hydroxylation.", "The purified enzymes, isolated from native sources or recombinantly produced may be used in vitro.", "Alternatively and as noted, glycosylation may be effected intracellularly using endogenous or recombinantly produced intracellular glycosylases.", "In addition, synthetic chemical methods may be employed.", "The antibiotic modular polyketides may contain any of a number of different sugars, although D-desosamine, or a close analog thereof, is most common.", "Erythromycin, picromycin, narbomycin and methymycin contain desosamine.", "Erythromycin also contains L-cladinose (34-methyl mycarose).", "Tylosin contains mycaminose (4-hydroxy desosamine), mycarose and 6-deoxy-D-allose.", "2-acetyl-1-bromodesosamine has been used as a donor to glycosylate polyketides by Masamune et al., 1975, J.", "Am.", "Chem.", "Soc.", "97: 3512-3513.Other, apparently more stable donors include glycosyl fluorides, thioglycosides, and trichloroacetimidates; see Woodward et al., 1981, J.", "Am.", "Chem.", "Soc.", "103: 3215; Martinet al., 1997, J.", "Am.", "Chem.", "Soc.", "119: 3193; Toshima et al., 1995, J.", "Am.", "Chem.", "Soc.", "117: 3717; Matsumoto et al., 1988, Tetrahedron Lett.", "29: 3575.Glycosylation can also be effected using the polyketide aglycones as starting materials and using Saccharopolyspora erythraea or Streptomyces venezuelae to make the conversion, preferably using mutants unable to synthesize macrolides.", "To provide an illustrative hybrid PKS of the invention as well as an expression vector for that hybrid PKS and host cells comprising the vector and producing the hybrid polyketide, a portion of the narbonolide PKS gene was fused to the DEBS genes.", "This construct also allowed the examination of whether the TE domain of the narbonolide PKS (pikTE) could promote formation of 12-membered lactones in the context of a different PKS.", "A construct was generated, plasmid pKOS039-18, in which the pikTE ORF was fused with the DEBS genes in place of the DEBS TE ORF (see FIG.", "5).", "To allow the TE to distinguish between substrates most closely resembling those generated by the narbonolide PKS, the fusion junction was chosen between the AT and ACP to eliminate ketoreductase activity in DEBS extender module 6 (KR6).", "This results in a hybrid PKS that presents the TE with a β-ketone heptaketide intermediate and a β-(S)-hydroxy hexaketide intermediate to cyclize, as in narbonolide and 10-deoxymethynolide biosynthesis.", "Analysis of this construct indicated the production of the 14-membered ketolide 3,6-dideoxy-3-oxo-erythronolide B (FIG.", "5, compound 6).", "Extracts were analyzed by LC/MS.", "The identity of compound 6 was verified by comparison to a previously authenticated sample (see PCT publication No.", "98/49315, incorporated herein by reference).", "The predicted 12-membered macrolactone, (8R,9S)-8,9-dihydromethyl-9-hydroxy-10-deoxymethynolide (see Kao et al, 1995, J.", "Am.", "Chem.", "Soc.", "127, incorporated herein by reference) was not detected.", "This result, along with others reported herein, suggests that protein interactions between the narbonolide PKS modules play a role in formation of the 12 and 14-membered macrolides.", "The above example illustrates also how engineered PKSs can be improved for production of novel compounds.", "Compound 6 was originally produced by deletion of the KR6 domain in DEBS to create a 3-ketolide producing PKS (see U.S. patent application Ser.", "No.", "09/073,538, filed 6 May 1998, and PCT publication No.", "WO 98/49315, each of which is incorporated herein by reference).", "Although the desired molecule was made, purification of compound 6 from this strain was hampered by the presence of 2-desmethyl ketolides that could not be easily separated.", "Extracts from Streptomyces lividans KS 114/pKOS039-18, however, do not contain the 2-desmethyl compounds, greatly simplifying purification.", "Thus, the invention provides a useful method of producing such compounds.", "The ability to combine the narbonolide PKS with DEBS and other modular PKSs provides a significant advantage in the production of macrolide antibiotics.", "Two other hybrid PKSs of the invention were constructed that yield this same compound.", "These constructs also illustrate the method of the invention in which hybrid PKSs are constructed at the protein, as opposed to the module, level.", "Thus, the invention provides a method for constructing a hybrid PKS which comprises the coexpression of at least one gene from a first modular PKS gene cluster in a host cell that also expresses at least one gene from a second PKS gene cluster.", "The invention also provides novel hybrid PKS enzymes prepared in accordance with the method.", "This method is not limited to hybrid PKS enzymes composed of at least one narbonolide PKS gene, although such constructs are illustrative and preferred.", "Moreover, the hybrid PKS enzymes are not limited to hybrids composed of unmodified proteins; as illustrated below, at least one of the genes can optionally be a hybrid PKS gene.", "In the first construct, the eryAI and eryAI genes were coexpressed with picAIV and a gene encoding a hybrid extender module 5 composed of the KS and AT domains of extender module 5 of DEBS3 and the KR and ACP domains of extender module 5 of the narbonolide PKS.", "In the second construct, the picAIV coding sequence was fused to the hybrid extender module 5 coding sequence used in the first construct to yield a single protein.", "Each of these constructs produced 3-deoxy-3-oxo-6-deoxyerythronolide B.", "In a third construct, the coding sequence for extender module 5 of DEBS3 was fused to the picAIV coding sequence, but the levels of product produced were below the detection limits of the assay.", "A variant of the first construct hybrid PKS was constructed that contained an inactivated DEBS1 extender module 1 KS domain.", "When host cells containing the resultant hybrid PKS were supplied the appropriate diketide precursor, the desired 13-desethyl-13-propyl compounds were obtained, as described in the examples below.", "Other illustrative hybrid PKSs of the invention were made by coexpressing the picAI and picAII genes with genes encoding DEBS3 or DEBS3 variants.", "These constructs illustrate the method of the invention in which a hybrid PKS is produced from coexpression of PKS genes unmodified at the modular or domain level.", "In the first construct, the enjAIII gene was coexpressed with the picAI and picAII genes, and the hybrid PKS produced 10-desmethyl-10,11-anhydro-6-deoxyerythronolide B in Streptomyces lividans.", "Such a hybrid PKS could also be constructed in accordance with the method of the invention by transformation of S. venzuelae with an expression vector that produces the enyAIII gene product, DEBS3.In a preferred embodiment, the S. venezuelae host cell has been modified to inactivate the picAIII gene.", "In the second construct, the DEBS3 gene was a variant that had an inactive KR in extender module 5.The hybrid PKS produced 5,6-dideoxy-5-oxo-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans.", "In the third construct, the DEBS3 gene was a variant in which the KR domain of extender module 5 was replaced by the DH and KR domains of extender module 4 of the rapamycin PKS.", "This construct produced 5,6-dideoxy-5-oxo-10-desmethyl-10,11-anhydroerythronolide B and 5,6-dideoxy-4,5-anhydro-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH and KR domains functioned only inefficiently in this construct.", "In the fourth construct, the DEBS3 gene was a variant in which the KR domain of extender module 5 was replaced by the DH, KR, and ER domains of extender module 1 of the rapamycin PKS.", "This construct produced 5,6-dideoxy-5-oxo-10-desmethyl-10,11-anhydroerythronolide B as well as 5,6-dideoxy-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH, KR, and ER domains functioned only inefficiently in this construct.", "In the fifth construct, the DEBS3 gene was a variant in which the KR domain of extender module 6 was replaced by the DH and KR domains of extender module 4 of the rapamycin PKS.", "This construct produced 3,6-dideoxy-2,3-anhydro-10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans.", "In the sixth construct, the DEBS3 gene was a variant in which the AT domain of extender module 6 was replaced by the AT domain of extender module 2 of the rapamycin PKS.", "This construct produced 2,10-didesmethyl-10,11-anhydro-6-deoxyerythronolide B in Streptomyces lividans.", "These hybrid PKSs illustrate the wide variety of polyketides that can be produced by the methods and compounds of the invention.", "These polyketides are useful as antibiotics and as intermediates in the synthesis of other useful compounds, as described in the following section.", "Section VI: Compounds The methods and recombinant DNA compounds of the invention are useful in the production of polyketides.", "In one important aspect, the invention provides methods for making ketolides, polyketide compounds with significant antibiotic activity.", "See Griesgraber et al., 1996, J. Antibiot.", "49: 465-477, incorporated herein by reference.", "Most if not all of the ketolides prepared to date are synthesized using erythromycin A, a derivative of 6-dEB, as an intermediate.", "While the invention provides hybrid PKSs that produce a polyketide different in structure from 6-dEB, the invention also provides methods for making intermediates useful in preparing traditional, 6-dEB-derived ketolide compounds.", "Because 6-dEB in part differs from narbonolide in that it comprises a 10-methyl group, the novel hybrid PKS genes of the invention based on the narbonolide PKS provide many novel ketolides that differ from the known ketolides only in that they lack a 10-methyl group.", "Thus, the invention provides the 10-desmethyl analogues of the ketolides and intermediates and precursor compounds described in, for example, Griesgraber et al., supra; Agouridas et al., 1998, J. Med.", "Chem.", "41: 4080-4100, U.S. Pat.", "Nos.", "5,770,579; 5,760,233; 5,750,510; 5,747,467; 5,747,466; 5,656,607; 5,635,485; 5,614,614; 5,556,118; 5,543,400; 5,527,780; 5,444,051; 5,439,890; 5,439,889; and PCT publication Nos.", "WO 98/09978 and 98/28316, each of which is incorporated herein by reference.", "Because the invention also provides hybrid PKS genes that include a methylmalonyl-specific AT domain in extender module 2 of the narbonolide PKS, the invention also provides hybrid PKS that can be used to produce the 10-methyl-containing ketolides known in the art.", "Thus, a hybrid PKS of the invention that produces 10-methyl narbonolide is constructed by substituting the malonyl-specific AT domain of the narbonolide PKS extender module 2 with a methylmalonyl specific AT domain from a heterologous PKS.", "A hybrid narbonolide PKS in which the AT of extender module 2 was replaced with the AT from DEBS extender module 2 was constructed using boundaries described in PCT publication No.", "98/49315, incorporated herein by reference.", "However, when the hybrid PKS expression vector was introduced into Streptomyces venezuelae, detectable quantities of 10-methyl picromycin were not produced.", "Thus, to construct such a hybrid PKS of the invention, an AT domain from a module other than DEBS extender module 2 is preferred.", "One could also employ DEBS extender module 2 or another methylmalonyl specific AT but utilize instead different boundaries than those used for the substitution described above.", "In addition, one can construct such a hybrid PKS by substituting, in addition to the AT domain, additional extender module 2 domains, including the KS, the KR, and the DH, and/or additional extender module 3 domains.", "Although modification of extender module 2 of the narbonolide PKS is required, the extent of hybrid modules engineered need not be limited to module 2 to make 10-methyl narbonolide.", "For example, substitution of the KS domain of extender module 3 of the narbonolide PKS with a heterologous domain or module can result in more efficient processing of the intermediate generated by the hybrid extender module 2.Likewise, a heterologous TE domain may be more efficient in cyclizing 10-methyl narbonolide.", "Substitution of the entire extender module 2 of the narbonolide PKS with a module encoding the correct enzymatic activities, i.e., a KS, a methylmalonyl specific AT, a KR, a DH, and an ACP, can also be used to create a hybrid PKS of the invention that produces a 10-methyl ketolide.", "Modules useful for such whole module replacements include extender modules 4 and 10 from the rapamycin PKS, extender modules 1 and 5 from the FK506 PKS, extender module 2 of the tylosin PKS, and extender module 4 of the rifamycin PKS.", "Thus, the invention provides many different hybrid PKSs that can be constructed starting from the narbonolide PKS that can be used to produce 10-methyl narbonolide.", "While 10-methyl narbonolide is referred to in describing these hybrid PKSs, those of skill recognize that the invention also therefore provides the corresponding derivatives produces by glycosylation and hydroxylation.", "For example, if the hybrid PKS is expressed in Streptomyces narbonensis or S. venezuelae, the compounds produced are 10-methyl narbomycin and picromycin, respectively.", "Alternatively, the PKS can be expressed in a host cell transformed with the vectors of the invention that encode the desosamine biosynthesis and desosaminyl transferase and picK hydroxylase genes.", "Other important compounds provided by the invention are the 6-hydroxy ketolides.", "These compounds include 3-deoxy-3-oxo erythronolide B, 6-hydroxy narbonolide, and 6-hydroxy-10-methyl narbonolide.", "In the examples below, the invention provides a method for utilizing EryF to hydroxylate 3-ketolides that is applicable for the production of any 6-hydroxy-3-ketolide.", "Thus, the hybrid PKS genes of the invention can be expressed in a host cell that contains the desosamine biosynthetic genes and desosaminyl transferase gene as well as the required hydroxylase gene(s), which may be either picK (for the C12 position) or eryK (for the C12 position) and/or eryF (for the C6 position).", "The resulting compounds have antibiotic activity but can be further modified, as described in the patent publications referenced above, to yield a desired compound with improved or otherwise desired properties.", "Alternatively, the aglycone compounds can be produced in the recombinant host cell, and the desired glycosylation and hydroxylation steps carried out in vitro or in vivo, in the latter case by supplying the converting cell with the aglycone.", "The compounds of the invention are thus optionally glycosylated forms of the polyketide set forth in formula (2) below which are hydroxylated at either the C6 or the C12 or both.", "The compounds of formula (2) can be prepared using the loading and the six extender modules of a modular PKS, modified or prepared in hybrid form as herein described.", "These polyketides have the formula: including the glycosylated and isolated stereoisomeric forms thereof; wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-4C; each of R1-R6 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted; each of X1-X5 is independently two H, H and OH, or ═O; or each of X1-X5 is independently H and the compound of formula (2) contains a double-bond in the ring adjacent to the position of said X at 2-3, 4-5, 6-7,8-9 and/or 10-11; with the proviso that: at least two of R1-R6 are alkyl (1-4C).", "Preferred compounds comprising formula 2 are those wherein at least three of R1-R5 are alkyl (1-4C), preferably methyl or ethyl; more preferably wherein at least four of R1-R5 are alkyl (1-4C), preferably methyl or ethyl.", "Also preferred are those wherein X2 is two H, ═O, or H and OH, and/or X3 is H, and/or X1 is OH and/or X5 is OH and/or X5 is OH.", "Also preferred are compounds with variable R* when R1-R5 is methyl, X2 is ═O, and X1, X4 and X5 are OH.", "The glycosylated forms of the foregoing are also preferred.", "The invention also provides the 12-membered macrolides corresponding to the compounds above but produced from a narbonolide-derived PKS lacking extender modules 5 and 6 of the narbonolide PKS.", "The compounds of the invention can be produced by growing and fermenting the host cells of the invention under conditions known in the art for the production of other polyketides.", "The compounds of the invention can be isolated from the fermentation broths of these cultured cells and purified by standard procedures.", "The compounds can be readily formulated to provide the pharmaceutical compositions of the invention.", "The pharmaceutical compositions of the invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid, or liquid form.", "This preparation will contain one or more of the compounds of the invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application.", "The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.", "The carriers which can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquefied form.", "In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.", "For example, the compounds of the invention may be utilized with hydroxypropyl methylcellulose essentially as described in U.S. Pat.", "No.", "4,916,138, incorporated herein by reference, or with a surfactant essentially as described in EPO patent publication No.", "428,169, incorporated herein by reference.", "Oral dosage forms may be prepared essentially as described by Hondo et al., 1987, Transplantation Proceedings XIX, Supp.", "6: 17-22, incorporated herein by reference.", "Dosage forms for external application may be prepared essentially as described in EPO patent publication No.", "423,714, incorporated herein by reference.", "The active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the disease process or condition.", "For the treatment of conditions and diseases caused by infection, a compound of the invention may be administered orally, topically, parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvant, and vehicles.", "The term parenteral, as used herein, includes subcutaneous injections, and intravenous, intramuscular, and intrasternal injection or infusion techniques.", "Dosage levels of the compounds of the invention are of the order from about 0.01 mg to about 50 mg per kilogram of body weight per day, preferably from about 0.1 mg to about 10 mg per kilogram of body weight per day.", "The dosage levels are useful in the treatment of the above-indicated conditions (from about 0.7 mg to about 3.5 mg per patient per day, assuming a 70 kg patient).", "In addition, the compounds of the invention may be administered on an intermittent basis, i.e., at semi-weekly, weekly, semi-monthly, or monthly intervals.", "The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.", "For example, a formulation intended for oral administration to humans may contain from 0.5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material, which may vary from about 5 percent to about 95 percent of the total composition.", "Dosage unit forms will generally contain from about 0.5 mg to about 500 mg of active ingredient.", "For external administration, the compounds of the invention may be formulated within the range of, for example, 0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, and most preferably from about 0.005% to 0.8% by weight.", "It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors.", "These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the particular disease or condition for which therapy is sought.", "A detailed description of the invention having been provided above, the following examples are given for the purpose of illustrating the invention and shall not be construed as being a limitation on the scope of the invention or claims.", "EXAMPLE 1 General Methodology Bacterial strains, plasmids, and culture conditions.", "Streptomyces coelicolor CH999 described in WO 95/08548, published 30 Mar.", "1995, or S. lividans K4-114, described in Ziermann and Betlach, Jan. 99, Recombinant Polyketide Synthesis in Streptomyces: Engineering of Improved Host Strains, BioTechniques 26:106-110, incorporated herein by reference, was used as an expression host.", "DNA manipulations were performed in Escherichia coli XL1-Blue, available from Stratagene.", "E. coli MC1061 is also suitable for use as a host for plasmid manipulation.", "Plasmids were passaged through E. coli ET12567 (dam dcm hsdS Cmr) (MacNeil, 1988, J. Bacteriol.", "170: 5607, incorporated herein by reference) to generate unmethylated DNA prior to transformation of S. coelicolor.", "E. coli strains were grown under standard conditions.", "S. coelicolor strains were grown on R2YE agar plates (Hopwood et al., Genetic manipulation of Streptomyces.", "A laboratory manual.", "The John Innes Foundation: Norwich, 1985, incorporated herein by reference).", "Many of the expression vectors of the invention illustrated in the examples are derived from plasmid pRM5, described in WO 95/08548, incorporated herein by reference.", "This plasmid includes a colEI replicon, an appropriately truncated SCP2* Streptomyces replicon, two act-promoters to allow for bidirectional cloning, the gene encoding the actII-ORF4 activator which induces transcription from act promoters during the transition from growth phase to stationary phase, and appropriate marker genes.", "Engineered restriction sites in the plasmid facilitate the combinatorial construction of PKS gene clusters starting from cassettes encoding individual domains of naturally occurring PKSs.", "When plasmid pRM5 is used for expression of a PKS, all relevant biosynthetic genes can be plasmid-borne and therefore amenable to facile manipulation and mutagenesis in E. coli.", "This plasmid is also suitable for use in Streptomyces host cells.", "Streptomyces is genetically and physiologically well-characterized and expresses the ancillary activities required for in vivo production of most polyketides.", "Plasmid pRM5 utilizes the act promoter for PKS gene expression, so polyketides are produced in a secondary metabolite-like manner, thereby alleviating the toxic effects of synthesizing potentially bioactive compounds in vivo.", "Manipulation of DNA and organisms.", "Polymerase chain reaction (PCR) was performed using Pfu polymerase (Stratagene; Taq polymerase from Perkin Elmer Cetus can also be used) under conditions recommended by the enzyme manufacturer.", "Standard in vitro techniques were used for DNA manipulations (Sambrook et al.", "Molecular Cloning: A Laboratory Manual (Current Edition)).", "E. coli was transformed using standard calcium chloride-based methods; a Bio-Rad E. coli pulsing apparatus and protocols provided by Bio-Rad could also be used.", "S. coelicolor was transformed by standard procedures (Hopwood et al.", "Genetic manipulation of Streptomyces.", "A laboratory manual.", "The John Innes Foundation: Norwich, 1985), and depending on what selectable marker was employed, transformants were selected using 1 mL of a 1.5 mg/mL thiostrepton overlay, 1 mL of a 2 mg/mL apramycin overlay, or both.", "EXAMPLE 2 Cloning of the Picromycin Biosynthetic Gene Cluster from Streptomyices venezuelae Genomic DNA (100 μg) isolated from Streptomyces venezuelae ATCC15439 using standard procedures was partially digested with Sau3AI endonuclease to generate fragments ˜40 kbp in length.", "SuperCosI (Stratagene) DNA cosmid arms were prepared as directed by the manufacturer.", "A cosmid library was prepared by ligating 2.5 μg of the digested genomic DNA with 1.5 μg of cosmid arms in a 20 μL reaction.", "One microliter of the ligation mixture was propagated in E. coli XL1-Blue MR (Stratagene) using a GigapackIII XL packaging extract kit (Stratagene).", "The resulting library of ˜3000 colonies was plated on a 10×150 mm agar plate and replicated to a nylon membrane.", "The library was initially screened by direct colony hybridization with a DNA probe specific for ketosynthase domain coding sequences of PKS genes.", "Colonies were alkaline lysed, and the DNA was crosslinked to the membrane using UV irradiation.", "After overnight incubation with the probe at 42° C., the membrane was washed twice at 25° C. in 2×SSC buffer+0.1% SDS for 15 minutes, followed by two 15 minute washes with 2×SSC buffer at 55° C. Approximately 30 colonies gave positive hybridization signals with the degenerate probe.", "Several cosmids were selected and divided into two classes based on restriction digestion patterns.", "A representative cosmid was selected from each class for further analysis.", "The representative cosmids were designated pKOS023-26 and pKOS023-27.These cosmids were determined by DNA sequencing to comprise the narbonolide PKS genes, the desosamine biosynthesis and transferase genes, the beta-glucosidase gene and the picK hydroxylase gene.", "These cosmids were deposited with the American Type Culture Collection in accordance with the terms of the Budapest Treaty.", "Cosmid pKOS023-26 was assigned accession number ATCC 203141, and cosmid pKOS023-27 was assigned accession number ATCC 203142.To demonstrate that the narbonolide PKS genes had been cloned and to illustrate how the invention provides methods and reagents for constructing deletion variants of narbonolide PKS genes, a narbonolide PKS gene was deleted from the chromosome of Streptomyces venezuelae.", "This deletion is shown schematically in FIG.", "4, parts B and C. A ˜2.4 kb EcoRI-KpnI fragment and a ˜2.1 kb KpnI-XhoI fragment, which together comprise both ends of the picAI gene (but lack a large portion of the coding sequence), were isolated from cosmid pKOS023-27 and ligated together into the commercially available vector pLitmus 28 (digested with restriction enzymes EcoRI and XhoI) to give plasmid pKOS039-07.The ˜4.5 kb HindIII-SpeI fragment from plasmid pKOS039-07 was ligated with the 2.5 kb HindIII-NheI fragment of integrating vector pSET152, available from the NRRL, which contains an E. coli origin of replication and an apramycin resistance-conferring gene to create plasmid pKOS039-16.This vector was used to transform S. venezuelae, and apramycin-resistant transformants were selected.", "Then, to select for double-crossover mutants, the selected transformants were grown in TSB liquid medium without antibiotics for three transfers and then plated onto non-selective media to provide single colony isolates.", "The isolated colonies were tested for sensitivity to apramycin, and the apramycin-sensitive colonies were then tested to determine if they produced picromycin.", "The tests performed included a bioassay and LC/MS analysis of the fermentation media.", "Colonies determined not to produce picromycin (or methymycin or neomethymycin) were then analyzed using PCR to detect an amplification product diagnostic of the deletion.", "A colony designated K3903 was identified, providing confirmation that the narbonolide PKS genes had been cloned.", "Transformation of strain K39-03 with plasmid pKOS039-27 comprising an intact picA gene under the control of the ermE* promoter from plasmid pWHM3 (see Vara et al., 1989, J. Bact.", "171: 5872-5881, incorporated herein by reference) was able to restore picromycin production.", "To determine that the cosmids also contained the picK hydroxylase gene, each cosmid was probed by Southern hybridization using a labeled DNA fragment amplified by PCR from the Saccharopolyspora erythraea C12-hydroxylase gene, eryK.", "The cosmids were digested with BamHI endonuclease and electrophoresed on a 1% agarose gel, and the resulting fragments were transferred to a nylon membrane.", "The membrane was incubated with the enyK probe overnight at 42° C., washed twice at 25° C. in 2×SSC buffer with 0.1% SDS for 15 minutes, followed by two 15 minute washes with 2×SSC buffer at 50° C. Cosmid pKOS023-26 produced an ˜3 kb fragment that hybridized with the probe under these conditions.", "This fragment was subcloned into the PCRscript™ (Stratagene) cloning vector to yield plasmid pKOS023-28 and sequenced.", "The ˜1.2 kb gene designated picK above was thus identified.", "The picK gene product is homologous to eryK and other known macrolide cytochrome P450 hydroxylases.", "By such methodology, the complete set of picromycin biosynthetic genes were isolated and identified.", "DNA sequencing of the cloned DNA provided further confirmation that the correct genes had been cloned.", "In addition, and as described in the following example, the identity of the genes was confirmed by expression of narbomycin in heterologous host cells.", "EXAMPLE 3 Heterologous Expression of the Narbonolide PKS and the Picromycin Biosynthetic Gene Cluster To provide a preferred host cell and vector for purposes of the invention, the narbonolide PKS was transferred to the non-macrolide producing host Streptomyces lividans K4-114 (see Ziermann and Betlach, 1999, Biotechniques 26, 106-110, and U.S. patent application Ser.", "No.", "09/181,833, filed 28 Oct. 1998, each of which is incorporated herein by reference).", "This was accomplished by replacing the three DEBS ORFs on a modified version of pCK7 (see Kao et al., 1994, Science 265, 509-512, and U.S. Pat.", "No.", "5,672,491, each of which is incorporated herein by reference) with all four narbonolide PKS ORFs to generate plasmid pKOS039-86 (see FIG.", "5).", "The pCK7 derivative employed, designated pCK7‘Kan’, differs from pCK7 only in that it contains a kanamycin resistance conferring gene inserted at its HindIII restriction enzyme recognition site.", "Because the plasmid contains two selectable markers, one can select for both markers and so minimize contamination with cells containing rearranged, undesired vectors.", "Protoplasts were transformed using standard procedures and transformants selected using overlays containing antibiotics.", "The strains were grown in liquid R5 medium for growth/seed and production cultures at 30° C. Transformed strains produced two compounds in similar yield (˜5-10 mg/L each).", "Polyketides produced in the host cells were analyzed by bioassay against Bacillus subtilis and by LC/MS analysis.", "Analysis of extracts by LC/MS followed by 1H-NMR spectroscopy of the purified compounds established their identity as narbonolide (FIG.", "5, compound 4; see Kaiho et al., 1982, J. Org.", "Chem.", "47: 1612-1614, incorporated herein by reference) and 10-deoxymethynolide (FIG.", "5, compound 5; see Lambalot et al., 1992, J.", "Antibiotics 45, 1981-1982, incorporated herein by reference), the respective 14 and 12-membered polyketide aglycones of YC17, narbomycin, picromycin, and methymycin.", "The production of narbonolide in Streptomyces lividans represents the expression of an entire modular polyketide pathway in a heterologous host.", "The combined yields of compounds 4 and 5 are similar to those obtained with expression of DEBS from pCK7 (see Kao et al., 1994, Science 265: 509-512, incorporated herein by reference).", "Furthermore, based on the relative ratios (˜1:1) of compounds 4 And 5 produced, it is apparent that the narbonolide PKS itself possesses an inherent ability to produce both 12 and 14-membered macrolactones without the requirement of additional activities unique to S. venezuelae.", "Although the existence of a complementary enzyme present in S. lividans that provides this function is possible, it would be unusual to find such a specific enzyme in an organism that does not produce any known macrolide.", "To provide a heterologous host cell of the invention that produces the narbonolide PKS and the picB gene, the picB gene was integrated into the chromosome of Streptomyces lividans harboring plasmid pKOS039-86 to yield S. lividans K39-18/pKOS039-86.To provide the integrating vector utilized, the picB gene was cloned into the Streptomyces genome integrating vector pSET152 (see Bierman et al., 1992, Gene 116, 43, incorporated herein by reference) under control of the same promoter (Pact1) as the PKS on plasmid pKOS039-86.A comparison of strains K39-18/pKOS39-86 and K4-114/pKOS039-86 grown under identical conditions indicated that the strain containing TEII produced 47 times more total polyketide.", "Each strain was grown in 30 mL of R5 (see Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory Manual; John Innes Foundation: Norwich, UK, 1985, incorporated herein by reference) liquid (with 20 μg/mL thiostrepton) at 30° C. for 9 days.", "The fermentation broth was analyzed directly by reverse phase HPLC.", "Absorbance at 235 nm was used to monitor compounds and measure relative abundance.", "This increased production indicates that the enzyme is functional in this strain.", "As noted above, because the production levels of compound 4 and 5 from K39-18/pKOS03986 increased by the same relative amounts, TEII does not appear to influence the ratio of 12 and 14-membered lactone ring formation.", "To express the glycosylated counterparts of narbonolide (narbomycin) and 10-deoxymethynolide (YC17) in heterologous host cells, the desosamine biosynthetic genes and desosaminyl transferase gene were transformed into the host cells harboring plasmid pKOS039-86 (and, optionally, the picB gene, which can be integrated into the chromosome as described above).", "Plasmid pKOS039-104, see FIG.", "6, comprises the desosamine biosynthetic genes, the beta-glucosidase gene, and the desosaminyl transferase gene.", "This plasmid was constructed by first inserting a polylinker oligonucleotide, containing a restriction enzyme recognition site for PacI, a Shine-Dalgarno sequence, and restriction enzyme recognition sites for NdeI, BglII, and HindIII, into a pUC19 derivative, called pKOS2447, to yield plasmid pKOS039-98.An ˜0.3 kb PCR fragment comprising the coding sequence for the N-terminus of the desI gene product and an ˜0.12 kb PCR fragment comprising the coding sequence for the C-terminus of the desR gene product were amplified from cosmid pKOS23-26 (ATCC 203141) and inserted together into pLitmus28 treated with restriction enzymes NsiI and EcoRI to produce plasmid pKOS039-101.The ˜6 kb SphI-PstI restriction fragment of pKOS23-26 containing the desI, desII, desIII, desIV, and desV genes was inserted into plasmid pUC19 (Stratagene) to yield plasmid pKOS039-102.The ˜6 kb SphI-EcoRI restriction fragment from plasmid pKOS039-102 was inserted into pKOS039-101 to produce plasmid pKOS039-103.The ˜6 kb BglII-PstI fragment from pKOS23-26 that contains the desR, desVI, desVII, and desVIII genes was inserted into pKOS39-98 to yield pKOS39-100.The ˜6 kb PacI-PstI restriction fragment of pKOS39-100 and the 6.4 kb NsiI-EcoRI fragment of pKOS39-103 were cloned into pKOS39-44 to yield pKOS39-104.When introduced into Streptomyces lividans host cells comprising the recombinant narbonolide PKS of the invention, plasmid pKOS39-104 drives expression of the desosamine biosynthetic genes, the beta-glucosidase gene, and the desosaminyl transferase gene.", "The glycosylated antibiotic narbomycin was produced in these host cells, and it is believed that YC17 was produced as well.", "When these host cells are transformed with vectors that drive expression of the picK gene, the antibiotics methymycin, neomethymycin, and picromycin are produced.", "In similar fashion, when plasmid pKOS039-18, which encodes a hybrid PKS of the invention that produces 3-deoxy-3-oxo-6-deoxyerythronolide B was expressed in Streptomyces lividans host cells transformed with plasmid pKOS39-104, the 5-desosaminylated analog was produced.", "Likewise, when plasmid pCK7, which encodes DEBS, which produces 6-deoxyerythronolide B, was expressed in Streptomyces lividans host cells transformed with plasmid pKC639-104, the 5-desosaminylated analog was produced.", "These compounds have antibiotic activity and are useful as intermediates in the synthesis of other antibiotics.", "EXAMPLE 4 Expression Vector for Desosaminyl Transferase While the invention provides expression vectors comprising all of the genes required for desosamine biosynthesis and transfer to a polyketide, the invention also provides expression vectors that encode any subset of those genes or any single gene.", "As one illustrative example, the invention provides an expression vector for desosaminyl transferase.", "This vector is useful to desosaminylate polyketides in host cells that produce NDP-desosamine but lack a desosaminyl transferase gene or express a desosaminyl transferase that does not function as efficiently on the polyketide of interest as does the desosaminyl transferase of Streptomyces venezuelae.", "This expression vector was constructed by first amplifying the desosaminyl transferase coding sequence from pKOS023-27 using the primers: N3917: 5′-CCCTGCAGCGGCAAGGAAGGACACGACGCCA-3′; and N3918: 5′-AGGTCTAGAGCTCAGTGCCGGGCGTCGGCCGG-3′, to give a 1.5 kb product.", "This product was then treated with restriction enzymes PstI and XbaI and ligated with HindIII and XbaI digested plasmid pKOS039-06 together with the 7.6 kb PstI-HindIII restriction fragment of plasmid pWHM1104 to provide plasmid pKOS039-14.Plasmid pWHM1104, described in Tang et al., 1996, Molec.", "Microbiol.", "22(5): 801-813, incorporated herein by reference, encodes the ermE* promoter.", "Plasmid pKOS039-14 is constructed so that the desosaminyl transferase gene is placed under the control of the ermE* promoter and is suitable for expression of the desosaminyl transferase in Streptomyces, Saccharopolyspora erythraea, and other host cells in which the ermE* promoter functions.", "EXAMPLE 5 Heterologous Expression of the picK Gene Product in E. coli The picK gene was PCR amplified from plasmid pKOS023-28 using the oligonucleotide primers: N024-36B (forward): 5′-TTGCATGCATATGCGCCGTACCCAGCAGGGAACGACC; and N024-37B (reverse): 5′-TTGAATTCTCAACTAGTACGGCGGCCCGCCTCCCGTCC.", "These primers alter the Streptomyces GTG start codon to ATG and introduce a SpeI site at the C-terminal end of the gene, resulting in the substitution of a serine for the terminal glycine amino acid residue.", "The blunt-ended PCR product was subcloned into the commercially available vector pCRscript at the SrfI site to yield plasmid pKOS023-60.An ˜1.3 kb NdeI-XhoI fragment was then inserted into the NdeI/XhoI sites of the T7 expression vector pET22b (Novagen, Madison, Wis.) to generate pKOS023-61.Plasmid pKOS023-61 was digested with restriction enzymes SpeI and EcoRI, and a short linker fragment encoding 6 histidine residues and a stop codon (composed of oligonucleotides 30-85a: 5′-CTAGTATGCATCATCATCATCATCATTAA-3′; and 30-85b: 5′-AATTTTAATGATGATGATGATGATGCATA-3′) was inserted to obtain plasmid pKOS023-68.Both plasmid pKOS023-61 and pKOS023-68 produced active PicK enzyme in recombinant E. coli host cells.", "Plasmid pKOS023-61 was transformed into E. coli BL21-DE3.Successful transformants were grown in LB-containing carbenicillin (100 μg/ml) at 37° C. to an OD600 of 0.6.Isopropyl-beta-D-thiogalactopyranoside (G) was added to a final concentration of 1 mM, and the cells were grown for an additional 3 hours before harvesting.", "The cells were collected by centrifugation and frozen at −80° C. A control culture of BL21-DE3 containing the vector plasmid pET21c (Invitrogen) was prepared in parallel.", "The frozen BL21-DE3/pKOS023-61 cells were thawed, suspended in 2 μL of cold cell disruption buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris/HCl, pH 8.0) and sonicated to facilitate lysis.", "Cellular debris and supernatant were separated by centrifugation and subjected to SDSPAGE on 10-15% gradient gels, with Coomassie Blue staining, using a Pharmacia Phast Gel Electrophoresis system.", "The soluble crude extract from BL21-DE3/pKOS023-61 contained a Coomassie stained band of Mr˜46 kDa, which was absent in the control strain BL21-DE3/pET21c.", "The hydroxylase activity of the picK protein was assayed as follows.", "The crude supernatant (20 μL) was added to a reaction mixture (100 μL total volume) containing 50 mM Tris/HCl (pH 7.5), 20 μM spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+oxidoreductase, 0.8 Unit of glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6 phosphate, and 20 mmol of narbomycin.", "The narbomycin was purified from a culture of Streptomyces narbonensis, and upon LC/MS analysis gave a single peak of [M+H]+=510.The reaction was allowed to proceed for 105 minutes at 30° C. Half of the reaction mixture was loaded onto an HPLC, and the effluent was analyzed by evaporative light scattering (ELSD) and mass spectrometry.", "The control extract (BL21-DE3/pET21c) was processed identically.", "The BL21-DE3/pKOS023-61 reaction contained a compound not present in the control having the same retention time, molecular weight and mass fragmentation pattern as picromycin ([M+H]+=526).", "The conversion of narbomycin to picromycin under these conditions was estimated to be greater than 90% by ELSD peak area.", "The poly-histidine-linked PicK hydroxylase was prepared from pKOS023-68 transformed into E. coli BL21 (DE3) and cultured as described above.", "The cells were harvested and the PicK protein purified as follows.", "All purification steps were performed at 4° C. E. coli cell pellets were suspended in 32 μL of cold binding buffer (20 mM Tris/HCl, pH 8.0, 5 mM imidazole, 500 mM NaCl) per mL of culture and lysed by sonication.", "For analysis of E. coli cell-free extracts, the cellular debris was removed by low-speed centrifugation, and the supernatant was used directly in assays.", "For purification of PicK/6-His, the supernatant was loaded (0.5 mL/min.)", "onto a 5 mL HiTrap Chelating column (Pharmacia, Piscataway, N.J.), equilibrated with binding buffer.", "The column was washed with 25 μL of binding buffer and the protein was eluted with a 35 μL linear gradient (5-500 mM imidazole in binding buffer).", "Column effluent was monitored at 280 nm and 416 nm.", "Fractions corresponding to the 416 nm absorbance peak were pooled and dialyzed against storage buffer (45 mM Tris/HCl, pH 7.5, 0.1 mM EDTA, 0.2 mM DTT, 10% glycerol).", "The purified 46 kDa protein was analyzed by SDSPAGE using Coomassie blue staining, and enzyme concentration and yield were determined.", "Narbomycin was purified as described above from a culture of Streptomyces narbonensis ATCC19790.Reactions for kinetic assays (100 μL) consisted of 50 mM Tris/HCl (pH 7.5), 100 μM spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 U glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6-phosphate, 20-500 μM narbomycin substrate, and 50-500 nM of PicK enzyme.", "The reaction proceeded at 30° C., and samples were withdrawn for analysis at 5, 10, 15, and 90 minutes.", "Reactions were stopped by heating to 100° C., for 1 minute, and denatured protein was removed by centrifugation.", "Depletion of narbomycin and formation of picromycin were determined by high performance liquid chromatography (HPLC, Beckman C-18 0.46×15 cm column) coupled to atmospheric pressure chemical ionization (APCI) mass spectroscopic detection (Perkin Elmer/Sciex API 100) and evaporative light scattering detection (Alltech 500 ELSD).", "EXAMPLE 6 Expression of the picK Gene Encoding the Hydroxalase in Streptomyces narbonensis To produce picromycin in Streptomyces narbonensis, a host that produces narbomycin but not picromycin, the methods and vectors of the invention were used to express the picK gene in this host.", "The picK gene was amplified from cosmid pKOS023-26 using the primers: N3903: 5′-TCCTCTAGACGTTTCCGT-3′; and N3904: 5′-TGAAGCTTGAATTCAACCGGT-3′ to obtain an ˜1.3 kb product.", "The product was treated with restriction enzymes XbaI and HindIII and ligated with the 7.6 kb XbaI-HindIII restriction fragment of plasmid pWHM1104 to provide plasmid pKOS039-01, placing the picK gene under the control of the ermE* promoter The resulting plasmid was transformed into purified stocks of S. narbonensis by protoplast fusion and electroporation.", "The transformants were grown in suitable media and shown to convert narbomycin to picromycin at a yield of over 95%.", "EXAMPLE 7 Construction of a Hybrid DEBS/Narbonolide PKS This example describes the construction of illustrative hybrid PKS expression vectors of the invention.", "The hybrid PKS contains portions of the narbonolide PKS and portions of rapamycin and/or DEBS PKS.", "In the first constructs, pKOS039-18 and pKOS039-19 the hybrid PKS comprises the narbonolide PKS extender module 6 ACP and thioesterase domains and the DEBS loading module and extender modules 1-5 as well as the KS and AT domains of DEBS extender module 6 (but not the KR domain of extender module 6).", "In pKOS039-19, the hybrid PKS is identical except that the KS1 domain is inactivated, i.e., the ketosynthase in extender module 1 is disabled.", "The inactive DEBS KS1 domain and its construction are described in detail in PCT publication Nos.", "WO 97/02358 and 99/03986, each of which is incorporated herein by reference.", "To construct pKOS039-18, the 2.33 kb BamHI-EcoRI fragment of pKOS023-27, which contains the desired sequence, was amplified by PCR and subcloned into plasmid pUC9.The primers used in the PCR were: N3905: 5′-TTTATGCATCCCGCGGGTCCCGGCGAG-3′; and N3906: 5′-TCAGAATTCTGTCGGTCACTTGCCCGC-3′.", "The 1.6 kb PCR product was digested with PstI and EcoRI and cloned into the corresponding sites of plasmid pKOS015-52 (this plasmid contains the relevant portions of the coding sequence for the DEBS extender module 6) and commercially available plasmid pLitmus 28 to provide plasmids pKOS039-12 and pKOS039-13, respectively.", "The BglII-EcoRI fragment of plasmid pKOS039-12 was cloned into plasmid pKOS011-77, which contains the functional DEBS gene cluster and into plasmid pJRJ2, which contains the mutated DEBS gene that produces a DEBS PKS in which the KS domain of extender module I has been rendered inactive.", "Plasmid pJRJ2 is described in PCI publication Nos.", "99/03986 and 97/02358, incorporated herein by reference.", "Plasmids pKOS039-18 and pKOS039-19, respectively, were obtained.", "These two plasmids were transformed into Streptomyces coelicolor CH999 by protoplast fusion.", "The resulting cells were cultured under conditions such that expression of the PKS occurred.", "Cells transformed with plasmid pKOS039-18 produced the expected product 3-deoxy-3-oxo-6-deoxyerythronolide B.", "When cells transformed with plasmid pKOS039-19 were provided (2S,3R)-2-methyl-3-hydroxyhexanoate NACS, 13-desethyl-13-propyl-3-deoxy-3-oxo-6-deoxyerythronolide B was produced.", "EXAMPLE 8 6-Hydroxylation of 3,6-dideoxy-3-oxoerythronolide B Using the eryF Hydroxylase Certain compounds of the invention can be hydroxylated at the C6 position in a host cell that expresses the eryF gene.", "These compounds can also be hydroxylated in vitro, as illustrated by this example.", "The 6-hydroxylase encoded by eryF was expressed in E. coli, and partially purified.", "The hydroxylase (100 pmol in 10 μL) was added to a reaction mixture (100 μl total volume) containing 50 mM Tris/HCl (pH 7.5), 20 μM spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 Unit of glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6-phosphate, and 10 nmol 6-deoxyerythronolide B.", "The reaction was allowed to proceed for 90 minutes at 30° C. Half of the reaction mixture was loaded onto an HPLC, and the effluent was analyzed by mass spectrometry.", "The production of erythronolide B as evidenced by a new peak eluting earlier in the gradient and showing [M+H]+=401.Conversion was estimated at 50% based on relative total ion counts.", "Those of skill in the art will recognize the potential for hemiketal formation in the above compound and compounds of similar structure.", "To reduce the amount of hemiketal formed, one can use more basic (as opposed to acidic) conditions or employ sterically hindered derivative compounds, such as 5-desosaminylated compounds.", "EXAMPLE 9 Measurement of Antibacterial Activity Antibacterial activity was determined using either disk diffusion assays with Bacillus cereus as the test organism or by measurement of minimum inhibitory concentrations (MIC) in liquid culture against sensitive and resistant strains of Staphylococcus pneumoniae.", "Example 10 Construction of Desosamine Containing Polyketide Libraries Using a Glycosyltransferase with Broad Substrate Specificity Desosamine is an important deoxyaminosugar present on a number of structurally related macrolide antibiotics such as erythromycin and is the only glycoside present on picromycin, methymycin, and the highly potent semisynthetic ketolides.", "In this example, a set of nine deoxysugar biosynthetic and auxiliary genes from the picromycin/methymycin (pik) cluster was integrated in the chromosome of Streptomyces lividans to create a host that synthesizes TDP-D-desosamine and can be used in combination with PKS expression plasmids to generate libraries of desosaminylated polyketides.", "The versatility of the DesVII desosaminyltransferase is demonstrated by formation of desosaminylated macrolides from more than twenty different 14-membered lactones.", "The attachment of desosamine is sufficient to confer antibiotic activity to each of the otherwise inactive aglycones, reinforcing the belief that this sugar plays a critical role in the molecular binding properties of erythromycin and related macrolides.", "This host and others that can be engineered to produce deoxysugar and polyketide tailoring pathways in accordance with the methods of the invention are valuable tools for expanding the size and diversity of polyketides that can be generated by combinatorial biosynthesis.", "References cited in this example are indicated by a reference number; the numbered list of references is located at the end of this example.", "All references cited are incorporated herein by reference.", "Much of the structural diversity and complexity among polyketides can be attributed to the chemistry performed by PKSs (1), and the modular architecture of catalytic domains within PKSs has been exploited by different rational and combinatorial engineering approaches to create polyketide diversity (24).", "However, structural variability among polyketides can also result from post-PKS biosynthetic steps, including oxidation and/or glycosylation with unique deoxy and amino sugars.", "Such modifications are often necessary to impart or enhance the specific biological activity of the molecule.", "For example, erythromycin A contains two deoxysugar moieties, L-cladinose and D-desosamine, that are required for antibacterial activity and the absence of either carbohydrate results in loss of potency.", "Although some chemical modifications to erythromycin have been discovered that can ameliorate the loss of the cladinose residue (5-7), there has been no substitution found for desosamine.", "This important deoxyaminosugar is also present in other macrolide antibiotics, such as oleandomycin and megalomicin, and is the only glycoside necessary to confer antibacterial activity to picromycin, methymycin, and the semisynthetic ketolide pharmacophores.", "Polyketide libraries generated by genetic modification of macrolide PKSs in which enzymatic domains and entire protein subunits were removed, added, or exchanged in various combinations have been produced (3, 4, 8).", "Because these libraries were constructed in heterologous hosts lacking glycosylation pathways, only the corresponding aglycones were produced.", "The methods and reagents of the present invention can be used to expand the capabilities of the combinatorial biosynthesis strategies described to incorporate post-PKS tailoring steps, in particular the addition of deoxysugar components.", "Some experiments have been performed in which structurally modified macrolactones are subsequently glycosylated in their native hosts (9-13), and also in bioconversion experiments in which a modified aglycone is fed to a PKS blocked mutant strain (14).", "These experiments indicate that glycosyltransferases are able to accept polyketide substrates with some amount of structural alteration.", "However, neither of these approaches is well-suited for the production and biological screening of large numbers of compounds, because most polyketide host organisms are difficult to manipulate genetically and the bioconversion of aglycones requires a tedious initial purification step.", "A more practical approach is the heterologous expression of deoxysugar biosynthetic pathways in hosts that have been developed for library expression.", "Although the effort to clone entire deoxysugar biosynthetic pathways in a heterologous organism can be a significant initial investment (most deoxysugars require six or more enzymatic steps whose genes are typically scattered within a polyketide gene cluster), these expression vectors, once made, can be easily combined with those containing PKSs to engineer glycosylated libraries rapidly.", "Olano et al.", "recently utilized a two-plasmid system to produce L-daunosamine, the deoxyaminosugar of daunorubicin and doxorubicin, in Streptomyces lividans (15).", "Here we report the development of a single expression vector for the production of desosaminylated macrolides in Streptomyces.", "Desosamine was selected as the sugar constituent, because it was believed that addition of this single deoxysugar would be sufficient to confer antibacterial activity upon macrolactones to which it was attached.", "The expression vector was combined with a library of existing PMS expression plasmids to produce several novel glycosylated macrolide compounds in S. lividans, providing the first examples in which both polyketide and deoxysugar pathways have been placed in a single heterologous host.", "A.", "Material and Methods (i) Strains, Culture Conditions, and DNA Manipulation DNA manipulation was performed in Escherichia coli XL1-Blue (Stratagene) using standard protocols (16).", "Bacillis subtilis was grown in LB at 37° C. PCR was performed with Pfu polymerase (Stratagene) under conditions recommended by the manufacturer.", "S. lividans K4-114 (17) was used as the host for expression of engineered PKS and desosamine genes.", "S. lividans strains were maintained on R2YE agar plates (18) with appropriate antibiotic selection.", "S. lividans protoplasts were transformed by the standard procedure (18) and transformants were selected using 1 ml of a 1 mg/ml thiostrepton and/or 1 ml of a 2 mg/ml apramycin overlay on R2YE regeneration plates.", "(ii) Construction of Expression Plasmids Expression plasmid pKOS39-104 was constructed as follows.", "The 6.0 kb Bgl II-Pst I fragment containing the picromycin des VIII, des VII, desVI and desR (partial) genes from cosmid pKOS23-26 (19) was subcloned into the Bgl II-Pst I sites of pKOS39-98, a pUC19 derivative with a redesigned multiple cloning site.", "The resulting plasmid, pKOS39-100, contains a Pac I site upstream of the Bgl II site which is used in a later cloning step.", "The 6 kb Sph I-Pst I fragment containing the desI (partial), desII, desIII, desIV and des V genes from pKOS23-26 was subcloned into the Sph I-Pst I of pUC19 to make pKOS39-102.The remaining 3′-end of the desR gene and 5′-end of the desI gene were PCR amplified from cosmid pKOS23-26 with the following oligonucleotides (restriction sites shown in italics): desR gene: forward 5′-AGATGCATTTCTGGGATGCCGCCACGGA; and reverse 5′-CGTCTAGACGTCACCAGACGTTGACCGTG; desI gene: forward 5′-TTTCTAGACGGTGGCCCGGAGGGAACATC; and reverse 5′-CGGAATTCCGCAGCTGGTCGGCGGCGCA.", "The two PCR fragments were digested with Nsi I-Xba I and Xba I-EcoR I, respectively, and ligated with Nsi I-EcoR I digested Litmus 28 (New England Biolabs) to obtain pKOS39-101B.", "The 6 kb Sph I-EcoR I fragment of pKOS39-102 was inserted into pKOS39-101B to make pKOS39-103.The 6.4 kb Nsi I-EcoR I fragment of pKOS39-103 and the 6 kb Pac I-Pst I fragment of pKOS39-100 were then ligated together with the 8.5 kb Pac I-EcoR I fragment of pKOS39-44 (20), yielding the final expression plasmid pKOS39-104.A restriction site and function map of this plasmid is shown below.", "(iii) Production and Analysis of Compounds All stains were grown in 5 ml liquid R2YE medium at 30° C. and analyzed following 5 days growth.", "For bioconversion experiments, aglycones (˜10 mg/liter) were fed at the start of fermentation.", "Fermentation broth was analyzed directly by liquid chromatography/mass spectrometry (LC/MS) and evaporative light scattering detection (ELSD) as previously described (20).", "An authentic sample of narbomycin prepared from Streptomyces narbonensis (19) was used to validate production of this compound.", "For LC/MS analysis of strains containing PKS expression plasmids the cultures were extracted twice with 5 ml of ethyl acetate/triethylamine (99:1), concentrated to dryness and resuspended in 0.5 ml of acetonitrile.", "(iv) Antibacterial Assays Extracts prepared from the culture broths as above were assayed for biological activity against.", "B. subtilis using an agar plate diffusion method (see Example 9).", "Samples (5 μl) from each of the extracts were pipetted to sterile filter disks, dried, and placed on an LB plate spread with 20 μl of an overnight culture of B. subtilis.", "The plates were incubated overnight at 37° C. to visualize zones of growth inhibition.", "B.", "Results (i) Construction and Validation of a Desosamine Expression System The picromycin/methymycin (pik) gene cluster from Streptomyces venezuelae (21) was chosen as the source of desosamine biosynthetic genes rather than other available clusters (i.e.", "erythromycin, oleandomycin, or megalomicin) for several reasons.", "First, all of the genes required for biosynthesis of TDP desosamine from glucose-1-phosphate, a primary metabolite, as well as the desosaminyl transferase are present in the pik cluster whereas one or more of the genes are missing or not yet identified in each of the other clusters.", "Second, the genes from the pik cluster are comprised in a single contiguous segment of DNA (the des cluster), compared to those in other clusters which are dispersed among other genes, facilitating cloning and plasmid construction.", "The organization of these genes in the picromycin biosynthetic gene cluster is shown below, followed by the depiction of the biosynthetic pathway.", "Third, the natural substrates for the desosaminyl transferase from the pik gene cluster, narbonolide and 10-deoxymethynolide, are themselves aglycones; in each of the other cases, desosamine is attached subsequent to addition of at least one other sugar.", "Furthermore, the difference in macrolactone ring sizes between narbonolide and 10-deoxymethynolide (14 and 12 atoms, respectively) suggests that the desosaminyl transferase from this cluster is somewhat forgiving towards its polyketide substrate.", "Seven genes in the des cluster, desI, desII, desIII, desIV, desV, desVI, and des VIII, are presumed to be responsible for the biosynthesis of TDP-D-desosamine (22).", "Also present is the des VII gene encoding the glycosyltransferase.", "In addition to catalyzing the transfer of desosamine to both 12- and 14-membered macrolactones, it has been shown that DesVII is able to incorporate non-natural deoxysugar substrates (22, 23).", "The desR gene encodes a β-glucosidase that removes a glucose residue attached to the C-2′ hydroxyl of desosamine (24).", "It is believed that the glucosylation of desosamine containing macrolides like methymycin, picromycin, and oleandomycin, causes inactivation and provides self-resistance to these compounds which are reactivated by a 13-glucosidase upon export (24, 25).", "S. lividans is known to possess at least two such glucosyltransferases which inactivate erythromycin and picromycin by the same mechanism (26).", "Therefore, it was important to include this gene for expression in S. lividans to produce desosaminylated compounds without the glucose modification.", "The expression system used here was adopted from the multi-vector system developed for separate expression of erythromycin PKS, or 6-deoxyerythronolide B synthase (DEBS), subunits in Streptomyces (4, 27; see also U.S. Pat.", "No.", "6,033,883).", "Plasmid pKOS39-104 contains the des genes cloned in a single orientation under control of the actI promoter and actII-44 activator.", "Since pKOS39-104 is a derivative of pSET152 (28), it contains the phiC31-int-attP loci for chromosomal integration in Streptomyces and can be used in conjunction with the pRM5-based PKS expression plasmid library (3; see also U.S. Pat.", "No.", "5,672,491).", "S. lividans K4114 was transformed with pKOS39-104 and designated K39-22.Confirmation that this strain produced TDP-D-desosamine was performed by feeding aglycones to the strain and looking for the presence of desosaminylated compounds by LC/MS analysis.", "Four aglycones (10 mg/liter each) were fed to liquid fermentations of S. lividans K39-22: narbonolide and 10-deoxymethynolide, the natural substrates for DesVII, 3-keto-6-deoxyerythronolide B (dEB), and 6-dEB.", "Fermentation broth from all four aglycone fed strains displayed antibacterial activity against B. subtilis whereas S. lividans K39-22 alone produced no detectable activity.", "LC/MS analysis demonstrated that each of the corresponding desosaminylated compounds narbomycin, 10-deoxymethymycin (YC17), 3-keto-5-O-desosaminyl-6-dEB, and 5-O-desosaminyl-6-dEB were produced.", "In each case, the parent ion (M+H+) of the expected compound was detected in addition to a characteristic ion at 158 amu produced by the desosamine fragment.", "Production of narbomycin in the narbonolide fed strain was further confirmed by comparison to authentic narbomycin obtained from S. narbonensis.", "LC/MS also revealed that a significant amount (50-90%) of the aglycone remained unconverted in each of the samples.", "These results established that the des expression vector was functional and that the DesVII glycosyltransferase was able to glycosylate non-natural macrolactone substrates.", "The bioassay results also confirmed that desosamine is sufficient to confer antibacterial activity to these macrolactones.", "There were no 2′-O-glucosyl derivatives detected, which indicates that the DesR glucosidase included in pKOS39-104 was also operational, although minor glucosylated products were putatively found in subsequent experiments with the strain (see below).", "(ii) Co-Expression of Desosamine and Aglycone Pathways in S. lividans.", "Although expression of both a modular polyketide pathway and a deoxysugar pathway together in a heterologous host has not been reported, the bioconversion results suggested that transformation of S. lividans K39-22 with plasmids encoding macrolide PKSs would lead to production of desosaminylated compounds.", "Plasmids encoding the PKSs that, in S. lividans, produce the same four aglycones used in the bioconversion studies were therefore transformed into S. lividans K39-22.Plasmid pKOS39-86 contains the picromycin/methymycin PKS and produces both narbonolide and 10-deoxymethynolide (20).", "Plasmid pKAO127 contains DEBS and produces 6-dEB (17).", "Plasmid pKOS39-18 contains DEBS with a modified terminal module that produces 3-keto dB (20).", "Culture broth from each of the transformed strains displayed activity against B. subtilis.", "LC/MS analysis as above confirmed the presence of each of the expected desosaminylated compounds as well as their aglycone precursors and minor amounts of the corresponding 2′-O-glucosyl derivatives.", "The total yield of narbomycin and 10-deoxymethymycin in S. lividans K39-22/pKOS3986 was approximately 1 mg/liter each and represents about a 20% conversion of the total aglycone produced.", "Thus, although both PKS and deoxysugar pathways function as expected, complete glycosylation of even the natural substrates for DesVII did not occur under these conditions.", "S. lividans K39-22 contains a copy of the ermE macrolide resistance gene, and no obvious growth defects were observed with production of the biologically active compounds.", "These results suggest that a limiting amount of TDP-desosamine is being produced by the strain under these conditions.", "(iii) Production and Biological Screening of a Glycosylated Macrolide Library Over 50 PKS expression plasmids have been constructed and tested in using DEBS and other macrolide PKS genes (3, 8, 20).", "These PKSs produce a variety of 14-membered macrolactones in which single or multiple carbon centers have been altered.", "Each plasmid contains the same pRM5-based vector as above, providing a convenient opportunity to expand and diversify any existing aglycone library by routine transformation of S. lividans K39-22.Because a C-5 hydroxyl would be necessary for glycosylation, a subset of 19 additional plasmids encoding PKSs that produce compounds containing this functional group was selected and tested.", "The desired desosaminylated polyketides would theoretically possess antibiotic activity, and the transformed strains can therefore be readily analyzed in a simple bioassay for production of glycosylated macrolides.", "All of the strains transformed and tested displayed antimicrobial activity against B. subtilis.", "The presumed structures of the desosamine containing compounds, based on the structures of the aglycones produced by the PKS on each plasmid, are shown below.", "Culture extracts from six of these stains (those containing plasmids pKOS15-22, pKOS15-106, pKOS39-20, pKOS1142, pKOS15-30, and pKOS2415) were examined by LC/MS and, in each case, the expected parent ion was found along with the 158 amu desosamine fragment.", "Two compounds were detected in the strain containing pKOS15-106 with molecular weights corresponding to 3-hydroxy and 3-keto derivatives.", "This is consistent with both aglycones being produced by plasmid pKOS15-109 in S. lividans.", "Two compounds were also detected in the strain with pKOS112, the predicted molecule, 5-O-desosaminyl-10-desmethyl-6-dEB, and a putative dehydrated derivative at carbons C-10 and C-11.Both aglycones were also produced when the plasmid was originally analyzed in S. lividans K4-114 (3), although only the former was reported at that time.", "As with the first set of plasmids tested, small amounts of 2′-O-glucosylated derivatives could also be detected in some of the culture extracts.", "The yields of the desosamine containing compounds were too low to determine absolute titers (<1 mg/L) and, therefore, the relative antibacterial activity of the compounds could not be determined from these assays.", "C. Discussion This example demonstrates that a minimal set of seven genes (desI, II, III, IV, V, VI, VIII) is sufficient for biosynthesis of TDP-desosamine from glucose-1-phosphate in S. lividans.", "The apparent low abundance of TDP-desosamine in the engineered host could be due either to the availability of glucose-1-phosphate in this host or to poor expression of the sugar biosynthesis and/or transferase genes.", "Alternatively, it is interesting to note that narbonolide and 10-deoxymethynolide are present in the natural picromycin/methymycin producing organism, S. venezuelae, and could therefore reflect that one or more of the enzymes from the des cluster is relatively inefficient.", "One can increase the amount of TDP-desosamine either by increasing expression levels of these genes and/or by complementing one or more of the enzymes in the pathway with homologs from other clusters such as erythromycin or oleandomycin.", "Expression of the minimal desosamine biosynthesis genes together with the DesVII desosaminyltransferase in S. lividans has enabled the production of more than 20 glycosylated macrolides with detectable antibacterial activity.", "The structures of the macrolides that were glycosylated highlight both the remarkable substrate tolerance of the DesVII glycosyltransferase as well as the ability of desosamine to impart biological activity to structurally diverse macrolactones.", "In addition to their antibacterial properties the desosamine containing compounds presented here may possess additional biological properties that are associated with erythromycin and other macrolides, including motilin antagonism and anti-inflammatory activities.", "Furthermore, the demonstration by others that DesVII and other glycosyltransferases can also tolerate modifications of the sugar substituent (22, 23, 29) opens the door to manipulation of both polyketide and deoxysugar pathways for the production of ‘unnatural’ natural product libraries.", "REFERENCES 1.O'Hagan, D. 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(1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, N.Y.).", "17.Ziermann, R. & Betlach, M. C. (1999) Biotechniques 26, 106-110.18.Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M. & Schrempf, H. (1985) Genetic Manipulation of Streptomyces: A Laboratory Manual (The John Innes Foundation, Norwich, UK).", "19.Betlach, M. C., Kealey, J. T., Betlach, M. C., Ashley, G. A.", "& McDaniel, R. (1998) Biochemistry 37, 14937-14942.20.Tang, L., Fu, H., Betlach, M. C. & McDaniel, R. (1999) Chem.", "& Biol.", "6, 553-558.21.Xue, Y., Zhao, L., Liu, H. -w. & Sherman, D. H. (1998) Proc.", "Natl.", "Acad.", "Sci.", "USA 95, 12111-12116.22.Zhao, L., Sherman, D. H. & Liu, H. -w. (1998) J.", "Am.", "Chem.", "Soc.", "120, 10256-10257.23.Zhao, L., Ahlert, J., Xue, Y., Thorson, J. S., Sherman, D. H. & Liu, H. -w. (1999) J.", "Am.", "Chem.", "Soc.", "121, 9881-9882.24.Zhao, L., Sherman, D. H. & Liu, H. -w. (1998) J.", "Am.", "Chem.", "Soc.", "120, 9374-9375.25.Quiros, L. M., Aguirrezabalaga, I., Olano, C., Mendez, C. & Salas, J.", "A.", "(1998) Mol.", "Microbiol.", "28, 1177-1185.26.Jenkins, G. & Cundliffe, E. (1991) Gene 108, 55-62.27.Ziermann, R. & Betlach, M. (2000) J. Ind.", "Microbiol.", "Biotech.", "24, 4650.28.Bierman, M., Logan, R., O'Brien, K., Seno, E. T., Nagaraja, R. & Schoner, B. E. (1992) Gene 116, 47-49.29.Gaisser, S., Reather, J., Wirtz, G., Kellenberger, L., Staunton, J.", "& Leadlay, P. F. (2000) Mol.", "Microbiol.", "36, 391-401.The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims." ] ]	Patent_10468828
[ [ "Method of manufacturing metallic composite material", "Disclosed is a method of manufacturing a metallic composite material for use as building material, the metallic composite material including an aluminium core sheet and a titanium clad layer on at least one side of the core sheet.", "The aluminium core sheet and titanium clad layer are bonded to one another by roll-bonding wherein only the aluminium core sheet prior to roll bonding is preheated to a temperature in the range of 50 to 200° C. The use of such metallic composite material and to a metallic composite material as such are also disclosed." ], [ "1.A method of manufacturing a metallic composite material for use as building material, said metallic composite material comprising an aluminium core sheet and a titanium cladding layer on at least one side of the core sheet, wherein the aluminium core sheet and titanium cladding layer are bonded to one another by roll-bonding wherein only the aluminium core sheet prior to roll bonding is preheated to a temperature in the range of 50 to 200° C. 2.A method according to claim 1, wherein the aluminium core sheet and titanium cladding layer are bonded to one another by roll-bonding wherein only the aluminium core sheet prior to roll bonding is preheated to a temperature in the range of 110 to 200° C. 3.A method according to claim 1, wherein the roll-bonding is carried out with a total rolling degree of not more than 50%.", "4.A method according to claim 3, wherein the roll-bonding is carried out with a total rolling degree of not more than 45%.", "5.A method according to claim 3, wherein the roll-bonding is carried out with a total rolling degree of not more than 40%.", "6.A method according to claim 1, wherein the roll-bonding is carried out with a total rolling degree of at least 25%.", "7.A method according to claim 6, wherein the roll-bonding is carried out with a total rolling degree of at least 30%.", "8.A method according to claim 1, wherein the roll-bonding is carried out in one single rolling step.", "9.A method according to claim 1, wherein the method includes pre-treatment of the surface of the titanium cladding layer facing the aluminium core sheet said pre-treatment comprises the step of brushing of said surface before the bonding step.", "10.A method according to claim 9 wherein the pre-treatment is carried out in a substantially dry atmosphere.", "11.A method according to claim 9 wherein the pre-treatment is carried out in an inert gas atmosphere.", "12.A method according to claim 1, wherein the aluminium core sheet has a composition within a range selected from of AA1000, AA6000, or AA3000-series aluminium alloys.", "13.A method according to claim 1, wherein the aluminium core sheet is made of an AA3004-series aluminium alloy.", "14.A method according to claim 1, wherein the titanium cladding layer has a composition, in weight percent, comprising: Fe 0.35 max.", "O 0.35 max.", "N 0.05 max.", "C 0.06 max.", "H 0.015% max.", "impurities each 0.1% max., total 0.4% max., balance titanium.", "15.A method according to claim 14, wherein the titanium cladding layer has a composition, in weight percent, comprising Fe of 0.15% max.", "16.A method according to claim 14, wherein the titanium cladding layer has a composition, in weight percent, comprising O of 0.12% max.", "17.A method according to claim 1, wherein the titanium cladding layer has a composition, in weight percent, consisting of: Fe 0.35 max.", "O 0.35 max.", "N 0.05 max.", "C 0.06 max.", "H 0.015% max.", "impurities each 0.1% max., total 0.4% max., balance titanium.", "18.A method according to claim 1, wherein said aluminium core sheet has a thickness after roll-bonding in the range of 0.7 to 1.5 mm.", "19.A method according to claim 1, wherein said aluminium core sheet has a thickness after roll-bonding in the range of 0.9 to 1.25 mm.", "20.A method according to claim 1, wherein one or both of said titanium cladding layer or layers have each a thickness after roll-bonding in the range of 0.05 to 0.4 mm.", "21.A method according to claim 1, wherein one or both of said titanium cladding layer or layers have each a thickness after roll-bonding in the range of 0.05 to 0.3 mm.", "22.A method according to claim 1, wherein following the roll-bonding of the sheet products into a composite material, the composite material is subjected to a final annealing by holding the composite material for 2 to 16 hours at a temperature in a range of 350 to 550° C. 23.A method according to claim 1, wherein following the roll-bonding of the sheet products into a composite material, the composite material is subjected to a final annealing by holding the composite material for 2 to 16 hours at a temperature in a range of 400 to 540° C. 24.Metallic composition in sheet form, comprising a sheet of aluminium alloy and a titanium cladding layer applied on one side of the aluminium alloy sheet, wherein said titanium cladding layer is applied on the core sheet by means of roll-bonding and wherein only the aluminium core sheet prior to roll bonding has been preheated to a temperature in a range of 50 to 200° C., the thickness of the aluminium core sheet is in the range of 0.7 to 1.5 mm and the thickness of the titanium cladding layer is in the range of 0.05 to 0.3 mm, the aluminium sheet has a composition within the ranges of AA3000-series aluminium alloys, and the titanium cladding layer has a composition, in weight percent, comprising: Fe 0.35 max.", "O 0.35max.", "N 0.05 max.", "C 0.06 max.", "H 0.015% max.", "impurities each 0.1% max., total 0.4% max., balance titanium.", "25.Metallic composite sheet according to claim 24, wherein the metallic composite sheet is a building sheet and the aluminium alloy is made of the AA3004-series aluminium alloy.", "26.Metallic composite sheet according to claim 24, consisting of the sheet of aluminium alloy and the titanium cladding layer applied on one side of the aluminium alloy sheet." ], [ "The invention relates to a method of manufacturing a metallic composite material, in particular for use as a building material, said metallic composite material comprising an aluminium core sheet and a titanium clad layer on one or both sides of the core sheet.", "The invention further relates to the use of such metallic composite material and to a metallic composite material as such.", "Within the scope of this invention roll forming, also known as contour roll forming or cold roll forming, is understood to be a continuous process for forming metal sheet, strip, or coiled stock into desired shapes of essentially uniform thickness by feeding the stock through a series of roll stations equipped with contoured rolls, see also Metals Handbook, 9th.", "edition, Vol.", "14, ASM International, 1988, pp.", "624-635.Metallic composite materials comprising a core sheet bonded with a titanium or titanium alloy clad layer to one surface or both surfaces of the core sheet are known in the art.", "For example thick carbon steel sheet or plate clad with titanium are employed in various constructions, in particular for use in the chemical industry and heat-exchangers, in which applications the very good corrosive performance of the titanium cladding is exploited.", "Typically copper or copper alloys are used as an intermediate layer between the carbon steel and the titanium clad layer.", "In another example aluminium core sheet clad with titanium can be used as building material, such as roofs and external walls.", "In this application the good anti-corrosive properties of the titanium are used.", "In addition titanium has a high-tech and very appealing appearance.", "The Japanese laid open patent publication JP-A-09-57465 discloses a method of manufacturing a metallic composite material, said metallic composite material comprising an aluminium core sheet and a titanium clad layer on one or both sides of the core sheet.", "The essence of the disclosed method lies in that the aluminium and titanium are metallurgically bonded by an explosive bonding method.", "In the disclosed example illustration the invention a 50 mm thick aluminium plate has been bonded on one side by means of explosive bonding to a 5 mm thick titanium plate.", "The resultant product is a 55 mm composite plate material.", "The plate material was subsequently cold rolled at room temperature to a final gauge of 1.1 mm.", "The other dimensions of the final sheet product were 3 m×4 m. After 2 years exposure of the resultant metallic composite sheet product to a marine coastal environment, no corrosion on the titanium surface occurred.", "A draw-back of the known method is that the metallic materials are bonded to one another by means of explosive bonding, which is a rather complex technique.", "A further disadvantage of explosive bonding is that it can only be applied batch-wise, and thereby limiting the dimensions of the resultant composite sheet product.", "A further disadvantage of the disclosed method is that in order to obtain of sheet product a 98% cold rolling degree needs to be applied.", "This cold rolling degree can only be obtained in multiple rolling steps, which require intermediate heat-treatment of the product to improve workability.", "And yet a further disadvantage of the disclosed method is that the resultant sheet product is that the formability, e.g.", "by means of roll forming, is limited due to the strain hardening as a result from the cold rolling.", "U.S. Pat.", "No.", "3,711,937 discloses a process of cladding aluminium sheet with a sheet of titanium by bringing the surfaces of the sheets into momentary contact under a pressure by means of hot rolling, and whereby both the aluminium sheet and the titanium sheet are being pre-heated to a temperature of 500 to 1000° F. (260 to 538° C.), preferably in a range of 600 to 850° F. (315 to 454° C.), and post-heating the composite sheet for a period of 0.1 to 1.0 hours at a temperature of 500 to 1150° F. (260 to 620° C.) to develop the bond.", "The composite metal produced by the disclosed process find their major application in electrochemical processing and in structural applications in the aircraft and aerospace industries.", "The Japanese laid open patent publication JP-A-08-336929 discloses a titanium-aluminium clad product for building purposes, whereby the ratio r1 of the thickness of the titanium layer to the thickness of the whole plate is expressed by 0.05≦r1≦0.6.This ratio is said to improve on the springback behaviour of the clad product.", "The clad product has been manufactured by means of roll-bonding and whereby at least the aluminium sheet has been pre-heated to a temperature of 450° C. It is an object of the present invention to provide a method of manufacturing a metallic composite material for use as a building material, said metallic composite material comprising an aluminium core sheet and a titanium clad layer on one or both sides of the core sheet, which method requires less complex process steps compared to the prior art described above.", "It is another object of the present invention to provide a method of manufacturing a metallic composite material for use as a building material, said metallic composite material comprising an aluminium core sheet and a titanium clad layer on one or both sides of the core sheet, which method requires a reduced number of rolling steps for obtaining a final gauge composite product.", "And it is another object of the present invention to provide a method of manufacturing a metallic composite material for use as a building material, said metallic composite material comprising an aluminium core sheet and a titanium clad layer on one or both sides of the core sheet, which method results in a metallic composite material which has an excellent formability, in particular by means of roll-forming.", "Yet another object of the present invention is to provide a method of manufacturing a metallic composite material for use as building material, ideally suitable as a roofing material.", "According to the invention in one aspect there is provided a method of manufacturing a metallic composite material, ideally suitable for use as building material, said metallic composite material comprising an aluminium core sheet and a titanium clad layer on at least one side of the core sheet, characterised in that the aluminium core sheet and titanium clad layer are bonded to one another by roll-bonding or roll-cladding and wherein only the aluminium core sheet prior to roll bonding is being preheated to a temperature in the range of 50 to 200° C., and preferably in the range of 110 to 200° C. The method of the invention achieves the effect that explosive bonding is not required to manufacture the metallic composite material.", "By means of roll-bonding an excellent metallurgical bond is obtained between the aluminium core sheet and the titanium clad layer.", "By preheating only the aluminium in the given range the metallurgical bond between the two different metal sheets is further improved significantly, and which allows the material to be subsequently formed by means of roll-forming.", "The skilled person will not designate the preheat temperature range for the aluminium cores sheet just prior to roll-bonding as hot rolling.", "For the aluminium core sheet preheat temperatures above 200° C. should be avoided, in order to sufficiently retain in particular the formability properties of the aluminium alloy in the resultant roll-bonded composite material.", "Roll-bonding allows for the production of end-less sheet material, e.g.", "coiled sheet material, whereas explosive bonding puts significant restrictions on the dimensions of the final composite sheet product.", "An advantage of having coiled metallic composite material is that it can be transported very cost efficiently to a construction site, on which construction site it may be rolled formed immediately, into for example roofing material, having dimensions of 50 to 100 cm wide and a length as desired, e.g.", "7 to 15 m or even more.", "It has been found that there is no mandatory need to increase the temperature of the titanium clad sheet also just prior to bonding via roll-bonding.", "In order to avoid oxidation of the surface of the titanium clad layer facing the aluminium core sheet it is even preferred that the titanium clad layer does not reach a temperature significantly above ambient temperature.", "In an industrial line the aluminium core sheet can be heated by known means to the increased temperature, and whereby a typical line-speed would be in the range of 10-25 m/s.", "The location of heating could be 2 to 5 m before it comes into line contact with the titanium clad layer.", "With such line-speeds the temperature of the aluminium core sheet remains almost unchanged prior to facing the titanium clad layer.", "In an embodiment of the method according to the invention, the roll-bonding is carried out with a total rolling degree, being the total reduction in thickness direction, of not more than 50%.", "By increasing the temperature of the aluminium core sheet in the given range, it is possible to achieve a very good bonding between the aluminium core sheet and the titanium clad layer without the requirement of a high deformation or rolling degree.", "In a preferred embodiment of the method according to the invention, the roll-bonding is carried out with a total rolling degree of not more than 45%, and more preferably of not more than 40%.", "In order to establish a good metallurgical bond between the aluminium and the titanium sheet a suitable lower limit for the rolling degree would be about 25%, and more preferably 30%.", "By increasing the temperature of the aluminium core sheet in the given range, it is possible to achieve a very good bonding between the aluminium core sheet and the titanium clad layer without the requirement of a high deformation of rolling degree.", "By applying the rolling degree in the given range the surfaces of the resultant metallic composite material have significantly less strain hardening, which allows for the material to have a desirable high degree of formability, in particular by means of roll-forming.", "In a preferred embodiment of the method according to the invention the roll-bonding is carried out in one single rolling step.", "By applying the rolling degree in the given range in one single rolling step an excellent metallurgical bond between the aluminium core sheet and the titanium clad layer is obtained, and the surfaces of the resultant metallic composite material have significantly less strain hardening, which allows for the material to have a desirable high degree of formability, in particular by means of roll-forming.", "Furthermore, by means of roll-bonding in one single rolling step there is no need for intermediate annealing treatments at elevated temperature to recover workability of the intermediate product for further cold rolling to final gauge.", "In an embodiment the method according to the invention is characterised in that it includes a pre-treatment of the surface of the titanium clad layer facing the aluminium core sheet, said pre-treatment comprises the step of brushing of said surface before the roll-bonding step.", "The brushing operation achieves the effect of removing at least part of the oxide-layer present on said surface.", "It has been found that the surface of the aluminium core sheet facing the titanium clad layer does not require any special pre-treatment, but it may be treated optionally by means of a brushing operation also.", "Preferably said brushing as pre-treatment is carried out in an essentially dry atmosphere, preferably using a normal atmosphere environment with no special precautionary measures taken or an inert gas atmosphere.", "It is preferred that no cooling fluids or the similar are applied in order to avoid re-oxidation of the brushed surface.", "By properly pre-treating the titanium clad layer by means of brushing, e.g.", "mechanically brushing, the resultant metallurgical bond between the aluminium core sheet and the titanium clad layer is further improved significantly.", "Following the roll-bonding of sheet products into the composite material, the resultant composite material may be subjected to a final annealing treatment in one or in multiple heating steps at temperatures in a range of 350 to 550° C., preferably in the range of 400 to 540° C., for a period of 2 to 16 hours in order to improve the roll-formability of said composite material, and also to further improve the metallurgical bond between the aluminium core sheet and the titanium clad layer or layers.", "Although various aluminium alloys may be employed in the method according of the invention, including those of the Aluminium Association (AA)1000 and AA6000-series, in a preferred embodiment of the method of the invention the aluminium core sheet has a composition within the ranges of the AA3000-series alloys, and a particular useful aluminium alloy is the AA3004-series alloy.", "It has been found that this aluminium alloy can be roll-bonded very good into titanium clad aluminium sheets, while maintaining an excellent roll-formability in the resultant composite material.", "Having an excellent roll-formability is an important characteristic of the composite material when the material is being used in building systems, in particular in standing-seam roofing and wall-cladding structures.", "Although various titanium-alloy sheets may be employed in the method according to the invention, in a preferred embodiment of the method according to the invention non-alloyed titanium sheets are used, in particular those having a chemical composition within the ranges, in weight percent, comprising: Fe 0.35 max., preferably 0.15 max., O 0.35 max., preferably 0.12 max., N 0.05 max., C 0.06 max., H 0.015% max., preferably 0.013% max., impurities each 0.1% max., total 0.4% max., balance titanium.", "It has been found that this type of non-alloyed titanium can be easily roll-bonded with aluminium alloys, while maintaining an excellent roll-formability in the resultant composite material, in particular when used in combination with aluminium alloys within the AA3000-series, and preferably within the AA3004-series ranges.", "In addition it has been found that in particular the combination of AA3004 aluminium core sheet in combination with a non-alloyed titanium clad layer allows for a cold rolling degree of up to 50% in one single rolling step during the roll-bonding operation.", "In an embodiment of the method according to the invention the aluminium core sheet has a thickness after roll-bonding in the range of 0.7 to 1.5 mm, and preferably in the range of 0.9 to 1.25 mm.", "Each titanium clad layer has after roll-bonding a thickness in the range of 0.05 to 0.4 mm, and preferably in the range of 0.05 to 0.3 mm.", "The thickness of each titanium clad layer is such that the thickness is in the range of 5 to 15% of the thickness of the aluminium core sheet.", "This means that the thickness of the aluminium core sheet prior to roll bonding is in the range of less than 3.0 mm, which is a material readily available in endless form on a coil.", "Also titanium sheet having a thickness of up to 0.5 mm are readily commercially-available.", "In another aspect of the invention metallic composite material obtained by the method, may be used as a building systems such as facade and roofing structures.", "Aluminium-titanium clad products suitable for application in building systems such as those known under the trade names KAL-ZIP and KAL-BAU may be obtained by the invention.", "In another aspect of the invention there is provided a metallic composite material in sheet form, in particular a roll-formable building sheet, comprising a sheet of aluminium or aluminium alloy and a titanium clad layer applied on one side of the aluminium sheet, and wherein said titanium clad layer is applied on the core sheet by means of roll-bonding, the thickness of the aluminium core sheet is in the range of 0.7 to 1.5 mm, and preferably in the range of 0.9 to 1.25 mm, and the thickness of each titanium clad layer is in the range of 0.05 to 0.3 mm, and preferably in the range of 0.05 to 0.15 mm, the aluminium sheet has a composition within the ranges of AA3000-series aluminium alloys, and preferably within the ranges of AA3004, and the titanium clad layer has a composition, in weight percent, comprising: Fe 0.35 max., preferably 0.15 max., O 0.35 max., preferably 0.12 max., N 0.05 max., C 0.06 max., H 0.015% max., preferably 0.013% max., impurities each 0.1% max., total 0.4% max., balance titanium.", "The invention will know be illustrated by a non-limitative example.", "On an industrial scale a metallic composite product has been manufactured consisting of an aluminium sheet roll-bonded on one side with titanium sheet, whereby 0.5 mm gauge titanium sheet has been roll-bonded to 2.5 mm gauge aluminium sheet.", "The titanium sheet was made from TRIKUTAN RT12 (3.7025 gem DIN 17860) in the II-B condition (cold rolled, annealed and pickled) and the aluminium sheet was an AA3004-series aluminium alloy in the 0-temper condition.", "The facing faces of both metal sheets had been pre-treated by means of brushing in order to remove at least partly the oxide-surface present.", "The titanium sheet was at room temperature and the aluminium sheet had been heated to a temperature of 120° C. in order to facilitate the bonding.", "Both metal sheets had been bonded to each other in one single cold-rolling step in a quatro cladding mill with a rolling degree of 47%, and whereby in the resultant metallic composite product the aluminium and titanium sheet had a thickness of 1.13 mm and 0.36 mm respectively.", "Sub-samples of the roll-bonded product had been annealed for 5 hours at 535° C. in a furnace with normal atmospheric conditions, and where after the samples were taken out of the annealing furnace and allowed to cool to room temperature.", "Following the annealing treatment the resultant bonding between the two metal sheets was excellent.", "Furthermore, the tensile properties before and after annealing had been determined in accordance with DIN-1624, incorporated herein by reference.", "The results are listed in Table 1, average over 6 specimen tested, and whereby “Rp 0.2” stands for the yield strength, “Rm” for the tensile strength, “Ag” for the uniform elongation and “A80” for the total elongation.", "From the results of Table 1 it can be seen that the metallic composite product following the annealing has a very high elongation and relatively low yield strength enabling it to be successfully rolled formed in panels for example standing-seam roofs.", "Due to the good mechanical bonding the roll-forming operation can be carried out without delamination of the clad product.", "TABLE 1 Before annealing After annealing Rp 0.2 [MPa] 260 127 Rm [MPa] 305 194 Ag [%] 2.4 14.6 A80 [%] 5.3 27.8 Having now fully described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made without departing from the spirit or scope of the invention as herein described." ] ]	Patent_10468851
[ [ "Under reamer", "An under reamer for expanding a borehole through an earthen formation is taught.", "The under reamer includes: a mandrel including an end for connection into a drill string and an opposite end and a housing including an inner bore, telescopically disposed over the mandrel and axially moveable relative to the mandrel but rotationally moveable with the mandrel.", "A plurality of under reamer arms are carried on the housing, the under reamer arms are formed as blocks including cutter-supporting portions in which poly-crystalline diamond compact cutters are mounted.", "The under reamer arms are moveable by driving the housing axially over the mandrel between a retracted position extending into the bore of the housing and an expanded position wherein the under reamers arms are pivoted out of the housing inner bore and supported therebehind by the mandrel such that the cutter-supporting portions are exposed for use to enlarge a borehole.", "The under reamer can include one or more lock assemblies for releasably locking the mandrel and housing against relative axial movement.", "The under reamer can also or alternately include a restriction nozzle to facilitate hydraulic operation thereof." ], [ "1.An under reamer for expanding a borehole through an earthen formation, the under reamer comprising: a mandrel including an end for connection into a drill string and an opposite end; a housing including an inner bore, telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; and a plurality of under reamer arms carried on the housing, the under reamer arms formed as blocks including cutter-supporting portions in which poly-crystalline diamond compact cutters are mounted, the under reamer arms being moveable by driving the housing axially over the mandrel between a retracted position extending into the bore the housing and an expanded position wherein the under reamer arms are pivoted out of the housing inner bore and supported by the mandrel such that the cutter-supporting portions are exposed for use to enlarge a borehole.", "2.The under reamer of claim 1 further comprising a hydraulic pressure chamber between the housing and the mandrel for driving the housing axially relative to the mandrel.", "3.The under reamer of claim 1 further comprising slots in the housing extending between the housing outer surface and the housing inner bore and the under reamer arms being pivotally mounted therein.", "4.The under reamer of claim 3 wherein each slot includes side, top and bottom surfaces that extend between the housing outer surface and the housing inner bore and the under reamer arms each include a outer facing surface and a rear surface and top, bottom and side surfaces extending between the outer facing surface and the rear surface and wherein the cutter supporting portions of the arm is a protrusion on the outer facing surface of the arm.", "5.The under reamer of claim 4 wherein in the expanded position, at least a portion of the arm top, bottom and side surfaces remain in the slot with the cutter supporting portions extending outwardly beyond the housing outer surface.", "6.The under reamer of claim 1 wherein the under reamer arms each include a rear surface, and a portion of the rear surfaces being wedge-shaped to permit the under reamer arms to fit together in the housing inner bore about the inner bore center axis.", "7.The under reamer of claim 6 wherein the mandrel includes a contour on its surface which supports the under reamer arms in the expanded position and the under reamer arms each include another portion of their rear surface that is curved to substantially conform to the contour of the mandrel surface.", "8.The under reamer of claim 1, further comprising a releasable lock between at least one under reamer arm and its slot to releasably lock the arm in the slot when in the retracted position.", "9.The under reamer of claim 1, further comprising an operational lock assembly operable to releasably lock the mandrel relative to the housing in an axial position corresponding to the expanded position.", "10.The under reamer of claim 1, further comprising a tripping lock assembly operable to releasably lock the mandrel relative to the housing in an axial position corresponding to the retracted position.", "11.The under reamer of claim 10, wherein the tripping lock assembly is operable to unlock by application of a selected fluid pressure to the mandrel.", "12.An under reamer for expanding a borehole through an earthen formation, the under reamer comprising: a mandrel including an end for connection into a drill string and an opposite end; a housing telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; a plurality of under reamer arms carried on the housing, the under reamer arms each including a cutter-supporting portion; and a slot for each under reamer arm, the slots extending from the outer surface of the housing to the housing inner bore; the under reamer arms each being pivotally moveable within their slots between a stored position in the slot and extending into the inner bore and an expanded position wherein the cutter-supporting portions extend beyond the outer surface of the housing for use to enlarge a borehole.", "13.The under reamer of claim 12 wherein the slots include sides and the under reamer arms include sides and the sides of the slots are formed to substantially conform to the sides of the arms.", "14.The under reamer of claim 12 wherein the slots each include an upper end and the under reamer arms each include an upper surface and the upper ends of the slots are formed to limit the pivotal movement of the arms into their extended position by abutment of the arms upper surfaces against the upper ends of the slots.", "15.The under reamer of claim 14, wherein the upper ends of the slots substantially conform to the shape of the upper surfaces of the under reamer arms.", "16.The under reamer of claim 12 wherein the mandrel includes an outer surface and the under reamer arms each include a rear surface and the rear surfaces of the under reamer arms are shaped to substantially conform to the outer surface of the mandrel.", "17.The under reamer of claim 16 wherein the under reamer arms each include a wedge shaped portion on their rear surfaces to permit an interfit between the arms in the stored position.", "18.The under reamer of claim 12 further comprising a releasable lock between at least one under reamer arm and the slot in which it is mounted to releasably lock the under reamer arm in the stored position within the slot.", "19.An under reamer for expanding a borehole through an earthen formation, the under reamer comprising: a mandrel including an end for connection into a drill string and an opposite end; a housing telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; a plurality of under reamer arms carried on the housing, the under reamer arms including cutter-supporting portions and being moveable by driving the housing axially over the mandrel between a stored position against the housing and an expanded position wherein the cutter-supporting portions are exposed for use to enlarge a borehole; and a lock for releasably locking the housing in relative axial position on the mandrel.", "20.The under reamer of claim 19 wherein the lock releasably locks the housing in a position on the mandrel to maintain the under reamer arms in the stored position.", "21.The under reamer of claim 19 wherein the lock releasably locks the housing in an operational position on the mandrel to maintain the under reamer arms in the expanded position.", "22.An under reamer for expanding a borehole through an earthen formation, the under reamer comprising: a mandrel including an uphole end for connection into a drill string and an opposite end and an inner bore extending between the uphole end and the opposite end; a housing including an inner bore, telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; a plurality of under reamer arms carried on the housing, the under reamer arms each including a cutter-supporting portion in which cutters are mounted, the under reamer arms being moveable by driving the housing axially over the mandrel between a stored position extending into the bore the housing and an expanded position wherein the under reamers arms are pivoted out of the housing inner bore and supported by the mandrel such that the cutter-supporting portions are exposed for use to enlarge a borehole; a fluid pressure chamber positioned between the housing and the mandrel and formed to accept pressurized fluid therein to drive the axial movement of the housing relative to the mandrel, a port from the mandrel inner bore to the pressure chamber to permit flow of pressurized fluid therethrough; and a restriction nozzle in the mandrel inner bore between the port and the opposite end of the mandrel to increase fluid pressure thereabove.", "23.The under reamer of claim 22, wherein the housing inner bore is formed as a fluid diffuser." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>When drilling a borehole through an earthen formation a pilot hole is drilled by a pilot bit and the hole can be enlarged by an under reamer.", "Under reamers have arms with cutters thereon that cut into the formation to enlarge the borehole to its intended gauge.", "Under reamers are useful in casing drilling, wherein the pilot bit must be of a size to pass through the bore of the casing and is, therefore, not sized to drill a borehole of a gauge that the casing can pass therethrough.", "Therefore, the pilot bit drills the pilot hole into the formation and under reamers enlarge the hole behind the pilot bit to a gauge greater than the casing outer diameter to permit advancement of the casing into the borehole.", "Under reamers are also useful when extending a borehole below installed casing.", "In such embodiments, the under reamer arms are collapsible to permit the under reamer to be moved through the bore of the casing and are expandable downhole to permit drilling of a borehole to a gauge greater than the outer diameter of the casing." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>An under reamer has been invented, which facilitates tripping through the casing inner bore and facilitates deployment of the under reamer arms down hole.", "In accordance with one broad aspect of the present invention there is provided an under reamer for expanding a borehole through an earthen formation, the under reamer comprising: a mandrel including an end for connection into a drill string and an opposite end; a housing including an inner bore, telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; and a plurality of under reamer arms carried on the housing, the under reamer arms formed as blocks including cutter-supporting portions in which poly-crystalline diamond compact cutters are mounted, the under reamer arms being moveable by driving the housing axially over the mandrel between a stored position extending into the inner bore of the housing and an expanded position wherein the under reamer arms are pivoted out of the housing inner bore and supported by the mandrel such that the cutter-supporting portions are exposed for use to enlarge a borehole.", "The axial movement of the housing relative to the mandrel can be driven by weight on bit or by fluid pressure inside the drill string.", "In one embodiment, the under reamer includes a hydraulic pressure chamber between the housing and the mandrel for driving the housing axially relative to the mandrel.", "The under reamer can include slots in the housing extending between the housing outer surface and the housing inner bore wherein the under reamer arms are pivotally mounted.", "The slots permit the rear surfaces of the under reamer arms to be open for driving contact with the mandrel.", "A portion of the rear surfaces can be wedge-shaped to permit the under reamer arms to fit together in the housing inner bore about the inner bore center axis.", "In addition or alternately, the under reamer rear surfaces can be formed to substantially conform to the outer surface contour of the portion of the mandrel which is positioned behind the arms when they are in the expanded position.", "This enhances the support provided by the mandrel for the arms, when the arms are in the expanded position.", "In accordance with another aspect of the present invention, there is provided an under reamer for enlarging a borehole through an earthen formation, the under reamer comprising: a mandrel including an end for connection into a drill string and an opposite end; a housing telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; a plurality of under reamer arms carried on the housing, the under reamer arms each including a cutter-supporting portion; and a slot for each under reamer arm, the slots extending from the outer surface of the housing to the housing inner bore; the under reamer arms each being pivotally moveable within their slots between a stored position in the slot and extending into the inner bore and an expanded position wherein the cutter-supporting portions extend beyond the outer surface of the housing for use to enlarge a borehole.", "The sides of the slots can be formed to substantially conform to the shape of the sides of the arms, so that the arms are supported by the slots.", "Preferably, the arms are formed such that, in the expanded position, they remain at least in part in the slot with their cutter supporting portions extending from the slot.", "The upper ends of the slots can be formed to limit the pivotal movement of the arms into their extended position by abutment of the arm upper surfaces against the upper ends of the slots.", "The upper ends of the slots can substantially conform to the shape of the upper surfaces of the under reamer arms, to enhance transfer of shock to the housing.", "The under reamer can include a releasable lock between each under reamer arm and the slot in which it is mounted to releasably lock the under reamer arm in the stored position within the slot.", "In accordance with another aspect of the present invention, there is provided an under reamer for enlarging a borehole through an earthen formation, the under reamer comprising: a mandrel including an end for connection into a drill string and an opposite end; a housing telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; a plurality of under reamer arms carried on the housing, the under reamer arms including cutter-supporting portions and being moveable by driving the housing axially over the mandrel between a stored position against the housing and an expanded position wherein the cutter-supporting portions are exposed for use to enlarge a borehole; and a lock for releasably locking the housing in relative axial position on the mandrel.", "The lock can releasably lock the housing in a position on the mandrel to maintain the under reamer arms in the stored position or, alternately, the lock can releasably lock the housing in an operational position on the mandrel to maintain the under reamer arms in the expanded position.", "In one embodiment, the operational lock is actuated to unlock by application of fluid pressure to the under reamer, the fluid pressure being selected to be greater than that present during tripping of the under reamer.", "In accordance with another aspect of the present invention, there is provided an under reamer for expanding a borehole through an earthen formation, the under reamer comprising: a mandrel including an uphole end for connection into a drill string and an opposite end and an inner bore extending between the uphole end and the opposite end; a housing including an inner bore, telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; a plurality of under reamer arms carried on the housing, the under reamer arms each including a cutter-supporting portion in which cutters are mounted, the under reamer arms being moveable by driving the housing axially over the mandrel between a stored position extending into the inner bore of the housing and an expanded position wherein the under reamers arms are pivoted out of the housing inner bore and supported by the mandrel such that the cutter-supporting portions are exposed for use to enlarge a borehole; a fluid pressure chamber positioned between the housing and the mandrel and formed to accept pressurized fluid therein to drive the axial movement of the housing relative to the mandrel, a port from the mandrel inner bore to the pressure chamber to permit flow of pressurized fluid therethrough; and a restriction nozzle in the mandrel inner bore between the port and the opposite end of the mandrel to increase fluid pressure thereabove.", "The housing inner bore can formed as a fluid diffuser to restore fluid pressure passing through to the pilot bit." ], [ "FIELD OF THE INVENTION This invention is directed to an under reamer for operating behind a pilot bit.", "BACKGROUND OF THE INVENTION When drilling a borehole through an earthen formation a pilot hole is drilled by a pilot bit and the hole can be enlarged by an under reamer.", "Under reamers have arms with cutters thereon that cut into the formation to enlarge the borehole to its intended gauge.", "Under reamers are useful in casing drilling, wherein the pilot bit must be of a size to pass through the bore of the casing and is, therefore, not sized to drill a borehole of a gauge that the casing can pass therethrough.", "Therefore, the pilot bit drills the pilot hole into the formation and under reamers enlarge the hole behind the pilot bit to a gauge greater than the casing outer diameter to permit advancement of the casing into the borehole.", "Under reamers are also useful when extending a borehole below installed casing.", "In such embodiments, the under reamer arms are collapsible to permit the under reamer to be moved through the bore of the casing and are expandable downhole to permit drilling of a borehole to a gauge greater than the outer diameter of the casing.", "SUMMARY OF THE INVENTION An under reamer has been invented, which facilitates tripping through the casing inner bore and facilitates deployment of the under reamer arms down hole.", "In accordance with one broad aspect of the present invention there is provided an under reamer for expanding a borehole through an earthen formation, the under reamer comprising: a mandrel including an end for connection into a drill string and an opposite end; a housing including an inner bore, telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; and a plurality of under reamer arms carried on the housing, the under reamer arms formed as blocks including cutter-supporting portions in which poly-crystalline diamond compact cutters are mounted, the under reamer arms being moveable by driving the housing axially over the mandrel between a stored position extending into the inner bore of the housing and an expanded position wherein the under reamer arms are pivoted out of the housing inner bore and supported by the mandrel such that the cutter-supporting portions are exposed for use to enlarge a borehole.", "The axial movement of the housing relative to the mandrel can be driven by weight on bit or by fluid pressure inside the drill string.", "In one embodiment, the under reamer includes a hydraulic pressure chamber between the housing and the mandrel for driving the housing axially relative to the mandrel.", "The under reamer can include slots in the housing extending between the housing outer surface and the housing inner bore wherein the under reamer arms are pivotally mounted.", "The slots permit the rear surfaces of the under reamer arms to be open for driving contact with the mandrel.", "A portion of the rear surfaces can be wedge-shaped to permit the under reamer arms to fit together in the housing inner bore about the inner bore center axis.", "In addition or alternately, the under reamer rear surfaces can be formed to substantially conform to the outer surface contour of the portion of the mandrel which is positioned behind the arms when they are in the expanded position.", "This enhances the support provided by the mandrel for the arms, when the arms are in the expanded position.", "In accordance with another aspect of the present invention, there is provided an under reamer for enlarging a borehole through an earthen formation, the under reamer comprising: a mandrel including an end for connection into a drill string and an opposite end; a housing telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; a plurality of under reamer arms carried on the housing, the under reamer arms each including a cutter-supporting portion; and a slot for each under reamer arm, the slots extending from the outer surface of the housing to the housing inner bore; the under reamer arms each being pivotally moveable within their slots between a stored position in the slot and extending into the inner bore and an expanded position wherein the cutter-supporting portions extend beyond the outer surface of the housing for use to enlarge a borehole.", "The sides of the slots can be formed to substantially conform to the shape of the sides of the arms, so that the arms are supported by the slots.", "Preferably, the arms are formed such that, in the expanded position, they remain at least in part in the slot with their cutter supporting portions extending from the slot.", "The upper ends of the slots can be formed to limit the pivotal movement of the arms into their extended position by abutment of the arm upper surfaces against the upper ends of the slots.", "The upper ends of the slots can substantially conform to the shape of the upper surfaces of the under reamer arms, to enhance transfer of shock to the housing.", "The under reamer can include a releasable lock between each under reamer arm and the slot in which it is mounted to releasably lock the under reamer arm in the stored position within the slot.", "In accordance with another aspect of the present invention, there is provided an under reamer for enlarging a borehole through an earthen formation, the under reamer comprising: a mandrel including an end for connection into a drill string and an opposite end; a housing telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; a plurality of under reamer arms carried on the housing, the under reamer arms including cutter-supporting portions and being moveable by driving the housing axially over the mandrel between a stored position against the housing and an expanded position wherein the cutter-supporting portions are exposed for use to enlarge a borehole; and a lock for releasably locking the housing in relative axial position on the mandrel.", "The lock can releasably lock the housing in a position on the mandrel to maintain the under reamer arms in the stored position or, alternately, the lock can releasably lock the housing in an operational position on the mandrel to maintain the under reamer arms in the expanded position.", "In one embodiment, the operational lock is actuated to unlock by application of fluid pressure to the under reamer, the fluid pressure being selected to be greater than that present during tripping of the under reamer.", "In accordance with another aspect of the present invention, there is provided an under reamer for expanding a borehole through an earthen formation, the under reamer comprising: a mandrel including an uphole end for connection into a drill string and an opposite end and an inner bore extending between the uphole end and the opposite end; a housing including an inner bore, telescopically disposed over the mandrel and axially moveable relative to the mandrel, the housing being rotationally moveable with the mandrel; a plurality of under reamer arms carried on the housing, the under reamer arms each including a cutter-supporting portion in which cutters are mounted, the under reamer arms being moveable by driving the housing axially over the mandrel between a stored position extending into the inner bore of the housing and an expanded position wherein the under reamers arms are pivoted out of the housing inner bore and supported by the mandrel such that the cutter-supporting portions are exposed for use to enlarge a borehole; a fluid pressure chamber positioned between the housing and the mandrel and formed to accept pressurized fluid therein to drive the axial movement of the housing relative to the mandrel, a port from the mandrel inner bore to the pressure chamber to permit flow of pressurized fluid therethrough; and a restriction nozzle in the mandrel inner bore between the port and the opposite end of the mandrel to increase fluid pressure thereabove.", "The housing inner bore can formed as a fluid diffuser to restore fluid pressure passing through to the pilot bit.", "BRIEF DESCRIPTION OF THE DRAWINGS A further, detailed, description of the invention, briefly described above, will follow by reference to the following drawings of specific embodiments of the invention.", "These drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope.", "In the drawings: FIG.", "1A is an axial sectional view through an under reamer in accordance with the present invention, with the under reamer arms in the expanded position; FIG.", "1B is an axial sectional view of the under reamer of FIG.", "1A with the under reamer arms in the retracted position; FIG.", "2 is a transverse sectional view through the under reamer of FIG.", "1A taken along line 2-2; FIG.", "3 is a transverse sectional view through the under reamer of FIG.", "1A taken along line 3-3; FIG.", "4 is a transverse sectional view through the under reamer of FIG.", "1A taken along line 4-4; FIG.", "5 is a transverse sectional view through the under reamer of FIG.", "1B taken along line 5-5; FIG.", "6A is an axial sectional view through another under reamer in accordance with the present invention, with the under reamer arms in the retracted position; FIG.", "6B is an axial sectional view of the under reamer of FIG.", "6A with the under reamer arms in the expanded position; FIG.", "6C is a perspective view of the under reamer of FIG.", "6A; FIG.", "6D is a perspective view of the under reamer of FIG.", "6B; FIG.", "7A is a transverse sectional view through the under reamer of FIG.", "6A taken along line 7A-7A; FIG.", "7B is a transverse sectional view through the under reamer of FIG.", "6A taken along line 7B-7B; FIG.", "7C is a transverse sectional view through the under reamer of FIG.", "6A along line 7C-7C; FIG.", "8 is a perspective view of an under reamer arm useful in the under reamer of FIG.", "6A; FIG.", "9A is an axial sectional view through another under reamer in accordance with the present invention with the under reamer arms in the retracted position.", "Reference is made to the sectional orientation indicated by line 9A-9A of FIG.", "10A; FIG.", "9B is an axial sectional view corresponding with FIG.", "9A with the under reamer arms in the expanded position; FIG.", "10A is a transverse sectional view through the under reamer of FIG.", "9B taken along line 10A-10A; FIG.", "10B is a transverse sectional view through the under reamer of FIG.", "9B taken along line 10B-10B; FIG.", "11A is an enlarged axial sectional view of the tripping lock assembly of the under reamer of FIG.", "9A; FIG.", "11B is a view corresponding to FIG.", "11A with the tripping lock assembly in the process of being unlocked; FIG.", "11C is a view corresponding to FIG.", "11A and following from FIG.", "11B, showing the tripping lock in the unlocked position, while the housing is moving up; FIG.", "11D is a transverse sectional view through the under reamer of FIG.", "9A taken along line 11D-11D; FIG.", "12A is a schematic sectional view of the under reamer arm section of an under reamer according to the present invention with the under reamers releasably locked in the retracted position; FIG.", "12B is a transverse sectional view through the under reamer of FIG.", "12A along line 12B-12B; FIG.", "12C is a schematic sectional view of the under reamer of FIG.", "12A with the under reamer arms in the expanded position; FIG.", "12D is a transverse sectional view along line 12D-12D of FIG.", "12C; FIG.", "13A is an enlarged axial sectional view of another tripping lock assembly useful in the present invention; FIG.", "13B is a view corresponding to FIG.", "13A with the tripping lock assembly in the process of being unlocked; FIG.", "13C is a view corresponding to FIG.", "13A and following from FIG.", "13B, showing the tripping lock in the unlocked position, while the housing is moving up.", "DETAILED DESCRIPTION OF THE PRESENT INVENTION The Figures show under reamers according to the present invention.", "As will be appreciated, under reamers are useful to act behind a pilot bit to enlarge a borehole.", "The under reamer is engaged at the lower end of a drill string.", "If the under reamer is used when drilling with casing, the drill string is a string of casing and the under reamer is releasably locked to the string of casing by, for example, a drilling lock assembly.", "The under reamer and pilot bit are rotated either by rotation of the casing string from surface or by use of a mud motor that is positioned between the under reamer and the drilling lock assembly.", "The under reamer of FIGS.", "1 to 5 includes a mandrel 2 and a housing 4, which carries a plurality of under reamer arms 5.The under reamer arms are mounted in slots 4e on the housing and are moveable between an expanded position (FIG.", "1A) wherein they are exposed for use to enlarge the well bore and a retracted position (FIG.", "1B), wherein they are retracted against or into the housing.", "The under reamer arms are moveable between the stored position and the expanded position by relative axial movement of the mandrel and the housing.", "In particular, when housing 4 is in a lower position relative to the mandrel, under reamer arms 5 are in or can be driven into the stored position and when housing 4 is moved upwardly over mandrel 2, such that the mandrel is inserted further into the housing, the under reamer arms are driven out to the expanded position.", "Mandrel 2 includes a lower end 2a and an upper end 2b.", "In the illustrated embodiment, upper end 2b is formed as a threaded pin for connection into the drill string.", "Of course, end 2b can be formed in other ways, as desired.", "Mandrel 2 also includes an inner bore 3.Housing 4 includes an upper end 4a and a lower end 4b formed as a threaded box for connection directly or indirectly to the pilot bit.", "End 4b can be formed in other ways for connection to the pilot bit.", "Housing 4 further includes an upper inner bore 6a, a middle inner bore 6b, a lower inner bore 6c, which together form an axial bore from end 4a to end 4b.", "A shoulder 7 is positioned between middle inner bore 6b and lower inner bore 6c.", "Mandrel 2 is positioned in the upper and the middle inner bores of housing 4 and the housing 4 is slidably mounted to move axially over mandrel 2, as limited by abutment of a shoulder 2c on the mandrel against end 4a of the housing and abutment of shoulder 4d on the housing against a shoulder formed by a split ring 8 mounted on the mandrel.", "When the under reamer is not in operation, housing 4 is normally in a lower position relative to the mandrel wherein shoulder 4d abuts against split ring 8.In this position, the mandrel is pulled up out of middle inner bore 4b with end 2a spaced from shoulder 7.For operation of the under reamer, however, housing 4 is driven to an upper position on the mandrel wherein end 4a abuts against shoulder 2c and end 2a is positioned close to shoulder 7.Housing 4 is driven upwardly by applying weight on the pilot bit, wherein the housing will be held stationary while the mandrel and the attached drill string is moved down.", "When end 2a of mandrel is positioned adjacent shoulder 7, inner bore 3 of mandrel is in communication with lower inner bore 6c of the housing.", "Seals 9 are provided to seal between end 2a and housing 4 when end 2a of mandrel is positioned adjacent shoulder 7, to seal against fluid flow between the mandrel and the housing past shoulder 7.While housing 4 and mandrel 2 can move axially with respect to each other, they are restricted from relative rotational movement by an interlock arrangement such as a hex-shaped area 2d, or other arrangement, on the mandrel that mates with a similarly shaped area on the housing.", "The interlock arrangement ensures that torque applied to the mandrel is transferred to the housing and thereby to the plurality of under reamer arms 5 mounted in slots 4e on the housing.", "Under reamer arms 5 have cutters 10 mounted in cutter supporting portions 5a at their outer ends and are pivotally connected at their opposite ends by pins 5b to housing 4.The pivotal connection via pins 5b permits arms 5 to move between a stored position within slots 4e and a radially expanded position wherein cutter supporting portions 5a extend out from slots 4e past the outer surface of housing 4.In the radially expanded position cutters 10 on portions 5a are exposed for enlarging the well bore.", "The position of each under reamer arm in the radially expanded position is limited by abutment of it the arm against a side of or catch on the slot.", "Under reamer arms 5 are mounted to extend into middle inner bore 6b of the housing in the space between shoulder 7 and end 2a of the mandrel, when the mandrel is drawn up out of the housing bore.", "As such, to reduce the effective outer radius, r, of the under reamer in the region of the arms in the stored position, arms 5 preferably are formed to abut against one another at or about the center axis of the middle inner bore 6b.", "To further reduce the effective outer radius, r, of the arms 5 in the stored position, their rear surfaces can be formed to fit closely together.", "In operation, it is advantageous that arms 5 not retract every time there is a pressure drop.", "Thus, in one embodiment, there is provided a releasable operational lock assembly to lock mandrel 2 and housing 4 in the operating position.", "In the illustrated embodiment of FIGS.", "1A and 1B, the operational releasable lock assembly includes a plurality of spring-biased detents 66 arranged in the housing to act inwardly to engage in a groove 68 formed in an outer surface of the mandrel.", "The spring force of detents 66 is selected to be greater than the force generated by the weight of housing 4, arms 5, the pilot bit (not shown) and other components below the housing which would tend to pull the housing down over the mandrel.", "However, the spring force of the detents 66 can be overcome to move the housing down and allow retraction of under reamer arms 5 when a selected force is applied, such as the force generated by pulling the under reamer arms against the bottom end of the casing when the under reamer is pulled up into the casing to be tripped to surface.", "Other operational locks can be used such as for example one in which the detents are positioned in the mandrel to engage a groove in the housing, one in which the detents are replaced by a c-ring or one including knurled areas positioned on the mandrel and the housing to interengage.", "In the illustrated embodiments of FIGS.", "1 to 5, the under reamer also includes a releasable tripping lock assembly 80 for releasably locking the housing in position on the mandrel with the mandrel pulled up out of middle inner bore 4b.", "This is useful during tripping the under reamer through the casing.", "For example, this tripping lock assembly prevents the mandrel and housing from moving axially relative to each other during tripping, thereby, for example, avoiding the arms from being driven out while the under reamer is inside the casing.", "The tripping lock assembly 80 includes a plurality of spring-biased plugs 11 mounted in the housing that engage in recesses 11a formed on the mandrel surface.", "Plugs 11 are normally biased by springs 11d against mandrel and will drop into recesses 11a, when the recesses are aligned therebelow, thus locking the position of the housing relative to the mandrel.", "Recesses 11a are positioned on the mandrel such that they align below plugs 11, when the housing is in the tripping position on the mandrel.", "The plugs can be moved against the force in springs 11d to drive the plugs out of the recesses by pressure actuated pins 11c disposed in holes extending from the mandrel inner bore 3 to recesses 11a on the mandrel.", "Pins 11c can be pushed inwardly toward inner bore 3, as limited by return 11e, and can be pushed outwardly into the recesses, as limited by abutment against the housing or plugs 11.Seals 11f seal against passage of fluid about pins 11c and ensure that fluid pressure acts against the bore-facing surfaces of the pins.", "In operation of the tripping lock assembly, during tripping of the under reamer, housing 4 is positioned such that plugs 11 are biased into recesses 11a.", "During tripping there is insufficient fluid pressure to drive pins 11c against plugs 11 such that they remain in the recesses.", "However, when drilling is desired to be initiated, fluid is pumped into the drill string, which is in communication with bore 3.Eventually there is sufficient pressure in bore 3 such that pins 11c are driven out against plugs 11 so that they no longer engage in recesses 11a.", "This releases the lock between the mandrel and the housing such that the housing can move upwardly over the mandrel when weight is applied to the pilot bit.", "The force in springs 11d is selected to limit the ability of pins 11c to push plugs 11 out of the recesses.", "The force is selected such that springs 11d will not collapse against the fluid pressures present in the drill string during tripping, but can be collapsed by fluid pressures present in the mandrel just below those pressure generated during drilling.", "To facilitate assembly, plugs 11 can be mounted in the housing by use of threaded keepers 11b.", "To assist in the generation of sufficient pressures, bore 3 has formed or positioned therein a restriction nozzle 34 to increase the pressure in the mandrel inner bore above the nozzle and against the inner bore-facing surfaces of pins 11c.", "Restriction nozzle 34 acts as to increase the pressure differential between the fluid pressure in the mandrel bore and the pressure external to the tool in the borehole in which the under reamer is operated.", "Referring to FIGS.", "6A to 8, another under reamer according to the present invention is shown.", "The illustrated under reamer can be actuated both by application of weight on bit, as in the under reamer described hereinabove, and by application of fluid pressure to the mandrel bore.", "The under reamer includes a mandrel 12 and a housing 14, which carries a plurality of under reamer arms 15.The under reamer arms are moveable between a retracted position (FIG.", "6A), wherein they are retracted against or into the housing and an expanded position (FIG.", "6B) wherein they are exposed for use to enlarge the well bore.", "The under reamer arms are moveable between the retracted position and the expanded position by relative axial movement of the mandrel and the housing.", "In particular, when housing 14 is in a lower position relative to the mandrel, under reamer arms 15 are in, or can be driven into, the retracted position and when housing 14 is moved upwardly over mandrel 12, such that the mandrel is inserted further into the housing, the under reamer arms are driven out to the expanded position.", "Mandrel 12 includes a lower end 12a and an upper end 12b, which in the illustrated embodiment is formed as a threaded pin for connection to the drilling lock assembly or another component for eventual connection to the drill string.", "Mandrel 12 further includes an inner bore 13.Housing 14 includes an upper end and a lower end 14b formed as a threaded box for connection directly or indirectly to the pilot bit.", "Housing 14 further includes an upper inner bore 16a, a middle inner bore 16b, a lower inner bore 16c and a shoulder 17 between the middle and lower inner bores.", "Mandrel 12 is positioned in the upper inner bore of housing 14 and housing 14 is slidably mounted to move axially over mandrel 12 as limited by abutment of shoulder 18 on housing with shoulder 22 on the mandrel.", "When the under reamer is not in operation, housing 14 is normally in a lower position relative to mandrel wherein shoulder 18 abuts against shoulder 22 and end 12a is spaced from shoulder 17.However, for operation of the under reamer, housing 14 is driven to an upper position on mandrel 12, wherein the mandrel extends into middle inner bore 16b and end 12a is adjacent shoulder 17.Housing 14 can be driven upwardly in two ways, first by injection of hydraulic fluid into a chamber 26 formed between the housing and the mandrel and/or, second, by applying weight on the pilot bit.", "In particular, in chamber 26 a piston face 28 is formed on the housing against which fluid pressure can act to drive the housing along mandrel 12.Piston face 28 is in communication with inner bore 13 of the mandrel via ports 30, such that fluid pressure applied from surface can be communicated through bore 13 and into chamber 26.Seals 32, 33 act between the housing and the mandrel to contain fluid pressure within the chamber.", "To enhance operation of chamber 26 to move housing 14 upwardly over the mandrel, bore 13 has formed or positioned therein a restriction nozzle 34 to increase the pressure in the mandrel inner bore above the nozzle, in ports 30 and in chamber 26.Restriction nozzle 34 acts as to increase the pressure differential between the fluid pressure in these areas and the pressure external to the tool in the borehole in which the under reamer is operated.", "When end 12a of the mandrel is in close proximity to shoulder 17, inner bore 13 of mandrel is in communication with lower inner bore 16c of the housing.", "Since it is desirable to achieve jetting into the housing lower inner bore 16c, seals 35 are provided at the interface to seal against fluid flow therebetween when the mandrel is positioned close to the shoulder 17.Lower inner bore 16c is formed as a diffuser, having an increasing inner diameter from shoulder 17 toward end 14b, to recover the pressure loss created by restriction nozzle 34.Thus, even though nozzle 34 is installed in the under reamer, the fluid pressure is not compromised at the pilot bit nozzles.", "While housing 14 and mandrel 12 can move axially with respect to each other, they are restricted from relative rotational movement by an interlock arrangement such as a splined surface 36 on the mandrel that mates with a correspondingly shaped area on the housing.", "The interlock arrangement ensures that torque applied to the mandrel is transferred to the housing and thereby to the plurality of under reamer arms 15 mounted in slots 42 on the housing.", "Under reamer arms 15 include cutter supporting portions 43 at their outer ends and are pivotally connected at their opposite ends by pins 46 to housing 14.The pivotal connections permit arms 15 to move between a stored position within slots 42 and a radially expanded position wherein cutter supporting portions 43 extend out from slots 42 past the outer surface of housing 14.In the radially expanded position cutters 44 on portions 43 are exposed for enlarging the well bore.", "The position of each under reamer arm in the radially expanded position is limited by abutment of surface 50 against upper end 52 of the slot in which it is mounted.", "Under reamer arms 15 are mounted to extend into middle inner bore 16b of the housing and, in particular, are positioned on the housing in the space between shoulder 17 and end 12a of the mandrel, when housing 14 is in the lower position on the mandrel.", "As such, arms 15 when in the stored position abut against one another at the center axis of the upper inner bore 16a.", "To reduce the effective outer radius, r, of the under reamer at the arms in the stored position, rear surfaces 56 of the arms can be formed to fit closely together.", "Under reamer arms 15, as noted hereinbefore, are driven radially outwardly when the housing is moved upwardly over the mandrel.", "In particular, the arms are driven outwardly initially by the force of fluid jetting through nozzle 34 and then by abutment of end 12a of mandrel against the rear surfaces of the arms.", "In the expanded position, the mandrel is positioned behind the arms and the arms are held out by the mandrel.", "To increase the strength of the arms, they are supported about their sides and rear surfaces.", "In particular, slots 42 are formed at their upper ends 52 and sides 53 to substantially conform to the sides of the arms and at least a portion 57 of the rear surfaces of the arms are formed to substantially conform to the outer surface of the mandrel.", "In addition, preferably the arms are formed with respect to the slots and the mandrel such that when they are in the expanded position, they are supported along their length within the slot with substantially only cutter supporting portions 43 extending out from the slot.", "The housing about slots 42 has formed thereon raised surfaces 62 to ream out or engage the hole ahead of the cutters 44.The housing can also include protrusions 63 above the slot which act as centralizers/spacers spacing the under reamer arms from surfaces such as the ID of the casing during tripping.", "In operation, it is advantageous that arms 15 not retract every time there is a pressure drop.", "Thus, in one embodiment, there is provided a releasable operational lock assembly to lock mandrel 12 and housing 14 in the operating position.", "In the illustrated embodiment of FIGS.", "6 and 7, the releasable lock assembly is a spring-biased detent arrangement including a plurality of spring-biased detents 66 in the housing which engage in a groove 68 in the mandrel.", "The spring force of detents 66 is selected to be greater than the force generated by the weight of housing 14, arms 15, the pilot bit (not shown) and other components below the housing which would tend to pull the housing down over the mandrel.", "However, the spring force of the detents 66 can be overcome to move the housing down and allow retraction of under reamer arms 15 when a selected force is applied, such as the force generated by pulling the under reamer arms against the end of the casing to trip the under reamer to surface.", "Upper surfaces 15a of the arms are ramped to facilitate folding down of the under reamer arms when they are butted against the casing.", "To facilitate manufacture and assembly, mandrel 12 and housing 14 can be formed in sections as shown and threaded together or secured in some other way.", "Nozzle 34 and a section of the housing around shoulder 17 are advantageously removable since they are subject to wear by fluid jetting therethrough.", "Pins 46 are conveniently installed through bores in the housing and secured between shoulders 70 and screws 72.Detents 66 are installed in ports 74 by threaded caps 76.An under reamer arm is shown in FIG.", "8 that is useful in under reamers according to the present invention.", "The under reamer arm includes a bore 78 for accepting pin 46 and at its opposite end a cutter-supporting portion 43 in which are installed a plurality of cutters 44a, 44b, 44c.", "Arm 15 includes those cutters 44a positioned for initially extending the borehole diameter, those cutters 44b for maintaining the gauge of the borehole and those cutters 44c for use in back reaming, should the under reamer be pulled up hole during operation.", "In the illustrated embodiment, the cutters are formed of polycrystalline diamond compact (PDC) and the arms are formed as blocks of material.", "The use of PDC cutters permits the arms to be rugged and the under reamer to be compact and rigid, with significant support for the arms.", "Hard facing can be used about the cutters to increase the durability of the arm.", "Referring to FIGS.", "9A to 12D, there is shown another under reamer according to the present invention.", "The under reamer is generally as shown in FIGS.", "6A to 7C but additionally includes a releasable tripping lock assembly 80 for releasably locking the housing to the mandrel during tripping the under reamer through the casing and a releasable under reamer arm lock 92 for locking the under reamer arms into the stored position.", "Generally, the under reamer includes a mandrel 12, a housing 14 and a plurality of under reamer arms 15, such as those shown in FIG.", "8 or according to other embodiments.", "Mandrel 12 includes a lower end 12a and an inner bore 13 with a venturi nozzle 34 formed therein.", "Housing 14 includes a lower end 14b formed as a threaded box for connection directly or indirectly to the pilot bit.", "Housing 14 includes an upper inner bore 16a and a middle inner bore 16b in which mandrel 12 is telescopically disposed.", "A hydraulic chamber 26 is formed between the housing and the mandrel and includes a piston face 28 formed on housing 14.Housing 14 further includes a shoulder 17 and therebelow a lower inner bore 16c formed as a diffuser.", "An interlock arrangement (FIG.", "10B) is provided between the mandrel and the housing.", "The interlock arrangement includes splines 36a on the mandrel that are formed to interlock with longitudinal grooves 36b in the upper inner bore of the housing.", "The under reamer arms include cutter-supporting portions at their outer ends, which have supported therein cutters 44.The under reamer arms 15 are pivotally mounted by pins 46 in slots 42 in a region of the housing adjacent the middle inner bore from which mandrel 12 is retracted when the housing is in the lower position on the mandrel.", "The under reamer includes a releasable operational lock assembly including a plurality of spring-biased detents 66 and a groove 68.The under reamer also includes a releasable tripping lock assembly 80 for releasably locking the housing to the mandrel during tripping the under reamer and a releasable under reamer arm lock 92 for locking the under reamer arms into the stored position.", "The tripping lock assembly 80 includes a collet 81 mounted on housing 14 in hydraulic chamber 26 and an annular piston 85.Piston 85 forms the upper end of chamber 26 and is biased into chamber 26 by springs 86 acting between the piston and the housing.", "Seals 87, such as o-rings, seal on either side of the piston to prevent loss of fluid from chamber 26.Fluid pressure can pass through collet 81 and act against face 85a of the piston to drive the piston against the force of springs 86.The piston includes a stepped wall 85b extending into the chamber along the housing such that a space is formed between mandrel 12 and wall 85b of the piston.", "Stepped wall 85b includes a first level 88a and a second level 88b, wherein the first level 88a defines an inner radius greater than that of the second level 88b.", "Collet 81 includes a plurality of lugs 82 biased inwardly by an elastomeric ring 83.The lugs are each mounted, with allowance for pivotal movement at their base end into an annular return 84 fixed on the housing.", "The opposite end of each lug are positioned between and can move radially inwardly and outwardly, relative to their base end mounting between mandrel 12 and stepped wall 85b.", "Since lugs 82 are mounted at their bases to housing 14, movement of piston 85 in the chamber drives wall 85b past lugs 82 so that the lugs can move between a position contacting first level 88a and a position contacting second level 88b.", "Collet 81 and piston 85 move along with the housing as it rides along mandrel 12.Collet 81 is formed to lock against a shoulder 89 on the mandrel, as will be more fully described hereinafter.", "Releasable under reamer arm lock 90 includes a plurality of spring-biased detents 92 mounted to engage between slots 42 and arms 15.In particular, in the illustrated embodiment, a spring-biased detent 92 is positioned in each slot 42 to engage into a recess 94 formed on the arm in that slot, the recess and detent are positioned to align when the arm is in the stored position in the slot.", "The spring force in each detent is sufficient to hold its associated under reamer arm against expanding out of its slot by gravity but can be easily overcome by application of force greater than gravity to the arm.", "It is to be understood that although one releasable under reamer arm lock has been described and illustrated, other under reamer arm locks can be used in accordance with the present invention.", "Preferably, an under reamer arm lock according to the present invention can be repeatedly locked and unlocked without manual adjustment.", "In particular, the arm lock can be unlocked to permit expansion of the arms, then relocked once the arms are retracted and then unlocked again, should the expansion of the arms be again desirable.", "Other under reamer arm locks can include, for example, a spring biased catch that engages over the upper surface of the arms, but can be overcome to provide for expansion or resilient, for example of rubber, pins, that can be deformed to permit passage thereby of the arms but regain their form to extend in front of the arm when the arm is retracted.", "For use, the under reamer mandrel is connected to a drill string, drilling lock assembly or other component for use downhole and a pilot bit is connected directly or indirectly to box end 14b of the housing.", "The under reamer when downhole, is in fluid communication with surface through bore 13 of the mandrel and rotation of the mandrel is transferred to the housing to rotate the under reamer arms and the pilot bit.", "In use, under reamer arms 15 are pivotally moveable between a stored position (FIG.", "9A) and an expanded position (FIG.", "9B).", "In the stored position, the housing is in a lower position relative to the mandrel wherein there is a space between end 12a of the mandrel and shoulder 17.Under reamer arms 15 in the stored position fit together along the center axis of the upper inner bore 16a.", "In the illustrated embodiment, the arms include wedge shaped ends 15b (FIG.", "12B) that permit the arms to fit closely together, thereby reducing their stored outer diameter.", "The under reamer is tripped through the borehole in this stored condition.", "It is desirable for control and ease of tripping that the under reamer arms remain in the stored position and the housing be in a fixed position on the mandrel.", "Thus, in this embodiment, detents 92 are biased into recesses 94 on the arms to prevent them from pivoting out from the housing by gravity when in a deviated hole.", "In addition, mandrel 12 and housing 14 are locked in relative position by locking assembly 80.In particular, in tripping locked position piston 85 is biased by springs 86 into chamber 26 and lugs 82 are engaged between second level 88b of wall 85b and mandrel 12 under shoulder 89.Since lugs are pivotally mounted at their bases on housing 14 and wall 85b is biased behind lugs 82, the lugs cannot bias outwardly past shoulder 89 and, thus, mandrel 12 cannot move relative to housing 14.Weight on bit would not be sufficient to move the housing relative to the mandrel.", "For operation of the under reamer, cutter-supporting portions 43 of the arms must be extended beyond the outer surface of the housing.", "Under reamer arms 15 are driven radially outwardly when the housing is moved upwardly over the mandrel.", "In particular, the arms are driven outwardly by initially the force of fluid jetting through nozzle 34 and then by abutment of the rear surfaces 56 of the arms against end 12a of the mandrel.", "Thus, when it is desired to expand under reamer arms 15 radially outwardly fluid pressure is applied or increased from surface such that restriction nozzle 34 creates a pressure differential between bore 13 and the borehole about the under reamer.", "In so doing, fluid pressure acts against piston face 85a of lock 80 and piston face 28 of housing 14.Preferably, springs 86 are selected to permit actuation of piston 85 at a lower pressure than movement of housing through piston face 28 such that lock 80 is released before pressure is sufficient to move the housing.", "As fluid pressure acts against face 85a, piston 85 is driven against springs 86.This causes wall 85b to move relative to lugs 82 such that the lugs drop onto first level 88a where there is sufficient space for the lugs to be driven radially outwardly by fluid pressure.", "As fluid pressure is further increased, housing 14 begins to be drawn upwardly over the mandrel, driving the lugs past shoulder 89.When housing 14 is forced upwardly, the mandrel is forced against arms 15.While, the arms may already have been released from detents 92 by the pressure of fluid jetting through nozzle 34, mandrel 12 will force the arms fully into their expanded position and support them in this expanded position.", "Housing 14 is drawn up over mandrel until end 12a is in close proximity to shoulder 17 (FIG.", "9B).", "In this position, detents 66 land in groove 68 and releasably lock the housing relative to the mandrel until the force driving the detents into engagement with groove 68 is overcome.", "In the expanded position, the arms are held out by the mandrel.", "Slots 42 and under reamer arms 15 are formed with consideration to each other such that the slots limit the radial outward pivotal expansion of the arms and such that the slots closely surround and support the arms along their sides.", "In addition, mandrel 12 and rear surfaces 56 of the arms are similarly curved such that the arms are supported by the mandrel.", "Should the pressure drop during operation, the arms will remain in the operational outwardly expanded position provided detents 66 remain engaged in groove 68.Should it be desirable to return the under reamer arms to the stored position, force is applied to drive the detents out of engagement with groove 68, as by reducing fluid pressure and pulling the under reamer upwardly to engage the arms against the casing shoe, so that mandrel 12 can be retracted from the housing.", "Another tripping lock assembly is shown in FIGS.", "13A to 13B.", "This tripping lock assembly is similar to that of FIG.", "11, except that rather than collet 81, lock pins 102 and floating ring 104 are used.", "Ring 104 floats in chamber 26 below piston 85 and fluid pressure can by pass the ring to act on piston 85, as in FIG.", "11.Ring 104 carries a plurality of pins 102 that extend through bores in the ring, but are moveable along their axis to move in and out, as permitted by the space about them.", "When housing 14 is locked by the tripping lock in a lower position on the mandrel (FIG.", "13A), ring 104 is positioned up against piston 85 with pins 102 jammed between second level 88b and mandrel 12 under shoulder 89.In this position, pins 102 butt against shoulder 89 to prevent the mandrel from moving further into the housing.", "However, when fluid pressure is applied to bore 13 and communicated to chamber 26, the piston is driven against springs 86 to move upwardly.", "This moves the wall 85b relative to ring 104 so that the pins become positioned adjacent the first level 88a.", "This provides room for pins 102 to move axially away from the mandrel so that the shoulder can move past the pins.", "Although preferred embodiments of the present invention have been described in some detail hereinabove, those skilled in the art will recognise that various substitutions and modifications may be made to the invention without departing from the scope and spirit of the appended claims." ] ]	Patent_10468856
[ [ "Therapeutic methods using herbal compositions", "Compositions derived from traditional Chinese herbal medicines, medicinal plants and extracts thereof, are provided for the prevention and treatment of cancers especially lung cancer, prostate cancer, liver cancer, breast cancer and leukemia.", "The composition is also useful for treating Helicobacter pylori infection The composition is also useful for treating or preventing a chronic inflammatory condition.", "The composition is also useful for treating or preventing cardiovascular disease.", "The composition is also useful for treating or preventing cerebral vascular disease.", "The compositions are useful as adjuncts to conventional surgery, chemotherapy or radiotherapy treatments in patients with cancer.", "Preferred compositions of the invention contain the herbal ingredients Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera." ], [ "1-50.", "(canceled) 51.A method of preventing or treating cancer in a patient in need thereof, said method comprising administering an effective amount of a composition comprising a mixture of Sophora tonkinensis, Polygonum bistorta and Prunella vulgaris; wherein the cancer is selected from the group consisting of lung cancer, prostate cancer, bladder cancer, liver cancer, breast cancer and leukemia.", "52.The method of claim 51, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris and Dictamnus dasycarpus Turcz.", "53.The method of claim 52, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus and Dictamnus dasycarpus Turcz.", "54.The method of claim 53, wherein the composition comprises a mixture of the following herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera.", "55.A method of treating Helicobacter pylori infection in a patient in need thereof, said method comprising administering an effective amount of a composition comprising a mixture of Sophora tonkinensis, Polygonum bistorta and Prunella vulgaris.", "56.The method of claim 55, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris and Dictamnus dasycarpus Turcz.", "57.The method of claim 56, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus and Dictamnus dasycarpus Turcz.", "58.The method of claim 57, wherein the composition comprises a mixture of the following herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera.", "59.A method of treating or reducing the reoccurrence of cancer in a patient in need, said method comprising administering an effective amount of a composition comprising a mixture of Sophora tonkinensis, Polygonum bistorta and Prunella vulgaris.", "60.The method of claim 59, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris and Dictamnus dasycarpus Turcz.", "61.The method of claim 60, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus and Dictamnus dasycarpus Turcz.", "62.The method of claim 61, wherein the composition comprises a mixture of the following herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera.", "63.The method of claim 59, wherein said composition is used as an adjunct to convention surgery, chemotherapy or radiation therapy treatments.", "64.A method of treating or preventing a chronic inflammatory condition in a patient in need thereof, said method comprising administering an effective amount of a composition comprising a mixture of Sophora tonkinensis, Polygonum bistorta and Prunella vulgaris.", "65.The method of claim 64, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris and Dictamnus dasycarpus Turcz.", "66.The method of claim 65, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus and Dictamnus dasycarpus Turcz.", "67.The method of claim 66, wherein the composition comprises a mixture of the following herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera.", "68.A method of treating or preventing cardiovascular disease in a patient in need thereof, said method comprising administering an effective amount of a composition comprising a mixture of Sophora tonkinensis, Polygonum bistorta and Prunella vulgaris.", "69.The method of claim 68, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris and Dictamnus dasycarpus Turcz.", "70.The method of claim 69, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus and Dictamnus dasycarpus Turcz.", "71.The method of claim 70, wherein the composition comprises a mixture of the following herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera.", "72.A method treating or preventing cerebral vascular disease in a patient in need thereof, said method comprising administering an effective amount of a composition comprising a mixture of Sophora tonkinensis, Polygonum bistorta and Prunella vulgaris.", "73.The method of claim 72, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris and Dictamnus dasycarpus Turcz.", "74.The method of claim 73, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus and Dictamnus dasycarpus Turcz.", "75.The method of claim 74, wherein the composition comprises a mixture of the following herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera.", "76.A method of preventing the occurrence of or treating precancerous cells in a patient in need thereof, said method comprising administering an effective amount of a composition comprising a mixture of Sophora tonkinensis, Polygonum bistorta and Prunella vulgaris.", "77.The method of claim 76, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris and Dictamnus dasycarpus Turcz.", "78.The method of claim 77, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus and Dictamnus dasycarpus Turcz.", "79.The method of claim 78, wherein the composition comprises a mixture of the following herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera.", "80.The method of claim 76, wherein the composition is used for the prevention or treatment of bronchial dysplasia.", "81.The method of claim 80, wherein the composition is used for the prevention or treatment of bronchial dysplasia wherein the method is for the prevention or treatment of chronic obstructive pulmonary disease.", "82.A method of treating or preventing atrophic gastritis in a patient in need thereof, said method comprising administering an effective amount of a composition comprising a mixture of Sophora tonkinensis, Polygonum bistorta and Prunella vulgarism.", "83.The method of claim 82, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris and Dictamnus dasycarpus Turcz.", "84.The method of claim 83, wherein the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus and Dictamnus dasycarpus Turcz.", "85.The method of claim 84, wherein the composition comprises a mixture of the following herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera.", "86.A composition consisting essentially of a mixture of at least three different herbs selected from the group consisting of: Herb A, Herb B, Herb C, Herb D and Herb E, wherein Herb A is Sophora tonkinensis (Sophora subprostrata) Herb B is Polygonum bistorta Herb C is Prunella vulgaris Herb D is selected from the group consisting of: Sonchus brachyotus Patrinia scabiosaefolia Fisch.", "Patrinia villosa Juss.", "Sonchus arvensis L. Thlaspi arvense L. Portulaca oleracea L. and Pulsatilla chinensis Reg.", "and Herb E is selected from the group consisting of: Dictamnus dasycarpus Kochia scoparia (L.) Schard.", "Sophora flavescens Ait.", "and Heydyotis diffusa (Willd.)", "Roxb; wherein, when the composition comprises three herbs, they are present in an amount from about 9% to about 57%; when the composition comprises four herbs, they are present in an amount from about 6% to about 43% or when the composition comprises four herbs, the first three are selected from the group consisting of Herb A, Herb B, Herb C and Herb D and are present in an amount from about 7% to about 57% and the fourth is selected from Herb E and is present in an amount from about 3.5% to about 28.5%; and when the composition comprises five herbs they are present in an amount from about 5% to about 35% or the first four herbs are selected from the group consisting of Herb A, Herb B, Herb C and Herb D and are present in an amount from about 6% to about 38% and the fifth herb is selected from Herb E and is present in an amount from about 3% to about 19%.", "87.The composition of claim 86 further comprising a sixth herb, Herb F, selected from the group consisting of: Dioscorea bulbifera Panax notoginseng (Burk) F. H. Chen.", "Bletilla striata (Thunb.)", "Reichb.f.", "Nelumbo nucifera Gaertn.", "Polygonum bistorta L. Cephalanoplos segetum (Boge) Kitam.", "Cirsium japonicum DC.", "Sophora japonica L. Typha angustifolia L. and Rubia cordifolia L.; wherein the first five herbs are selected from the group consisting of Herb A, Herb B, Herb C, Herb D and Herb E wherein the herbs are present in an amount from about 5% to about 33% and the sixth herb is selected from Herb F and is present in an amount from about 1.0% to about 9.5% or the first four herbs are selected from the group consisting of Herb A, Herb B, Herb C or Herb D and is present in an amount from about 5% to about 38% and the fifth is selected from Herb E and is present in an amount from about 2.5% to about 19% and the sixth herb is selected from Herb F and is present in an amount from about 1% to about 9.5%.", "88.The composition of claim 87, wherein Herb F is present in an amount of up to about 3.0% 89.The composition of claim 88, wherein Herb F is present in an amount of up to about 1.4%.", "90.The composition of claim 89, wherein Herb F is present in an amount of about 1.4%." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>For the past twenty-five years there has been significant progress in the field of cancer research; however, in spite of these positive results, the mortality rate for the most common cancers still remains high.", "Indeed, the goal of the National Cancer Institute of a fifty percent reduction in overall cancer mortality by the year 2000 has not been met.", "The term “cancer” is a general one referring to more than 100 forms of the disease which may manifest itself in almost every tissue type of the body.", "Of the myriad forms of cancer, lung cancer is the most common cause of death worldwide, followed by stomach cancer.", "Other common forms of cancer include cancers of the colon, rectum, breast, prostate, mouth and esophagus.", "Lung cancer imposes an enormous burden on health care.", "The World Health Report 2000 estimates that lung cancer has resulted in 860,000 deaths among men and 333,000 deaths among women per year, and it is the leading cause of cancer deaths worldwide.", "In the United States and Canada, more people are dying from lung cancer than from breast cancer, colorectal cancer and prostate cancer combined.", "In addition, the incidence of lung cancer in women is rising at an average annual rate of 7% which is the most rapid rate of increase for any cancer.", "The most dominant cause of lung cancer is tobacco use, but occupational and environmental exposure to various other carcinogenic substances can also influence disease development.", "In long-term smokers, the risk of lung cancer never returns to the “baseline” level of a never-smoker, even years after smoking cessation.", "With a large reservoir (100 million in the United States and Canada alone) of current and former smokers, who are at risk, lung cancer will continue to be a major health problem for at least several more decades even if current efforts to curb tobacco smoking were successful.", "The overall five-year survival rate of lung cancer is less than 15%.", "Despite advances in modern medicine, the survival rate has not improved substantially over the last two decades.", "A different approach is therefore needed to control lung cancer.", "Prostate cancer is the most commonly diagnosed male malignancy in the United States and the second leading cause of cancer death.", "It is estimated that in the year 2000, there will be 180,400 new cases and 31,900 deaths caused by prostate cancer.", "Although prostate cancer responds effectively to orchitectomy or antiandrogen therapy when detected at an early stage, over time, the residual androgen-insensitive cells recolonize, expand, and ultimately establish a hormone-resistant state that often results in fatality.", "It would be useful, therefore, to have a cancer treatment which could prevent the proliferation of prostate cancer and maintain it in a dormant state.", "The incidence of gastric cancer has fallen in most countries but it is still the most common form of cancer in many countries of East Asia, including China.", "Globally, gastric cancer is the second most frequent cause of cancer death and it is estimated that in 1990, there were almost 800,000 new cases and about 630,000 deaths.", "Similarly, esophageal cancer, the eighth most common cancer worldwide and responsible for 316,000 new cases and 286,000 deaths in 1990, is also very common in China and other Asian countries.", "Both of these cancers, and especially esophageal cancer, have low survival rates and thus it would be beneficial to have an alternative treatment approach for these types of cancers.", "Traditionally, the focus of cancer research has been in developing therapies and treatments for patients already afflicted with the disease.", "However, over the last few decades, new insights into the development of cancer as a disease have been gained.", "It is now understood that cancer is not the result of a single initiation event but of a gradual, multi-step process characterized by a period of several years between the initiation event and the onset of invasive or metastatic disease.", "In general, the process of carcinogenesis can be divided into three phases: initiation, promotion, and progression.", "In initiation, a fixed genetic mutation results from the interaction of a carcinogen with DNA.", "The extent of the molecular change depends on a number of factors including the nature of the carcinogen, the rate and type of carcinogenic metabolism and the response of the DNA repair systems.", "The next phase, promotion, may occur over extended periods of time and is characterized by the proliferation of the altered cells.", "This phase may be affected by agents that alter growth rates.", "During the final phase, progression, genetic and phenotypic changes occur which ultimately cause the development of premalignant lesions into invasive cancer.", "The multi-step nature of carcinogenesis suggests the possibility of intervention at a precancerous state.", "This is the basis for chemoprevention, which refers to the use of natural or synthetic agents to prevent the development of cancer, either by blocking the DNA damage that initiates the carcinogenesis process or by arresting/regressing existing pre-malignant lesions.", "Since the mid-1950s, research has been directed at finding compounds with potential chemopreventive properties.", "The search for these agents has demonstrated a unique challenge.", "Chemopreventive agents must have low toxicity and be relatively free of side effects because they are intended for administration to healthy people over long periods of time.", "This is in direct contrast to chemotherapy drugs, such as cis platinum or paclitaxel (Taxol™), which are used as chemotherapeutic agents to treat people already afflicted with cancer.", "Chemotherapeutic agents are chosen for their ability to kill tumor cells but because they are also toxic to healthy cells, they usually cause harmful side effects.", "One of the major sources of potential chemopreventive agents is plants.", "For example, consumption of cruciferous vegetables, such as broccoli, cauliflower and cabbage is associated with a lower risk of various cancers.", "Fruits and vegetables contain a number of potentially active chemopreventive compounds, such as carotenoids, dithiolthiones and isothiocyanates.", "They are capable of inhibiting the development of tumors of the lungs, colon, mammary glands and bladder in laboratory animals.", "Three proof-of-principle clinical trials suggest that chemoprevention might be an effective strategy to control lung cancer.", "A study by Hong and co-workers in the United States showed that in patients with cured head and neck cancer, high dose 13-cis-retinoic acid for 12 months was more effective than placebo in preventing second primary cancers in the upper aerodigestive tract.", "However, 13-cis-retinoic acid at this dosage carries unacceptable toxicity for use in the general population.", "Another study by Pastorino and co-workers in Europe showed that retinol palmitate in a dose of 300,000 Units per day for 12 months was more effective than placebo in preventing second primary lung cancer.", "A third study in China found that daily doses of a combination of vitamins and minerals consisting of betaarotene, vitamin E and selenium resulted in a 21% decrease in stomach cancer deaths in high-risk people in China.", "However, subsequent phase III clinical trials using beta-carotene or retinal (the Alpha-Tocopherol, Beta-Carotene Trial, the Beta-Carotene and Retinol Efficacy Trial, and the EUROSCAN study) failed to show a reduction in lung cancer incidence in high-risk individuals, such as heavy smokers with or without exposure to asbestos, compared to placebo.", "In fact, the use of beta-carotene in those who continued to smoke during the study was found to increase the risk of lung cancer.", "Several reasons were postulated to explain why chemopreventive treatment with retinoids was ineffective or even harmful in active smokers.", "There may be adverse interactions between tobacco carcinogens and the chemopreventive agent.", "Beta-carotene, for example, is a pro-oxidant at high arterial oxygen tension.", "It can enhance conversion of benzo[A]pyrene to the ultimate carcinogen as well as inducing cytochrome P450.Another reason for the lack of effect in active smokers is that ongoing tobacco carcinogen exposure may counteract the effect of the chemopreventive agent.", "A number of chemopreventive agents are currently under clinical investigation.", "Examples of these include fenretinide, selenium, inhaled budesonide, COX-2 inhibitors, farnesyl transferase inhibitors, lipoxygenase inhibitors, EGF-kinase inhibitors and green tea.", "Although promising, it remains to be shown if these agents can be shown to be useful in ongoing clinical trials.", "The best example to-date that chemoprevention can prevent cancer is Tamoxifen.", "Tamoxifen is an estrogen antagonist.", "In women at high risk of breast cancer, the Breast Cancer Prevention Trial showed a 49% decrease in invasive cancer and a 50% decrease in non-invasive breast cancer with Tamoxifen versus placebo (J Natl Cancer Inst 1998; 90:1371-88).", "In addition, there was a decrease in the incidence of fractures due to osteoporosis.", "However, there was a slight increase in the risk of endometrial cancer, deep venous thrombosis and pulmonary embolism.", "More selective estrogen-receptor modulators such as Raloxifene, are being tested against Tamoxifen to determine if these agents may have similar chemopreventive effect but fewer side effects than Tamoxifen.", "A variety of Chinese herbs have been used for centuries to treat different diseases.", "The great majority of these are empirical, open, non-randomized studies without placebo control groups.", "Although some of these herbs have been used to treat patients with cancer, they are not considered to be disease specific.", "Rather, they are used for “dispersing heat, detoxification, improving stasis and removing mass”.", "Herbs such as Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz , and Dioscorea bulbifera are known to have properties that may be useful for the prevention and treatment of cancers.", "One such composition is known as ZSP or Zeng Sheng Ping; however, the exact formulation of the composition is not known.", "European Patent application 93 121109.8 describes a composition comprising Sophora tonkinensis Gapnep 42 mg, Polygonum bistorta L. 42 mg, Prunella vulgaris L. 42 mg, Sonchus brachyotus DC 42 mg, Dictamnus dasycarpus Turcz 21 mg, and Dioscorea bulbifera 10 mg which was useful for the treatment of patients with mouth, esophagus or digestive tract cancers.", "It is well known that a chemopreventive agent that works in one organ may not work in the other organ.", "For example Tamoxifen, as discussed above, has been used in the treatment of breast cancer, but has not been found to be effective in any other cancers.", "Likewise, there is no evidence that other promising agents for preventing breast cancer, such as raloxifene and anastrozole, prevent other cancers.", "Therefore, persons would not predict that the composition described in the above referenced European Patent application, which was effective for treating esophagus cancer, would also show activity in other organs such as lung, prostate or breast.", "There is, thus, a need to determine other uses for this herbal composition and other related herbal compositions." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Thus, according to the present invention there is provided a composition comprising herbs for uses in treating or preventing a variety of medical conditions.", "Thus, according to this embodiment of the invention, there is provided a composition comprising a mixture of at least three different herbs selected from the group consisting of herbs identified in this invention as: Herb A, Herb B, Herb C, Herb D, Herb E and Herb F; wherein Herb A is selected from the group consisting of: Sophora tonkinensis ( Sophora subprostrata ) Belamcanda chinensis Scrophularia ningpoensis Isatis tinctoria Isatis indigotica and Baphicacanthus cusia; Herb B is selected from the group consisting of: Polygonum bistorta Polygonum lapidosum Polygonum viviparum Polygonum manshuriense Polygonum alopecuroides Polygonum sphaerostachyum Andrographis paniculata Taraxacum mongolicum and Chrysanthemun indicum; Herb C is selected from the group consisting of: Prunella vulgaris Artemissia capillaris Gardenia jasminoides Rosa rugosa and Lophatherum gracile; Herb D is selected from the group consisting of: Sonchus brachyotus Patrinia scabiosaefolia Patrinia villosa Sonchus arvensis Thlaspi arvense Portulaca oleracea and Pulsatilla chinensis; Herb E is selected from the group consisting of: Dictamnus dasycarpus Kochia scoparia Sophora flavescens and Heydyotis diffusa and Herb F is selected from the group consisting of: Dioscorea bulbifera Panax notoginseng Bletilla striata Nelumbo nucifera Polygonum bistorta Cephalanoplos segetum Cirsium japonicum Sophora japonica Typha angustifolia , and Rubia cordifolia ; wherein said compostions is used as chemopreventive and therapeutic agents.", "In one example of this invention, there is provided a method of preventing or treating cancer by administering to a patient in need thereof an effective amount of a composition as defined above.", "In a further example of this embodiment of the invention, there is provided a method of treating Helicobacter pylori infection by administering to a patient in need thereof an effective amount of a composition as defined above.", "In yet a further example of this embodiment of the invention, there is provided a method of treating or reducing the reoccurrence of cancer by administering to a patient in need thereof an effective amount of a composition as defined above.", "In yet a further example of this embodiment of the invention, there is provided a method of treating or preventing cancer by administering to a patient in need thereof an effective amount of a composition as defined above, as adjuncts to conventional surgery, chemotherapy or radiotherapy treatments.", "In a further example of this invention, there is provided a method of treating atrophic gastritis by administering to a patient in need thereof an effective amount of a composition as defined above.", "In a further example of this embodiment of the invention, there is provided a method of treating or preventing a chronic inflammatory condition by administering to a patient in need thereof an effective amount of a composition as defined above.", "In yet a further example of this embodiment of the invention, there is provided a method of treating or preventing cardiovascular disease by administering to a patient in need thereof an effective amount of a composition as defined above.", "In yet a further example of this embodiment of the invention, there is provided a method of treating or preventing cerebral vascular disease by administering to a patient in need thereof an effective amount of a composition as defined above." ], [ "The present invention relates to compositions comprising herbs derived from traditional Chinese medicine and their use as chemopreventive and therapeutic agents.", "BACKGROUND OF THE INVENTION For the past twenty-five years there has been significant progress in the field of cancer research; however, in spite of these positive results, the mortality rate for the most common cancers still remains high.", "Indeed, the goal of the National Cancer Institute of a fifty percent reduction in overall cancer mortality by the year 2000 has not been met.", "The term “cancer” is a general one referring to more than 100 forms of the disease which may manifest itself in almost every tissue type of the body.", "Of the myriad forms of cancer, lung cancer is the most common cause of death worldwide, followed by stomach cancer.", "Other common forms of cancer include cancers of the colon, rectum, breast, prostate, mouth and esophagus.", "Lung cancer imposes an enormous burden on health care.", "The World Health Report 2000 estimates that lung cancer has resulted in 860,000 deaths among men and 333,000 deaths among women per year, and it is the leading cause of cancer deaths worldwide.", "In the United States and Canada, more people are dying from lung cancer than from breast cancer, colorectal cancer and prostate cancer combined.", "In addition, the incidence of lung cancer in women is rising at an average annual rate of 7% which is the most rapid rate of increase for any cancer.", "The most dominant cause of lung cancer is tobacco use, but occupational and environmental exposure to various other carcinogenic substances can also influence disease development.", "In long-term smokers, the risk of lung cancer never returns to the “baseline” level of a never-smoker, even years after smoking cessation.", "With a large reservoir (100 million in the United States and Canada alone) of current and former smokers, who are at risk, lung cancer will continue to be a major health problem for at least several more decades even if current efforts to curb tobacco smoking were successful.", "The overall five-year survival rate of lung cancer is less than 15%.", "Despite advances in modern medicine, the survival rate has not improved substantially over the last two decades.", "A different approach is therefore needed to control lung cancer.", "Prostate cancer is the most commonly diagnosed male malignancy in the United States and the second leading cause of cancer death.", "It is estimated that in the year 2000, there will be 180,400 new cases and 31,900 deaths caused by prostate cancer.", "Although prostate cancer responds effectively to orchitectomy or antiandrogen therapy when detected at an early stage, over time, the residual androgen-insensitive cells recolonize, expand, and ultimately establish a hormone-resistant state that often results in fatality.", "It would be useful, therefore, to have a cancer treatment which could prevent the proliferation of prostate cancer and maintain it in a dormant state.", "The incidence of gastric cancer has fallen in most countries but it is still the most common form of cancer in many countries of East Asia, including China.", "Globally, gastric cancer is the second most frequent cause of cancer death and it is estimated that in 1990, there were almost 800,000 new cases and about 630,000 deaths.", "Similarly, esophageal cancer, the eighth most common cancer worldwide and responsible for 316,000 new cases and 286,000 deaths in 1990, is also very common in China and other Asian countries.", "Both of these cancers, and especially esophageal cancer, have low survival rates and thus it would be beneficial to have an alternative treatment approach for these types of cancers.", "Traditionally, the focus of cancer research has been in developing therapies and treatments for patients already afflicted with the disease.", "However, over the last few decades, new insights into the development of cancer as a disease have been gained.", "It is now understood that cancer is not the result of a single initiation event but of a gradual, multi-step process characterized by a period of several years between the initiation event and the onset of invasive or metastatic disease.", "In general, the process of carcinogenesis can be divided into three phases: initiation, promotion, and progression.", "In initiation, a fixed genetic mutation results from the interaction of a carcinogen with DNA.", "The extent of the molecular change depends on a number of factors including the nature of the carcinogen, the rate and type of carcinogenic metabolism and the response of the DNA repair systems.", "The next phase, promotion, may occur over extended periods of time and is characterized by the proliferation of the altered cells.", "This phase may be affected by agents that alter growth rates.", "During the final phase, progression, genetic and phenotypic changes occur which ultimately cause the development of premalignant lesions into invasive cancer.", "The multi-step nature of carcinogenesis suggests the possibility of intervention at a precancerous state.", "This is the basis for chemoprevention, which refers to the use of natural or synthetic agents to prevent the development of cancer, either by blocking the DNA damage that initiates the carcinogenesis process or by arresting/regressing existing pre-malignant lesions.", "Since the mid-1950s, research has been directed at finding compounds with potential chemopreventive properties.", "The search for these agents has demonstrated a unique challenge.", "Chemopreventive agents must have low toxicity and be relatively free of side effects because they are intended for administration to healthy people over long periods of time.", "This is in direct contrast to chemotherapy drugs, such as cis platinum or paclitaxel (Taxol™), which are used as chemotherapeutic agents to treat people already afflicted with cancer.", "Chemotherapeutic agents are chosen for their ability to kill tumor cells but because they are also toxic to healthy cells, they usually cause harmful side effects.", "One of the major sources of potential chemopreventive agents is plants.", "For example, consumption of cruciferous vegetables, such as broccoli, cauliflower and cabbage is associated with a lower risk of various cancers.", "Fruits and vegetables contain a number of potentially active chemopreventive compounds, such as carotenoids, dithiolthiones and isothiocyanates.", "They are capable of inhibiting the development of tumors of the lungs, colon, mammary glands and bladder in laboratory animals.", "Three proof-of-principle clinical trials suggest that chemoprevention might be an effective strategy to control lung cancer.", "A study by Hong and co-workers in the United States showed that in patients with cured head and neck cancer, high dose 13-cis-retinoic acid for 12 months was more effective than placebo in preventing second primary cancers in the upper aerodigestive tract.", "However, 13-cis-retinoic acid at this dosage carries unacceptable toxicity for use in the general population.", "Another study by Pastorino and co-workers in Europe showed that retinol palmitate in a dose of 300,000 Units per day for 12 months was more effective than placebo in preventing second primary lung cancer.", "A third study in China found that daily doses of a combination of vitamins and minerals consisting of betaarotene, vitamin E and selenium resulted in a 21% decrease in stomach cancer deaths in high-risk people in China.", "However, subsequent phase III clinical trials using beta-carotene or retinal (the Alpha-Tocopherol, Beta-Carotene Trial, the Beta-Carotene and Retinol Efficacy Trial, and the EUROSCAN study) failed to show a reduction in lung cancer incidence in high-risk individuals, such as heavy smokers with or without exposure to asbestos, compared to placebo.", "In fact, the use of beta-carotene in those who continued to smoke during the study was found to increase the risk of lung cancer.", "Several reasons were postulated to explain why chemopreventive treatment with retinoids was ineffective or even harmful in active smokers.", "There may be adverse interactions between tobacco carcinogens and the chemopreventive agent.", "Beta-carotene, for example, is a pro-oxidant at high arterial oxygen tension.", "It can enhance conversion of benzo[A]pyrene to the ultimate carcinogen as well as inducing cytochrome P450.Another reason for the lack of effect in active smokers is that ongoing tobacco carcinogen exposure may counteract the effect of the chemopreventive agent.", "A number of chemopreventive agents are currently under clinical investigation.", "Examples of these include fenretinide, selenium, inhaled budesonide, COX-2 inhibitors, farnesyl transferase inhibitors, lipoxygenase inhibitors, EGF-kinase inhibitors and green tea.", "Although promising, it remains to be shown if these agents can be shown to be useful in ongoing clinical trials.", "The best example to-date that chemoprevention can prevent cancer is Tamoxifen.", "Tamoxifen is an estrogen antagonist.", "In women at high risk of breast cancer, the Breast Cancer Prevention Trial showed a 49% decrease in invasive cancer and a 50% decrease in non-invasive breast cancer with Tamoxifen versus placebo (J Natl Cancer Inst 1998; 90:1371-88).", "In addition, there was a decrease in the incidence of fractures due to osteoporosis.", "However, there was a slight increase in the risk of endometrial cancer, deep venous thrombosis and pulmonary embolism.", "More selective estrogen-receptor modulators such as Raloxifene, are being tested against Tamoxifen to determine if these agents may have similar chemopreventive effect but fewer side effects than Tamoxifen.", "A variety of Chinese herbs have been used for centuries to treat different diseases.", "The great majority of these are empirical, open, non-randomized studies without placebo control groups.", "Although some of these herbs have been used to treat patients with cancer, they are not considered to be disease specific.", "Rather, they are used for “dispersing heat, detoxification, improving stasis and removing mass”.", "Herbs such as Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera are known to have properties that may be useful for the prevention and treatment of cancers.", "One such composition is known as ZSP or Zeng Sheng Ping; however, the exact formulation of the composition is not known.", "European Patent application 93 121109.8 describes a composition comprising Sophora tonkinensis Gapnep 42 mg, Polygonum bistorta L. 42 mg, Prunella vulgaris L. 42 mg, Sonchus brachyotus DC 42 mg, Dictamnus dasycarpus Turcz 21 mg, and Dioscorea bulbifera 10 mg which was useful for the treatment of patients with mouth, esophagus or digestive tract cancers.", "It is well known that a chemopreventive agent that works in one organ may not work in the other organ.", "For example Tamoxifen, as discussed above, has been used in the treatment of breast cancer, but has not been found to be effective in any other cancers.", "Likewise, there is no evidence that other promising agents for preventing breast cancer, such as raloxifene and anastrozole, prevent other cancers.", "Therefore, persons would not predict that the composition described in the above referenced European Patent application, which was effective for treating esophagus cancer, would also show activity in other organs such as lung, prostate or breast.", "There is, thus, a need to determine other uses for this herbal composition and other related herbal compositions.", "SUMMARY OF THE INVENTION Thus, according to the present invention there is provided a composition comprising herbs for uses in treating or preventing a variety of medical conditions.", "Thus, according to this embodiment of the invention, there is provided a composition comprising a mixture of at least three different herbs selected from the group consisting of herbs identified in this invention as: Herb A, Herb B, Herb C, Herb D, Herb E and Herb F; wherein Herb A is selected from the group consisting of: Sophora tonkinensis (Sophora subprostrata) Belamcanda chinensis Scrophularia ningpoensis Isatis tinctoria Isatis indigotica and Baphicacanthus cusia; Herb B is selected from the group consisting of: Polygonum bistorta Polygonum lapidosum Polygonum viviparum Polygonum manshuriense Polygonum alopecuroides Polygonum sphaerostachyum Andrographis paniculata Taraxacum mongolicum and Chrysanthemun indicum; Herb C is selected from the group consisting of: Prunella vulgaris Artemissia capillaris Gardenia jasminoides Rosa rugosa and Lophatherum gracile; Herb D is selected from the group consisting of: Sonchus brachyotus Patrinia scabiosaefolia Patrinia villosa Sonchus arvensis Thlaspi arvense Portulaca oleracea and Pulsatilla chinensis; Herb E is selected from the group consisting of: Dictamnus dasycarpus Kochia scoparia Sophora flavescens and Heydyotis diffusa and Herb F is selected from the group consisting of: Dioscorea bulbifera Panax notoginseng Bletilla striata Nelumbo nucifera Polygonum bistorta Cephalanoplos segetum Cirsium japonicum Sophora japonica Typha angustifolia, and Rubia cordifolia; wherein said compostions is used as chemopreventive and therapeutic agents.", "In one example of this invention, there is provided a method of preventing or treating cancer by administering to a patient in need thereof an effective amount of a composition as defined above.", "In a further example of this embodiment of the invention, there is provided a method of treating Helicobacter pylori infection by administering to a patient in need thereof an effective amount of a composition as defined above.", "In yet a further example of this embodiment of the invention, there is provided a method of treating or reducing the reoccurrence of cancer by administering to a patient in need thereof an effective amount of a composition as defined above.", "In yet a further example of this embodiment of the invention, there is provided a method of treating or preventing cancer by administering to a patient in need thereof an effective amount of a composition as defined above, as adjuncts to conventional surgery, chemotherapy or radiotherapy treatments.", "In a further example of this invention, there is provided a method of treating atrophic gastritis by administering to a patient in need thereof an effective amount of a composition as defined above.", "In a further example of this embodiment of the invention, there is provided a method of treating or preventing a chronic inflammatory condition by administering to a patient in need thereof an effective amount of a composition as defined above.", "In yet a further example of this embodiment of the invention, there is provided a method of treating or preventing cardiovascular disease by administering to a patient in need thereof an effective amount of a composition as defined above.", "In yet a further example of this embodiment of the invention, there is provided a method of treating or preventing cerebral vascular disease by administering to a patient in need thereof an effective amount of a composition as defined above.", "BRIEF DESCRIPTION OF THE DRAWINGS These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein: FIG.", "1 shows the effect of ACAPHA on the activity of mouse spenocytes to produce IL-2.FIG.", "2 shows the effect of ACAPHA on the growth of S-180 tumor in mice after radiotherapy.", "FIG.", "3 is a bar graph showing the growth suppressive effect of 72 hours of treatment of 3 μg/ml of ACAPHA 1 on androgen responsive LNCAP and androgen-refractory JCA-1, PC-3 and DU-145 prostate cancer cells.", "FIG.", "4 shows the effect of concentration (A, assessed after 72 hours of treatment) and duration of treatment (B) of ACAPHA 1 on androgen refractory JCA-1 prostate cancer cells in vitro.", "FIG.", "5 shows the comparison of effects of ACAPHA 1 (A1), ACAPHA 2 (A2), and ACAPHA 3 (A3) on colony formation using androgen-refractory (DU-145, JCA-1 and PC-3) and androgen-responsive (LNCaP) cells.", "FIG.", "6 compares the effects of 72 hours of treatment with various concentrations of ACAPHA 1 (A1), ACAPHA 2 (A2) and ACAPHA 3 (A3), respectively, on changes in the distribution of cell cycle phase progression in the androgen-refractory human prostate JCA-1 cell line.", "FIG.", "7 compares effects of 72 hours of treatment with various concentrations of ACAPHA 1 A1), ACAPHA 2 (A2) and ACAPHA 3 (A3), respectively on changes in the distribution of cell cycle phase progression in the androgen-responsive human prostate LNCAP cells.", "FIG.", "8 shows the effects of various concentrations of ACAPHA 1 (A1), ACAPHA 2 (A2), and ACAPHA 3 (A3), respectively, on time-dependent increase in secreted PSA in media of androgen-responsive human prostate LNCAP cells.", "Cells were treated with ACAPHA for 1, 2, or 3 days.", "PSA in the culture media was determined using the TANDEM E kit from Beckman Coulter.", "FIG.", "9 shows the Western blot analysis of effects of various concentrations of ACAPHA 1 (A1), ACAPHA 2 (A2), and ACAPHA 3 (A3), respectively, on expression of secreted PSA in media of androgen-responsive human prostate LNCAP cells.", "Cells were treated with ACAPHA for 3 days.", "PSA in the culture media was determined using PSA cognate antibody and visualized by color reaction.", "FIG.", "10 shows the cell growth profiles of Jurkat and K562 leukemia cells treated with different concentrations of ACAPHA2 for 48 hours.", "FIG.", "11 shows the cell growth profiles of MCF7 and MDA-MG468 breast cancer cells treated with different concentrations of ACAPHA2 for 96 hours.", "FIG.", "12 shows the cell growth profiles of Jurkat, K562, MCF7 and MDA-MB-468 cells treated with different concentrations of ACAPHA2.FIG.", "13 shows the cell cycle distribution of Jurkat leukemia cells after 48 hours of treatment with different concentrations of ACAPHA2.FIG.", "14 shows the cell cycle distribution of K562 leukemia cells after 48 hours of treatment with different concentrations of ACAPHA2.FIG.", "15 shows the cell cycle distribution of MCF7 breast cancer cells after 96 hours of treatment with different concentrations of ACAPHA2.FIG.", "16 shows the cell cycle distribution of MDA-MB-468 breast cancer cells after 96 hours of treatment with different concentrations of ACAPHA2.FIG.", "17 is a plot comparing the survival rates of patients treated with ACAPHA 1 as an adjunct therapy to radiotherapy compared to radiotherapy alone in patients with esophageal cancer.", "FIG.", "18 is a plot comparing the survival rates of patients treated with ACAPHA 1 as an adjunct therapy to surgery compared to surgery alone in patients with esophageal cancer.", "FIG.", "19 is a bar graph comparing the survival rates of patients treated with ACAPHA 1 as an adjunct to conventional surgery and radiotherapy 12, 24 and 36 months post-treatment.", "FIG.", "20 is a bar graph showing the clinical response of individuals having bronchial dysplasia to treatment with ACAPHA 1 and other putative chemopreventive agents six months post-treatment analyzed using site specific analysis.", "The bars represent complete regression of dysplasia (CR), no response (NR) and progression of disease (PD)/or appearance of new dysplastic lesions under treatment.", "Retinol=Vitamin A; Sialor=Anetholtrithione; Pulmicort=Inhaled Budesonide.", "FIG.", "21 is a bar graph of the data of FIG.", "20 analyzed using Person specific analysis.", "FIG.", "22 is a bar graph showing the clinical response of individuals having bronchial dysplasia to treatment with ACAPHA 1 six months post-treatment and six months off ACAPHA 1 analyzed using site specific analysis.", "FIG.", "23 is a bar graph of the data of FIG.", "22 analyzed using person specific analysis.", "DETAILED DESCRIPTION OF PREFERRED EMBODIMENT The present invention relates to compositions comprising herbs and their use as chemopreventive and therapeutic agents.", "In one embodiment of the present invention the composition comprises a mixture of at least three different herbs, known hereinafter as Herb A, Herb B, Herb C, Herb D, Herb E and Herb F. According to the present invention Herb A is selected from the group consisting of: Sophora tonkinensis (Sophora subprostrata) Belamcanda chinensis Scrophularia ningpoensis Isatis tinctoria Isatis indigotica Fort, and Baphicacanthus cusia.", "According to the present invention Herb B is selected from the group consisting of: Polygonum bistorta Polygonum tapidosum Polygonum viviparum Polygonum manshuriense Polygonum alopecuroides Polygonum sphaerostachyum Andrographis paniculata Taraxacum mongolicum and Chrysanthemun indicum.", "According to the present invention Herb C is selected from the group consisting of: Prunella vulgaris Artemissia capillaris Gardenia jasminoides Rosa rugosa and Lophatherum gracile.", "According to the present invention Herb D is selected from the group consisting of: Sonchus brachyotus Patrinia scabiosaefolia Patrinia villosa Sonchus arvensis Thlaspi arvense Portulaca oleracea and Pulsatilla chinensis.", "According to the present invention Herb E is selected from the group consisting of: Dictamnus dasycarpus Kochia scoparia Sophora flavescens and Heydyotis diffusa.", "According to the present invention Herb F is selected from the group consisting of: Dioscorea bulbifera Panax notoginseng Bletilla striata Nelumbo nucifera Polygonum bistorta Cephalanoplos segetum Cirsium japonicum Sophora japonica Typha angustifolia and Rubia cordifolia.", "When, according to the present invention, the composition comprises three different herbs, one of which is selected from each of Herb A, Herb B, Herb C, Herb D or Herb E, each of the three herbs is present in an amount from about 9% to about 57%.", "In a further example of this embodiment, the three herbs are each present in an amount from about 15% to 50%.", "In a further example of this embodiment, each of the three herbs are present in an amount from 25% to 40%.", "In a further example of this embodiment, each of the three herbs are present in an amount of about 33%.", "In one embodiment of this example the composition comprises a mixture of the following herb: Sophora tonkinensis, Polygonum bistorta and Prunella vulgaris.", "When the composition comprises four different herbs, one of which is selected from each of Herb A, Herb B, Herb C, Herb D or Herb E, each of the four herbs are present in an amount from about 6% to about 43%.", "In a further example of this embodiment, each of the four herbs are present in an amount from about 15% to about 35%.", "In a further example of this embodiment, each of the four herbs are present in an amount of about 25%.", "In a further example of the present invention, when the composition comprises four herbs, three of which are selected from the group consisting of Herb A, Herb B, Herb C, Herb D and the fourth herb is selected from the group consisting of Herb E. The composition of the first three herbs are each present in an amount from about 7% to about 57% and the fourth herb is present in an amount from about 3.5% to about 28.5%.", "In a further example of this embodiment, each of the first three herbs are present in an amount from about 15% to about 48% and the fourth herb is present in an amount from about 7.5% to about 24%.", "In a further example of this embodiment, the first three herbs are present in a composition in an amount of about 20% to about 43%, and the fourth herb is present in an amount from about 10% to about 21.5%.", "In a further example of this embodiment, the first three herbs are present in an amount of about 28% and the fourth herb is present in an amount of about 14%.", "In one further example of this embodiment, the composition comprises a mixture of the following herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris and Dictamnus dasycarpus.", "In yet a further embodiment of this invention, the composition comprises five different herbs, one of which is each selected from Herb A, Herb B, Herb C, Herb D or Herb E, each of the five herbs are present in an amount from about 5% to about 35%.", "In a further example of this embodiment, each of the five herbs are present in an amount from about 10% to about 30%.", "In yet a further example of this embodiment, each of the five herbs are present in an amount from about 15% to about 25%.", "In a further example of this embodiment, each of the five herbs are present in an amount of about 20%.", "In yet a further embodiment of this invention, the first four herbs are selected one from each of the group Herb A, Herb B, Herb C and Herb D whereas the fifth herb is selected from Herb E and the first four herbs are present in an amount from about 6% to about 38%, whereas the fifth herb is present at an amount from about 3% to about 19%.", "In a further example of this embodiment, the first four herbs are present in the composition at an amount from about 12% to about 32%, and the fifth herb is present in an amount from about 6% to about 16%.", "In yet a further example of this embodiment, the first four herbs are present in an amount from about 18% to about 26% and the fifth herb is present in an amount from about 9% to about 13%.", "In yet a further example of this embodiment, the first four herbs are present at an amount of about 22%, whereas the fifth herb is present at an amount of about 11%.", "In one example of this embodiment the composition comprises a mixture of the following herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus and Dictamnus dasycarpus Turcz.", "In yet a further embodiment of this invention, the composition comprises six different herbs, wherein the first five herbs are selected from the group consisting of Herb A, Herb B, Herb C, Herb D and Herb E, and the sixth herb is selected from Herb F and the first five herbs are present in an amount from about 5% to about 31% and the sixth herb is present in an amount from about 1.0% to about 17.5%.", "In a further example of this embodiment, each of the first five herbs are present in an amount from about 10% to about 26%, and the sixth herb is present in an amount from about 5% to about 15%.", "In a further example of this embodiment, the first five herbs are each present in an amount from about 15% to about 21% and the sixth herb is present in an amount from about 7.5 to about 12.5%.", "In a further example of this embodiment, the first five herbs are present in an amount of about 18% and the sixth herb is present in an amount of about 10%.", "In yet a further example of this embodiment, the first four herbs are selected from the group of Herb A, Herb B, Herb C or Herb D. The fifth herb is selected from Herb E and the sixth herb is selected from Herb F, wherein the first three herbs are each present in an amount of about 5% to about 38%, the fifth herb is present in an amount of about 2.5% to about 19% and the sixth herb is present in an amount from about 1.0 to about 9.5%.", "In a further example of this embodiment, the first four herbs are present in an amount of about 10% to about 33%.", "The fifth herb is present in an amount of about 5% to about 16.5% and the sixth herb is present in an amount from about 3% to about 8.25%.", "In yet a further example of this embodiment, the first four herbs are present in an amount of about 15% to about 28%; herb five is present in an amount of about 7.5% to about 14.5% and the sixth herb is present in an amount from about 4.25% to about 7%.", "In yet a further example of this embodiment, the first four herbs are present in an amount of about 21%.", "The fifth herb is present in an amount of about 10.5% and the sixth herb is present in the amount of about 5.5%.", "In one example of this embodiment the composition comprises a mixture of the following herbs: Sophora tonkinesis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera.", "Thus one example of the present invention is a combination of Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera in an amount of about 21%, 21%, 21%, 21%, 10.5%, and 5.5%, respectively, and is referred to in the present application as ACAPHA 1 or ACAPHA-A1.In a further embodiment of the present invention Herb F is reduced by about 75%.", "As will be shown in one of the examples which follow, some patients treated with a composition of ACAPHA 1 showed elevated liver enzymes.", "According to the present invention, a composition was provided wherein Dioscorea bulbifera was reduced by about 75%, which resulted in a corresponding reduction in high liver enzyme levels of patients being treated with this new composition.", "In one example of this embodiment there is provided a composition comprising a combination of Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera in an amount of about 21.9%, 21.9%, 21.9%, 21.9%, 11.0%, 1.4% and is referred to in the present application as ACAPHA 2 or ACAPHA-A2.In yet a further embodiment of the present invention the Herb F is eliminated entirely from the composition.", "Thus, according to this embodiment the composition comprises a combination of Herb A, Herb B, Herb C, Herb D and Herb E in an amount of from 6%-38%, 6%-38%, 6%-38%, 6%-38%, and 3%-19%, respectively.", "In one example of this embodiment there is provided a composition comprising a combination of Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus and Dictamnus dasycarpus Turcz in an amount of about 22%, 22%, 22%, 22%, and 11%, respectively and is referred to in the present application as ACAPHA 3 or ACAPHA - A3.According to the present invention, it was found that in clinical trials with ACAPHA 1 that certain percentage of patients liver enzymes, specifically AST (Aspartate Aminotransferase) was elevated.", "As a result, the ACAPHA 2 or 3 formulations, which have a lower concentration of the herb, designated herein as Herb F, for example Dioscorea bulbifera, as in ACAPHA 2 or a complete elimination of Herb F from the composition, as in ACAPHA 3, are less toxic and thus are preferred over the ACAPHA 1 composition.", "It is important to ensure that the herbs which are used according to the present invention are selected such that they contain only acceptable levels of contaminants such as metals or pesticides.", "Various regions of China have been surveyed and it was found that the component herbs may be harvested from the Guangxi, Hunan, Liaoning, Anhui, Hebei and Jiangsu regions of China during the summer and autumn months and no pesticides are used.", "The plants are purchased dried and whole or parts of these herbs are used in manufacturing.", "The general manufacturing scheme is as follows: the herbs are either subjected to an aqueous extraction, the aqueous extract is then filtered if necessary to remove large particles, the aqueous extract is then dried to a powder.", "Alternatively, it is possible to use the herbs directly by grinding to a powder.", "The powdered herbs are then used in the production of the therapeutic in a variety of forms for administration.", "Any suitable mode of delivery can be used according to the present invention.", "For example the composition of the present invention can be delivered orally by a pill, tablet, drink, candy or paste.", "The composition can also be delivered as a transdermal patch, by inhalation or suppository.", "Delivery of the composition as an injectable is also possible, according to the present invention.", "Therefore, the composition can be administered as a therapeutic agent or as a dietary supplement.", "As previously noted, in one embodiment of the present invention, the tablets are formulated into 0.3 g tablets.", "A typical daily dosage is about 1.2 to 6 g/day based on the body weight and/or severity of the condition.", "Generally the composition is given two to three times daily.", "According to the present invention, the compositions are useful for the prevention and treatment of cancers.", "In one embodiment of the present invention, the cancers which are found to be treated or prevented are selected from the group consisting of lung cancer, liver cancer, bladder cancer, breast cancer, leukemia and prostate cancer.", "In yet a further embodiment of the present invention, the compositions have been found to be useful as adjuncts to conventional surgery, chemotherapy or radiotherapy treatments in patients with cancer selected from but not limited to lung cancer, esophagus cancer, stomach cancer, oral cavity cancer, liver cancer, bladder cancer, breast cancer and prostate cancer and leukemia.", "In the treatment of cancer, either alone or in combination with conventional cancer treatments including surgery, chemotherapy and radiotherapy, it has been found that the weight loss normally associated with cancer treatment did not occur in patients who were also treated with ACAPHA.", "An improvement in the quality of life therefore was found in the patients treated with ACAPHA.", "Furthermore, patients treated with ACAPHA showed a reduced level of cancer relapse or reoccurrence than patients who were not treated with ACAPHA.", "Further, according to the present invention, the compositions are useful for the treatment or prevention of bronchial dysplasia.", "Bronchial dysplasia is a pre-malignant lesion of the bronchial epithelial.", "Currently, there is no standard treatment for bronchial dysplasia.", "Because tobacco use is on of the major causes of lung cancer, and former heavy smokers retain an elevated risk for lung cancer, even years after they have stopped smoking, the use of the compositions of the present invention for the treatment or prevention of bronchial dysplasia will have wide reaching benefits.", "In a further embodiment of the present invention, the compositions have been found to be useful for the treatment of Helicobacter pylori infection.", "Experimental results have shown broad immunostimulating activity of ACAPHA in enhancing cellular, hormonal and macrophage function.", "This activity would represent a potential mechanism for the anti-infective effect associated with ACAPHA in the treatment of Helicobacter pylori associated with atrophic gastritis as well as for the enhanced immune surveillance associated with ACAPHA effect in chemo prevention of tumor development of dysplasia.", "Thus, according to the present invention, the compositions have also been found useful for the treatment of atrophic gastritis.", "In a further embodiment of the present invention, the composition has been found to be useful for preventing or treating a chronic inflammatory condition.", "Although not wishes to be bound by any particular theory, it is possible that the anti-anti-inflammatory activity is a result of ACAPHA's anti oxidant capacity.", "In yet a further embodiment of the present invention, the composition of the present invention has been found to be useful in the treatment or prevention of cardiovascular disease or cerebral vascular disease.", "The present invention will now be illustrated by the following examples, which should not be considered limiting.", "EXAMPLES Example 1 Antimutagenic and Anticancer-Promoting Properties A) Antimutagenic Properties Ames tests demonstrated that ACAPHA1 has no mutagenic activity.", "ACAPHA 1 however, has antimutagenic activity.", "At 5, 25, 100 mg/plate, it inhibited the reverse mutation of TA100 induced by MNNG (N-methyl-N′-nitro-N′-nitrosoguanidine) at 0.5 μg/plate (Table 1), AFB (Aflatoxin B) at 0.25 μg/plate (Table 2), Bap (Benzo(a)pyrene) at 0.5 μg/plate (Table 3), and that of TA98 induced by AFB at 0.25 μg/plate (Table 4).", "TABLE 1 Inhibition of ACAPHA on reverse mutation of TA100 induced by MNNG ACAPHA mg/plate MNNG Colony Inhibition rate mg/disc S-9 mg/disc X ± SD % 100 — — 157 ± 8.0 0 — — 146 ± 10.4 0 — 0.5 298 ± 19.6 5 — 0.5 222 ± 23.0 25.5 25 — 0.5 201 ± 61.7 32.6 100 — 0.5 189 ± 17.0 36.6 TABLE 2 Inhibition of ACAPHA on reverse mutation of TA100 induced by AFB ACAPHA MNNG Colony Inhibition rate mg/plate S-9 mg/disc X ± SD % 100 −9 — 155 ± 16.5 0 0 — 144 ± 9.2 0 0 0.25 1239 ± 66.0 5 0 0.25 1030 ± 63.2 16.9 25 0 0.25 550 ± 65.4 55.6 100 0 0.25 352 ± 16.7 71.6 TABLE 3 Inhibition of ACAPHA on reverse mutation of TA100 induced by BaP ACAPHA mg/plate MNNG Colony Inhibition rate mg/disc S-9 mg/disc X ± SD % 0 −9 — 144 ± 9.2 0 0 0.5 514 ± 52.9 5 0 0.5 465 ± 86.4 10.0 25 0 0.5 254 ± 15.2 50.6 100 0 0.5 258 ± 19.6 49.8 TABLE 4 Inhibition of ACAPHA on reverse mutation of TA98 induced by AFB ACAPHA mg/plate MNNG Colony Inhibition rate mg/disc S-9 mg/disc X ± SD % 0 — — 26 ± 2.5 0 −9 — 48 ± 3.1 100 — — 34 ± 5.3 100 0 — 46 ± 8.3 0 0 0.25 785 ± 65.6 5 0 0.25 628 ± 83.6.20.0 25 0 0.25 468 ± 41.2 40.4 100 0 0.25 69 ± 8.1 91.2 B) Antimicronucleus Properties ACAPHA 1 inhibited micronucleus in mouse bone marrow induced by cyclophosphamide (CTX).", "The results in Tables 5 and 6 show that the quantity of micronucleus in polychromatic erythrocytes in the mouse bone marrow induced by CTX were significantly reduced by ACAPHA.", "The effects in 4 different dose groups were proportional to the dosage.", "The differences were very significant (p<0.001) comparing to the control CTX group.", "They also indicate that ACAPHA can protect the chromosomes from the injury.", "TABLE 5 Effects of ACAPHA on micronucleus rate of mouse bone marrow induced by CTX Micronucleus Group Dose(LD50) Cells Rate(%0) P value ACAPHA + CTX ½ 10000 6.9 ± 0.4 <0.001 ACAPHA + CTX ¼ 10000 9.1 ± 0.6 <0.001 ACAPHA + CTX ⅛ 10000 9.4 ± 0.5 <0.001 ACAPHA + CTX {fraction (1/10)} 10000 10.3 ± 0.4 <0.001 ACAPHA ½ 8000 1.4 ± 0.2 NS NS 0.5 ml 8000 2.0 ± 0.3 CTX 30 mg/kg 8000 17.3 ± 1.1 <0.001 TABLE 6 Inhibitory effect of ACAPHA on the formation of micronucleus in bone marrow of mice induced by CTX.", "Group Dose(LD-50) Inhibition rate(%) Results ACAPHA + CTX ½ 60.79 0 ACAPHA + CTX ¼ 48.29 0 ACAPHA + CTX ⅛ 46.59 0 ACAPHA + CTX {fraction (1/10)} 40.47 ± C) Anti-promoting effect of ACAPHA i) The Inhibitory Effects of TPA ACAPHA has significant inhibitory effects on the skin edema in the mouse ear (as shown in Table 7) and hyperplasia of the skin in the mouse back (as shown in Table 8).", "All the results indicate that ACAPHA has inhibitory effects on TPA (12-O-tetradecanoylphorbol-13-acetate), which means ACAPHA has the anti-promoting effect.", "TABLE 7 The inhibitory effect of ACAPHA on edema of mouse ear skin induced by TPA TPA ACAPHA Weight of ear mg mg (mg) X ± SE P value 0 0 6.36 ± 0.22 <0.001 0.4 0 13.24 ± 0.56 <0.001 0.4 0 16.22 ± 0.59 <0.02 0.4 2 9.87 ± 1.37 <0.02 0.4 0 17.56 ± 1.51 <0.01 0.4 4 10.48 ± 0.23 <0.01 TABLE 8 The Inhibitory of ACAPHA on hyperplasia mouse back skin induced by TPA TPA ACAPHA Thickness of the skin μg Mg X ± SE P 0 0 20.00 ± 1.69 <0.001 1 0 42.29 ± 2.37 1 0 32.27 ± 0.99 <0.001 1 10 15.63 ± 0.61 1 0 39.17 ± 1.67 <0.001 1 10 20.00 ± 1.29 ii The Inhibition of ACAPHA 1 on the Elevation of ODC Activity in Mouse Epidermal Cells Induced by Croton Oil Thirty mice were randomized into four groups, that is the control, croton oil, croton oil plus ACAPHA(5 g/kg) and (10 g/kg).", "The above doses were given at the day of and two days before the test of ODC (Ornithine decarboxylase) activity.", "The control and croton oil group received saline orally.", "According to O'Brien et al (O'Brien TG et al.", "Prog Clin Biol Res 1989; 298:213-13), ODC was taken from epithelial cells of the mice and its activity was tested.", "Table 9 shows that ACAPHA has significant inhibitory effects on the increase in ODC activity of mouse epidermal cells induced by croton oil.", "The effect was related to the dosage.", "It suggests that ACAPHA tablet has anti-promoting effects.", "TABLE 9 The Inhibitory effect of ACAPHA on the activity, of ODC of epidermal cells of mice induced by croton oil ODC activity (pmol4CO2/30 min/mg Group protein) Control 35.4 ± 5.35 Croton oil 101.7 ± 1.80 Croton oil + ACAPHA 5 g/kg 82.5 ± 10.56 Croton oil + ACAPHA 10 g/kg 61.2 ± 9.89 Example 2 The Anti-Tumor and Tumor Prophylactic Effects of ACAPHA in Animals A) ACAPHA 1 and ACAPHA+5FU's Inhibiting Effect on Induced Epithelial Carcinoma BW Swiss female mice (22 to 28 g) were selected for the experiment.", "N-Nitrososarcosine ethylester (2 g/kg) were used to induce the dysplasia and carcinogenesis.", "Following the induction of dysplasia in the fore stomach, the carcinogen was discontinued.", "After 35 days ACAPHA and ACAPHA+5FU (5-fluorouracil) were then given at a dosage of 8 tablets/kg body weight, 3 times/wk, to a total of 20 times.", "The mice were weighed each week for the purpose of dose adjustment.", "After 11 weeks the mice were killed by cervical dislocation.", "After gross examination, the whole stomach was fixed in formalin and paraffin sections were stained with H&E (Hematoxylin & eosin).", "The lesions were classified into dysplasia, precancerous lesion, early stage and late stage of carcinoma.", "Table 10 shows that during the experiment, the weight growth of mice in ACAPHA group was similar to that of the control group.", "The dysplasia and carcinoma rates were significantly reduced by ACAPHA treatment, P<0.05.The dysplasia carcinoma rates were reduced even further when ACAPHA and 5FU were used together.", "TABLE 10 The inhibitory effect of ACAPHA and ACAPHA + 5FU on epithelial carcinoma in the fore stomach of mice Pulmonary Inhibition # of metastasis rate GROUP Animals Case % % p Control Group 1 6 6 100.0 Prevent Group 2 6 5 83.3 17.7 >0.05 Group 3 8 4 50.0 50 <0.01 B) The Effect of ACAPHA 1 on Induced Dysplasia and Canceration of Hamster Oral Mucous Membrane Syria hamsters (43) provided by Beijing Biological Products Institute, ages were 4 to 8 weeks, 60 to 100 g were used.", "DMBA (7,12-dimethylbenz(a)anthracene)-acetone (0.5%) was rubbed in the middle of cheek pouch (close to the buccoanterior vein) of both sides of hamster three times per week for six weeks.", "Biopsies (13) were performed randomly after finishing the treatment course.", "About 50% of the hamster developed mild dysplasia in the oral cavity.", "ACAPHA was provided by Cancer Institute of Chinese Academy of Medical Sciences.", "Animals were divided to two groups: 17 animal (34 lesions) were fed ACAPHA 2 tablets per animal per day for 10 weeks.", "After that, the animals were monitored for 8 more weeks.", "Twenty-six animals (52 lesions) received no medications.", "Macroscopic observations include the appearing time, area, color, texture of the injury as well as the general condition.", "The tissues in the most conspicuous injuries of oral pouch were biopsied at 10 h week of medication and 8 weeks after stoppage of medication.", "The specimens were fixed in formalin and sections were stained with H&E.", "The results were classified into normal, simple hyperplasia, mild dysplasia, severe dysplasia and carcinoma based on Smith and Pindborg's classification.", "The results were analyzed statistically.", "Changes in the mucous membrane of cheek pouch of hamster were detected after rubbing of DMBA for three weeks.", "Different leukoplakia could be seen with naked eyes.", "Plain leukoplakia comprised up to 65.3%, granular, 31.3%; pappilonodular, 3.2% and carcinoma, 0.5%.", "13 biopsies were conducted randomly and the histology examination showed 7 with mild dysplasia(53.8%) and 6 with hyperplasia(46.2%).", "Changes in mucous membrane of cheek pouch of hamster after administration of ACAPHA for 10 weeks and after stoppage of ACAPHA for 8 weeks by microscope: After treatment for 10 weeks, the canceration rate in control group was 96.2% and treatment, 35.3%(Table 11).", "After fasting ACAPHA for 8 weeks, all the animals in control group became cancerous and the canceration rate in treatment group was 47.1%(Table 12).", "There were a few cases that regressed to the normal mucous membrane after medication.", "They are 23.5% and 20.6% in each group.", "The cases that remained at dysplasia were 41.2% and 32.4% respectively in two groups.", "TABLE 11 Changes in mucous membrane of cheek pouch of hamster after administration of ACAPHA for 10 weeks Simple Severe No.", "of Normal Hyperplasia Mild Dysplasia Dysplasia Cancer Canceration Group Cases case % case % case % case % case % % ACAPHA 34 2 5.9 6 17.6 14 41.2 2 5.9 10 29.4 35.3 Control 52 1 1.9 1 1.9 3 5.8 47 90.4 96.2 TABLE 12 Changes in mucous membrane of cheek pouch of hamster after stoppage of ACAPHA for 8 weeks Simple Severe No.", "of Normal Hyperplasia Mild Dysplasia Dysplasia Cancer Canceration Group Cases case % case % case % case % case % % ACAPHA 34 4 11.8 3 8.8 11 32.4 2 5.9 14 41.2 49.1 Control 52 52 100 52 100 100 The canceration rates (including cancer and severe dysplasia) and improvement rates (including hyperplasia and normal mucous membrane) were used as the indices of the efficacy with which statistical analyses were performed.", "There was very significant difference in the canceration rates between treatment and control groups (P<0.01).", "Similar results were observed after medication for 10 weeks and after stoppage for 8 weeks.", "There was no significant difference between two groups in the improvement rate.", "The results suggest that ACAPHA had some inhibitory effect on the canceration of precancerous lesions of oral mucous membrane.", "It could inhibit part of the canceration and the others remained at the stage of dysplasia with the possibility of canceration.", "It could prolong the stage of the precancerous stage.", "ACAPHA could not regress all the precancerous lesions to normal mucous membrane.", "DMBA (5%) was rubbed on the oral mucous membrane of the hamsters three times per week for six weeks to induce dysplasia and then the hamsters were randomized into two groups.", "Forty-one hamsters in the control group received no further treatment.", "Sixteen hamsters received 2 crushed tablets of ACAPHA by gavage, three times per week for 12 weeks.", "Macroscopic observations show that the mucous membrane in most parts of the dysplastic lesion (up to 3A of the total) changed from smooth to coarse then to smooth.", "Tumors with pedicle in the size of a rice grain were seen in minor part (¼ of the total).", "in the control group, progressive changes were seen from smooth to coarse to nodular to tumor.", "Comparing with the animals treated with ACAPHA for 12 weeks, half of the dysplasia progressed to cancer.", "The cancer grew rapidly in size and the largest was up to 1 cubic centimeter.", "Table 13 shows the Microscopic observations after ACAPHA treatment for 12 weeks.", "TABLE 13 Microscopic observations after ACAPHA treated precancerous lesion of oral mucous membrane of hamster for 12 weeks No.", "of GROUP Cases Normal Hyperplasia Mild dysplasia Severe dysplasia Carcinoma Control 20 0 3 4 6 7 ACAPHA 6 3 — 2 1 Microscopic changes are more objective for diagnosis.", "Two microscopic criteria were used for judging the effect of ACAPHA on the development of dysplasia.", "The rate of canceration (including severe epidermal dysplasia) and the rate of improvement (including normal epithelium and epithelial hyperplasia) were analyzed with statistics.", "The U-test results are shown in Table 14.TABLE 14 The two rates after ACAPHA treated precancerous lesion of oral mucous membrane of hamster for 12 weeks GROUP Improvement rate U test Canceration Rate U test Control 15% P < 0.05 65% P > 0.05 ACAPHA 50% 50% There was significant difference in improvement rate between the ACAPHA treated and control group.", "It suggests that ACAPHA can reverse the canceration of dysplasia in oral mucous membrane of the hamster.", "No significant difference in canceration rate indicates that ACAPHA has no obvious effects on the canceration in the short term.", "Animals treated with ACAPHA for 12 weeks were reassessed macroscopically 3 months later.", "Of the 10 cases with dysplasia, 3 had tumors of 2-3 mm3 in a limited area and the rest of them had a smooth mucous membrane.", "In the control group, all hamsters except two had a coarse mucous membrane with multiple and dispersed tumors.", "Most tumors were more than 1 cm3.Animals treated with ACAPHA for 12 weeks were reassessed microscopically 3 months later.", "Tables 15 and 16 show that there was significant difference in improvement rates between the ACAPHA treatment and control groups (p<0.05).", "TABLE 15 Microscopic observations 3 month after ACAPHA treatment No.", "of GROUP Cases Normal Hyperplasia Mild dysplasia Severe dysplasia Carcinoma Control 21 0 6 2 1 12 ACAPHA 10 5 2 0 0 3 TABLE 16 U-test for post-treatment observations GROUP Improvement rate U Canceration rate U test Control 28.6% P < 0.05 61.9% P < 0.05 ACAPHA 70.0% 30.0% The long-term observation verifies that ACAPHA has a positive effect on the regression of dysplasia of the oral muccous membrane of the hassters.", "Significant difference is seen in canceration rates too (p<0.05).", "It indicates that ACAPHA tablet has inhibitory effect on canceration on the dysplasia of the oral mucous membrane of the hamster.", "Table 17 shows that measurements by multifunction morphometry shows that the nuclei in ACAPHA group are almost the same as those of the normal mucous membrane.", "TABLE 17 Nuclear measurements after administration of ACAPHA Maximum Minimum Diameter GROUP area (μm) Circle (μm) diameter (μm) Morph Factor (μm) ACAPHA 33.25 ± 11.30 21.67 ± 3.95 8.39 ± 1.77 0.67 ± 0.11 5.50 ± 1.12 Control 40.40 ± 17.23 23.95 ± 5.30 9.17 ± 2.16 0.60 ± 0.14 6.13 ± 1.42 Normal 32.29 ± 12.19 22.15 ± 3.91 8.61 ± 1.49 0.49 ± 0.15 5.55 ± 1.33 Table 18 shows that there were significant morphological changes towards the normal values in the nuclei of dysplastic cells after ACAPHA treatment.", "The nuclei in the untreated control group show multimorphic and heteromorphic changes.", "TABLE 18 U-test for the measurement of nuclei Item ACAPHA vs Control ACAPHA vs normal Area P < 0.01 P > 0.05 Circle P < 0.01 P > 0.05 Max diameter P < 0.01 P > 0.05 Morph Factor P < 0.01 P < 0.01 Min Diameter P < 0.01 P > 0.05 The measurement of nuclear DNA in both groups is shown in Table 19.TABLE 19 Nuclear DNA content of mucous membrane cells of hamster oral cavity after ACAPHA treatment.", "About 2C 2.5C-5C >5C GROUP DNA total Cells (%) Cells (%) cells (%) Normal 8.15 ± 2.84 86, 41 13, 59 0 ACAPHA treatment 9.91 ± 3.54 68, 03 31, 71 0, 26 Control no treatment 12.50 ± 5.61 45, 94 48, 69 5, 38 U-test for the results in Table 19 is shown in Table 20.TABLE 20 U-test for nuclear DNA.", "Item ACAPHA vs Control ACAPHA vs Normal DNA Total P < 0.01 P < 0.01 2C cells (%) P < 0.01 P < 0.01 2.5C-5C cells (%) P < 0.01 P < 0.01 >5C cells (%) P < 0.01 — The main peak in the bar chart of the DNA in ACAPHA group is 2C cells group, which is similar to the normal.", "The main peak in the control untreated group is 2.5-C4.5C multiples.", "There are very significant differences in the DNA quantity and multiples (>5C) between two groups.(p<0.01).", "The above results suggested that ACAPHA has obvious short term and long-term inhibitory effects on the development of dysplasia and canceration of the oral mucous membrane of the hamster induced by DMBA.", "The epithelium after treatment has significant differences in both morphology and nuclear DNA content from the dysplasia tissue and is similar to the normal tissue.", "Example 3 The Effect of ACAPHA 1 on Carcinoma of Nasopharynx in Rats Induced with Nitrosamine DNP (N N′-dinitrosopiperazine) DNP (0.5%) was injected SC in the axilla of each rat twice a week for seven weeks to induce nasopharyngeal carcinoma.", "The Prevention Group began to receive ACAPHA one day before the DNP and treatment group began to receive ACAPHA one day after fourteen DNP injections.", "Table 21 shows that the canceration rate in the control group was 60%, significantly higher than the prevention group of 36.5% (p<0.05).", "The results indicated that ACAPHA has preventive effect on the development of nasopharyngeal carcinoma.", "ACAPHA treatment also has inhibitory effect.", "The data show that ACAPHA significantly reduced the development of precancerous lesions and canceration rates (P<0.01).", "TABLE 21 The prevention and the treatment with ACAPHA on precancerous lesion of nasopharynx in rats induced by DNP Number of Precancerous Lesions Canceration GROUP feeding N I II III IV % P Control — 60 60 57 23 13 36(60.0) Prevention 60 52 52 50 8 11 19(36.5) <0.05 Treatment 5 29 29 25 1 6 1(24.1) <0.01 Example 4 The Inhibitory Effect of ACAPHA 1 on Dysplasia of Bladder Mucous Membranes of Rats Iduced by Nitrosamine BBN (N-butyl-N-(4hydroxy-butyl)nitrosamine) BBN was given three times a week for 24 weeks to induce bladder mucous dysplasia in rats.", "Animals in the treatment group received 4 ACAPHA tablets orally, three times per week until the end of the experiment.", "The control animals did not receive any treatment.", "The bladder wall of the animals in the control group thickened following BBN treatment.", "Tumors were seen in all bladders in this group.", "The bladder was filled with tumor nodules in some animals.", "The diameter of the tumors ranged from 0.5 cm to 2 cm, some with central necrosis.", "In the ACAPHA treatment group, the bladder mucous membrane was only slightly thickened.", "Large tumor nodules were only seen in a few rats.", "Most of them had small tumors or had no tumors.", "There was papilloma in parts of the bladder wall of animals in all groups.", "The proliferation of epithelium was less than six layers and lined with papilla.", "Differentiation of the cells was good and little mitosis could be seen.", "Carcinomas were seen too in part of the animals in all groups.", "The epithelium proliferation in the carcinoma was more than six layers.", "The nucleus fission was seen frequently.", "The papilla was not clear or disappeared.", "The carcinoma cells disperse between the normal cells like a nest.", "TABLE 22 The inhibitory effect of ACAPHA on canceration of dysplasia rat rats bladder induced by BBN Papilloma Cystocarcinoma Inhibition rate GROUP Animal Case % Case % % Control 20 20 100.0 12 60.0 ACAPHA 18 12 66.7* 1 5.6* 90.7 P < 0.05 P < 1 Papilloma was found in all 20 rats of the control group.", "Carcinoma was seen in the bladders of 12 rats (60%).", "Twelve of the eighteen rats in the ACAPHA groups had papilloma (66.7%) and only one had carcinoma.(5.6%).", "It suggests very significant difference from the control group(p<0.01).", "The inhibitory rate was up to 90% (Table 22).", "The results suggest that ACAPHA has significant inhibitory effects on the canceration of the epithelium dysplasia of the rat bladder induced by the BBN.", "Example 5 The Effect of ACAPHA 1 on Lung Metastasis in Mouse Tumor Model DBA/2 mouse no.2 spontaneous lung cancer was transplanted to the mouse SC.", "The latent period was 5 to 8 days and the metastatic rate was 100%.", "The lung metastatic carcinoma was a transparent node.", "The survival time of mouse with the tumor was around 35 days.", "Mice were divided into three groups: the control group with 6 mice.", "The second group: 6 mice fed with ACAPHA twice before the transplant and 8 doses after.", "The third group 8 mice treated with 9 doses of ACAPHA before the transplant and 8 doses after.", "The survival rate of the transplant in both the control and the prevention group was 100%.", "It was easier to remove the metastatic carcinoma in the prevention group than in the control group.", "The metastatic carcinoma was bigger and adhered closely to the skin in the control group.", "Results in Table 23 shows that ACAPHA had significant inhibitory effects on metastatic carcinoma.", "TABLE 23 Inhibitory effect of ACAPHA on lung metastasis Average In- # of weight Min- hibition GROUP Animals of tumor (g) Max (g) rate (%) p Control Group 1 6 3.04 2.35-3.50 Prevention Group 2 6 2.17 1.4-2.78 28.6 <0.01 Group 3 8 1.55 0.65-2.85 49 <0.01 Total 14 1.81 0.65-2.85 40.5 <0.01 Macroscopic metastatic carcinoma was seen in the lungs of all 6 mice in the control group, but only 3 in the second group and 1 in the third group.", "The inhibition rates were 50% and 87.5% respectively.", "(Table 24).", "TABLE 24 The inhibitory effect of ACAPHA on Lung metastasis of mice (1) # of Lung metastasis Inhibition GROUP Animals Cases % rate (%) P Control Group (1) 6 6 100.0 Prevent Group (2) 6 3 50.0 50.0 <0.01 Group (3) 8 1 12.5 87.5 <0.01 Total 14 4 28.6 71.4 <0.01 There were differences in the number and the size of metastatic carcinoma between the control group and the prevention group.", "In the lungs of all 6 mice in the control group, there were dispersing transparent metastatic tumor nodules of the size of millet were dispersed throughout the lung.", "In the prevention groups, the numbers and size were smaller than those in the control group.", "Of the 3 mice with metastasis in the group 2, one mouse only had 2 nodules the size of a pinhead.", "In the lung of the only mouse in group 3, 5 pinhead-sized nodules were seen.", "The average of the weight of lung over body weight of mice in the control group was 0.0143 and the average of 4 mice in the two treatment groups was 0.011.The difference was 23.1%.", "(Table 25).", "TABLE 25 The inhibitory effect of ACAPHA on Lung metastasis in mice (2) Lung + tumor weight/ Inhibition GROUP # of Animals Metastasis body weight (%) Control (1) 6 6 0.0143 Prevent (2 + 3) 14 4 0.0110 23.1 The pathological results are shown in Table 26.Lung metastasis was seen in all 6 mice in control group, and 5 of 6 in group 2 mice treated with ACAPHA.", "Two of these were detected microscopically.", "Of the 8 mice in group 3 treated with ACAPHA, 4 mice has lung metastasis but the rate was 50%, significantly lower than the control group.", "3 of these four were detected microscopically.", "TABLE 26 The inhibitory effects of ACAPHA on microscopic pulmonary metastasis in mice Pulmonary Inhibition metastasis rate GROUP # of Animals Case % % p Control Group 1 6 6 100.0 Prevent Group 2 6 5 83.3 17.7 >0.05 Group 3 8 4 50.0 50 <0.01 The above results suggest that ACAPHA has significant inhibitory effect on lung metastasis and the longer ACAPHA was fed before the transplant, the more effective the treatment.", "Example 6 Effect of ACAPHA 1 on Mice Exposed to Lethal Dose of X-Ray Mice in the treatment group received ACAPHA for 7 days (3 g/kgX14) before and after the exposure.", "Control animals received 0.4 ml tap water orally.", "X-ray was originated from a Phillips linear accelerator at 8MEV/20×20 cm/100 cm/250GY.", "The LD50 of total body irradiation in mouse was 400- 600 GY.", "700GY X-ray was used for the test and the survival rate in 30 days was recorded.", "TABLE 27 Effect of ACAPHA on mice exposed to lethal dose of X-ray Survival Weight (g) ACAPHA x-ray 30 Day rate No Group N X ± SD administration (GY) survival (%) P value 1 Control 10 23.75 ± 0.93 Water0.4 ml/each 700 1 10 P. 0 × 14 ACAPHA 10 23.45 ± 0.91 3 g/kg P.0 × 14 700 6 60 <0.02 2 Control 10 19.80 ± 1.75 Water0.4 ml/each 750 0 0 P. 0 × 14 ACAPHA 10 19.70 ± 1.85 3 g/kg P. 0 × 14 750 3 30 <0.05 Table 27 shows that at 700GY exposure, the survival rate in ACAPHA group was 60%, significantly improved over the 10% in the control group (p<0.02).", "At exposure dose of 750GY, the survival rate of ACAPHA group was 30% and the control 0 (p<0.05).", "To further test the effects of ACAPHA on the sensitivity of tumors to X-ray exposure, 30 S180 mice were randomly into 3 groups.", "The control group received no treatment after the tumor transplant.", "For the exposure only group, mice were exposed to X-ray at 2400GY locally once when the tumor grew to 4 mm3.The same dose was given to mice in the third group, but 3 g/kg of ACAPHA was fed for 10 days staring on the same day of the exposure.", "FIG.", "2 shows that tumors in the control group grew well while the curves for the other two groups were low and parallel.", "The results indicate that ACAPHA has no effects on the sensitivity of the tumor to X-ray.", "Example 7 Effect of ACAPHA on Human Prostate Cancer Cell Growth Prostate cancer is an increasing public health problem among men in the United States.", "If caught at an early stage, prostate cancer often responds to orchitectomy or antiandrogen therapy.", "However, over time residual androgen-insensitive cells will recolonize, expand and establish a hormone-resistant state, leading to a recurrence of the prostate cancer.", "In the following example the effect of ACAPHA on androgen responsive and androgen refractory human prostate cell lines was investigated.", "The results showed that ACAPHA is an effective inhibitor of human prostate cell growth.", "Human prostate cell lines (DU-145, LNCAP, and PC-3) were obtained from American Type Culture Collection (Rockville, Md.).", "These cell lines were derived from various sites to which primary tumor had metastasized.", "The JCA-1 cells were established at the New York Medical College from the primary prostate cancer site prior to the administration of hormone.", "Androgen-responsive LNCAP cells may be representative of latent prostate cancer, whereas androgen-refractory JCA-1, PC-3, and DU-145 cells are considered to better represent advanced prostate cancer.", "Cells were maintained in RPMI 1640 media containing 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and supplemented with 10% heat-inactivated fetal calf serum.", "ACAPHA solutions for the in vitro studies were prepared by stirring ACAPHA powder or tablets in 70% ethanol (340 mg/ml) at 150 rpm for one hour at room temperature.", "Insoluble material was removed by centrifugation.", "The ethanolic extracts were sterilized by passing through 0.22 μm filters and kept in aliquots in a refrigerator.", "Final concentrations of ACAPHA used for different experiments were prepared by diluting the stock with RPMI1640.Control cultures received the carrier solvent.", "Two different lots of ACAPHA 1 tablets and three different formulations of ACAPHA in powder form ACAPHA 1, ACAPHA 2 and ACAPHA 3 were prepared in an identical manner.", "Effect of ACAPHA on Growth of Prostate Cancer Cells.", "Prostate cancer cell lines were treated with different concentrations of ACAPHA and after 24, 48 and 72 hours, cells were harvested.", "Cell number was determined by hemocytometer and cell viability was assessed by trypan dye exclusion assay.", "Ethanolic extracts of ACAPHA 1 inhibited cell proliferation in both hormone-dependent and hormone-independent prostate cancer cells.", "At 3 μg/ml of ACAPHA 1, hormone-dependent LNCAP cells was more sensitive to the treatment than hormone-refractory cell lines.", "The growth suppression is shown as follows: LNCAP (67.4%)>JCA-1 (45.7%)>PC-3 (37.5%)>DU-145 (31.1%) (FIG.", "3).", "Further studies showed that growth suppression of JCA-1 cells was both concentration and time dependent.", "This cell line has been shown to be refractory to inhibition by resveratrol and other agents currently used for treatment of prostate cancer.", "With 3 μg/ml of ACAPHA 1 treatment it reached its lC50, and dramatic growth inhibition accompanying with decreasing in cell viability was found at 5 and 10 μg/ml of ACAPHA 1 (FIG.", "4A) and more significant effect was found in day 3 of treatment (FIG.", "4B).", "The study demonstrates that ACAPHA 1 has potent inhibitory activity on both androgen-responsive and androgen-refractory human prostate cells.", "Therefore, ACAPHA 1 may be useful for patients who have failed to respond to conventional therapy.", "Effect of ACAPHA on Clonogenicity of Prostate Cancer Cells.", "The growth suppressive effect of three different formulations (compositions) of ACAPHA was tested with the clonogenic assay.", "FIG.", "5 shows that dose-dependent inhibition was observed whereas formulation variation was not seen.", "ACAPHA1, 2 and 3 effectively suppressed colony formation against all four human prostate cancer cell lines.", "Effect of ACAPHA on Cell Cycle Progression.", "Cultures were exposed to varying concentrations of ACAPHA 1 (peripheral blood lymphocytes) or ACAPHA 1, 2 and 3 (JCA-1 and LNCAP cells).", "Cells were harvested, stained with DAPI fluorochrome and analyzed with a flow cytometer.", "ACAPHA 1 did not have a significant effect on cell cycle distribution of unstimulated and mitogen-stimulated peripheral blood lymphocytes.", "These results are presented in Tables 28 and 29.FIGS.", "6 and 7 similarly show that ACAPHA 1 did not have a significant effect on cell cycle distribution of androgen-refractory human JCA-1 and androgen-responsive LNCaP cell lines.", "Further results are also shown in Tables 30 and 31.Using Jurkat (human T cell leukemia cell line) T cells and JCA-1 prostate cancer cells, ACAPHA 1 displayed differential growth suppressive effects on these two cell types, exerting minimal effects on T cell growth while substantially reducing growth of prostate cancer cells.", "This data suggests that ACAPHA has no apparent toxic effects.", "It was also demonstrated that the growth suppressive effect is unrelated to changes in p53 and p21 expression (Table 32).", "TABLE 28 Distribution of the various phases of the cell cycle Time ACAPHA 1 Cell Cycle Distribution PBL (hr) (μl/ml) G1 S G2/M −PHA 48 0 93.6 0.7 5.6 −PHA 48 1 95.3 0.5 4.2 −PHA 48 3 92.8 0.8 6.4 −PHA 48 5 96.6 0.7 2.7 +PHA 48 0 62.4 30 7.6 +PHA 48 1 63.1 29.6 7.3 +PHA 48 3 71.2 19.7 9.1 +PHA 48 5 73.8 19.5 6.7 TABLE 29 Distribution of the various phases of the cell cycle Time ACAPHA 1 Cell Cycle Distribution PBL (hr) (μl/ml) G1 S G2/M −PHA 72 0 91.9 1.6 6.5 −PHA 72 1 91.7 1.4 6.9 −PHA 72 3 90.8 1.7 7.5 −PHA 72 5 89.7 1.8 8.5 +PHA 72 0 60.6 29.4 10 +PHA 72 1 53.8 35.3 10.8 +PHA 72 3 52.8 36.1 11.1 +PHA 72 5 56.8 31.8 11.4 TABLE 30 Shows the quantitation of the effects of various concentrations of ACAPHA 1 (A1), ACAPHA 2 (A2) and ACAPHA 3 (A3) respectively on changes in the distribution of cell cycle phase progression in the androgen-refractory human prostate JCA-1 cell line.", "Cells were treated with ACAPHA for 72 hours.", "Treatment JCA-1 (μl/ml) G1 S G2/M Apoptosis control 61.3 30.2 8.5 ACAPHA-A1 1 60.9 32.9 6.1 ACAPHA-A1 3 54.8 38.2 7 ACAPHA-A1 5 50.3 41.1 8.6 ACAPHA-A2 1 55.4 35.6 9 ACAPHA-A2 3 55.9 26 18.1 ACAPHA-A2 5 58 31.7 10.2 ACAPHA-A3 1 59.2 31.7 9.1 ACAPHA-A3 3 61.9 28.9 9.2 ACAPHA-A3 5 52.6 34.9 12.4 TABLE 31 The Quantitation of the effects of various concentrations of ACAPHA 1 (A1) and ACAPHA 2 (A2) respectively on changes in the distribution of cell cycle phase progression in the androgen-responsive human prostate LNCaP cells.", "Cells were treated with ACAPHA for 72 hours.", "Treatment LNCaP (μl/ml) G1 S G2/M Apoptosis control 81.7 13.6 4.7 ACAPHA-A1 1 76.4 18 5.6 ACAPHA-A1 3 75.3 16.9 7.8 ACAPHA-A1 5 74.6 17.9 7.5 ACAPHA-A2 1 73.6 16.7 9.7 ACAPHA-A2 3 75 17.9 7.1 ACAPHA-A2 5 75.9 18 6.2 TABLE 32 Effect of ACAPHA on p53 and p12 Levels Treatment p21 p53 Control 17.8 16.2 ACAPHA-1 (3 μl/ml) 18.1 16.6 ACAPHA-2 (3 μl/ml) 18.1 16.1 Effect of ACAPHA on Secreted Form of Prostate Specific Antigen (PSA).", "PSA has been identified as a marker to aid in the diagnosis of prostate cancer and for monitoring their responses to therapy.", "FIG.", "8 illustrates the effect of three formulations of ACAPHA, ACAPHA 1 (A1), ACAPHA 2 (A2) and ACAPHA 3 (A3), on time and dose-dependent PSA secretion in androgen-responsive LNCaP cells.", "The figure illustrates that increasing concentrations of ACAPHA significantly inhibited the secretion of PSA in both a dose and time-dependent manner.", "These results were confirmed by western blot analysis (FIG.", "9).", "Since PSA is a commonly accepted marker for human prostate cancer, these findings suggest that ACAPHA may be potentially useful in alleviating some of the symptoms of prostate carcinogenesis.", "Since the expression of PSA is downstream of changes in the androgen receptor, cellular androgen receptors and PSA in both control and ACAPHA treated cell extracts were evaluated with immunoblot analysis.", "Intracellular androgen receptors and PSA expression was down-regulated after treatment with 5 μg/ml of ACAPHA extract (Figure not shown).", "In summary, this cell line study indicates that ACAPHA 1 (A1), ACAPHA 2 (A2) and ACAPHA 3(A3) appear to inhibit the growth of both androgen sensitive and androgen refractory prostate cancer cells in vitro.", "The inhibitory effect does not appear to be related to changes in cell cycle progression, changes in p53 and p21 tumor suppressor gene expression or cellular apoptosis.", "Further ACAPHA 1 (A1), ACAPHA2 (A2) and ACAPHA 3(A3) inhibited the secretion of PSA tumor marker in vitro.", "ACAPHA is relatively benign and is not cytotoxic to normal cells.", "The mechanism of ACAPHA has not yet been elucidated, but it is likely related to genes that are directly or indirectly under the control of NFkB.", "Example 8 In Vitro Inhibition of Proliferation of Human Leukemia Cells and Breast Cancer Cells by ACAPHA 2 Human Jurkat and K562 leukemia cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum, glutamine and gentamycin.", "Human MCF-7 and MDA-MB468 breast cancer cells were obtained from the American Type Culture Collection (Rockville, Md.).", "MCF-7 cells were cultured as monolayers in Dulbecco's MEM supplemented with 10% fetal bovine serum, glutamine and gentamycin.", "MDA-MB468 cells were maintained in L15 medium supplemented with 10% fetal bovine serum, L-glutamine, sodium pyruvate, 2-mercaptoethanol and gentamycin.", "Jurkat, K562 and MCF-7 cells were cultured in 5% CO2 humidified incubator at 37° C. without C02.ACAPHA 2 tablets were dissolved in 70% ethanol (340 mg/ml) and stirred with intermittent mixing at 150 rpm for 60 minutes at room temperature.", "The insoluble material was removed by centrifugation and the soluble supernatant was sterilized by passing through at 0.22 mm filter and kept in aliquots in a refrigerator.", "Before use, the stock was diluted further with culture medium to give the final indicated concentrations.", "Cell Growth Quadruplicate or triplicate samples of cells in 96 well plate were treated with ACAPHA 2 prepared in culture medium at different concentrations.", "Cell growth was enumerated by counting viable cells in a hemacytometer by trypan blue dye exclusion on days 1, 2, 3 and 4.In the initial experiments it was established that Jurkat and K562 cells have logarithmic growth at days 2 and 3 while MCF-7 and MDA-MB-468 cells at day 4.In subsequent experiments Jurkat and K562 cells were assessed on day 2 while MCF-7 and MDA-MB468 cells on day 4.Cell growth data from treated cells were normalized to percent of control.", "ACAPHA 2 at 1/100 dilution was growth inhibitory, causing an >50% of growth inhibition of both human leukemia (Jurkat and K5652; FIG.", "10) and breast cancer (MCF-7 and MDA-MB-468; FIG.", "11) cell lines.", "At higher dilutions, ACAPHA 2 showed differential growth inhibition on the cell lines.", "At 1/200 dilution, it caused 50% inhibition on K562 and MCF-7 cells but 93 and 87% inhibition on Jurkat and MDA-MB-468 cells respectively.", "At 1/500 dilution, ACAPHA 2 treatment still resulted in 63% and 72% growth inhibition on Jurkat and MDA-MB468 cells respectively (FIGS.", "12).", "Cell Cycle Analysis with Flow Cytometry For Jurkat and K562 cells, at the end of the ACAPHA 2 treatment period, the cells were washed twice with Ca2+ and Mg2+ free Hanks' balanced salt solution, fixed in ice-cold 70% methanol overnight at −20° C. Fixed cells were washed free of methanol and treated with 2 mg/ml propidium iodide.", "Cell samples were passed through a 40 micron nylon mesh and a 27 g needle before they were analyzed with a BD Bioscience Facscalibur flow cytometer.", "For adherent MCF-7 and MDA-MB468 cells, they were detached from culture vessel with trypsin-EDTA solution before treated with the above procedure for cell cycle analysis.", "In Jurkat cells, ACAPHA 2 at 1/100 dilution caused an increase of cells in SubG0/G1 phase (apoptotic/necrotic cells), cells arrested in G0/G1 phase and reduced G2/M fraction.", "At 1/200 and 1/500 dilutions, ACAPHA 2 treatment still resulted in an increased number of cells arrested in G0/G1 phase and reduced of cells in S phase (FIG.", "13).", "In K562 cells, ACAPHA 2 at 1/100 and 1/200 dilutions caused an increase in the number of cells in SubG0/G1 phase, reduction of cells in G0/G1 and G2/M phases, and increase of cells in S phase.", "The cell cycle profile of cells treated with 1/500 of ACAPHA 2 was similar to the control (FIG.", "14).", "In MCF-7 cells, ACAPHA 2 at 1/100 dilution caused a slight increase of Sub0/G1 fraction, significant reduction of cells in G0/G1 and G2/M phases and significant increase of cells in S phase.", "At 1/200 and 1/500 dilutions, a slight increase of cells in S phase and decrease of cells in G2/M phase was observed (FIG.", "15).", "In MDA-MB468 cells, ACAPHA at 1/100 was cytotoxic.", "At 1/200 dilution, ACAPHA caused a significant increase of cells in SubG0/G1 and S phases, and a significant reduction of cells in G0/G1 and G2/M phases.", "At 1/1,000 dilution, the distribution of cells in the different phases was similar to the control (FIG.", "16).", "C. DNA Fragmentation Analysis DNA strand fragmentation was examined by extracting cellular DNA in the cell samples and subjecting them to 1% agarose gel electrophoresis in 40 mM Tris-acetate, 1 mM EDTA buffer pH 7.2 at 10 V/cm.", "The results showed that ACAPHA did not cause DNA fragmentation in Jurkat and MCF-7 cells at 1/100 dilution by 1% agarose gel electrophoresis suggesting ACAPHA did not induce apoptosis in these cells (Figures not shown).", "Example 9 Use of ACAPHA 1 as an Adjunctive Therapy for Treatment of Esophageal Cancer A five year trial in China commenced in January, 1997 to assess the efficacy of ACAPHA 1 as an adjunctive therapy for treatment of esophageal carcinoma.", "According to this trial, patients diagnosed with esophageal carcinoma were either treated by surgery or radiotherapy in conjunction with ACAPHA 1 administration, at a dose of 300 mg tablets given 6 to 8 times per day.", "Historical data of patients treated for esophageal cancer by surgery or radiotherapy at the same cancer hospital was used for comparison.", "Survival rates in patient subjects twelve, twenty-four and thirty-six months after treatment commencement are summarized in FIGS.", "17, 18 and 19 and in Tables 33 and 34.As shown in the Figures, the highest survival rate was in patients receiving ACAPHA 1 treatment in combination with surgery.", "Patients receiving ACAPHA 1 treatment in combination with radiotherapy also had a significantly higher survival rate as compared to patients receiving radiotherapy alone.", "The difference in survival rate between patients receiving conventional treatment and patients receiving ACAPHA 1 adjunctive therapy becomes more pronounced the longer the time period elapsed.", "For example, three years post-surgery, the survival rate of the 104 patients receiving surgery only was only 35.8%.", "By comparison, 82.9% of the 123 patients receiving a combination of surgery and ACAPHA 1 adjunctive therapy survived to the three year milestone.", "TABLE 33 Summary of clinical data comparing esophageal cancer staging in patients treated with ACAPHA 1 as an adjunct to conventional surgery or radiotherapy Stage I Stage II Stage III Stage IV Group N N % N % N % N % Statistical Analysis Surgery + 123 7 5.7 47 38.2 63 51.2 6 4.9 x2 = 7.72 p > 0.05 ACAPHA 1 Surgery Alone 104 3 2.9 51 49.0 50 48.1 — — Radiotherapy + 9487 2 2.1 37 39.4 48 52.1 6 6.4 X2 = 1.41 p > 0.05 ACAPHA 1 Radiotherapy 7 8.1 27 31.0 53 60.9 — — Alone TABLE 34 Comparison of survival rates of esophageal cancer patients treated with ACAPHA 1 as an adjunct to conventional surgery and radiotherapy 36 months post-treatment Survival Rate (%) Treatment Months Post-Rx Plus ACAPHA 1 U-Test Surgery 122436 97.2 94.2 U = 1.09 p > 0.05 88.9 66.1 U = 3.82 p < 0.01 82.9 35.8 U = 6.04 p < 0.01 Radiotherapy 122436 91.3 94.2 U = 0.74 p > 0.05 67.2 51.3 U = 2.56 p < 0.05 52.3 26.4 U = 2.71 p < 0.01 In a further clinical trial started in January 1995, the survival rate after five years of post-radiotherapy patients with esophageal cancer also treated with ACAPHA was determined.", "Patients were randomized into two equal groups; 140 patients after radiotherapy for esophageal cancer were randomized to the treatment group and 140 patients after radiotherapy esophageal cancer at the same time were randomized to the control group.", "For patients in the ACAPHA treatment group, 6 to 8 tablets of ACAPHA were given twice a day.", "The effect of ACAPHA on the survival rate of the post-surgery patient with esophageal cancer is shown in Table 35.The one, two, three and four year survival rate in the treatment group were 76.12%, 45.52%, 39.55% and 29.1% respectively, which were 13.3%, 16.9%, 32.9% and 52.5% higher than the 67.18, 38.93%, 29.77% and 19.08% in the control group.", "Though there was no significant difference between each group (P>0.05, Table 35), the survival rate was increasing yearly.", "After 5 years, the survival rate in treatment group was 26.87%, which was 85.3% higher than the 14.5% in the control group (p<0.01).", "It indicates that ACAPHA can increase the 5 year survival rate of post radiotherapy patients with esophageal cancer and has good results as an adjuvant to radiotherapy for patients with esophageal cancer.", "TABLE 35 Effect of ACAPHA on post radiotherapy survival rate of esophageal cancer patients.", "ACAPHA Control Increase in Survival Survival Survival Survival Rate Year Rate SD Rate SD U Value (%) 1 0.7612 0.0368 0.6718 0.0410 1.6227 p > 0.05 13.3 2 0.4552 0.0430 0.3893 0.0426 1.0887 p > 0.05 16.9 3 0.3955 0.0422 0.2977 0.0400 1.6820 p > 0.05 32.9 4 0.2910 0.0392 0.1908 0.0343 1.9237 p > 0.05 52.5 5 0.2687 0.0383 0.1450 0.0308 2.5169 p < 0.01 85.3 The data of the two studies of chemoprevention with ACAPHA after radiotherapy were combined.", "The results indicated that the distribution of the samples were comparable.", "Table 36 shows that one, two, three, four and five year survival rate in the treatment group were 78.93%, 51.68%, 44.93%, 33.23% and 30.67%, which were 14.7%, 35.7%, 59.7%, 74.1% and 111.3% higher than the 68.81%, 38.07%, 28.14%, 19.09% and 14.51% in the control group (p<0.01).", "The survival rates increased yearly.", "TABLE 36 Effect of ACAPHA on post radiotherapy survival rate of esophageal cancer patients.", "ACAPHA Control Increase in Survival Survival Survival Survival Rate Year Rate SD Rate SD U Value (%) 1 0.7893 0.0279 0.6881 0.0314 2.4093 p < 0.01 14.7 2 0.5168 0.0354 0.3807 0.0329 2.8162 p < 0.01 35.7 3 0.4493 0.0367 0.2814 0.0306 3.2138 p < 0.01 59.7 4 0.3323 0.0383 0.1909 0.0287 2.9545 p < 0.01 74.1 5 0.3067 0.0381 0.1451 0.0272 3.4401 p < 0.01 111.3 Based on the above results, it can be concluded that ACAPHA could increase the survival rate of the post treatment (radiotherapy or surgery) patients with esophageal cancer.", "Example 10 Effect of ACAPHA 1 on Helicobacter pylon Infection Associated Polyp Type Gastritis Polyp type gastritis is a special form of chronic gastritis.", "It can be present as the only problem, or it can exist with other gastric disorders.", "The special feature is the presence of pathological umbilicus shaped swellings of the gastric mucosa.", "In China, it comprises about 15% of chronic gastritis and 85% of these patients have H. pylori (HP) infection.", "H. pylori is important in the etiology of gastric cancer.", "It has recently been recognized by the International Agency for Research on Cancer as a human carcinogen.", "HP is assumed to cause gastric cancer indirectly because it provokes gastritis, which is a precursor of gastric atrophy, metaplasia and dysplasia.", "In the following example, the effect of ACAPHA 1 on HP infection associated polyp type gastritis was investigated.", "A total of 16 patients, ages ranging from 35 to 62 years old, were used in this study.", "Disease duration ranged from 4 to 12 months, with a mean of 7 months.", "Selection criteria of polyp type gastritis was based on diagnostic criteria and HP infection was tested using urease test and biopsy sections.", "An experienced endoscopist examined each patient prior to selection.", "Detailed descriptions were made during the endoscopic assessment and photos were taken.", "In addition, a minimum of 2 mucosal specimens in areas with pathological changes were collected and examined by an experienced pathologist.", "Patients that fulfilled the criteria for polyp type gastritis and tested positive for HP infection were included in the study group.", "The detailed clinical condition of the test subjects were then recorded and WBC counts and liver and kidney function tests were performed.", "Patients in the study group were instructed to take 8 tablets of ACAPHA 1, twice a day for 3 months (a total of 16 tablets/day).", "During the treatment, all other medications used for treatment of gastric conditions were stopped.", "At the end of the treatment period, side effects of ACAPHA 1 and changes in the disease states were recorded.", "WBC, liver and kidney function tests were repeated.", "The same endoscopist repeated the gastric endoscopic examinations, recording any observable changes and taking photos.", "Biopsy samples were taken in the same mucosal areas as before and the same pathologists assessed the histological sections.", "The presence of HP infection was again tested.", "None of the 16 patients had adverse reactions or side effects during treatment with ACAPHA 1.Among the treated patients, 13 cases (81.3%) had obvious improvement in the original clinical symptoms of gastric distention, intestinal pain, heart burn due to hypersecretion of gastric acid and vomiting after 1 month of ACAPHA 1 treatment.", "After 2 months of treatment, the original clinical symptoms disappeared in these patients.", "After 3 months of treatment, only 2 cases showed no significant improvement in clinical symptoms.", "The efficacy rate as assessed by clinical symptoms was 87.5%.", "After 3 months of ACAPHA 1 treatment, endoscopic examination revealed that in 12 of the 16 cases, the pathological features of mucosal umbilicus type swelling had totally disappeared.", "Histopathological assessment showed that there was regression from polyp type gastritis to a mild form of superficial gastritis.", "In addition, 11 cases tested negative for HP infection.", "There were 4 cases that did not respond to treatment but after an additional 3 months of ACAPHA 1 treatment, 3 patients no longer showed gastric mucosal umbilicus type swelling.", "Histopathological assessment of these patients showed a regression to a mild form of superficial gastritis.", "In 2 of the 4 cases, HP infection tested negative.", "The overall efficacy rate of ACAPHA 1 treatment as assessed by gastric endoscopy and histopathology was 93.8%.", "The HP infection curative rate was 81.3%.", "The results showed that ACAPHA 1 was an effective treatment for polyp type gastritis as measured by clinical assessment, endoscopic examination and histopathological assessment.", "In addition, ACAPHA 1 was effective in eradicating HP infection.", "The eradication rate of HP infection by ACAPHA 1 is similar to the rate achieved by the internationally recognized standard treatment of HP using a double drug regimen, consisting of bismuth compound and an antibiotic, or bismuth-based triple therapy.", "Example 11 Effect of ACAPHA 1 on Chronic Atrophic Gastritis Chronic atrophic gastritis is a common disease in middle and old age.", "The etiology and mechanism of the disease is still unclear.", "As a result, there is still no specific effective therapy and current treatment is mainly directed at treatment of symptoms.", "In chronic atrophic gastritis, gastric mucosal epithelial gland metaplasia and atypical hyperplasia are characteristic pathological features, which are also closely associated with the development of gastric carcinoma.", "Some scientists have even considered that these features are a precancerous pathological change.", "Intestinal gland metaplasia refers to the presence of intestinal glandular epithelium in the gastric mucosa.", "Based on the amount present, the disease can be classified into mild, moderate and severe categories.", "Atypical hyperplasia of gastric mucosal epithelium is also referred to as gastric dysplasia.", "The main features include atypical morphology of the cells, irregular architecture and atypical cellular distribution.", "According to the degree of tissue involvement the changes can also be classified into mild, moderate and severe categories.", "Due to the absence of specific effective drugs to improve or cure this condition, surgery is usually recommended to prevent the development of cancer in severe cases of dysplasia.", "Development of a new drug that can improve or eradicate intestinal gland metaplasia and dysplasia should greatly reduce the incidence of gastric carcinoma.", "In this example, the effect of ACAPHA 1 on chronic atrophic gastritis was investigated.", "A total of 26 subjects were used in this study conducted in China.", "All patients were diagnosed with chronic atrophic gastritis and had either intestinal gland epithelium metaplasia or dysplasia.", "Selection of patients was carried out according to strict international diagnostic criteria.", "The severity of gastric mucous gland epithelium metaplasia and dysplasia was also assessed according to strict international criteria: Before selection, an experienced endoscopist examined the gastric lining of each patient and obtained gastric mucosal biopsy samples at defined sites.", "An experienced pathologist then examined histological sections of the biopsy samples.", "Cases that fulfilled the selection criteria were selected for the study.", "Detailed case histories were then recorded for each patient and WBC, kidney and liver function tests were performed.", "Patients who were admitted into the study were told to take 3 ACAPHA 1 tablets, twice a day for 3 months.", "All other medications for the treatment of chronic atrophic gastritis were stopped.", "At the end of the treatment period, adverse reactions and side effects caused by ACAPHA 1 and clinical changes were recorded.", "WBC, liver and kidney function tests were once again performed.", "The same endoscopist examined the gastric lining of the patients and recorded any observable changes.", "A repeat sampling of gastric mucosal biopsies were performed in the same sites as before and the same pathologist examined histological sections from the biopsies.", "Finally, an independent experienced pathologist reexamined each histological section to confirm the accuracy of the findings.", "None of the study patients with chronic atrophic gastritis experienced adverse reactions or side effects after taking ACAPHA 1.In addition, there were no significant changes in the WBC, liver and kidney function tests after ACAPHA 1 therapy.", "In 16 cases (61.5%), clinical symptoms of abdominal pain, belching (eructation), and abdominal distention significantly improved after 1 month of ACAPHA 1 therapy.", "After 2 months, 22 cases (85.4%) showed significant improvement of clinical symptoms and after 3 months, only one patient did not have significant improvement.", "Therefore, the clinical symptom improvement rate was 96.2% after three months.", "Comparison of endoscopic examinations before and after ACAPHA 1 treatment indicated that 7 cases with focal necrosis had significant improvement or disappearance of lesion following treatment.", "There were no significant changes in other features of chronic atrophic gastritis revealed by endoscopy.", "Histopathological examination of biopsy sections before and after ACAPHA 1 treatment revealed significantly more than the endoscopic examinations.", "It was found that 7 cases of chronic atrophic gastritis regressed to chronic superficial gastritis.", "In particular, out of 12 cases of severe intestinal gland epithelium metaplasia, 5 regressed to moderate or mild forms of the metaplasia and 2 no longer showed any features of severe metaplasia.", "Out of 9 cases with moderate intestinal glandular epithelium metaplasia, 3 regressed to mild metaplasia and the remaining 6 no longer showed any features of moderate metaplasia.", "Finally, in all 5 cases of mild intestinal glandular epithelium metaplasia, all abnormal features disappeared after ACAPHA 1 treatment.", "In the cases of severe atypical hyperplasia, 4 cases regressed to mild atypical hyperplasia and in 2 cases the pathological changes disappeared.", "In all 8 cases with moderate atypical hyperplasia and 12 cases with mild atypical hyperplasia the abnormal features disappeared after ACAPHA 1 treatment.", "The results from this study indicate that ACAPHA 1 was effective against chronic atrophic gastritis and intestinal gland epithelium metaplasia and dysplasia.", "Example 12 The Effect of ACAPHA 1 on Oral Lichen Planus Oral Lichen Planus (OLP) is a chronic inflammatory condition associated with the immune system.", "Some reports have suggested that it has a tendency to become cancerous.", "Presently there is no effective treatment for this condition.", "In this example, the effects of ACAPHA 1 on patients with OLP were investigated.", "Ten patients with confirmed diagnoses of OLP and unresponsive to other forms of treatment were used in this study.", "One month prior to ACAPHA 1 treatment, all patients stopped taking medication that may affect the immune system.", "ACAPHA 1 was given at a dose of 3 tablets, 3 times a day for 4 to 12 months.", "The patients were given oral examinations every 2 months and had serum and urine tests for SGPT and BUN.", "Following ACAPHA 1 treatment, OLP in the patients was monitored for another 4 to 7 months.", "The overall effective rate was 90%, with 10% cured, 40% showing marked improvement, 40% showing improvement and 10% showing no change.", "The liver and kidney functions remained normal during the study indicating that ACAPHA 1 did not have any detectable side effects.", "The results suggest that ACAPHA 1 is an effective treatment for OLP.", "In view of the fact that the treatment is simple and inexpensive and long term treatment did not affect liver and kidney functions, this would suggest that ACAPHA 1 has potential as a treatment for OLP and to prevent it from progressing to oral cancerous lesions.", "Example 13 Effect of ACAPHA 2 on Breast Cystic Hyperplasia The breast cystic hyperlasia is common in clinical practice, especially in married women or those with child-bearing history.", "Based on ACAPHA 2's antiproliferative function on the epithelium, improving the immune response and regulating endocrine function, 121 patients were chosen for this study.", "The results of ACAPHA 2 on breast cystic hyperplasia are shown below in Table 37.TABLE 37 Effect of ACAPHA 2 on mammary cystic hyperplasia Symptom and Sign No.", "of Cases Cured (%) Improved (%) Ineffective (%) Pain 121 72(59.5) 36(29.6) 12(9.9) Node 121 68(56.2) 49(40.5) 4(3.3) Pathologically, proliferation can be seen in the fibrous tissue around the lobe of the mammary gland and some papillary proliferation can be seen in the epithelium.", "Symptoms are pains in the breast periodically and persistently, nodular mass with different size and unclear edge.", "After treatment with ACAPHA 2, the efficacy is satisfactory without any obvious side effects.", "Example 14 Long Term Effect of ACAPHA (8 Years) on Mortality & Morbidity of Cancers (1992-2001) Patients ages 40 to 65 diagnosed with severe epithelial dysplasia of esophagus by cytology, were randomized into 2 groups.", "The treatment group was given 8 tablets daily of ACAPHA 1 and the control group was given a placebo.", "The study was conducted for three years with a follow up after treatment for an additional five years.", "ACAPHA 1 was shown to have a significant positive effect on the incidence and mortality of esophageal cancer and other cancers.", "Table 38 shows the incidence of cancer in patients receiving ACAPHA 1 for three years after a five year follow up.", "TABLE 38 Control group Treatment group Sub-total Esophagus 207 55 262 Other cancers Lung 21 4 25 Liver 23 5 28 Stomach 10 3 13 Intestine 2 3 5 Larynx 2 2 4 Oral 1 0 1 Pancreas 1 0 1 Penis 2 0 2 Cervix 3 1 4 Ovary 1 0 1 Breast 2 0 2 Bone 0 1 1 Brain 4 0 4 Thyroid 1 0 1 Lymphoma 2 0 2 Unknown 0 1 1 Sub-Total 75 20 95 Table 39 shows the effect of ACAPHA 1 on mortality and morbidity.", "TABLE 39 Deceased case Diagnosed case Sub-total # of # of # of # of Group cases cases % RR(95% CI) cases % RR(95% CI) Cases % RR(95% CI) Control 2826 321 11.4 RR = 0.70 115 4.07 RR = 0.43 436 15.43 RR = 0.60 (0.55-0.87) (0.27-0.67) (0.49-0.75) Treatment 1242 101 8.13 22 1.77 123 9.9 Table 40 shows the effect of ACAPHA I on mortality and incidence of esophageal cancer.", "TABLE 40 Deceased case Diagnosed case Sub-total # of # of # of # of Group cases cases % RR(95% CI) cases % RR(95% CI) Cases % RR(95% CI) Control 2826 66 2.34 RR = 0.65 9 0.32 RR = 0.25 75 2.64 RR = 0.60 (0.39-1.09) (0.03-0.40) (0.37-0.99) Treatment 1242 19 1.53 1 0.1 20 1.61 Table 41 shows the effect of ACAPHA 1 on mortality of cancers other than esophageal cancer.", "TABLE 41 Deceased case Diagnosed case Sub-total # of # of # of # of Group cases cases % RR(95% CI) cases % RR(95% CI) Cases % RR(95% CI) Control 2826 66 2.34 RR = 0.65 9 0.32 RR = 0.25 75 2.64 RR = 0.60 (0.39-1.09) (0.03-0.40) (0.37-0.99) Treatment 1242 19 1.53 1 0.8 20 1.61 Table 42 shows the effect of ACAPHA 1 on incidence of mortality of cardiovascular diseases.", "TABLE 42 Deceased case Diagnosed case Sub-total # of # of # of # of Group cases cases % RR(95% CI) cases % RR(95% CI) Cases % RR(95% CI) Control 2826 33 1.17 RR = 0.69 21 0.74 RR = 0.22 54 1.91 RR = 0.50 (0.39-1.40) (0.05-0.92) (0.27-0.94) Treatment 1242 10 0.81 2 0.16 12 0.97 Table 43 shows the effect of ACAPHA 1 on the incidence and mortality of cerebral vascular diseases.", "TABLE 43 Deceased case Diagnosed case Sub-total # of # of # of # of Group cases cases % RR(95% CI) cases % RR(95% CI) Cases % RR(95% CI) Control 2826 48 1.7 RR = 0.71 12 0.43 RR = 0.76 60 2.12 RR = 0.72 (0.40-1.27) (0.24-2.35) (0.43-1.21) Treatment 1242 15 1.21 4 0.32 19 1.53 Note (for tables 38 to 43) 1.“RR = 1”: no protective & improvement effect 2.“RR < 1”: with protective effect 3.“RR > 1”: with improvement effect 4.“95% CI > 1.0”: without notable statistical difference Example 15 Long Term Effect of ACAPHA (18 Years) on Mortality & Morbidity of Cancers (1983-2001) Patients aged 40 to 65 were diagnosed with severe epithelial dysplasia of esophagus by cytology, were randomized into two groups.", "The treatment group received ACAPHA 1 at a dose of 8 tablets per day.", "The control group received a placebo.", "The treatment period was for five years with a follow up for an additional 13 years.", "ACAPHA 1 was shown to have significant positive effect on the incidence and mortality of esophageal cancer and other cancers.", "Table 44 shows the incidence of cancer in patients receiving ACAPHA 1 for five years and off for 13 years.", "TABLE 44 Control group Treatment group Sub-total Esophagus 121 77 198 Other cancers Lung 3 3 6 Liver 6 3 9 Stomach 17 10 27 Intestine 3 1 4 Larynx 1 0 1 Breast 2 1 3 Bone 0 1 1 Brain 0 1 1 Sub-Total 32 20 52 Table 45 shows the effect of ACAPHA 1 on mortality and morbidity.", "TABLE 45 Deceased case Diagnosed case Sub-total # of # of # of # of Group cases cases % RR(95% CI) cases % RR(95% CI) Cases % RR(95% CI) Control 523 199 38.05 RR = 0.60 74 14.15 RR = 0.38 273 52.20 RR = 0.45 (0.46-0.78) (0.24-0.59) (0.35-0.57) Treatment 528 142 26.89 31 5.87 173 32.77 Table 46 shows the effect of ACAPHA 1 on mortality and incidence of esophageal cancer.", "TABLE 46 Deceased case Diagnosed case Sub-total # of # of # of # of Group cases cases % RR(95% CI) cases % RR(95% CI) Cases % RR(95% CI) Control 523 87 16.63 RR = 0.70 34 6.50 RR = 0.33 121 23.14 RR = 0.57 (0.50-1.00) (0.17-0.65) (0.41-0.78) Treatment 528 65 12.31 12 2.27 77 14.58 Table 47 shows the effect of ACAPHA 1 on mortality of cancers other than esophageal cancer TABLE 47 Deceased case Diagnosed case Sub-total # of # of # of # of Group cases cases % RR(95% CI) cases % RR(95% CI) Cases % RR(95% CI) Control 523 26 4.97 RR = 0.68 6 1.15 RR = 0.33 32 6.12 RR = 0.60 (0.37-1.25) (0.07-1.63) (0.34-1.07) Treatment 528 18 3.41 2 0.38 20 3.79 Table 48 shows the effect of ACAPHA 1 on incidence of mortality of cardiovascular diseases.", "TABLE 48 Deceased case Diagnosed case Sub-total # of # of # of # of Group cases cases % RR(95% CI) cases % RR(95% CI) Cases % RR(95% CI) Control 523 29 5.55 RR = 0.71 15 2.87 RR = 0.65 44 8.41 RR = 0.68 (0.40-1.25) (0.29-1.06) (0.42-1.09) Treatment 528 21 3.98 10 1.89 31 5.87 Table 49 shows the effect of ACAPHA 1 on incidence of mortality of cerebral vascular diseases.", "TABLE 49 Deceased case Diagnosed case Sub-total # of # of # of # of Group cases cases % RR(95% CI) cases % RR(95% CI) Cases % RR(95% CI) Control 523 39 7.46 RR = 0.62 13 2.49 RR = 0.38 52 9.94 RR = 0.55 (0.37-1.04) (0.13-1.06) (0.34-0.87) Treatment 528 25 4.74 5 0.95 30 5.68 Note (for tables 44 to 49) 1.“RR = 1”: no protective & improvement effect 2.“RR < 1”: with protective effect 3.“RR > 1”: with improvement effect 4.“95% CI > 1.0”: without notable statistical difference Example 16 Effect of ACAPHA 1 on Bronchial Dysplasia Bronchial dysplasia is a premalignant lesion of the bronchial epithelium.", "Currently there is no standard treatment for bronchial dysplasia.", "The prevalence of bronchial intraepithelial neoplasia (dysplasia and carcinoma in-situ) has been studied using sputum cytology examination and flourescence bronchoscopy.", "In smokers with chronic obstructive pulmonary disease (COPD) and a smoking history of more than 40 packs per year, the prevelence of mild, moderate, or severe atypia and canconoma in-situ in sputum cytology examinations was found to be 48%, 25%, 0.8% and 0.9% respectively.", "In current and former smokers over 40 years of age with a smoking history of more than 30 packs per year, the prevelance of mild, moderate or severe dysplasia and carcinoma in-situ on flourescence bronchoscopy and biopsy, was found to be 44%, 14%, 4.3% and 1.2% respectively.", "Because tobacco use is one of the major causes of lung cancer, and former heavy smokers retain an elevated risk for lung cancer even years after they have stopped smoking, the effects of ACAPHA 1 on bronchial dysplasia in former smokers was investigated.", "Twenty current and former smokers over 40 years of age with a smoking history of at least 30 pack-years (i.e.", "1 pack per day for 30 years or more) and one or more sites of bronchial dysplasia on fluorescence bronchoscopy directed bronchial biopsies were recruited to take part in the study.", "Participants from the placebo arm of two concurrent chemoprevention trials with identical inclusion and exclusion criteria and similar clinical protocols aside from the chemopreventive agent were used for comparison purposes.", "Following an initial interview, which included a questionnaire to document the smoking history, fluorescence bronchoscopy using the LIFE-Lung device (Xillix Technologies Corp., Richmond, B.C., Canada) was performed on each patient.", "Biopsies were taken from areas with abnormal fluorescence.", "In addition, at least two control biopsies were taken from an upper or lower lobe.", "A minimum of four biopsies were taken per individual.", "The participants were instructed to take ACAPHA 1 at a dose of 8 tablets, twice a day for 6 months.", "They were seen monthly for examination of drug related adverse effects.", "Complete blood counts, AST, LD, bilirubin, alkaline phosphatase, calcium, phosphate, electrolytes, BUN, creatinine, triglycerides and cholesterol were measured at baseline and monthly for 6 months while on ACAPHA 1 for toxicity monitoring.", "Fluorescence bronchoscopy was repeated after 6 months of ACAPHA 1 treatment.", "All previously biopsied sites were identified and re-biopsied under fluorescence bronchoscopy.", "Biopsies were also taken from new areas that appeared to be dysplastic under fluorescence examination.", "The biopsies were fixed in buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin.", "All the biopsies were systematically reviewed by an experienced pathologist.", "All biopsies were classified into one of eight groups.", "The normal group was represented by pseudostratified ciliated columnar epithelium.", "The basal cell hyperplasia group was represented by an increase in the number and stratification of normal-appearing basal cells still covered with normal ciliated or mucin secreting cells.", "The metaplasia group was represented by a stratified epithelium and cytoplasmic changes consistent with squamoid differentiation.", "Mild, moderate or severe dysplasia and carcinoma in-situ were classified according to WHO criteria.", "The final group was classified as invasive, metastatic carcinoma.", "The primary end-point of the study (i.e.", "surrogate end-point biomarker) was change in the histopathology grade based on the risk of progression to invasive cancer.", "From the study by Frost and co-workers (J. Occp.", "Med 1986; 28:692-703), the risk of progression of mild, moderate and severe dysplasia to invasive lung cancer within nine years were 4%, 10%, and 40%, respectively.", "On a site by site analysis, complete response (CR) was defined by regression of the dysplastic lesion to hyperplasia/normal.", "Progressive disease (PD) was defined as appearance of lesions that were mild dysplasia or worse irrespective of whether the site was biopsied at baseline or worsening of the dysplastic lesions present at baseline by two grades or more (i.e.", "mild dysplasia to severe dysplasia or worse, moderate/severe dysplasia to carcinoma in-situ/invasive cancer).", "No response (NR) referred to sites that were not a CR or PD.", "On a participant by participant basis, CR was defined as regression of all dysplastic lesions found at baseline to no higher than hyperplasia as defined by the site by site analysis at six months and no appearance of new dysplastic lesions that were mild dysplasia or worse.", "PD was defined as progression of one or more sites to a higher grade as defined by the site by site analysis referred to above or appearance of new dysplastic lesions that were mild dysplasia or worse at six months.", "Partial response (PR) was defined as regression of some but not all of the dysplastic lesions but no appearance of new lesions that were mild dysplasia or worse.", "No response (NR) referred to subjects who did not have a CR, PR or PD.", "Descriptive statistics were used to summarize subject characteristics and pathologic evaluations of the bronchial biopsy examinations.", "Comparison between groups was done with the Mann-Whitney test.", "Pearson's Chi-square test was applied to a contingency table analysis to determine the association of bronchial dysplasia with treatment (ACAPHA 1 or placebo).", "All P values are two-sided.", "A two-sided P value of less than 0.05 was considered to be statistically significant.", "To adjust for the effect of various factors on the likelihood of regression of preinvasive lesions, a multiple logistic regression analysis was used.", "The analysis included the variables: age, sex, and the smoking intensity (pack-years).", "All analyses were unconditional and tests of statistical significance and confidence intervals (CIs) for odds ratios (ORs) were based on the log-likelihood test.", "The characteristics of the participants are shown in Table 50.There was no significant differences in age, sex and smoking history between the ACAPHA 1 and placebo groups.", "There was also no significant difference in the number of biopsies taken, the proportion of dysplastic lesions or the severity of the dysplastic lesions between the two groups.", "A total of 421 biopsies were taken in the placebo group (average 5.8 biopsies per subject).", "Twenty-four of the 421 biopsies (5.7%) were moderate/severe dysplasia and 168 biopsies (39.9%) showed mild dysplasia.", "In the ACAPHA 1 group, a total of 121 biopsies were taken (average 6.1 per subject).", "Ten of the 121 biopsies (8.3%) were moderate/severe dysplasia and 36 biopsies (29.8%) showed mild dysplasia.", "TABLE 50 Characteristics of participants in the Phase IIA Trial of the Effect of ACAPHA 1 on Bronchial Dysplasia ACAPHA 1 Placebo* Number of Participants 20 73 Age (Years) Median 58 55 Range 46-71 43-72 Gender Male 12 44 Female 8 29 Smoking Former 6 11 Status Current 14 62 Pack-Years Mean 65 46 Range 30-85 30-72 Placebo data are drawn from two concurrent Phase IIb placebo-controlled trials evaluating an identical cohort treated under the identical protocol at the same institution.", "With reference to FIGS.", "20 and 21, at six months, the complete regression rate of areas with dysplasia was significantly higher after taking ACAPHA 1 than placebo (63% versus 33%, P<0.001).", "The NR and PD rates were also significantly lower (NR 37% versus 66%, P<0.001; PD 5% versus 18%, P=0.008).", "There were 8 new lesions that were moderate/severe dysplasia and 32 new lesions with mild dysplasia in the placebo group but only 1 new lesion with mild dysplasia in the ACAPHA 1 group.", "This response is higher than any known chemopreventative agent that has been tested in Phase II/I clinical trials thus far.", "In the person specific analysis, the CR rate was 50% in those who took ACAPHA 1 versus 13% in those who were on placebo (p<0.001).", "The PD rate was also significantly lower in those who were on ACAPHA 1 (20% versus 61%, P=0.003).", "Multiple logistic regression analysis was used to determine the simultaneous effects of age, gender, smoking intensity (pack-years) and the effect of treatment on the CR rate.", "ACAPHA 1 had a strong effect on the CR rate (P=0.001).", "Gender had a strong association with CR (P=0.004) suggesting that women had, on average 6.5 times higher odds of CR (95% CI=1.8 to 23.1 times).", "None of the remaining variables had a significant association with CR.", "Seventeen subjects were available for the twelve month follow-up assessment after being off ACAPHA for six months.", "FIG.", "22 shows, on a site-by-site analysis, the CR, NR and PD rates were 80%, 17% and 3% respectively.", "FIG.", "23 shows, on a person specific analysis, the CR, PR and PD rates were 71%, 24% and 5% respectively.", "Thus, the effects of ACAPHA 1 were sustained even after the participants were taken off the treatment for six months.", "All the participants tolerated the ACAPHA 1 well.", "There was no symptomatic adverse event.", "Sub-clinical elevation of AST was observed in 7 of the 20 participants.", "One of them had preexisting hepatitis C. Alcohol consumption was thought to be the reason for the AST elevation in another participant.", "The AST level returned to normal after reducing the dose of ACAPHA 1 to 8 tablets once daily in 4 subjects and discontinuation of the ACAPHA 1 in two subjects.", "The seventh subject was able to complete the six months course at the full dose with mild elevation of AST only.", "LD levels were also elevated in a number of patients.", "The liver enzyme results from some patients are shown below in Table 51.Following these results it is suggested that the ACAPHA 2 or 3 formulation be considered for further application as these formulations are less toxic but are as therapeutically effective.", "The results show that in both the subject specific and the lesion specific analyses, there was a significantly better complete response rate in the treatment group than in the control group after 6 months of ACAPHA 1 treatment.", "There was also a significant reduction in the progression rate in the lesion specific analysis when the ACAPHA 1 treatment group was compared to the placebo group.", "This response is better than any known chemopreventive agent that has been tested thus far.", "FIGS.", "21 and 22 compare the efficacy of ACAPHA 1 with placebo, Retinol (Vitamin A), Sialor (Anetholtrithione) and Pulmicort™ (Inhaled budesonide).", "As indicated above, the proportion of subjects having a complete response (CR) was significantly higher than other potential chemopreventive agents.", "Furthermore, the effects of ACAPHA were sustained even after the participants were taken off the treatment for six months.", "TABLE 51 ACAPHA 1: Liver Results Month Patient 1 2 3 3A 4 4A 4B 4C 5 5A 1 AST: 20 AST: 27 AST: 24 AST: AST: 23 AST: AST: AST: AST: 28 AST: LD*: 103 LD: 122 LD: 127 LD: LD: 126 LD: LD: LD: LD: 117 LD: 2 AST: 27 AST: 28 AST: 27 AST: AST: 22 AST: AST: AST: AST: 24 AST: LD: 150 LD: 149 LD: 168 LD: LD: 156 LD: LD: LD: LD: 168 LD: 3 AST: 34 AST: AST: +44 AST: AST: +58 AST: AST: AST: AST: +68 AST: +72 LD: 140 LD: LD: 175 LD: LD: 589 LD: LD: LD: LD: 644 LD: 591 4 AST: 20 AST: 23 AST: 24 AST: AST: 20 AST: AST: AST: AST: 22 AST: LD: 123 LD: 128 LD: 128 LD: LD: 142 LD: LD: LD: LD: 124 LD: 5 AST: 29 AST: 66 AST: 29 AST: AST: 27 AST: AST: AST: AST: 32 AST: LD: 132 LD: 170 LD: 140 LD: LD: 145 LD: LD: LD: LD: 175 LD: 6 AST: 20 AST: +11 AST: +16 AST: AST: +18 AST: AST: AST: AST: 20 AST: LD: 369 LD: 138 LD: 138 LD: LD: 142 LD: LD: LD: LD: 154 LD: 7 AST: 16 AST: 18 AST: AST: AST: 17 AST: AST: AST: AST: 21 AST: LD: 147 LD: 174 LD: LD: LD: 168 LD: LD: LD: LD: 181 LD: 8 AST: 28 AST: 30 AST: 33 AST: AST: +43 AST: AST: AST: AST: +191 AST: +542 LD: 135 LD: 150 LD: 149 LD: LD: 157 LD: LD: LD; LD: +222 LD: +1060 9 AST: 20 AST: 21 AST: 31 AST: AST: +45 AST: +45 AST: AST: AST: +48 AST: LD: 103 LD: 117 LD: 127 LD: LD: 133 LD: 133 LD: LD: LD: +124 LD: 10 AST: 23 AST: 25 AST: 25 AST: AST: AST: AST: AST: AST: +68 AST: LD: 118 LD: 110 LD: 123 LD: +301 +131 +92 +73 LD: 151 LD: LD: 193 LD: 164 LD: 165 LD: 493 11 AST: 20 AST: 22 AST: 27 AST: AST: 29 AST: AST: AST: AST: 32 AST: LD: 97 LD: 116 LD: 134 LD: LD: 110 LD: LD: LD: LD: 121 LD: 12 AST: 22 AST: 19 AST: +18 AST: AST: +17 AST: AST: AST: AST: +17 AST: LD: 146 LD: 168 LD: 173 LD: LD: 158 LD: LD: LD: LD: 158 LD: 13 AST: 28 AST: 30 AST: 25 AST: AST: 25 AST: AST: AST: AST: 24 AST: LD: 161 LD: 151 LD: 155 LD: LD: 136 LD: LD: LD: LD: 146 LD: 14 AST: 21 AST: 25 AST: 27 AST: AST: 26 AST: AST: AST: AST: 24 AST: LD: 154 LD: 144 LD: 154 LD: LD: 148 LD: LD: LD: LD: 138 LD: 15 AST: +17 AST: +15 AST: 34 AST: AST: +15 AST: AST: AST: AST: 19 AST: LD: 130 LD: 147 LD: 145 LD: LD: 139 LD: LD: LD: LD: 154 LD: 16 AST: 30 AST: 30 AST: 30 AST: AST: 31 AST: AST: AST: AST: 31 AST: LD: 163 LD: 189 LD: 189 LD: LD: 158 LD: LD: LD: LD: 158 LD: 17 AST: 19 AST: 21 AST: AST: AST: 18 AST: AST: AST: AST: +15 AST: LD: 113 LD: 132 LD: LD: LD: 124 LD: LD: LD: LD: 130 LD: 18 AST: 24 AST: 25 AST: 26 AST: AST: 27 AST: AST: AST: AST: 27 AST: LD: 137 LD: 140 LD: 147 LD: LD: 147 LD: LD: LD: LD: 150 LD: 19 AST: 21 AST: +16 AST: +16 AST: AST: +17 AST: AST: AST: AST: +16 AST: LD: 113 LD: 120 LD: 129 LD: LD: 125 LD: LD: LD: LD: 143 LD: 20 AST: +43 AST: AST: AST: AST: +65 AST: AST: AST: AST: +179 AST: +323 LD: 141 LD: 146 +161 +113 LD: 132 LD: LD: LD: LD: 209 LD: +687 LD: 146 LD: 478 21 AST: +43 AST: AST: AST: AST: +65 AST: AST: AST: AST: +179 AST: +323 LD: 141 LD: 146 +161 +113 LD: 132 LD: LD: LD: LD: 209 LD: +687 LD: 146 LD: 478 Month 9-1 YR Patient 5B 6 6A 7 7A 7B 7C 8 F/U 1 AST: AST: 29 AST: AST: +83 AST: +171 AST: +295 AST: +73 AST: AST: 22 LD: LD: 120 LD: LD: 138 LD: LD: 575 LD: 388 LD: LD: 109 2 AST: AST: 20 AST: AST: 24 AST: AST: AST: AST: AST: 27 LD: LD: 157 LD: LD: 156 LD: LD: LD: LD: LD: 187 3 AST: AST: +59 AST: AST: +73 AST: AST: AST:.", "AST: AST: +71 LD: LD: 521 LD: LD: 194 LD: LD: LD: LD: LD: 204 4 AST: AST: 20 AST: AST: 20 AST: AST: AST: AST: AST: 22 LD: LD: 123 LD: LD: 117 LD: LD: LD: LD: LD: 161 5 AST: AST: +146 AST: +64 AST: +51 AST: +38 AST: AST: AST: AST: 27 LD: LD: +212 LD: 533 LD: 167 LD: 157 LD: LD: LD: LD: 165 6 AST: AST: 19 AST: AST: +18 AST: AST: AST: AST: AST: 22 LD: LD: 131 LD: LD: 140 LD: LD: LD: LD: LD: 147 7 AST: AST: 20 AST: AST: 21 AST: AST: AST: AST: AST: +16 LD: LD: 166 LD: LD: 189 LD: LD: LD: LD: LD: 192 8 AST: +262 AST: +68 AST: +111 AST: +234 AST: +220 AST: +56 AST: 29 AST: AST: 28 LD: +668 LD: 157 LD: 521 LD: +216 LD: 641 LD: 479 LD: 125 LD: LD: 139 9 AST: AST: +60 AST: AST: +102 AST: AST: AST: AST: AST: 24 LD: LD: 131 LD: LD: 151 LD: LD: LD: LD: LD: 112 10 AST: AST: +49 AST: AST: +70 AST: +50 AST: AST: AST: AST: 19 LD: LD: 120 LD: LD: 137 LD: 415 LD: LD: LD: LD: 123 11 AST: AST: 25 AST: AST: 25 AST: AST: AST: AST: AST: 33 LD: LD: 114 LD: LD: 117 LD: LD: LD: 22 LD: 118 LD: 113 12 AST: AST: +18 AST: AST: 21 AST: AST: AST: AST: AST: 22 LD: LD: 155 LD: LD: 176 LD: LD: LD: LD: LD: 532 13 AST: AST: 28 AST: AST: 22 AST: AST: AST: AST: AST: 27 LD: LD: 147 LD: LD: 123 LD: LD: LD: LD: LD: 155 14 AST: AST: 25 AST: AST: 19 AST: AST: AST: AST: AST: 30 LD: LD: 167 LD: LD: 454 LD: LD: LD: LD: LD: 178 15 AST: AST: 23 AST: AST: +16 AST: AST: AST: AST: AST: +17 LD: LD: 127 LD: LD: 102 LD: LD: LD: LD: LD: 148 16 AST: AST: 26 AST: AST: 29 AST: AST: AST: AST: AST: 31 LD: LD: 150 LD: LD: 162 LD: LD: LD: LD: LD: 166 17 AST: AST: 19 AST: AST: 17 AST: AST: AST: AST: AST: 20 LD: LD: 129 LD: LD: 401 LD: LD: LD: LD: LD: 413 18 AST: AST: 20 AST: AST: 27 AST: AST: AST: AST: AST: 31 LD: LD: 132 LD: LD: 154 LD: LD: LD: LD: LD: 153 19 AST: AST: +15 AST: AST: 20 AST: AST: AST: AST: AST: LD: LD: 133 LD: LD: 125 LD: LD: LD: LD: LD: 20 AST: AST: +83 AST: +50 AST: 32 AST: AST: AST: AST: AST: 20 LD: LD: 136 LD: 361 LD: 139 LD: LD: LD: LD: LD: 129 21 AST: AST: +83 AST: +50 AST: 32 AST: AST: AST: AST: AST: 20 LD: LD: 136 LD: 361 LD: 139 LD: LD: LD: LD: LD: 129 AST (Aspartate Aminotransferase) = U/L **LD (Lactic Dehydrogenase) = U/L As will be apparent to those skilled in the art in light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof.", "Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims." ] ]	Patent_10468896
[ [ "Stereophonic Device for Headphones and Audio Signal Processing Program", "A stereophonic device for headphones to which a monophonic signal or a stereophonic signal is inputted comprises an uncorrelating processing unit for reducing the correlation between two signals obtained by dividing the inputted monophonic signal into two channels or two signals constituting the inputted stereophonic signal, a reflected sound adding processing unit for adding a reflected sound, and a sound image localizing processing unit for controlling the position where a sound image is localized." ], [ "1.In a stereophonic device for headphones to which a monophonic signal or a stereophonic signal is inputted, a stereophonic device for headphones, comprising: an uncorrelating processing unit for reducing the correlation between two signals obtained by dividing the inputted monophonic signal into two channels or two signals constituting the inputted stereophonic signal; a reflected sound adding processing unit for adding a reflected sound; and a sound image localizing processing unit for controlling the position where a sound image is localized.", "2.An audio signal processing program used for a stereophonic device for headphones to which a monophonic signal or a stereophonic signal is inputted, wherein a computer is caused to perform: uncorrelating processing for reducing the correlation between two signals obtained by dividing the inputted monophonic signal into two channels or two signals constituting the inputted stereophonic signal; reflected sound adding processing for adding a reflected sound; and sound image localizing processing for controlling the position where a sound image is localized.", "3.In a stereophonic device for headphones to which front signals for two or more channels and surround signals for two or more channels are inputted, a stereophonic device for headphones, characterized in that there are provided, with respect to each of the inputted front signal and the inputted surround signal, an uncorrelating processing unit for reducing the correlation between the signals, a reflected sound adding processing unit for adding a reflected sound, and a sound image localizing processing unit for controlling the position where a sound image is localized.", "4.A sound signal processing program used for a stereophonic device for headphones to which front signals for two or more channels and surround signals for two or more channels are inputted, comprising a program for causing a computer to subject the inputted front signal to uncorrelating processing for reducing the correlation between the signals, reflected sound adding processing for adding a reflected sound, and sound image localizing processing for controlling the position where a sound image is localized, and a program for causing the computer to subject the inputted surround signal to uncorrelating processing for reducing the correlation between the signals, reflected sound adding processing for adding a reflected sound, and sound image localizing processing for controlling the position where a sound image is localized." ], [ "<SOH> BACKGROUND ART <EOH>When music is reproduced using normal headphones, a sound image is localized in the head of a listener (in-head localization), so that a sound field having a spreading feeling cannot be reproduced.", "An object of the present invention is to provide a stereophonic device for headphones in which a sound field having a spreading feeling can be reproduced and an audio signal processing program." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.", "1 is a block diagram showing the configuration of a stereophonic device for headphones to which a monophonic signal or a stereophonic signal is inputted.", "FIGS.", "2 a and 2 b are schematic views showing the filter characteristics of a first FIR digital filter constituting a left signal-uncorrelating processing unit 3 a and the filter characteristics of a second FIR digital filter constituting a right signal-uncorrelating processing unit 3 b.", "FIG.", "3 is a block diagram showing a conventional basic sound image localizing processing circuit.", "FIG.", "4 is a schematic view for explaining a method of calculating the characteristics of a sound image localization filter using a head related transfer function.", "FIG.", "5 is an electrical diagram showing the configuration of a stereophonic device for headphones to which front signals for three or more channels and surround signals for two channels are inputted.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to a stereophonic device for headphones for reproducing a sound field having a natural spreading feeling using the headphones and an audio signal processing program.", "BACKGROUND ART When music is reproduced using normal headphones, a sound image is localized in the head of a listener (in-head localization), so that a sound field having a spreading feeling cannot be reproduced.", "An object of the present invention is to provide a stereophonic device for headphones in which a sound field having a spreading feeling can be reproduced and an audio signal processing program.", "DISCLOSURE OF INVENTION In a stereophonic device for headphones to which a monophonic signal or a stereophonic signal is inputted, a first stereophonic device for headphones according to the present invention is characterized by comprising an uncorrelating processing unit for reducing the correlation between two signals obtained by dividing the inputted monophonic signal into two channels or two signals constituting the inputted stereophonic signal; a reflected sound adding processing unit for adding a reflected sound; and a sound image localizing processing unit for controlling the position where a sound image is localized.", "A first audio signal processing program according to the present invention is an audio signal processing program used for a stereophonic device for headphones to which a monophonic signal or a stereophonic signal is inputted, characterized in that a computer is caused to perform uncorrelating processing for reducing the correlation between two signals obtained by dividing the inputted monophonic signal into two channels or two signals constituting the inputted stereophonic signal; reflected sound adding processing for adding a reflected sound; and sound image localizing processing for controlling the position where a sound image is localized.", "In a stereophonic device for headphones to which front signals for two or more channels and surround signals for two or more channels are inputted, a second stereophonic device for headphones according to the present invention is characterized in that there are provided, with respect to each of the inputted front signal and the inputted surround signal, an uncorrelating processing unit for reducing the correlation between the signals, a reflected sound adding processing unit for adding a reflected sound, and a sound image localizing processing unit for controlling the position where a sound image is localized.", "A second audio signal processing program according to the present invention is a sound signal processing program used for a stereophonic device for headphones to which front signals for two or more channels and surround signals for two or more channels are inputted, characterized by comprising a program for causing a computer to subject the inputted front signal to uncorrelating processing for reducing the correlation between the signals, reflected sound adding processing for adding a reflected sound, and sound image localizing processing for controlling the position where a sound image is localized, and a program for causing the computer to subject the inputted surround signal to uncorrelating processing for reducing the correlation between the signals, reflected sound adding processing for adding a reflected sound, and sound image localizing processing for controlling the position where a sound image is localized.", "According to the present invention, a stereophonic device for headphones in which a sound field having a spreading feeling can be reproduced and an audio signal processing program.", "BRIEF DESCRIPTION OF DRAWINGS FIG.", "1 is a block diagram showing the configuration of a stereophonic device for headphones to which a monophonic signal or a stereophonic signal is inputted.", "FIGS.", "2a and 2b are schematic views showing the filter characteristics of a first FIR digital filter constituting a left signal-uncorrelating processing unit 3a and the filter characteristics of a second FIR digital filter constituting a right signal-uncorrelating processing unit 3b.", "FIG.", "3 is a block diagram showing a conventional basic sound image localizing processing circuit.", "FIG.", "4 is a schematic view for explaining a method of calculating the characteristics of a sound image localization filter using a head related transfer function.", "FIG.", "5 is an electrical diagram showing the configuration of a stereophonic device for headphones to which front signals for three or more channels and surround signals for two channels are inputted.", "BEST MODE FOR CARRYING OUT THE INVENTION Referring now to the drawings, an embodiment of the present invention will be described.", "[1] DESCRIPTION OF FIRST EMBODIMENT FIG.", "1 illustrates the configuration of a stereophonic device for headphones to which a monophonic signal and a stereophonic signal are inputted.", "The stereophonic device for headphones comprises two switches 1 and 2 for switching a monophonic signal Mono and a stereophonic signal (a left input signal Lin and a right input signal Rin), an uncorrelating processing unit 3 for subjecting the signal inputted from each of the switches 1 and 2 to uncorrelating processing, a reflected sound adding processing unit 4 provided in the succeeding stage of the uncorrelating processing unit 3, and a sound image localizing processing unit 5 provided in the succeeding stage of the reflected sound adding processing unit 4.At both the time of inputting the stereophonic signal and the time of inputting the monophonic signal, a left output signal Lout and a right output signal Rout are outputted from the stereophonic device for headphones.", "The uncorrelating processing unit 3, the reflected sound adding processing unit 4, and the sound image localizing processing unit 5 will be described.", "[2] DESCRIPTION OF UNCORRELATING PROCESSING UNIT 3 The uncorrelating processing unit 3 is for reducing the correlation between two input signals, and has been conventionally used when two pseudo stereophonic signals are generated from one signal which is a morphonic signal.", "The uncorrelating processing unit 3 shown in FIG.", "1 employs a band division system, and comprises a left signal-uncorrelating processing unit 3a provided in the succeeding stage of the switch 1 and a right signal-uncorrelating processing unit 3b provided in the succeeding stage of the switch 2.In the left signal-uncorrelating processing unit 3a, the input signal is delayed by a delay device DLa1 and is delayed by a delay device DLa2.A delay time period of the delay device DLa1 and a delay time period of the delay device DLa2 differ from each other.", "Multipliers MLa1, MLa2, and MLa3 are respectively provided with respect to the input signal and output signals of the delay devices DLa1 and DLa2.The input signal and the output signals of the delay devices DLa1 and DLa2 are respectively inputted to the corresponding multipliers MLa1, MLa2, and MLa3, and multiplied by coefficients.", "Output signals of the multipliers MLa1, MLa2, and MLa3 are added together by an adder ALa, and the result of the addition is outputted as a left signal L1.The configuration of the right signal-uncorrelating processing unit 3b is the same as the left signal-uncorrelating processing unit 3a, and comprises delay devices DRa1 and DRa2, multipliers MRa1, MRa2, and MRa3, and an adder ARa.", "The result of the addition by the adder ARa is outputted as a right signal R1.The left signal-uncorrelating processing unit 3a is composed by a first FIR digital filter, and the right signal-uncorrelating processing unit 3b is composed by a second FIR digital filter.", "The filter characteristics of the first FIR digital filter are shown in FIG.", "2a, and the filter characteristics of the second FIR digital filter are shown in FIG.", "2b.", "The filter characteristics of each of the FIR digital filters are such characteristics that the frequency band is divided into a plurality of bands, and a passage band and a prevention band alternately appear, as shown in FIGS.", "2a and 2b.", "The first FIR digital filter and the second FIR digital filter respectively have such characteristics that the passage bands and the prevention bands are opposite to each other such that their filter outputs L1 and R1 are unrelated to each other even if their input signals are the same signal such as a monophonic signal.", "[3] DESCRIPTION OF REFLECTED SOUND ADDING PROCESSING UNIT 4 A person perceives a soundscape by a reflected sound or a reverberant sound produced by the ceiling and the wall of a listening place.", "With headphones in which no reflected sound or reverberant sound in a room is produced, therefore, there is no soundscape.", "The reflected sound adding processing unit 4 produces a reflected sound or a reverberant sound in a room to give a soundscape to a listener even when the listener listens to music with the headphones.", "The reflected sound adding processing unit 4 comprises an adder 4a for calculating the difference between the output signal L1 of the left signal-uncorrelating processing unit 3a and the output signal R1 of the right signal-uncorrelating processing unit 3b, a left signal-reflected sound adding unit 4b, and a right signal-reflected sound adding unit 4c.", "In the left signal-reflected sound adding unit 4b, the input signal L1 is delayed by a predetermined time period by each of a plurality of delay devices DLb1 to DLbn connected in series.", "Multipliers MLb1 to MLbn are respectively provided with respect to output signals of the delay devices DLb1 to DLbn.", "The output signals of the delay devices DLb1 and DLbn are respectively inputted to the corresponding multipliers MLb1 to MLbn and multiplied by coefficients.", "Consequently, a plurality of types of reflected sounds are produced.", "The output signals of the multipliers MLb1 to MLbn are respectively added to the input signal L1 by adders ALb1 to ALbn, and the respective results of the addition are outputted as a left signal L2.Consequently, a plurality of types of reflected sounds are added to the input signal L1.The configuration of the right signal-uncorrelating processing unit 4c is the same as the left signal-uncorrelating processing unit 4b, and comprises a plurality of delay devices DRb1 and DRbn, a plurality of multipliers MRb1 to MRbn, and a plurality of adders ARb1 to ARbn.", "The result of the addition by the adder ARbn is outputted as a right signal R2.", "[4] DESCRIPTION OF SOUND IMAGE LOCALIZING PROCESSING UNIT 5 The sound image localizing processing unit 5 is for controlling the position where a sound image is localized.", "Before describing the sound image localizing processing unit 5 shown in FIG.", "1, a conventional basic sound image localizing processing circuit will be described.", "FIG.", "3 illustrates the conventional basic sound image localizing processing circuit.", "A left signal inputted to an input terminal P1 is fed to a first sound image localization filter 301 and a second sound image localization filter 302, where filter processing corresponding to a filter coefficient of each of the filters 301 and 302 is performed.", "A right signal inputted to an input terminal P2 is fed to a third sound image localization filter 303 and a fourth sound image localization filter 304, where filter processing corresponding to a filter coefficient of each of the filters 303 and 304 is performed.", "The characteristics of the first sound image localization filter 301 and the characteristics of the fourth sound image localization filter 304 are the same, and the characteristics of the second sound image localization filter 302 and the characteristics of the third sound image localization filter 303 are the same.", "An output of the first sound image localization filter 301 and an output of the third sound image localization filter 303 are added together by an adder 311, and the result of the addition is outputted as Lout.", "An output of the second sound image localization filter 302 and an output of the fourth sound image localization filter 304 are added together by an adder 312, and the result of the addition is outputted as Rout.", "Each of the sound image localization filters is found by a head related transfer function, described below.", "Generally used as each of the sound image localization filters is an FIR (Finite Impulse Response) digital filter having several hundred taps.", "Description is now made of a method of calculating the characteristics of the sound image localization filter using the head related transfer function.", "Let HLL, HLR, HRL, and HRR be respectively transfer functions for transfer paths from real speakers L and R arranged on the right and left sides ahead of a listener 300 to the right and left ears of the listener 300, as shown in FIG.", "4.Further, let WL and WR be transfer functions from a virtual sound source position P where a sound is desired to be localized to the right and left ears of the listener 100.The transfer functions are all described on the frequency axis.", "In order that the listener can listen to an audio as if the audio were outputted from the virtual sound source position P irrespective of the fact that the audio is outputted from the real speakers L and R, the following equation (1) must hold, letting X be an input signal and letting Lout and Rout be respectively output signals from the real speakers L and R: ( W L W R ) ⁢ X = ( H LL ⁢ H LR H RL ⁢ H RR ) ⁢ ( L out R out ) ( 1 ) Consequently, the respective signals Lout and Rout outputted from the real speakers L and R are found, as expressed by the following equation (2): ( L out R out ) = 1 H LL ⁢ H RR - H LR ⁢ H RL ⁢ ( H RR - H LR - H RL ⁢ H LL ) ⁢ ( W L W R ) ⁢ X ( 2 ) Furthermore, assuming that the real speakers L and R are set up symmetrically as viewed from the listener, the symmetrical transfer functions are the same, so that the following equations (3) and (4) hold.", "HTHR and HCRS are respectively substituted for the same transfer functions.", "HTHR=HLL=HRR (3) HCRS=HLR=HRL (4) Consequently, the foregoing equation (2) can be rewritten, as expressed by the following equation (5): ( L out R out ) = 1 H LL ⁢ H RR - H LR ⁢ H RL ⁢ ( H RR - H LR - H RL ⁢ H LL ) ⁢ ( W L W R ) ⁢ X = 1 H THR 2 - H CRS 2 ⁢ ( H THR - H CRS - H CRS ⁢ H THR ) ⁢ ( W L W R ) ⁢ X = ( H THR ⁢ W L - H CRS ⁢ W R H THR 2 - H CRS 2 H THR ⁢ W R - H CRS ⁢ W L H THR 2 - H CRS 2 ) ⁢ X = ( H 1 H 2 ) ⁢ X ⁡ ( ∵ ( H 1 = H THR ⁢ W L - H CRS ⁢ W R H THR 2 - H CRS 2 H 2 = H THR ⁢ W R - H CRS ⁢ W L H THR 2 - H CRS 2 ) ) ( 5 ) Used as a filter obtained by converting H1 and H2 in the foregoing equation (5) into those in a time axis is an FIR digital filter having several hundred taps.", "The frequency characteristics of the first sound image localization filter 301 and the fourth sound image localization filter 302 in FIG.", "3 correspond to H1 in the foregoing equation (5), and the frequency characteristics of the second sound image localization filter 302 and the third sound image localization filter 303 correspond to H2 in the foregoing equation (5).", "Description is made of the sound image localizing processing unit 5 shown in FIG.", "1.The sound image localizing processing unit 5 shown in FIG.", "1 comprises two delay devices DLc and DRc, two multipliers MLc and MRc, and two adders ALc and ARc.", "The left signal L2 inputted from the left signal-reflected sound adding unit 4b is fed to the adder ALc, and is fed to a first processing circuit comprising the delay device DLc and the multiplier MLc.", "The right signal R2 inputted from the right signal-reflected sound adding unit 4c is fed to the adder ARc, and is fed to a second processing circuit comprising the delay device DRc and the multiplier MRc.", "In the adder ALc, the left signal L2 and an output signal of the second processing circuit are added together, and the result of the addition is outputted as the left output signal Lout.", "In the adder ARc, the right signal R2 and an output signal of the first processing circuit are added together, and the result of the addition is outputted as the right output signal Rout.", "The sound image localizing processing unit 5 shown in FIG.", "1 is one obtained by replacing the first sound image localization filter 301 and the fourth sound image localization filter 304 in the conventional basic sound image localizing processing circuit shown in FIG.", "3 with through processing which is one type of filter processing and replacing the second sound image localization filter 302 and the third sound image localization filter 304 in the conventional basic sound image localizing processing circuit with a processing circuit comprising a delay device and a multiplier.", "The filter characteristics of the first processing circuit comprising the delay device DLc and the multiplier MLc and the filter characteristics of the second processing circuit comprising the delay device DRc and the multiplier MRc are adjusted, thereby localizing a sound image outside the head.", "That is, the sound image is prevented from being localized in the head.", "[2] DESCRIPTION OF SECOND EMBODIMENT FIG.", "5 illustrates the configuration of a stereophonic device for headphones to which front signals for three or more channels and surround signals for two channels are inputted.", "A multiplier MC multiplies a center input signal Center by a coefficient.", "An adder AL1 adds an output signal of the multiplier MC to a front left input signal Lin.", "An adder AR1 adds an output signal of the multiplier MC to a front right input signal Rin.", "An uncorrelating processing unit 103, a reflected sound adding processing unit 104, and a sound image localizing processing unit 105, which are the same as those shown in FIG.", "1, are provided with respect to a front left signal obtained by the adder AL1 and a front right signal obtained by the adder AR1.Furthermore, an uncorrelating processing unit 203, a reflected sound adding processing unit 204, and a sound image localizing processing unit 205, which are the same as those shown in FIG.", "1, are provided with respect to a surround left input signal Surround Lin and a surround right input signal Surround Rin.", "An adder AL2 adds a surround left signal obtained from the sound image localizing processing unit 205 to a front left signal obtained from the sound image localizing processing unit 105, and the result of the addition is outputted as a left output signal Lout.", "An adder AR2 adds a surround right signal obtained from the sound image localizing processing unit 205 to a front right signal obtained from the sound image localizing processing unit 105, and the result of the addition is outputted as a right output signal Rout." ] ]	Patent_10468898
[ [ "Herbal compositions useful as chemopreventive and therapeutic agents", "Composition derived from traditional Chinese herbal medicines, medicinal plants and extracts thereof, are provided.", "These compositions can be used as chemopreventive and therapeutic agents." ], [ "1-8.", "(canceled) 9.A composition comprising a mixture of at least three different herbs selected from the group consisting of Herb A, Herb B, Herb C, Herb D, Herb E and Herb F, wherein Herb A is selected from the group consisting of: Sophora tonkinensis Belamcanda chinensis Scrophularia ningpoensis.", "Isatis tinctoria Isatis indigotica and Baphicacanthus cusia; Herb B is selected from the group consisting of: Polygonum bistorta Polygonum lapidosum.", "Polygonum viviparum Polygonum manshuriense Polygonum alopecuroides Polygonum sphaerostachyum Andrographis paniculata Taraxacum mongolicum and Chrysanthemun indicum; Herb C is selected from the group consisting of: Prunella vulgari Artemissia capillaris Gardenia jasminoides Rosa rugosa and Lophatherum gracile; Herb D is selected from the group consisting of: Sonchus brachyotus Patrinia scabiosaefolia Patrinia villosa Sonchus arvensis Thlaspi arvense Portulaca oleracea and Pulsatilla chinensis; Herb E is selected from the group consisting of: Dictamnus dasycarpus Kochia scoparia Sophora flavescens and Heydyotis diffusa; and Herb F is selected from the group consisting of: Dioscorea bulbifera Panax notoginseng Bletilla striats Nelumbo nucifers Polygonum bistorta Cephalanoplos segetum Cirsium japonicum.", "Sophora japonica.", "Typha angustfolia and Rubia cordifolia.", "; and wherein the composition does not comprise more than 5 herbs selected from the group consisting of Sophora tonkinesis (Sophora subprostata), Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus and Dioscorea bulbifera.", "10.The composition of claim 9, wherein one of each three herbs is different from at least one of: Herb A, Herb B, Herb C, Herb D, Herb E and Herb F. 11.The composition of claim 9, wherein Herb A is selected from the group consisting of: Belamcanda chinensis Scrophularia ningpoensis.", "Isatis tinctoria Isatis indigotica and Baphicacanthus cusia; Herb B is selected from the group consisting of: Polygonum lapidosum.", "Polygonum viviparum Polygonum manshuriense Polygonum alopecuroides Polygonum sphaerostachyum Andrographis paniculata Taraxacum mongolicum and Chrysanthemun indicum; Herb C is selected from the group consisting of: Artemissia capillaris Gardenia jasminoides Rosa rugosa and Lophatherum gracile; Herb D is selected from the group consisting of: Patrinia scabiosaefolia Patrinia villosa Sonchus arvensis Thlaspi arvense Portulaca oleracea and Pulsatilla chinensis; Herb E is selected from the group consisting of: Kochia scoparia Sophora flavescens and Heydyotis diffusa; and Herb F is selected from the group consisting of: Panax notoginseng Bletilla striats Nelumbo nucifers Cephalanoplos segetum Cirsium japonicum.", "Typha angustfolia and Rubia cordifolia.", "12.The composition of claim 9, wherein the composition comprises three herbs, one of which is selected from each of Herb A, Herb B, Herb C, Herb D or Herb E and wherein each is present in an amount from about 9% to about 57%.", "13.The composition of claim 9, wherein the composition comprises four herbs, one of which is selected from each of Herb A, Herb B, Herb C, Herb D or Herb E, and wherein each is present in an amount from about 6% to about 43%.", "14.The composition of claim 9, wherein the composition comprises four herbs, the first three are selected from the group consisting of Herb A, Herb B, Herb C and Herb D and are present in an amount from about 7% to about 57% and the fourth is selected from Herb E and is present in an amount from about 3.5% to about 28.5%.", "15.The composition of claim 9, wherein the composition comprises five herbs, one of which is selected from each of Herb A, Herb B, Herb C, Herb D or Herb E, and wherein each is present in an amount from about 5% to about 35%.", "16.The composition according to claim 9, wherein the composition comprises five herbs, and wherein the first four herbs are selected from the group consisting of Herb A, Herb B, Herb C and Herb D and are present in an amount from about 6% to about 38% and the fifth herb is selected from Herb E and is present in an amount from about 3% to about 19%.", "17.The composition according to claim 9, wherein the composition comprises six herbs, and wherein the first five herbs are selected from the group consisting of Herb A, Herb B, Herb C, Herb D and Herb E wherein the herbs are present in an amount from about 5% to about 31% and the sixth herb is selected from Herb F and is present in an amount from about 2.5% to about 17.5%.", "18.The composition according to claim 9, wherein the composition comprises six herbs, and wherein the first four herbs are selected from the group consisting of Herb A, Herb B, Herb C and Herb D and are present in an amount from about 5% to about 38% and the fifth is selected form Herb E and is present in an amount from about 2.5% to about 19% and the sixth herb is selected from Herb F and is present in an amount from about 1.75% to about 19%." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>For the past twenty-five years there has been significant progress in the field of cancer research; however, in spite of these positive results, the mortality rate for the most common cancers still remains high.", "Indeed, the goal of the National Cancer Institute of a fifty percent reduction in overall cancer mortality by the year 2000 has not been met.", "The term “cancer” is a general one referring to more than 100 forms of the disease which may manifest itself in almost every tissue type of the body.", "Of the myriad forms of cancer, lung cancer is the most common cause of death worldwide, followed by stomach cancer.", "Other common forms of cancer include cancers of the colon, rectum, breast, prostate, mouth and esophagus.", "Lung cancer imposes an enormous burden on health care.", "The World Health Report 2000 estimates that lung cancer has resulted in 860,000 deaths among men and 333,000 deaths among women per year, and it is the leading cause of cancer deaths worldwide.", "In the United States and Canada, more people are dying from lung cancer than from breast cancer, colorectal cancer and prostate cancer combined.", "In addition, the incidence of lung cancer in women is rising at an average annual rate of 7% which is the most rapid rate of increase for any cancer.", "The most dominant cause of lung cancer is tobacco use, but occupational and environmental exposure to various other carcinogenic substances can also influence disease development.", "In long-term smokers, the risk of lung cancer never returns to the “baseline” level of a never-smoker, even years after smoking cessation.", "With a large reservoir (100 million in the United States and Canada alone) of current and former smokers, who are at risk, lung cancer will continue to be a major health problem for at least several more decades even if current efforts to curb tobacco smoking were successful.", "The overall five-year survival rate of lung cancer is less than 15%.", "Despite advances in modern medicine, the survival rate has not improved substantially over the last two decades.", "A different approach is therefore needed to control lung cancer.", "Prostate cancer is the most commonly diagnosed male malignancy in the United States and the second leading cause of cancer death.", "It is estimated that in the year 2000, there will be 180,400 new cases and 31,900 deaths caused by prostate cancer.", "Although prostate cancer responds effectively to orchitectomy or antiandrogen therapy when detected at an early stage, over time, the residual androgen-insensitive cells recolonize, expand, and ultimately establish a hormone-resistant state that often results in fatality.", "It would be useful, therefore, to have a cancer treatment which could prevent the proliferation of prostate cancer and maintain it in a dormant state.", "The incidence of gastric cancer has fallen in most countries but it is still the most common form of cancer in many countries of East Asia, including China.", "Globally, gastric cancer is the second most frequent cause of cancer death and it is estimated that in 1990, there were almost 800,000 new cases and about 630,000 deaths.", "Similarly, esophageal cancer, the eighth most common cancer worldwide and responsible for 316,000 new cases and 286,000 deaths in 1990, is also very common in China and other Asian countries.", "Both of these cancers, and especially esophageal cancer, have low survival rates and thus it would be beneficial to have an alternative treatment approach for these types of cancers.", "Traditionally, the focus of cancer research has been in developing therapies and treatments for patients already afflicted with the disease.", "However, over the last few decades, new insights into the development of cancer as a disease have been gained.", "It is now understood that cancer is not the result of a single initiation event but of a gradual, multi-step process characterized by a period of several years between the initiation event and the onset of invasive or metastatic disease.", "In general, the process of carcinogenesis can be divided into three phases: initiation, promotion, and progression.", "In initiation, a fixed genetic mutation results from the interaction of a carcinogen with DNA.", "The extent of the molecular change depends on a number of factors including the nature of the carcinogen, the rate and type of carcinogenic metabolism and the response of the DNA repair systems.", "The next phase, promotion, may occur over extended periods of time and is characterized by the proliferation of the altered cells.", "This phase may be affected by agents that alter growth rates.", "During the final phase, progression, genetic and phenotypic changes occur which ultimately cause the development of premalignant lesions into invasive cancer.", "The multi-step nature of carcinogenesis suggests the possibility of intervention at a precancerous state.", "This is the basis for chemoprevention, which refers to the use of natural or synthetic agents to prevent the development of cancer, either by blocking the DNA damage that initiates the carcinogenesis process or by arresting/regressing existing pre-malignant lesions.", "Since the mid-1950s, research has been directed at finding compounds with potential chemopreventive properties.", "The search for these agents has demonstrated a unique challenge.", "Chemopreventive agents must have low toxicity and be relatively free of side effects because they are intended for administration to healthy people over long periods of time.", "This is in direct contrast to chemotherapy drugs, such as cis platinum or paclitaxel (Taxol™), which are used as chemotherapeutic agents to treat people already afflicted with cancer.", "Chemotherapeutic agents are chosen for their ability to kill tumor cells but because they are also toxic to healthy cells, they usually cause harmful side effects.", "One of the major sources of potential chemopreventive agents is plants.", "For example, consumption of cruciferous vegetables, such as broccoli, cauliflower and cabbage is associated with a lower risk of various cancers.", "Fruits and vegetables contain a number of potentially active chemopreventive compounds, such as carotenoids, dithiolthiones and isothiocyanates.", "They are capable of inhibiting the development of tumors of the lungs, colon, mammary glands and bladder in laboratory animals.", "Three proof-of-principle clinical trials suggest that chemoprevention might be an effective strategy to control lung cancer.", "A study by Hong and co-workers in the United States showed that in patients with cured head and neck cancer, high dose 13-cis-retinoic acid for 12 months was more effective than placebo in preventing second primary cancers in the upper aerodigestive tract.", "However, 13-cis-retinoic acid at this dosage carries unacceptable toxicity for use in the general population.", "Another study by Pastorino and co-workers in Europe showed that retinol palmitate in a dose of 300,000 Units per day for 12 months was more effective than placebo in preventing second primary lung cancer.", "A third study in China found that daily doses of a combination of vitamins and minerals consisting of beta-carotene, vitamin E and selenium resulted in a 21% decrease in stomach cancer deaths in high-risk people in China.", "However, subsequent phase III clinical trials using beta-carotene or retinal (the Alpha-Tocopherol, Beta-Carotene Trial, the Beta-Carotene and Retinol Efficacy Trial, and the EUROSCAN study) failed to show a reduction in lung cancer incidence in high-risk individuals, such as heavy smokers with or without exposure to asbestos, compared to placebo.", "In fact, the use of beta-carotene in those who continued to smoke during the study was found to increase the risk of lung cancer.", "Several reasons were postulated to explain why chemopreventive treatment with retinoids was ineffective or even harmful in active smokers.", "There may be adverse interactions between tobacco carcinogens and the chemopreventive agent.", "Beta-carotene, for example, is a pro-oxidant at high arterial oxygen tension.", "It can enhance conversion of benzo[A]pyrene to the ultimate carcinogen as well as inducing cytochrome P450.Another reason for the lack of effect in active smokers is that ongoing tobacco carcinogen exposure may counteract the effect of the chemopreventive agent.", "A number of chemopreventive agents are currently under clinical investigation.", "Examples of these include fenretinide, selenium, inhaled budesonide, COX-2 inhibitors, farnesyl transferase inhibitors, lipoxygenase inhibitors, EGF-kinase inhibitors and green tea.", "Although promising, it remains to be shown if these agents can be shown to be useful in ongoing clinical trials.", "The best example to-date that chemoprevention can prevent cancer is Tamoxifen.", "Tamoxifen is an estrogen antagonist.", "In women at high risk of breast cancer, the Breast Cancer Prevention Trial showed a 49% decrease in invasive cancer and a 50% decrease in non-invasive breast cancer with Tamoxifen versus placebo (J Natl Cancer Inst 1998; 90:1371-88).", "In addition, there was a decrease in the incidence of fractures due to osteoporosis.", "However, there was a slight increase in the risk of endometrial cancer, deep venous thrombosis and pulmonary embolism.", "More selective estrogen-receptor modulators such as Raloxifene, are being tested against Tamoxifen to determine if these agents may have similar chemopreventive effect but fewer side effects than Tamoxifen.", "A variety of Chinese herbs have been used for centuries to treat different diseases.", "The great majority of these are empirical, open, non-randomized studies without placebo control groups.", "Although some of these herbs have been used to treat patients with cancer, they are not considered to be disease specific.", "Rather, they are used for “dispersing heat, detoxification, improving stasis and removing mass”.", "Herbs such as Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz , and Dioscorea bulbifera are known to have properties that may be useful for the prevention and treatment of cancers.", "One such composition is known as ZSP or Zeng Sheng Ping; however, the exact formulation of the composition is not known.", "European Patent application 93 121109.8 describes a composition comprising Sophora tonkinensis Gapnep 42 mg, Polygonum bistorta L. 42 mg, Prunella vulgaris L. 42 mg, Sonchus brachyotus DC 42 mg, Dictamnus dasycarpus Turcz 21 mg, and Dioscorea bulbifera 10 mg which was reported to be useful for the treatment of patients with mouth, esophagus or digestive tract cancers.", "Thus, there is a need to develop other herbal remedies for the treatment and prophylaxis of common cancers, and for other therapeutic uses." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Thus, according to the present invention there is provided a composition comprising herbs.", "Thus, according to this embodiment of the invention, there is provided a composition comprising a mixture of at least three different herbs selected from the group consisting of herbs, which for the purpose of this invention have been designated as: Herb A, Herb B, Herb C, Herb D, Herb E and Herb F. In the composition defined in EPO 93121109.8 the herb corresponding to Herb A was Sophora tonkinensis ( Sophora subprostrata ).", "According to the present invention it has been found that any one of the following herbs can be used in place of Sophora tonkinensis: Belamcanda chinensis Scrophularia ningpoensis.", "Isatis tinctoria Isatis indigotica or Baphicacanthus cusia Bremek.", "In the composition defined in EPO 93121109.8 the herb corresponding to Herb B was Polygonum bistorta .", "According to the present invention it has been found that any one of the following herbs can be used in place of Polygonum bistorta: Polygonum lapidosum Polygonum viviparum Polygonum manshuriense Polygonum alopecuroides Polygonum sphaerostachyum Andrographis paniculata Taraxacum mongolicum or Chrysanthemun indicum.", "In the composition defined in EPO 93121109.8 the herb corresponding to Herb C was Prunella vulgaris .", "According to the present invention it has been found that any one of the following herbs can be used in place of Prunella vulgaris: Artemissia capillaris Gardenia jasminoides Rosa rugosa or Lophatherum gracile.", "In the composition defined in EPQ 93121109.8 the herb corresponding to Herb D was Sonchus brachyotus .", "According to the present invention it has been found that any one of the following herbs can be used in place of Sonchus brachyotus: Patrinia scabiosaefolia Patrinia villosa Sonchus arvensis Thlaspi arvense Portulaca oleracea or Pulsatilla chinensis.", "In the composition defined in EPO 93121109.8 the herb corresponding to Herb E was Dictamnus dasycarpus .", "According to the present invention it has been found that any one of the following herbs can be used in place of Dictamnus dasycarpus: Kochia scoparia Sophora flavescens or Heydyotis diffusa.", "In the composition defined in EPO 93121109.8 the herb corresponding to Herb F was Dioscorea bulbifera .", "According to the present invention it has been found that any one of the following herbs can be used in place of Dioscorea bulbifera: Panax notoginseng Bletilla striata Nelumbo nucifera Polygonum bistorta Cephalanoplos segetum Cirsium japonicum Sophora japonica Typha angustifolia .", "or Rubia cordifolia.", "Further according to the present invention there is provided a use of said composition as chemopreventive and therapeutic agents.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "The present invention relates to compositions comprising herbs and their use as chemopreventive and therapeutic agents.", "BACKGROUND OF THE INVENTION For the past twenty-five years there has been significant progress in the field of cancer research; however, in spite of these positive results, the mortality rate for the most common cancers still remains high.", "Indeed, the goal of the National Cancer Institute of a fifty percent reduction in overall cancer mortality by the year 2000 has not been met.", "The term “cancer” is a general one referring to more than 100 forms of the disease which may manifest itself in almost every tissue type of the body.", "Of the myriad forms of cancer, lung cancer is the most common cause of death worldwide, followed by stomach cancer.", "Other common forms of cancer include cancers of the colon, rectum, breast, prostate, mouth and esophagus.", "Lung cancer imposes an enormous burden on health care.", "The World Health Report 2000 estimates that lung cancer has resulted in 860,000 deaths among men and 333,000 deaths among women per year, and it is the leading cause of cancer deaths worldwide.", "In the United States and Canada, more people are dying from lung cancer than from breast cancer, colorectal cancer and prostate cancer combined.", "In addition, the incidence of lung cancer in women is rising at an average annual rate of 7% which is the most rapid rate of increase for any cancer.", "The most dominant cause of lung cancer is tobacco use, but occupational and environmental exposure to various other carcinogenic substances can also influence disease development.", "In long-term smokers, the risk of lung cancer never returns to the “baseline” level of a never-smoker, even years after smoking cessation.", "With a large reservoir (100 million in the United States and Canada alone) of current and former smokers, who are at risk, lung cancer will continue to be a major health problem for at least several more decades even if current efforts to curb tobacco smoking were successful.", "The overall five-year survival rate of lung cancer is less than 15%.", "Despite advances in modern medicine, the survival rate has not improved substantially over the last two decades.", "A different approach is therefore needed to control lung cancer.", "Prostate cancer is the most commonly diagnosed male malignancy in the United States and the second leading cause of cancer death.", "It is estimated that in the year 2000, there will be 180,400 new cases and 31,900 deaths caused by prostate cancer.", "Although prostate cancer responds effectively to orchitectomy or antiandrogen therapy when detected at an early stage, over time, the residual androgen-insensitive cells recolonize, expand, and ultimately establish a hormone-resistant state that often results in fatality.", "It would be useful, therefore, to have a cancer treatment which could prevent the proliferation of prostate cancer and maintain it in a dormant state.", "The incidence of gastric cancer has fallen in most countries but it is still the most common form of cancer in many countries of East Asia, including China.", "Globally, gastric cancer is the second most frequent cause of cancer death and it is estimated that in 1990, there were almost 800,000 new cases and about 630,000 deaths.", "Similarly, esophageal cancer, the eighth most common cancer worldwide and responsible for 316,000 new cases and 286,000 deaths in 1990, is also very common in China and other Asian countries.", "Both of these cancers, and especially esophageal cancer, have low survival rates and thus it would be beneficial to have an alternative treatment approach for these types of cancers.", "Traditionally, the focus of cancer research has been in developing therapies and treatments for patients already afflicted with the disease.", "However, over the last few decades, new insights into the development of cancer as a disease have been gained.", "It is now understood that cancer is not the result of a single initiation event but of a gradual, multi-step process characterized by a period of several years between the initiation event and the onset of invasive or metastatic disease.", "In general, the process of carcinogenesis can be divided into three phases: initiation, promotion, and progression.", "In initiation, a fixed genetic mutation results from the interaction of a carcinogen with DNA.", "The extent of the molecular change depends on a number of factors including the nature of the carcinogen, the rate and type of carcinogenic metabolism and the response of the DNA repair systems.", "The next phase, promotion, may occur over extended periods of time and is characterized by the proliferation of the altered cells.", "This phase may be affected by agents that alter growth rates.", "During the final phase, progression, genetic and phenotypic changes occur which ultimately cause the development of premalignant lesions into invasive cancer.", "The multi-step nature of carcinogenesis suggests the possibility of intervention at a precancerous state.", "This is the basis for chemoprevention, which refers to the use of natural or synthetic agents to prevent the development of cancer, either by blocking the DNA damage that initiates the carcinogenesis process or by arresting/regressing existing pre-malignant lesions.", "Since the mid-1950s, research has been directed at finding compounds with potential chemopreventive properties.", "The search for these agents has demonstrated a unique challenge.", "Chemopreventive agents must have low toxicity and be relatively free of side effects because they are intended for administration to healthy people over long periods of time.", "This is in direct contrast to chemotherapy drugs, such as cis platinum or paclitaxel (Taxol™), which are used as chemotherapeutic agents to treat people already afflicted with cancer.", "Chemotherapeutic agents are chosen for their ability to kill tumor cells but because they are also toxic to healthy cells, they usually cause harmful side effects.", "One of the major sources of potential chemopreventive agents is plants.", "For example, consumption of cruciferous vegetables, such as broccoli, cauliflower and cabbage is associated with a lower risk of various cancers.", "Fruits and vegetables contain a number of potentially active chemopreventive compounds, such as carotenoids, dithiolthiones and isothiocyanates.", "They are capable of inhibiting the development of tumors of the lungs, colon, mammary glands and bladder in laboratory animals.", "Three proof-of-principle clinical trials suggest that chemoprevention might be an effective strategy to control lung cancer.", "A study by Hong and co-workers in the United States showed that in patients with cured head and neck cancer, high dose 13-cis-retinoic acid for 12 months was more effective than placebo in preventing second primary cancers in the upper aerodigestive tract.", "However, 13-cis-retinoic acid at this dosage carries unacceptable toxicity for use in the general population.", "Another study by Pastorino and co-workers in Europe showed that retinol palmitate in a dose of 300,000 Units per day for 12 months was more effective than placebo in preventing second primary lung cancer.", "A third study in China found that daily doses of a combination of vitamins and minerals consisting of beta-carotene, vitamin E and selenium resulted in a 21% decrease in stomach cancer deaths in high-risk people in China.", "However, subsequent phase III clinical trials using beta-carotene or retinal (the Alpha-Tocopherol, Beta-Carotene Trial, the Beta-Carotene and Retinol Efficacy Trial, and the EUROSCAN study) failed to show a reduction in lung cancer incidence in high-risk individuals, such as heavy smokers with or without exposure to asbestos, compared to placebo.", "In fact, the use of beta-carotene in those who continued to smoke during the study was found to increase the risk of lung cancer.", "Several reasons were postulated to explain why chemopreventive treatment with retinoids was ineffective or even harmful in active smokers.", "There may be adverse interactions between tobacco carcinogens and the chemopreventive agent.", "Beta-carotene, for example, is a pro-oxidant at high arterial oxygen tension.", "It can enhance conversion of benzo[A]pyrene to the ultimate carcinogen as well as inducing cytochrome P450.Another reason for the lack of effect in active smokers is that ongoing tobacco carcinogen exposure may counteract the effect of the chemopreventive agent.", "A number of chemopreventive agents are currently under clinical investigation.", "Examples of these include fenretinide, selenium, inhaled budesonide, COX-2 inhibitors, farnesyl transferase inhibitors, lipoxygenase inhibitors, EGF-kinase inhibitors and green tea.", "Although promising, it remains to be shown if these agents can be shown to be useful in ongoing clinical trials.", "The best example to-date that chemoprevention can prevent cancer is Tamoxifen.", "Tamoxifen is an estrogen antagonist.", "In women at high risk of breast cancer, the Breast Cancer Prevention Trial showed a 49% decrease in invasive cancer and a 50% decrease in non-invasive breast cancer with Tamoxifen versus placebo (J Natl Cancer Inst 1998; 90:1371-88).", "In addition, there was a decrease in the incidence of fractures due to osteoporosis.", "However, there was a slight increase in the risk of endometrial cancer, deep venous thrombosis and pulmonary embolism.", "More selective estrogen-receptor modulators such as Raloxifene, are being tested against Tamoxifen to determine if these agents may have similar chemopreventive effect but fewer side effects than Tamoxifen.", "A variety of Chinese herbs have been used for centuries to treat different diseases.", "The great majority of these are empirical, open, non-randomized studies without placebo control groups.", "Although some of these herbs have been used to treat patients with cancer, they are not considered to be disease specific.", "Rather, they are used for “dispersing heat, detoxification, improving stasis and removing mass”.", "Herbs such as Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera are known to have properties that may be useful for the prevention and treatment of cancers.", "One such composition is known as ZSP or Zeng Sheng Ping; however, the exact formulation of the composition is not known.", "European Patent application 93 121109.8 describes a composition comprising Sophora tonkinensis Gapnep 42 mg, Polygonum bistorta L. 42 mg, Prunella vulgaris L. 42 mg, Sonchus brachyotus DC 42 mg, Dictamnus dasycarpus Turcz 21 mg, and Dioscorea bulbifera 10 mg which was reported to be useful for the treatment of patients with mouth, esophagus or digestive tract cancers.", "Thus, there is a need to develop other herbal remedies for the treatment and prophylaxis of common cancers, and for other therapeutic uses.", "SUMMARY OF THE INVENTION Thus, according to the present invention there is provided a composition comprising herbs.", "Thus, according to this embodiment of the invention, there is provided a composition comprising a mixture of at least three different herbs selected from the group consisting of herbs, which for the purpose of this invention have been designated as: Herb A, Herb B, Herb C, Herb D, Herb E and Herb F. In the composition defined in EPO 93121109.8 the herb corresponding to Herb A was Sophora tonkinensis (Sophora subprostrata).", "According to the present invention it has been found that any one of the following herbs can be used in place of Sophora tonkinensis: Belamcanda chinensis Scrophularia ningpoensis.", "Isatis tinctoria Isatis indigotica or Baphicacanthus cusia Bremek.", "In the composition defined in EPO 93121109.8 the herb corresponding to Herb B was Polygonum bistorta.", "According to the present invention it has been found that any one of the following herbs can be used in place of Polygonum bistorta: Polygonum lapidosum Polygonum viviparum Polygonum manshuriense Polygonum alopecuroides Polygonum sphaerostachyum Andrographis paniculata Taraxacum mongolicum or Chrysanthemun indicum.", "In the composition defined in EPO 93121109.8 the herb corresponding to Herb C was Prunella vulgaris.", "According to the present invention it has been found that any one of the following herbs can be used in place of Prunella vulgaris: Artemissia capillaris Gardenia jasminoides Rosa rugosa or Lophatherum gracile.", "In the composition defined in EPQ 93121109.8 the herb corresponding to Herb D was Sonchus brachyotus.", "According to the present invention it has been found that any one of the following herbs can be used in place of Sonchus brachyotus: Patrinia scabiosaefolia Patrinia villosa Sonchus arvensis Thlaspi arvense Portulaca oleracea or Pulsatilla chinensis.", "In the composition defined in EPO 93121109.8 the herb corresponding to Herb E was Dictamnus dasycarpus.", "According to the present invention it has been found that any one of the following herbs can be used in place of Dictamnus dasycarpus: Kochia scoparia Sophora flavescens or Heydyotis diffusa.", "In the composition defined in EPO 93121109.8 the herb corresponding to Herb F was Dioscorea bulbifera.", "According to the present invention it has been found that any one of the following herbs can be used in place of Dioscorea bulbifera: Panax notoginseng Bletilla striata Nelumbo nucifera Polygonum bistorta Cephalanoplos segetum Cirsium japonicum Sophora japonica Typha angustifolia.", "or Rubia cordifolia.", "Further according to the present invention there is provided a use of said composition as chemopreventive and therapeutic agents.", "DETAILED DESCRIPTION OF PREFERRED EMBODIMENT The present invention relates to compositions comprising herbs and their use as chemopreventive and therapeutic agents.", "In one embodiment of the present invention the composition comprises a mixture of at least three different herbs, known hereinafter as Herb A, Herb B, Herb C, Herb D, Herb E and Herb F. According to the present invention Herb A is selected from the group consisting of: Belamcanda chinensis Scrophularia ningpoensis Isatis tinctoria Isatis indigotica and Baphicacanthus cusia.", "According to the present invention Herb B is selected from the group consisting of: Polygonum lapidosum Polygonum viviparum Polygonum manshuriense Polygonum alopecuroides Polygonum sphaerostachyum.", "Andrographis paniculata Taraxacum mongolicum and Chrysanthemun indicum.", "According to the present invention Herb C is selected from the group consisting of: Artemissia capillaris Gardenia jasminoides Rosa rugosa and Lophatherum gracile.", "According to the present invention Herb D is selected from the group consisting of: Patrinia scabiosaefolia Patrinia villosa Sonchus arvensis Thlaspi arvense Portulaca oleracea and Pulsatilla chinensis.", "According to the present invention Herb E is selected from the group consisting of: Kochia scoparia Sophora flavescens and Heydyotis diffusa.", "According to the present invention Herb F is selected from the group consisting of: Panax notoginseng.", "Bletilla striata Nelumbo nucifera.", "Polygonum bistorta Cephalanoplos segetum.", "Cirsium japonicum.", "Sophora japonica Typha angustifolia and Rubia cordifolia.", "When, according to the present invention, the composition comprises three different herbs, one of which is selected from each of Herb A, Herb B, Herb C, Herb D or Herb E, each of the three herbs are present in an amount from about 9% to about 57%.", "In a further example of this embodiment, the three herbs are each present in an amount from about 15% to about 50%.", "In a further example of this embodiment, each of the three herbs are present in an amount from about 25% to about 40%.", "In a further example of this embodiment, each of the three herbs are present in an amount of about 33%.", "When the composition comprises four different herbs, one of which is selected from each of Herb A, Herb B, Herb C, Herb D or Herb E, each of the four herbs are present in an amount from about 6% to about 43%.", "In a further example of this embodiment, each of the four herbs are present in an amount from about 15% to about 35%.", "In a further example of this embodiment, each of the four herbs are present in an amount of about 25%.", "In a further example of the present invention, when the composition comprises four herbs, three of which are selected from the group consisting of Herb A, Herb B, Herb C, Herb D and the fourth herb is selected from the group consisting of Herb E. The composition of the first three herbs are each present in an amount from about 7% to about 57% and the fourth herb is present in an amount from about 3.5% to about 28.5%.", "In a further example of this embodiment, each of the first three herbs are present in an amount from about 15% to about 48% and the fourth herb is present in an amount from about 7.5% to about 24%.", "In a further example of this embodiment, the first three herbs are present in a composition in an amount of about 20% to about 43%, and the fourth herb is present in an amount from about 10% to about 21.5%.", "In a further example of this embodiment, the first three herbs are present in an amount of about 28% and the fourth herb is present in an amount of about 14%.", "In yet a further embodiment of this invention, the composition comprises five different herbs, one of which is each selected from Herb A, Herb B, Herb C, Herb D or Herb E, each of the five herbs are present in an amount from about 5% to about 35%.", "In a further example of this embodiment, each of the five herbs are present in an amount from about 10% to about 30%.", "In yet a further example of this embodiment, each of the five herbs are present in an amount from about 15% to about 25%.", "In a further example of this embodiment, each of the five herbs are present in an amount of about 20%.", "In yet a further embodiment of this invention, the first four herbs are selected one from each of the group Herb A, Herb B, Herb C and Herb D whereas the fifth herb is selected from Herb E and the first four herbs are present in an amount from about 6% to about 38%, whereas the fifth herb is present at an amount from about 3% to about 19%.", "In a further example of this embodiment, the first four herbs are present in the composition at an amount from about 12% to about 32%, and the fifth herb is present in an amount from about 6% to about 16%.", "In yet a further example of this embodiment, the first four herbs are present in an amount from about 18% to about 26% and the fifth herb is present in an amount from about 9% to about 13%.", "In yet a further example of this embodiment, the first four herbs are present at an amount of about 22%, whereas the fifth herb is present at an amount of about 11%.", "In yet a further embodiment, the composition comprises six herbs.", "The first five herbs are selected from the group consisting of Herb A, Herb B, Herb C, Herb D and Herb E, and the sixth herb is selected from Herb F and the first five herbs are present in an amount from about 5% to about 31% and the sixth herb is present in an amount from about 2.5% to about 17.5%.", "In a further example of this embodiment, each of the first five herbs are present in an amount from about 10% to about 26%, and the sixth herb is present in an amount from about 5% to about 15%.", "In a further example of this embodiment, the first five herbs are each present in an amount from about 15% to about 21% and the sixth herb is present in an amount from about 7.5 to about 12.5%.", "In a further example of this embodiment, the first five herbs are present in an amount from about 18% and the sixth herb is present in an amount from about 10%.", "In yet a further example of this embodiment, the first four herbs are selected from the group of Herb A, Herb B, Herb C or Herb D. The fifth herb is selected from Herb E and the sixth herb is selected from Herb F, wherein the first three herbs are each present in an amount from about 5% to about 38%, the fifth herb is present in an amount from about 2.5% to about 19% and the sixth herb is present in an amount from about 1.75% to about 9.5%.", "In a further example of this embodiment, the first four herbs are present in an amount from about 10% to about 33%.", "The fifth herb is present in an amount from about 5% to about 16.5% and the sixth herb is present in an amount from about 3% to about 8.25%.", "In yet a further example of this embodiment, the first four herbs are present in an amount from about 15% to about 28%; herb five is present in an amount from about 7.5% to about 14.5% and the sixth herb is present in an amount from about 4.25% to about 7%.", "In yet a further example of this embodiment, the first four herbs are present in an amount from about 21%.", "The fifth herb is present in an amount from about 10.5% and the sixth herb is present in the amount from about 5.5%.", "It is important to ensure that the herbs which are used according to the present invention are selected such that they contain only acceptable levels of contaminants such as metals or pesticides.", "Various regions of China have been surveyed and it was found that the component herbs can be harvested from the Guangxi, Hunan, Liaoning, Anhui, Hebei and Jiangsu regions of China during the summer and autumn months and no pesticides are used.", "The plants are purchased dried and whole or parts of these herbs are used in manufacturing.", "The general manufacturing scheme is as follows: the herbs are either subjected to an aqueous extraction, the aqueous extract is then filtered if necessary to remove large particles, the aqueous extract is then dried to a powder.", "Alternatively, it is possible to use the herbs directly by grinding to a powder.", "The powdered herbs are then used in the production of the therapeutic in a variety of forms for administration.", "Any suitable mode of delivery can be used according to the present invention.", "For example the composition of the present invention can be delivered orally by a pill, tablet, drink, candy or paste.", "The composition can also be delivered as a transdermal patch, by inhalation or suppository.", "Delivery of the composition as an injectable is also possible, according to the present invention.", "Therefore, the composition can be administered as a therapeutic agent or as a dietary supplement.", "In one embodiment of the present invention, tablets are formulated into 0.3 g tablets.", "A typical daily dosage is about 1.2 to 6 g/day based on the body weight and/or severity of the condition.", "According to the present invention the compositions are useful for a variety of therapeutic treatments.", "In one embodiment of the present invention the compositions are useful for the prevention or treatment of cancers.", "According to one embodiment of the present invention, the composition comprises additional therapeutic agents.", "Examples of such agents can include, but are not limited to, chemotherapeutic agents.", "As will be apparent to those skilled in the art in light of the foregoing disclosure, may alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof.", "Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims." ] ]	Patent_10468903
[ [ "Method for producing a coating and composition for crosslinkable coatings", "The invention concerns a method for producing a coating having high hardness, impact resistance and chemical attack resistance, characterised in that it consists in: applying on a substrate, simultaneously or successively: A) a polymer composition containing hydroxyl groups; B) an amine-formaldehyde type resin composition; C) a polyisocyanate composition containing at least 30 mol % of compounds comprising at least a biuret group; D) optionally the usual additives; and heating said substrate to a temperature enabling said constituents to be crosslinked." ], [ "1.A method for producing a coating having a high hardness, a high impact strength and a high resistance to chemical attack, which comprises the application, to a substrate, of a blend for simultaneous or successive addition, comprising: A) a composition of a polymer containing hydroxyl groups; B) an amine-formaldehyde-type resin composition; C) a polyisocyanate that include containing at least 30 mol % of compounds that include at least one biuret group; and D) optionally, standard additives; and the heating of said substrate to a temperature allowing said components to be crosslinked.", "2.The method as claimed in claim 1, wherein at least 10 mol % of the isocyanate functional groups are blocked by a monofunctional blocking agent.", "3.The method as claimed in claim 1, wherein the blocking agent comprises heterocyclic nitrogen compounds.", "4.The method as claimed in claim 3, wherein the blocking agent is a pyrazole, optionally substituted with one or more substituents.", "5.The method as claimed in claim 4, wherein the blocking agent is 3,5-dimethylpyrazole.", "6.The method as claimed in claim 1, wherein polymer (A) is an acrylic polyol having a hydroxyl content of between 1 and 5 g/100 g of polymer solids content.", "7.The method as claimed in claim 1, wherein polymer (A) is an acrylic polyol having a molecular weight of between 1 000 and 5 000.8.The method as claimed in claim 1, wherein constituent (B) is a melamine-formaldehyde resin.", "9.The method as claimed in claim 1, wherein component (C) is the product obtained from polycondensation of an aliphatic or cycloaliphatic diisocyanate or triisocyanate.", "10.The method as claimed in claim 7, wherein component (C) is the product obtained from polycondensation of a diisocyanate.", "11.The method as claimed in claim 1, wherein the solids content of component (A) is between 10 and 60% with respect to the weight of components (A), (B), (C) and (D).", "12.The method as claimed in claim 1, wherein the solids content of component (D) is between 0 and 65% with respect to the weight of components (A), (B), (C) and (D).", "13.The method as claimed in claim 1, wherein component (C) comprises at least 50% of polyisocyanate compounds that include at least one biuret group with respect to the total weight of the polyisocyanate compounds.", "14.The method as claimed in claim 1, wherein component (C) comprises at least 15% by weight of compounds of formula (I): in which R1, R2 and R3, which are identical or different, are linear, branched or cyclic C1-C20 hydrocarbon chains that optionally comprise an isocyanate functional group.", "15.The method as claimed in claim 1, wherein the ratio of isocyanate functional groups to hydroxyl functional groups is less than 3.16.A coating composition, which comprises: A) from 20 to 50% by weight of solids content of a polymer containing hydroxyl groups; B) from 3 to 20% by weight of solids content of a resin of the amine-formaldehyde type; C) from 5 to 20% by weight of solids content of a polyisocyanate composition comprising at least 50 mol % of one or more biuret groups, in which at least 10 mol % of the isocyanate groups are protected using a monofunctional blocking agent; D) from 0 to 30% by weight of solids content of various additives for the coating; and E) from 10 to 20% by weight of one or more solvents.", "17.The composition as claimed in claim 16, wherein the blocking agent comprises heterocyclic nitrogen compounds.", "18.The composition as claimed in claim 16, wherein the blocking agent is a pyrazole optionally substituted with one or more substituents.", "19.The composition as claimed in claim 16, wherein the blocking agent is 3,5-dimethylpyrazole.", "20.The composition as claimed in claim 16, wherein polymer (A) is an acrylic polyol having a hydroxyl content of between 1 and 5 g/100 g of polymer solids content.", "21.The composition as claimed in claim 16, wherein polymer (A) is an acrylic polyol having a molecular weight of between 30 000 and 5 000.22.The composition as claimed in claim 16, wherein component (B) is a melamine-formaldehyde resin.", "23.The composition as claimed in claim 16, wherein component (C) is the product obtained from polycondensation of an aliphatic or cycloaliphatic diisocyanate or triisocyanate.", "24.The composition as claimed in claim 23, wherein component (C) is the product obtained from polycondensation of a diisocyanate.", "25.The composition as claimed in claim 16, wherein component (C) comprises at least 50% of polyisocyanate compounds that include at least one biuret group with respect to the total weight of the polyisocyanate compounds.", "26.The composition as claimed in claim 16, wherein component (C) comprises at least 15% by weight of compounds of formula (I): in which R1, R2 and R3, which are identical or different, are linear, branched or cyclic C1-C20 hydrocarbon chains that optionally comprises an isocyanate functional group.", "27.The composition as claimed in claim 16, wherein the ratio of isocyanate functional groups to hydroxyl functional groups is less than 3." ], [ "The invention relates to crosslinkable coatings based on polymers comprising hydroxyl groups and on crosslinking agents based on polyisocyanates and amine-formaldehyde resins.", "Polyurethane coatings obtained by crosslinking between the hydroxyl functional groups of the polyol and the isocyanate functional groups of a polyisocyanate oligomer are widely used for their hardness, durability, chemical resistance and impact strength properties.", "Coating compositions are also known in which the crosslinking agent is formed by amine-formaldehyde resins that react with the hydroxyl functional groups of the polyol in order to form a network.", "However, the mechanical properties of the coatings obtained are often unsatisfactory.", "In addition, these coatings have a tendency to be sensitive to the curing temperature, and this is not without application problems.", "Moreover, FR 2 322 911 teaches compositions for curable varnishes comprising a blend of polymers containing hydroxyl groups, of amine-formaldehyde-type resins and of polyisocyanates, in which compositions the polyisocyanate may include compounds having a biuret group.", "However, the varnishes obtained have an unsatisfactory hardness.", "The object of the invention is to provide coating compositions, especially paints or varnishes, that have a high hardness.", "Another object of the invention is to provide coatings that have a high flexibility.", "Another object of the invention is to provide coatings that have a high impact strength.", "Another object is to provide coatings that have higher resistance to chemicals; more precisely, coatings that have a resistance with a rating of 3 or better (i.e.", "a rating of 3, 2, 1 or 0), for each of the attacks below: Sulfuric acid attack; acetic acid attack; aqueous ammonia attack; ethanol attack; and butanone attack (the rating system will be detailed later in the description and in the examples).", "Another object is to provide coatings and precursor binder compositions that have a resistance to butanone attack with a rating of 2 or preferably better (a resistance with a rating of better than 2 corresponding to a rating of less than 2).", "Yet another object is to provide coating compositions that have a long pot life and can be processed in one-component systems called “1K” systems, i.e.", "in which all of the coating components, especially the crosslinkable compounds, are present in one and the same system.", "The subject of the invention is a method for producing a coating having a high hardness, a high impact strength and a high resistance to chemical attack, characterized in that it comprises the application, to a substrate, of a blend, for simultaneous or successive addition, comprising: A) a composition of a polymer containing hydroxyl groups; B) an amine-formaldehyde-type resin composition; C) a polyisocyanate composition containing at least 30 mol % of compounds that include at least one biuret group and the isocyanate functional groups of which are free or temporarily blocked; D) optionally, standard additives; and the heating of said substrate to a temperature allowing said components to be crosslinked.", "The term “high hardness” is understood within the invention to mean a hardness of greater than 200 s (Persoz hardness), as measured one hour after the coating has been cured (minimum cure: 30 minutes at 140° C.).", "The term “high impact strength” is understood to mean a strength of greater than 8 (Erichsen cupping).", "Surprisingly, the inventors have furthermore discovered that coatings having the aforementioned properties could be obtained when, as crosslinking agent, a polyisocyanate composition is used in which at least some of the isocyanate groups are blocked by a blocking agent, the blocking agent, upon its release from the isocyanate group, reacting completely or partly with the amine-formaldehyde resin and thus participating in the formation of the network without causing defects in the coating film after the latter has been cured, and while limiting the amount of VOCs (volatile organic compounds).", "It is advantageous for at least 10 mol %, advantageously 20 mol %, preferably at least 50 mol % and up to 100 mol % of the isocyanate functional groups to be blocked by a blocking agent or a mixture of blocking agents, these preferably being monofunctional.", "Component (A) is advantageously a polyol having a hydroxyl content of between 1 and 5 g/l 00 g, advantageously between 3.5 and 4.5 g/100 g, expressed with respect to the solids content.", "For this purpose, it is possible to use polyacrylates containing hydroxyl groups, polyesters or alkyds, or mixtures thereof.", "Most particularly preferred are polyacrylates containing hydroxyl groups, the molecular weight M, of which ranging from 3 000 to 50 000, advantageously from 5 000 to 30 000.It is also preferable for the molecular weight M, to range from 2 000 to 20 000, preferably from 3 000 to 10 000.The molecular weight (Mw) is measured by gas chromatography (GC), polystyrene being used as reference.", "This method makes it possible at the same time to obtain Mw (weight-average molecular weight) and Mn (number-average molecular weight).", "The elution solvent is tetrahydrofuran (THF).", "These polyols are as described on pages 40 to 49 of “Waterborne & Solvent based surface coating resins and their application”, Vol.", "III; John Wiley & Sons, (1998).", "The polyol polymer is generally dissolved in an organic solvent.", "As solvent, mention may be made in particular of esters, aromatic hydrocarbons, ethers, ether esters or amides.", "It is also possible to use aqueous solutions, emulsions or dispersions of polyols or aqueous-organic formulations.", "According to one advantageous embodiment of the present invention, the polyol may be a polyol with a high solids content (SC), the SC of which is between 60 and 100%.", "Constituent A is advantageously present in the compositions according to the invention with a solids content of between 10 and 60%, advantageously 20 and 40%, with respect to the total weight of components (A), (B), (C) and (D).", "Constituent B is a resin of the amine-formaldehyde type, preferably an at least partially etherified precondensate of formaldehyde and of melamine or of urea.", "Solvent-free liquid products may be used for the purpose of applying varnishes or paints with a high solids content.", "However, it is preferred to use the standard amine-formaldehyde resins that contain solvents or aqueous or aqueous-organic solutions, dispersions or emulsions.", "As amines, it is possible to use triazines, triazoles, diazines, guanidines or guanamines, for example N,N′-dimethylurea, acetylenediurea, dicyanamide, benzoguanamine or alkyl-substituted melamines.", "At least some, preferably all, of all the hydroxyl functional groups of the amine-formaldehyde precondensates are etherified.", "These mixed ether functional groups therefore carry, on one side, the chain of the precondensate and, on the other side, a hydrocarbon chain.", "This hydrocarbon chain is advantageously connected to said ether functional group via a carbon of sp3 hybridization; advantageously, they have at most 10 carbon atoms, preferably at most 5 carbon atoms.", "As hydrocarbon chains, mention may especially be made of methyl, ethyl, propyls, butyls (especially n-butyl) or benzyl.", "The amine-formaldehyde resin is prepared in a known manner by acid-catalyzed condensation, aqueous formaldehyde preferably being used.", "Details may be found, for example, in the work by Houben-Weyl, “Methoden der Organischen Chemie” [Methods of Organic Chemistry], (1963), Volume 14/2, pages 319 et seq.", "It is preferable for said amine-formaldehyde precondensates to be produced with an excess (generally a slight excess) of formaldehyde, thereby making it possible in particular to maximize the hydroxyl functional end groups.", "In general, the residual formaldehyde after condensation lies within the range from 1 per thousand to 2 percent by mass of the precondensate.", "Thus, one particularly preferred melamine-formaldehyde resin is a resin having a functionality of at least 6, a dynamic viscosity (DIN 53177, 23° C.) of 500 to 2 500 mPa·s, a compatibility with n-heptane of between 2.0 and 5.0 ml/g, a free formaldehyde content of less than 1% and a density of about 1 g/mL.", "Mention may be most particularly made of the resins sold by Vianova Resins, now Solutia, under the name MAPRENAL®.", "Component (B) is advantageously used with a solids content of between 1 and 40%, advantageously 3 and 20%, by weight with respect to the total weight of components (A), (B), (C) and (D).", "Component (B) is generally dissolved in an organic solvent, especially an alcohol.", "Component (C) is a polyisocyanate composition comprising at least 30%, advantageously at least 50% by weight, preferably at least 60% by weight, of polyisocyanates that include at least one biuret group.", "Advantageously, the polyisocyanate composition is obtained from polycondensation, advantageously catalytic double condensation or triple condensation, of initial isocyanate monomers in the presence of water, an alcohol or an amine.", "The polyisocyanate composition may contain polyisocyanates the number of isocyanate functional groups (NCO) per molecule of which is greater than or equal to 2, such as 4-isocyanatomethyl-1,8-diisocyanato-octamethylene or the isocyanatoethyl ester of lysine diisocyanate.", "The polyisocyanate composition advantageously contains at least 15% by weight of a compound of formula (I): in which R1, R2 and R3, which are identical or different, represent a hydrogen atom, a linear, branched or cyclic C1-C20 hydrocarbon chain that is optionally substituted, preferably substituted with one or two isocyanate functional groups and/or optionally with a group derived from an isocyanate functional group, namely a carbamate, allophanate, isocyanurate, uretidione, iminooxadiazinedione or oxadiazinetrione group, with the proviso that at most only one of R1, R2 and R3 represents a hydrogen atom.", "The composition according to the invention advantageously comprises at least 15%, advantageously at least 30% and preferably at least 50% by weight of compounds of formula (I), in which R1, R2 and R3, which are identical or different, are linear, branched or cyclic C1-C20 hydrocarbon chains that advantageously include an isocyanate functional group.", "These compounds are also called “true biurets”.", "The polyisocyanate composition of the invention also advantageously contains from 10 to 70%, preferably 15 to 60%, by weight of compounds that include more than one biuret group, namely compounds of general formula (I) in which at least one of R1, R2 and R3 comprises a biuret group of formula: The polyisocyanate composition of the invention furthermore advantageously comprises from 0 to 80%, preferably from 0 to 50%, by weight of isocyanurate compounds that include one or more isocyanurate rings.", "Advantageously, the polyisocyanate composition contains from 0 to 50%, preferably from 0 to 30%, by weight of monoisocyanurate compounds.", "The polyisocyanate composition furthermore contains: from 0 to 50%, advantageously from 0.5 to 30%, by weight of compounds having a mono-uretidione group; from 0 to 50%, advantageously from 0 to 30%, by weight of compounds having an allophanate group; from 0 to 30%, advantageously from 0 to 5%, by weight of monomers that have not reacted.", "The content of isocyanate monomers depends on the volatility of the latter.", "It is preferable to work with a low monomer content (advantageously at most 1% by mass) when these monomers are volatile.", "When it is desired not to work with low isocyanate monomer contents, monomers of very low volatility may therefore be chosen, particularly triisocyanate monomers.", "The polyisocyanate compositions of the invention may be obtained by a method comprising the following steps: a) polycondensation, preferably tricondensation, of a mixture of initial isocyanate monomers in the presence of a tricondensation catalyst, of a compound chosen from water (water or compounds that release water in the reaction mixture being preferred), an alcohol or an amine, in the presence, or advantageously in the absence, of solvent, under the usual conditions; b) stopping of the reaction at the desired degree of conversion of the NCO functional groups, generally from 2 to 50%, advantageously from 5 to 40% and preferably from 10 to 30% of the NCO functional groups; and c) optionally, removal of the unreacted monomers.", "Reference may also be made to the methods described in EP-A-0 259 233 or U.S. Pat.", "No.", "5,103,045.The tricondensation catalyst is advantageously a catalyst for the formation of a biuret functional group and, in particular, moderately strong acids having a pKa of at least 1, advantageously at least 2 (for example, phosphoric or phosphonic acids) and preferably at least 3, or else a carboxylic acid as described in FR 86/12524.To obtain a blocked polyisocyanate composition, a blocking agent or mixture of blocking agents, these preferably being monofunctional, is added to the reaction mixture, before or during step a), after step b) or after step c), in an amount corresponding to the proportion of isocyanate functional groups that it is desired to block, and the reactants are left to react under the reaction conditions for reaction between the isocyanate functional group and the reactive hydrogen of the blocking agent according to the scheme: Am—H+ISO—N═C=O→Am—CO—NH—ISO, ISO being as defined above and Am representing the residue of the blocking agent after removal of the reactive hydrogen.", "The most commonly used blocking agents are those cited by M. Wicks in his article “Blocked isocyanates”, Progress in Organic Coatings (1975), Vol.", "3, p. 73, their deblocking temperatures advantageously being above 50° C., preferably of about 90° C. The blocking agents may be divided into three groups: those whose mobile hydrogen is carried by a chalcogen; those whose mobile hydrogen is carried by a nitrogen; and those whose mobile hydrogen is carried by a carbon.", "Among those whose mobile hydrogen is carried by a chalcogen (preferably a light chalcogen, namely sulfur and oxygen), those in which the chalcogen is an oxygen are most particularly used.", "Among the latter, mention may in particular be made of: products having an >N—OH sequence, such as, for example, oximes (═N—OH) or hydroxyimides ([—CO—]2N—OH); and phenols, most particularly those for which the aromatic nucleus is depleted in electrons, such as hydroxypicolines and hydroxybenzoates (for example, EP-A-680 984-and WO 98/4608).", "Mention may also be made of the compounds disclosed in application EP-A-0 661 278.Among those whose mobile hydrogen is carried by a nitrogen, mention may in particular be made of: monosubstituted amides, and particularly lactams (the most widely used one being caprolactam); imides ([—CO—]2N—H), most particularly cyclic imides such as succinimide; and unsaturated nitrogen heterocycles, especially those with a five-membered ring (advantageously doubly unsaturated), preferably comprising at least two heteroatoms (preferably nitrogen).", "Among the latter, mention may be made of diazoles (such as glyoxalines, imidazoles and pyrazoles), triazoles, or even tetrazoles, optionally including one or more substituents.", "This group of blocking agents is particularly preferred.", "Mention may most particularly be made of pyrazole and its derivatives having one or more substituents, especially alkyl groups, for example 3,5-dimethylpyrazole.", "Mention may also be made of the compounds disclosed in application EP-A-0 661 278.Among those whose mobile hydrogen is carried by a carbon, mention may essentially be made of compounds of malonic nature, that is to say an RCH< radical carrying two electron-withdrawing groups (such as carbonyl, nitrile, Rf or perfluoroalkyl).", "In this regard, mention may especially be made of the following pairs of blocking agents: methyl amyl ketoxime/2-hydroxypyridine and dimethylpyrazole/2-hydroxypyridine.", "The present invention is not limited to the nature of the isocyanate monomers employed.", "Thus, the isocyanate monomers may be aliphatic, including cycloaliphatic, diisocyanates or triisocyanates, such as: polymethylene diisocyanates, and especially hexamethylene diisocyanate (HDI), 2-methylpentamethylene diisocyanate, 4-isocyanato-methyloctamethylene diisocyanate and 2,4,4-trimethylhexamethylene diisocyanate; isophorone diisocyanate (IPDI), norbornane diisocyanate (NBDI), 1,3-bis(isocyanatomethyl)cyclohexane (BIC), 4,4′-diisocyanatodicyclohexyl-methane (H12-MDI) and cyclohexyl-1,4-diisocyanate (CHDI).", "The preferred isocyanates according to the invention are those in which at least one, advantageously two and preferably three of the above conditions are fulfilled: at least one, advantageously two, of the NCO functional groups are linked to a hydrocarbon backbone via a saturated carbon (sp3); at least one, advantageously two, of said saturated (Sp3) carbons carries at least one, advantageously two, hydrogen(s).", "In other words, it has been found that better results are obtained when the carbon carrying the isocyanate functional group carries a hydrogen, preferably two hydrogens; it is also preferable for said saturated (sp3) carbons to even be, at least in the case of one third, advantageously at least in the case of one half and preferably at least in the case of two thirds of them, linked to the backbone via a carbon atom that itself carries at least one hydrogen, more preferably two hydrogens; and all the intermediate carbons from which the isocyanate functional groups are linked to the hydrocarbon backbone are saturated (sp3) carbons, advantageously some, preferably all, of which carry a hydrogen, preferably two hydrogens.", "It is furthermore even preferable for said saturated (sp3) carbons to be, at least in the case of one third, advantageously at least in the case of one half and preferably at least in the case of two thirds of them, linked to said backbone via a carbon atom that itself carries at least one hydrogen, more preferably two hydrogens.", "In general, the preferred initial isocyanates (monomers) are those having at least one polymethylene chain sequence (comprising from 2 to 6 methylene chain links).", "Isocyanates, particularly aliphatic diisocyanates, especially C1-C10 alkyl isocyanates, are preferred in which the alkyl chain is linear or lightly branched.", "The term “lightly branched” is understood to mean the absence of any tertiary and/or neopentyl carbon.", "HDI, IPDI, NBDI, H12MDI and MPDI are particularly preferred.", "Component (C) is generally diluted in a solvent, so that the solids content of the composition is between 60 and 75% by weight.", "Component (D) may be chosen from one of more of the following compounds: cellulose esters, spreading agents, plasticizers, silicone oils, thixotropic agents, wetting agents and mar-resistance agents, pigments or fillers, for example titanium dioxide, carbon black, organic or mineral colored pigments, talc or barium sulfate, crosslinking catalysts, UV stabilizers, etc.", "Examples of suitable catalysts are, on the one hand, sulfonic acids, especially methanesulfonic acids or trifluoromethanesulfonic acids, for example para-toluenesulfonic acid, and, on the other hand, certain tin compounds, such as dibutyltin dilaurate or latent forms of these compounds, such as salts of para-toluenesulfonic acid, methanesulfonic acid or perfluoroalkanesulfonic acids.", "It is also possible to use latent forms of these catalysts, such as the amine salts of these strong acids.", "The crosslinking catalysts are present in a proportion ranging from 0 to 3%, preferably from 0 to 1%, by weight.", "Component (D) may be contained in the proportions of 0 to 65%, preferably 0 to 50%, by weight.", "Advantageously, the ratio of isocyanate functional groups to hydroxyl functional groups is less than 3, advantageously less than 2 and preferably between 0.3 and 1.5.The subject of the invention is also a coating composition, characterized in that it comprises: A) from 20 to 50% by weight of solids content of a polymer containing hydroxyl groups; B), from 3 to 20% by weight of solids content of a resin of the amine-formaldehyde type; C) from 5 to 20% by weight of solids content of a polyisocyanate composition containing at least 50 mol % of one or more biuret group(s), in which at least 10%, advantageously at least 20% and preferably at least 50% of the isocyanate groups are protected using a monofunctional blocking agent; D) from 0 to 30% by weight of solids content of various coating additives; and E) from 10 to 20% by weight of solvents.", "Components (A), (B), (C) and (D) are as defined above.", "Component (E) furthermore comprises the solvents described above for the formulation of components (A) and (B), optionally one or more other solvents for the formulation of the final coating composition, n-butyl acetate, ethylene glycol acetate, methyl ethyl ketone or xylene.", "The examples below illustrate the invention without however limiting it.", "EXAMPLE 1 Coating Formulation The following ingredients were mixed in order and homogenized: Compound 2 Compound 1 (comparative) VIACRYL SC 370/75 SNA* 50.35 50.35 n-BUTANOL 3.50 3.50 SOLVESSO 150 7.00 7.00 SOLVESSO 100 9.10 9.10 MAPRENAL VMF 3611/70B** 23.15 — XYLENE/BUTANOL (7/3) 5.30 5.30 polyol:SC = 75%; dry OH = 3.64%; melamine-formaldehyde resin.", "Various varnish formulations were prepared by adding to compound 1 or to compound 2 a polyisocyanate composition 1 (PIC 1) containing 100% isocyanate functional groups blocked by 3,5-dimethylpyrazole (3,5-DMP) having the following composition before blocking: % by weight PIC 1 composition (solids content) Residual HDI 0.25 HDI dimer 2.46 HDI biuret 33.0 HDI bis(biuret) 17.7 Heavy [tris(biuret)] fractions 21.6 At the same time, varnish formulations (comparative example) were prepared by adding, to compounds 1 and 2 above, a polyisocyanate composition PIC 2 having an identical composition except that the biuret was replaced with isocyanurate in the HDI biuret, the HDI bis(biuret) and the heavy fractions.", "VARNISH FORMULATIONS SC = 48% NCO/OH 1.05 1.05 1.05 1.05 Compound 1 50 50 — — Compound 2 — — 40 40 (comp.)", "1% PTSA 2.40 2.40 1.92 1.92 Xylene 3.83 1.94 5.84 4.33 PIC 2 9.05 — 7.24 — PIC 1 — 10.80 — 8.64 EXAMPLE 2 Properties of the Coatings Obtained with the Compositions Of the Invention The above varnish compositions were applied to glass or steel substrates using a 100 μm spiral filmograph in the case of the steel substrate and in the case of the glass plates.", "After 30 minutes of desolvation, the compositions were cured at 140° C. and 150° C. for 30 minutes.", "After 7 days at room temperature, the following properties were measured: hardness (Persoz); flexibility; impact strength.", "TABLE I Persoz hardness on a glass plate Compound 2 Compound 1 (comp.)", "PIC 2 PIC 1 PIC 2 PIC 1 NCO/OH 1.05 1.05 1.05 1.05 T0 + 1 h 140° C. 279 322 353 317 150° C. 337 357 T0 + 48 h 140° C. 303 331 350 321 150° C. 361 365 T0 + 7 d 140° C. 321 330 351 321 150° C. 358 366 TABLE II Persoz hardness on R46 steel plate Compound 2 Compound 1 (comp.)", "PIC 2 PIC 1 PIC 2 PIC 1 NCO/OH 1.05 1.05 1.05 1.05 T0 + 1 h 140° C. 244 263 273 268 150° C. 291 T0 + 48 h 140° C. 257 274 288 273 150° C. 299 T0 + 7 d 140° C. 249 271 284 270 150° C. 301 TABLE III Flexibility tests on R46 steel plates Compound 2 Compound 1 (comp.)", "PIC 2 PIC 1 PIC 2 PIC 1 NCO/OH 1.05 1.05 1.05 1.05 Thickness (μm) 140° C. 39 40 29 32 150° C. 39 Erichsen cup test 140° C. 8.4 8.3 8.1 8.5 150° C. 8.0 Impact test AFNOR 15 25 5 15 ASTM 24 14 6 8 Impact test AFNOR 20 50 <5 5 ASTM 18 46 <4 8 Cross-cutting test 30 min 0 0 0 0 140° C. *Values greater than or equal to “9” were not to be considered as the piston of the cup passed through the plate.", "TABLE IV Chemical resistance Compound 1 Compound 2 Hardener PIC 1 PIC 1 30 min at 140° C. NCO/OH 1.05 1.05 25% acetic acid, 1 h 0-1 5 10% sulfuric acid, 1 h 1- 5 10% aqueous ammonia, 1 h 1- 1- Ethanol, 1 h 1 1- MEK, 3 min 1 1 30 min at 150° C. NCO/OH 1.05 1.05 25% acetic acid, 1 h 1- 5 10% sulfuric acid, 1 h 0 5 10% aqueous ammonia, 1 h 1- 1 Ethanol, 1 h 1 1 MEK, 3 min 1 4 Ratings 0 to 5: 0: the film is not attacked and the solvent leaves no trace; 1: the film is very slightly attacked after evaporation of the solvent; a mark is observed around the perimeter of the drop.", "The inner part is identical to the rest of the film in appearance and to the touch (after complete evaporation, the film is slightly tacky to touch).", "2: the film is attacked after evaporation of the solvent; it is observed that the position of the drop is blurred and that the film is tacky even after evaporation of the solvent; 3: the film is wrinkled, a sign that crosslinking is neither complete nor uniform; 4: the film is dissolved after evaporation of the solvent; a rim is observed around the periphery of the drop.", "Inside, the film is sticky to touch and has no cohesion; 5: the substrate is seen after evaporation of the solvent; a rim is observed around the periphery of the drop.", "Inside, the film is destroyed and the substrate can be seen.", "It is clearly apparent that the results obtained with composition 1 are superior to the results obtained with composition 2.For compounds without the amine-formaldehyde resin, the isocyanate-based compositions (PIC 2) give better results, whereas when a melamine-formaldehyde resin is used the compositions containing biuret give, in all cases, results that are at least as good and are often better." ] ]	Patent_10468904
[ [ "Electro-optic modulator", "An electro-optic device includes a semiconducting layer in which is formed a waveguide, a modulator formed across the waveguide comprising a p-doped region to one side and an n-doped region to the other side of the waveguide, wherein at least one of the doped regions extends from the base of a recess formed in the semiconducting layer.", "In this way, the doped regions can extend further into the semiconducting layer and further hinder escape of charge carriers without the need to increase the diffusion distance of the dopant and incur an additional thermal burden on the device.", "In an SOI device, the doped region can extend to the insulating layer.", "Ideally, both the p and n-doped regions extend from the base of a recess, but this may be unnecessary in some designs.", "Insulating layers can be used to ensure that dopant extends from the base of the recess only, giving a more clearly defined doped region.", "The (or each) recess can have non-vertical sides, such as are formed by v-groove etches, A combination of a vertical sidewall at the base of the recess and a non-vertical sidewall at the opening could be used." ], [ "1.An electro-optic device including a semiconductor layer in which is formed a waveguide, a modulator formed across the waveguide comprising a doped region on either side, a lower confinement structure for charge carriers beneath the waveguide, lateral confinement structures for charge carriers on either side of the waveguide which extend to the lower confinement structure, at least one of the lateral confinement structures being the doped region to that side of the waveguide, the doped region extending to the lower confinement structure.", "2.An electro-optic device according to claim 1 in which both lateral confinement structures are provided by the doped regions on either side of the waveguide.", "3.An electro-optic device according to claim 1 in which the semiconducting layer is formed on an insulating layer supported on a substrate.", "4.An electro-optic device according to claim 1 in which the insulating layer is the lower confinement structure.", "5.An electro-optic device according to claim 4 in which the lateral confinement structures extend from the base of a recess in the semiconducting layer to the insulating layer.", "6.An electro-optic device according to claim 1 in which one or both doped regions extend from the base of a recess formed in the semiconducting layer.", "7.An electro-optic device according to claim 5, in which each lateral confinement structure and/or doped region extends from the base only of a said recess.", "8.An electro-optic device according to claim 1 in which the waveguide is covered by an insulating layer.", "9.An electro-optic device according to claim 8 in which the insulating layer extends to at least one side of a recess.", "10.An electro-optic device according to claim 5 in which at least part of the depth of the or each recess has non-vertical sides.", "11.An electro-optic device according to claim 1 in which the doped region to one side of the waveguide is p-type and the doped region to the other side of the waveguide is n-type.", "12.An electro-optic device including a semiconducting layer in which is formed a waveguide, a modulator formed across the waveguide comprising a doped region on either side, wherein at least one of the doped regions extends from a base only of a recess formed in the semiconducting layer.", "13.An electro-optic device according to claim 12 in which an insulating layer, is provided on side walls of the device to inhibit diffusion of dopant therethrough.", "14.An electro-optic device according to claim 12 in which the dopant is implanted in the semiconducting layer.", "15.An electro-optic device according to claim 1 in which the waveguide is a rib waveguide.", "16.An electro-optic device according to claim 1 which a plurality of such lateral modulators are formed along the length of the waveguide, each being a p-i-n modulator, adjacent modulators being mutually reversed in orientation.", "17.An electro-optic device according to claim 16 in which there are an even number of modulators.", "18.An electro-optic device according to claim 15 in which there are four modulators.", "19.An electro-optic device according to claim 1 in which the semiconducting layer comprises silicon, preferably crystalline silicon.", "20.An electro-optic device including a semiconductor layer in which is formed a waveguide, a modulator formed across the waveguide comprising a doped region on either side, a lower confinement structure for charge carriers beneath the waveguide, lateral confinement structures for charge carriers on either side of the waveguide, each of which extends from the base of a recess in the semiconducting layer to the lower confinement structure, at least one of the lateral confinement structures being the doped region to that side of the waveguide.", "21.An electro-optic device including a semiconductor layer in which is formed a waveguide, a modulator formed across the waveguide comprising a doped region on either side, a lower confinement structure for charge carriers beneath the waveguide, lateral confinement structures for charge carriers on either side of the waveguide which extend to the lower confinement structure, at least one of the lateral confinement structures being the doped region to that side of the waveguide, the doped region extending from the base of a recess formed in the semiconducting layer, to the lower confinement structure." ], [ "The present invention relates to an electro-optic modulator.", "Electro-optic modulators influence an optical mode and are understood to work by affecting the complex refractive index of a semiconductor waveguide and thereby affecting the transmission of light propagating therein.", "In such a device, a p-i-n diode normally is formed across the waveguide and injects charge carriers into the region in which the mode is propagating.", "The presence of significant numbers of charge carriers affects the local refractive index and thus the speed of light transmission.", "This effect can be used in switches, interferometers etc.", "In SOI (Silicon On Insulator) optical devices the mode propagates in a silicon layer over an insulating layer supported on a substrate.", "The insulating layer can then act as a confinement layer for the charge carriers in the vertical direction.", "However, it has been found that significant numbers of charge carriers still escape laterally, and this adversely affects both the performance of the device and its reproducibility.", "The present invention therefore provides, in its first aspect, an electro-optic device including a semiconductor layer in which is formed a waveguide, a modulator formed across the waveguide comprising a doped region on either side, a lower confinement structure for charge carriers beneath the waveguide, lateral confinement structures for charge carriers on either side of the waveguide which extend to the lower confinement structure, at least one of the lateral confinement structures being the doped region to that side of the waveguide, the doped region extending to the lower confinement structure.", "Preferably the semiconducting layer comprises silicon, more preferably crystalline silicon.", "The lower confinement structure preferably comprises an electrically insulating layer.", "The lower confinement structure for charge carriers may also comprise an optical confinement structure.", "It is preferred that the semiconducting layer is formed on an insulating layer supported on a substrate, e.g.", "an SOI device.", "In this case, the insulating layer can provide the lower confinement structure.", "It is naturally preferred that both lateral confinement structures are provided by respective p and n-doped regions either side of the waveguide.", "However, there may at times be no need or no motivation to do so on one particular side, for example if there are adjacent structures which themselves may provide electrical confinement.", "The lateral confinement structures and/or the doped regions (where the two are distinct) can extend from the base of a recess in the semiconducting layer to the insulating layer; preferably they extend from the base only of such a recess.", "The waveguide is normally covered by an insulating layer to ensure a distinct refractive index step, confining the optical mode, and to reduce unwanted electrical effects.", "This insulating layer can extend to and down at least one side of the recess, which if put in place prior to doping will ensure that dopant extends from the base of the recess only, giving a more clearly defined doped region.", "The (or each) recess can have non-vertical sides, such as are formed by v-groove etches, which method of manufacture has the advantage that metal contacts leading to the doped regions will be less prone to failure due to thinning at the edge of the recess.", "A combination of a vertical sidewall at the base of the recess and a non-vertical sidewall at the opening could be used.", "In a standard lateral p-i-n diode structure, the doped region to one side is p-type and the doped region to the other side of the waveguide is n-type.", "Other diode geometries exist, however, and other electrical structures such as transistors can be provided.", "Confining structures are used in the present invention to inhibit the escape of charge carriers from the region of interest.", "Physical structures such as deep trenches, insulating layers and the like will provide confinement.", "In addition, we have found that a doped region provides confinement.", "It is thought that opposite charges are confined by a recombination process and like charges are confined by the concentration gradient established by the dopant.", "In its second aspect, the present invention provides an electro-optic device including a semiconducting layer in which is formed a waveguide, a modulator formed across the waveguide comprising a doped region on either side, wherein at least one of the doped regions extends from the base only of a recess formed in the semiconducting layer.", "In this way, the doped regions can extend further into the semiconducting layer and further hinder escape of charge carriers laterally within the semiconductor layer.", "This is however achieved without the need to increase the diffusion distance of the dopant, which would incur an additional thermal burden on the device.", "Further, limitation of the dopant area to the base of the recess reduces the lateral spread of dopant and keeps the volume used for transmission of the optical mode clear of dopant, which if present is apt to cause losses and inefficiency.", "As before, an insulating layer, e.g.", "an oxide layer, can be provided on side walls of the device to inhibit diffusion of dopant therethrough.", "Alternatively, or in addition, the dopant can be implanted in the semiconducting layer.", "The waveguide is preferably a rib waveguide, although other designs of waveguide are known and can be used.", "A plurality of such lateral modulators can be formed along the length of the waveguide, each being a p-i-n modulator, adjacent modulators being mutually reversed in orientation.", "This gives a particularly efficient form of modulation.", "Ideally there will be an even number of modulators, e.g.", "two, four, six or eight.", "Embodiments of the present invention will now be described by way of example, with reference to the accompanying figures, in which; FIG.", "1 is a vertical section through a known lateral p-i-n diode modulator; FIG.", "2a is a model of the carrier density of the modulator of FIG.", "1; FIG.", "2b is a plot of the carrier concentration along line A-A of FIG.", "2a; FIGS.", "3 and 4 are vertical sections through p-i-n diode modulators being first and second embodiments of the present invention; FIG.", "5a is a model of the carrier density of the modulator of FIG.", "4; FIG.", "5b is a plot of the carrier concentration along line B-B of FIG.", "5a; FIG.", "5c is a model of the carrier density of the modulator of FIG.", "3; FIG.", "6 is a vertical section through a p-i-n diode modulator according to a third embodiment of the present invention; and FIG.", "7 compares the attenuation of the modulator of FIG.", "1 with the modulator of FIG.", "6.FIG.", "1 shows a standard SOI (silicon-on-insulator) rib waveguide with a lateral injection p-i-n diode made by diffusion into a semiconducting slab.", "In this arrangement, the waveguide 10 is in the form of a rib on the surface of the silicon epi layer 12.The epi layer 12 is formed on an insulating oxide layer 14 which is itself supported on a substrate 16.On either side of the rib waveguide 10 are a pair of doped regions.", "To one side there is an n+ doped area 18, and to the other there is a p+ doped area 20.Metal contacts, 22, 24 lead to the doped regions 18, 20 and a p-i-n diode is thus formed.", "An insulating layer 26, eg of silicon dioxide, is formed over the rib waveguide 10 to provide a more distinct refractive index step and thereby assist in optical confinement, and also beneath the metal contacts 22, 24 away from the doped regions 18, 20 to provide electrical insulation.", "FIG.", "2 shows a model of the carrier distribution for this device.", "In the upper region of the graph, the physical structure of the device can be seen in terms of the rib 10, the insulating layer 26 and the metal contacts 22, 24.Within the semi-conducting area, the concentration of holes is shown by way of contour lines; the peak hole concentration is 1020 at point 30 and the minimum is 1014 at 32.Contours in the intermediate regions are on a logarithmic scale.", "Details of the electron concentration are not shown as the device is symmetrical and therefore the hole and electron concentrations will be complementary.", "It can be seen that in the doped regions 30, 32 there is a significant working concentration of charge carriers.", "However, there is a distinct gap 34 beneath the doped regions but above the insulating layer 14 where the charge carrier concentration is at a high level, and almost equal to the carrier concentration in the core of the waveguide.", "This modulation structure is therefore inefficient due to the lack of carrier confinement and poor overlap between the generated high carrier concentration and the optical mode.", "FIG.", "2b shows the charge concentration in the area of the optical mode, and on the logarithmic scale it can be seen that the general charge carrier concentration is at approximately 5-6×1017 cm−3.FIG.", "3 shows the first embodiment of the invention.", "The rib waveguide 10 is again formed on the silicon epi layer 12 of the SOI structure.", "However, a pair of trenches 36, 38 are formed either side of the rib waveguide 10 and doping is carried out into these trenches to form an n+ doped area 18 and a p+ doped area 20.Metal contacts 22, 24 are again provided, as is a protective and insulating oxide layer 26.The increased depth from which the doping is carried out, due to the trenches 36, 38, means that the n+ and p+ doped areas 18, 20 reach the insulating layer 14 beneath the silicon epi layer 12.FIG.", "4 shows a similar structure according to a second embodiment of the invention.", "In the first embodiment of FIG.", "3, doping is carried out so as to extend from the base of the recesses 36, 38 only.", "In FIG.", "4, this limitation of the extent of doping is not present.", "These examples could be achieved (for example) by diffusion processes or by ion implantation.", "In FIG.", "3, the protective oxide layer 26a is extended to the sides of the recesses 36, 38 in order to assist in limiting the doped area to the base of the trench.", "The dopant profile of FIG.", "3 is very advantageous.", "As the dopant extends only from the base of the trench and not from the sides, the lateral spread of the dopant during drive-in is significantly limited.", "In addition, the use of the recess from which the dopant extends reduces the amount of drive-in needed, and thus further reduces the lateral spread.", "Thus, the lateral dimension is more reproducible and more accurately ascertainable.", "Dopant concentrations in the vicinity of the optical mode can be avoided, reducing losses and inefficiency.", "The doped regions can be safely moved closer to the optical mode, thereby reducing the dimensions of the device with the attendant advantages thereof.", "FIG.", "5a shows a model of the structure of FIG.", "4, displayed on the same basis as FIG.", "2a.", "It can be seen that the areas 30a, 32a of greatest carrier concentration are confined in the structure in that they extend to the underlying insulator layer 14.Thus, carrier escape is prevented in this arrangement and carrier loss must therefore be by recombination.", "FIG.", "5b shows carrier concentration in the area of the optical mode, and it can be seen that on the logarithmic scale this varies between 7 and 8×1018 cm−3, a detectably higher level than that of FIG.", "2b indicating the carriers are not escaping to the same extent.", "FIG.", "5c shows a similar model for the structure of FIG.", "3.It can be seen that there is little lateral escape of charge carriers, and little spread of dopant into the optical mode region.", "FIG.", "6 shows a third embodiment of the present invention.", "This corresponds generally to that of FIG.", "4 but the trench recesses 36a, 38a are formed with angled edges such as that at 40.This can be achieved via a v-groove etch, for example.", "This eases the deposition of the metal contacts (not shown in FIG.", "6) as they do not need to traverse a sharp corner and are therefore less prone to thinning.", "Dopant can be confined to the base of the trench if desired.", "In order to compare the performance advantage by employing the present invention, a device was fabricated according to FIG.", "6 with a 1.8 micron etch on a 2.6 micron slab.", "A second device was fabricated identically except that the diodes were placed on the surface of the slab region as per FIG.", "1.Doping was introduced into both devices using a diffusion process, thus creating two structures which are identical except that the vertical depth of the doping is greater in the novel device.", "The attenuation of light passing through the waveguide due to carrier injection was measured in a 2 mm long diode structure.", "Attenuation is directly proportional to the carrier concentration injected into the waveguide region by the forward biased diode.", "The results of this measurement are shown in FIG.", "7.Line 42 is for the novel structure whereas line 44 is for the comparative structure without recesses.", "It can be seen that for any given current, the attenuation achieved for the novel diode 42 is approximately 40% greater than for the standard structure of FIG.", "1.Therefore, it can be inferred that the carrier concentration in the waveguide must also be greater in the novel diode structure as generally predicated by the modeling shown in FIGS.", "2-5.Alternatively, to achieve the same level of attenuation a lower injection current is required thereby giving a lower power drain and less local heating.", "In a further alternative the same attenuation can be achieved for the same current using a physically smaller modulator structure.", "The above-described embodiments have all been SOI structures.", "It is to be expected that application of the invention to non-SOI devices will still give enhanced results although an alternative lower confinement structure will need to be provided.", "Further, the above embodiments concentrate on single p and single n junction devices, whereas other diode geometries and other electrical structures such as transistors could also be considered.", "It will be appreciated that many variations can be made to the above-described embodiments, without departing from the scope of the present invention." ] ]	Patent_10468938
[ [ "Immunological detection of prostate diseases and prostasome-related conditions", "The present invention relates to a diagnostic and/or prognostic reagent comprising a component which selectively binds to anti-prostasome autoantibodies, as well as an immunoassay using said reagent.", "Furthermore, the invention relates to an in vitro method for diagnosing and/or prognosticating a condition reflecting the prostasome presence in body fluids, comprising a) binding of anti-prostasome autoantibodies with a component which selectively binds to anti-prostasome autoantibodies; and b) detection of said binding.", "The conditions may be different forms of prostate cancer or prostasome-related diseases or other conditions where anti-prostasome autoantibodies are present in the body." ], [ "1.An agent which selectively binds to anti-prostasome auto antibodies.", "2.An agent according to claim 1, which is a diagnostic and/or prognostic reagent.", "3.An agent according to claim 1, comprising structures involved in the binding of prostasomes or prostasomal-like material and anti-prostasome autoantibodies.", "4.A kit for diagnosis/prognosis of prostasome-related diseases comprising a reagent according to claim 1.5.An immunoassay for detection of anti-prostasome autoantibodies in body fluids comprising the reagent according to claim 2.6.An immunoassay according to claim 5, wherein the body fluid is serum or plasma.", "7.An immunoassay according to claim 5 wherein the agent is bound to a solid support.", "8.An immunoassay according to claim 5, wherein the solid support is a microtitre plate.", "9.An immunoassay according to claim 5 which is an ELISA or flow cytometry.", "10.An in vitro method for diagnosing and/or prognosticating a condition reflecting the prostasome presence in body fluids, comprising: a) binding of anti-prostasome autoantibodies with a component which selectively binds to anti-prostasome autoantibodies; and b) detection of said binding.", "11.A method according to claim 10, wherein said component in a) comprises structures involved in the binding of prostasomes or prostasomal-like material and anti-prostasome autoantibodies.", "12.A method according to claim 10, wherein the condition is cancer.", "13.A method according to claim 10, wherein the condition is prostasome-related disease.", "14.A method according to claim 10, wherein the condition is prostate cancer.", "15.A method according to claim 10, wherein the condition is prostatic disease.", "16.A method according to claim 10, wherein the detection in step b) is by ELISA or flow cytometry techniques.", "17.", "(canceled) 18.A method according to claim 11, wherein the condition is cancer.", "19.A method according to claim 11, wherein the condition is prostasome-related disease.", "20.A method according to claim 11, wherein the condition is prostate cancer.", "21.A method according to claim 11, wherein the condition is prostatic disease." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Prostate cancer is the fourth most commonly diagnosed cancer in men worldwide and the most commonly diagnosed in Swedish men.", "The age-adjusted incidence of prostate cancer has increased by about 1.8% per year over the past decade.", "This increase may be partly due to the introduction of better diagnostic and therapeutic techniques.", "The prevalence of prostate cancer is comparatively low in men younger than 60 years in Sweden but is 4% in men 75-79 years of age and is as high as 5-6% in men over the age of 80 years.", "Autopsy studies have indicated a much higher prevalence of latent prostate cancer.", "Hence, a prevalence of 44% was found in men 50-59 years of age and 83% in those 70-79 years, which suggests that many prostate cancers do not reach a clinically significant stage.", "In Sweden, 6,610 new cases of prostate cancer were reported in 1998; 67% of these men were older than 70 years, and only 0.26% were younger than 50 years.", "In 1997, 2,448 men died of prostate cancer indicating substantial mortality from this cancer form.", "It has also been shown that the younger the patient is at the time of diagnosis, the higher is the mortality.", "The causes of prostate cancer are essentially unknown, although several factors have been shown to be associated with a higher risk for this type of cancer as increasing age, family history of prostate cancer, men in Western countries, and especially American men of African heritage.", "Nonbacterial prostatitis is a benign and common disease mostly among young men.", "The etiology of the disease is not fully understood.", "The symptoms include genital and pelvic pain, urgency to void and cold sensitivity.", "Although the symptoms are suggestive of an infection in the prostate gland, there is a lack of clear bacterial infectious etiology for a majority of men with these symptoms.", "Among alternative etiologic factors, it has been claimed that an autoimmune component to the disease might exist.", "This means that a self-reactivity directed against the prostate or a prostate component by the immune system could be involved in the etiology of the disease.", "Accordingly, a production of autoantibodies against some component of the prostatic secretion in patients with nonbacterial prostatitis could contribute to their symptoms.", "Autoantibodies can be produced in the body and cause severe diseases, like rheumatic artritis and a type of diabetes, by attacking cells carrying the corresponding antigens.", "No autoantibodies, which recognize or affect cancer metastases, have yet been reported.", "The prostate gland is one of the major accessory genital glands with mainly exocrine functions.", "Although being a unified organ, three pairs of lobes can be distinguished.", "Histologically the prostate is composed of a large series of independent branching ducts, all of which enter the prostatic urethra.", "Hence, the prostate gland and its secretion represent a closed secretory system and the secretory components will normally not be able to appear in the circulation.", "Human prostatic fluid contains high amounts of monovalent and divalent cations.", "It is also rich in enzymes involved in carbohydrate metabolism and protein degradation.", "Besides these soluble substances the prostate gland secretes the above-mentioned prostasomes which are prostatic secretory products coating the sperm cells.", "Prostasomes are surrounded by a bilayered and sometimes tri- or multi-layered membrane and they have a diameter of 40-500 nm.", "Prostasomes are secretory products of the prostate gland.", "The membrane architecture of these organelles is complex and two-dimensional gel electrophoresis of membrane material has revealed about 80 different protein entities.", "Also, an unusually high cholesterol/phospholipid ratio is inherent in this membrane.", "The prostasomes contain neuroendocrine and CD molecules and many different enzymes are part of the prostasome membrane mosaic.", "Prostasomes have been ascribed many different biologic activities, but their physiologic function is still unclear.", "They can interact with spermatozoa and promote their motility characteristics in different ways.", "They are also immunosuppressive and inhibit superoxide anion generation by neutrophil granulocytes.", "The prostasomes can modulate complement-mediated immune responses, and CD 59, an inhibitor of the membrane attack complex of complement, resides on prostasomes.", "The present inventors have previously isolated and purified prostasomes not only from seminal plasma and expressed prostatic fluid (exprimate) but also from the human prostate gland as well as from vertebrate metastases of prostatic cancer.", "They have also cultured human prostatic cancer cells of the PC3 and similar cell lines on plates in monolayer and found that they can produce prostasomes.", "These PC3 cells have been fractionated and the prostasomes purified C3 prostasomes).", "In addition, the present inventors and others have produced monoclonal (1, 3-4) and polyclonal (2) antibodies against some types of prostasomes.", "Historically, prostate cancer was most often diagnosed in men presenting with symptoms derived from a local tumour or metastatic spread of a tumour, such as dysfunctional voiding or bone pain, and the disease was at an advanced stage at the time of diagnosis.", "Occasionally, it was an accidental finding on digital rectal examination or upon histological examination of tissue obtained during surgery on men with benign prostatic hyperplasia.", "Accordingly, there is a need of improvement and intensified use of diagnostic procedures on men at risk, resulting in more extensive detection of nonlethal prostate cancers.", "Measurement of prostate specific antigen (PSA) in serum was initiated in the latter half of the 1980s in many Western countries.", "This measure changed the pattern of diagnosis of prostate cancer with more cases detected at an early stage and fewer cases at advanced stages.", "However, since PSA is not a prostate cancer specific marker in serum it is not the ideal diagnostic marker and therefore not accommodated for screening of prostate cancer.", "There is growing concern about the PSA-test, as it is employed at present.", "The test is hampered by its incapacity for discriminating in a proper way between benign prostatic hyperplasia and prostate cancer and, what is more, between prostate cancer with high metastasising potential (aggressive prostate cancer) and such cancer with no or weak aggressiveness.", "This inability of the test often will end up in truncating surgery, i.e.", "total prostatectomy.", "This type of overtreatment is a great inconvenience not only for patients but also for society due to extra costs." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based upon the demonstration of anti-prostasome autoantibodies in serum of patients with prostate cancer due to the border-breaking growth of neoplastic cells in the prostate.", "The invention also comprises the idea that prostasomes, due to their smallness, will appear in the circulation earlier than the much bigger (about 150 times) prostate cancer cells, which are spread via the blood.", "Accordingly, a time-window may be offered for the prostasomes, during which the anti-prostasome autoantibodies can be developed and detected by our test, before the bigger prostate cancer cells will be released into circulation and set their metastases.", "The anti-prostasome autoantibodies in body fluids (here represented by blood plasma/serum) may, for example, be detected with an ELISA technique (see below).", "Herewith, the invention presents a new way for diagnosing early metastasis and for discriminating between the dangerous (metastasis-prone) prostate cancer and the more or less harmless (not metastasis-prone) variant of prostate cancer.", "The anti-prostasome autoantibodies, which we have detected in serum from patients with a prostate cancer, do not attack the cancer cells and are therefore not recognized clinically.", "In a first aspect, the invention relates to an agent which selectively binds to anti-prostasome autoantibodies.", "In one embodiment, the agent is a diagnostic and/or prognostic reagent comprising a component which selectively binds to anti-prostasome autoantibodies.", "The component should comprise structures involved in the binding of prostasomes and anti-prostasome autoantibodies, for example prostasomes or epitope(s) exposed in prostasomes binding to anti-prostasome antibodies, i.e.", "with selective affinity to anti-prostasome autoantibodies.", "By the term ‘selective’ is also meant that the binding is mainly selective.", "The reagent (i.e.", "antigen) may comprise prostasomal-like material, for example, of natural, pathological, synthetic or recombinant origin.", "In a second aspect, the invention relates to a kit for diagnosis/prognosis of prostasome-related diseases comprising a reagent as defined above.", "In a third aspect, the invention relates to an immunoassay for detection of anti-prostasome autoantibodies in body fluids comprising the above reagent.", "The body fluid may be serum or plasma.", "Further alternatives are urine and semen.", "Optionally, the reagent (i.e.", "the antigen) is bound to a solid support, such as a microtitre plate which is commonly used in, for example, ELISA.", "The immunoassay according to the invention may be combined with a conventional PSA assay if desired.", "In a fourth aspect, the invention relates to an in vitro method for diagnosing and/or prognosticating a condition reflecting the prostasome presence in body fluids, comprising a) binding of anti-prostasome autoantibodies with a component which selectively binds to anti-prostasome autoantibodies; and b) detection of said binding.", "Preferably, the detection is by by ELISA or flow cytometry techniques.", "The condition may be cancer, such as prostate cancer, or prostatic diseases.", "For instance, the demonstration of anti-prostasome autoantibodies in body fluids may be important to better differentiate between bacterial and nonbacterial prostatitis (see above).", "An improved differential diagnosis between these two conditions would facilitate the position that should be taken to the question of proper treatment.", "In a fifth aspect, the invention relates to use, or method of using, of an agent which selectively binds to anti-prostasome autoantibodies for the production of a drug for prevention and/or treatment of prostasome-related diseases.", "For example, the drug may be formulated as a vaccine.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates to immunological detection of prostate diseases, among which prostate cancer holds a unique position, and of prostasome-related diseases with examples on clinical applications of the invention.", "BACKGROUND OF THE INVENTION Prostate cancer is the fourth most commonly diagnosed cancer in men worldwide and the most commonly diagnosed in Swedish men.", "The age-adjusted incidence of prostate cancer has increased by about 1.8% per year over the past decade.", "This increase may be partly due to the introduction of better diagnostic and therapeutic techniques.", "The prevalence of prostate cancer is comparatively low in men younger than 60 years in Sweden but is 4% in men 75-79 years of age and is as high as 5-6% in men over the age of 80 years.", "Autopsy studies have indicated a much higher prevalence of latent prostate cancer.", "Hence, a prevalence of 44% was found in men 50-59 years of age and 83% in those 70-79 years, which suggests that many prostate cancers do not reach a clinically significant stage.", "In Sweden, 6,610 new cases of prostate cancer were reported in 1998; 67% of these men were older than 70 years, and only 0.26% were younger than 50 years.", "In 1997, 2,448 men died of prostate cancer indicating substantial mortality from this cancer form.", "It has also been shown that the younger the patient is at the time of diagnosis, the higher is the mortality.", "The causes of prostate cancer are essentially unknown, although several factors have been shown to be associated with a higher risk for this type of cancer as increasing age, family history of prostate cancer, men in Western countries, and especially American men of African heritage.", "Nonbacterial prostatitis is a benign and common disease mostly among young men.", "The etiology of the disease is not fully understood.", "The symptoms include genital and pelvic pain, urgency to void and cold sensitivity.", "Although the symptoms are suggestive of an infection in the prostate gland, there is a lack of clear bacterial infectious etiology for a majority of men with these symptoms.", "Among alternative etiologic factors, it has been claimed that an autoimmune component to the disease might exist.", "This means that a self-reactivity directed against the prostate or a prostate component by the immune system could be involved in the etiology of the disease.", "Accordingly, a production of autoantibodies against some component of the prostatic secretion in patients with nonbacterial prostatitis could contribute to their symptoms.", "Autoantibodies can be produced in the body and cause severe diseases, like rheumatic artritis and a type of diabetes, by attacking cells carrying the corresponding antigens.", "No autoantibodies, which recognize or affect cancer metastases, have yet been reported.", "The prostate gland is one of the major accessory genital glands with mainly exocrine functions.", "Although being a unified organ, three pairs of lobes can be distinguished.", "Histologically the prostate is composed of a large series of independent branching ducts, all of which enter the prostatic urethra.", "Hence, the prostate gland and its secretion represent a closed secretory system and the secretory components will normally not be able to appear in the circulation.", "Human prostatic fluid contains high amounts of monovalent and divalent cations.", "It is also rich in enzymes involved in carbohydrate metabolism and protein degradation.", "Besides these soluble substances the prostate gland secretes the above-mentioned prostasomes which are prostatic secretory products coating the sperm cells.", "Prostasomes are surrounded by a bilayered and sometimes tri- or multi-layered membrane and they have a diameter of 40-500 nm.", "Prostasomes are secretory products of the prostate gland.", "The membrane architecture of these organelles is complex and two-dimensional gel electrophoresis of membrane material has revealed about 80 different protein entities.", "Also, an unusually high cholesterol/phospholipid ratio is inherent in this membrane.", "The prostasomes contain neuroendocrine and CD molecules and many different enzymes are part of the prostasome membrane mosaic.", "Prostasomes have been ascribed many different biologic activities, but their physiologic function is still unclear.", "They can interact with spermatozoa and promote their motility characteristics in different ways.", "They are also immunosuppressive and inhibit superoxide anion generation by neutrophil granulocytes.", "The prostasomes can modulate complement-mediated immune responses, and CD 59, an inhibitor of the membrane attack complex of complement, resides on prostasomes.", "The present inventors have previously isolated and purified prostasomes not only from seminal plasma and expressed prostatic fluid (exprimate) but also from the human prostate gland as well as from vertebrate metastases of prostatic cancer.", "They have also cultured human prostatic cancer cells of the PC3 and similar cell lines on plates in monolayer and found that they can produce prostasomes.", "These PC3 cells have been fractionated and the prostasomes purified C3 prostasomes).", "In addition, the present inventors and others have produced monoclonal (1, 3-4) and polyclonal (2) antibodies against some types of prostasomes.", "Historically, prostate cancer was most often diagnosed in men presenting with symptoms derived from a local tumour or metastatic spread of a tumour, such as dysfunctional voiding or bone pain, and the disease was at an advanced stage at the time of diagnosis.", "Occasionally, it was an accidental finding on digital rectal examination or upon histological examination of tissue obtained during surgery on men with benign prostatic hyperplasia.", "Accordingly, there is a need of improvement and intensified use of diagnostic procedures on men at risk, resulting in more extensive detection of nonlethal prostate cancers.", "Measurement of prostate specific antigen (PSA) in serum was initiated in the latter half of the 1980s in many Western countries.", "This measure changed the pattern of diagnosis of prostate cancer with more cases detected at an early stage and fewer cases at advanced stages.", "However, since PSA is not a prostate cancer specific marker in serum it is not the ideal diagnostic marker and therefore not accommodated for screening of prostate cancer.", "There is growing concern about the PSA-test, as it is employed at present.", "The test is hampered by its incapacity for discriminating in a proper way between benign prostatic hyperplasia and prostate cancer and, what is more, between prostate cancer with high metastasising potential (aggressive prostate cancer) and such cancer with no or weak aggressiveness.", "This inability of the test often will end up in truncating surgery, i.e.", "total prostatectomy.", "This type of overtreatment is a great inconvenience not only for patients but also for society due to extra costs.", "SUMMARY OF THE INVENTION The present invention is based upon the demonstration of anti-prostasome autoantibodies in serum of patients with prostate cancer due to the border-breaking growth of neoplastic cells in the prostate.", "The invention also comprises the idea that prostasomes, due to their smallness, will appear in the circulation earlier than the much bigger (about 150 times) prostate cancer cells, which are spread via the blood.", "Accordingly, a time-window may be offered for the prostasomes, during which the anti-prostasome autoantibodies can be developed and detected by our test, before the bigger prostate cancer cells will be released into circulation and set their metastases.", "The anti-prostasome autoantibodies in body fluids (here represented by blood plasma/serum) may, for example, be detected with an ELISA technique (see below).", "Herewith, the invention presents a new way for diagnosing early metastasis and for discriminating between the dangerous (metastasis-prone) prostate cancer and the more or less harmless (not metastasis-prone) variant of prostate cancer.", "The anti-prostasome autoantibodies, which we have detected in serum from patients with a prostate cancer, do not attack the cancer cells and are therefore not recognized clinically.", "In a first aspect, the invention relates to an agent which selectively binds to anti-prostasome autoantibodies.", "In one embodiment, the agent is a diagnostic and/or prognostic reagent comprising a component which selectively binds to anti-prostasome autoantibodies.", "The component should comprise structures involved in the binding of prostasomes and anti-prostasome autoantibodies, for example prostasomes or epitope(s) exposed in prostasomes binding to anti-prostasome antibodies, i.e.", "with selective affinity to anti-prostasome autoantibodies.", "By the term ‘selective’ is also meant that the binding is mainly selective.", "The reagent (i.e.", "antigen) may comprise prostasomal-like material, for example, of natural, pathological, synthetic or recombinant origin.", "In a second aspect, the invention relates to a kit for diagnosis/prognosis of prostasome-related diseases comprising a reagent as defined above.", "In a third aspect, the invention relates to an immunoassay for detection of anti-prostasome autoantibodies in body fluids comprising the above reagent.", "The body fluid may be serum or plasma.", "Further alternatives are urine and semen.", "Optionally, the reagent (i.e.", "the antigen) is bound to a solid support, such as a microtitre plate which is commonly used in, for example, ELISA.", "The immunoassay according to the invention may be combined with a conventional PSA assay if desired.", "In a fourth aspect, the invention relates to an in vitro method for diagnosing and/or prognosticating a condition reflecting the prostasome presence in body fluids, comprising a) binding of anti-prostasome autoantibodies with a component which selectively binds to anti-prostasome autoantibodies; and b) detection of said binding.", "Preferably, the detection is by by ELISA or flow cytometry techniques.", "The condition may be cancer, such as prostate cancer, or prostatic diseases.", "For instance, the demonstration of anti-prostasome autoantibodies in body fluids may be important to better differentiate between bacterial and nonbacterial prostatitis (see above).", "An improved differential diagnosis between these two conditions would facilitate the position that should be taken to the question of proper treatment.", "In a fifth aspect, the invention relates to use, or method of using, of an agent which selectively binds to anti-prostasome autoantibodies for the production of a drug for prevention and/or treatment of prostasome-related diseases.", "For example, the drug may be formulated as a vaccine.", "DETAILED DESCRIPTION OF THE INVENTION The invention will now be described more closely below in association with a non-limiting experimental part.", "The following procedures will be exemplified: 1.Preparation of prostasomes 2.Production of antibodies against prostasome components 3.Characterisation of prostasomes from seminal plasma, prostate tissues, prostate cancers, and PC3 cells 4.Functional similarities between seminal prostasomes and PC3 prostasomes 5.Anti-prostasome autoantibodies in serum from patients with metastasis of prostate cancer 6.Anti-prostasome autoantibodies in serum from patients with urological symptoms.", "Prostasomes were prepared from different sources, namely from seminal plasma, prostate tissues, bone metastases of prostate cancers, and the cancer cell line PC3 (p.1).", "Polyclonal chicken antibodies and monoclonal mouse antibodies were produced against some of these preparations (p.2).", "The monoclonal antibodies, which were directed against various components of the prostasomes, were applied to characterise differences among the types of prostasome (p.3).", "In addition, the differential expression of, for instance, enzymes and CD-factors were studied (p.3).", "Also functional properties, for instance, the prostasome ability to activate sperm cells were compared between prostasome types (p.4).", "The useful application of prostasome components in clinical practice was examined both for immunodiagnosis of metastasis of prostate cancer (p.5) and for immunodetection of anti-prostasome autoantibodies among patients with urological disturbances (p.6).", "1.Preparation of Prostasomes Seminal plasma: Semen samples were centrifuged for 20 minutes at 1 000×g to separate spermatozoa and other cells from the seminal plasma, which was pooled (12-15 samples) and ultracentrifuged at 10 000×g for 15 minutes to pellet possible cells and cell debris.", "The supernatant was subsequently subjected to another ultracentrifugation for 2 hours at 100 000×g to pellet the prostasomes.", "The prostasomes were resuspended in 30 mmol/L Tris-HCl, 130 mmol/L NaCl, pH 7.6 (isotonic Tris-HCl buffer).", "The pellet suspension was further purified on a Sephadex G 200 column (Pharmacia AB, Uppsala, Sweden), to separate them from an amorphous substance.", "The eluant was the isotonic Tris-HCl buffer, and the eluate was monitored at 260 and 280 nm.", "Those fractions (5-12) with initial elevated UV absorbance were pooled and ultracentrifuged at 100 000×g for 2 hours and diluted to a protein concentration of 2 mg/ml using a Protein Assay ESL method (Boehringer Mannheim, Germany).", "Prostate tissues and bone metastases: Tissues from two prostate glands and 12 bone metastases of prostate cancers (PAD verified) were homogenised in the isotonic Tris-HCl buffer.", "After homogenisation, the suspensions were first centrifuged 3 times at 3000×g, +4° C. for 15 minutes, then twice for 15 minutes at 10 000×g, +4° C. The supernatants obtained were then ultracentrifuged for 2 hours at 100 000×g.", "The pellets obtained were thereafter treated exactly in the same way as the seminal plasma samples described above.", "PC-3 cell line: The human prostatic carcinoma cell line PC-3 was obtained from the American Type Culture Collection (Rockville, Md., USA).", "The cells were maintained in RPMI 1640 cell culture medium supplemented with 10% heat-inactivated fetal calf serum and 2 mM L-glutamine (Sigma Chemicals, MO, USA.).", "The cells were grown in Falcon Petri dishes (100 mm) at 37° C. and each plate yielded 2-3×106 cells, which were removed by trypsin and carefully washed in the isotonic Tris-HCl buffer and centrifuged.", "They were again suspended in the Tris-HCl buffer and frozen at −70° C. The frozen PC-3 cells from about 8-10 plates were thawed and pooled and the suspension of disintegrated cells was centrifuged at 1500×g for 30 minutes and then at 10 000×g for 15 minutes to remove cell debris.", "The supernatant was ultracentrifuged at 100 000×g for 2 hours, and pelleted prostasomes were suspended in the isotonic Tris-HCl buffer.", "The dissociated prostasomes were run through the Sephadex G 200 column and treated as above.", "2.Production of Antibodies Against Prostasome Components The immunogenicity of the prostasomes has been demonstrated by the production of several types of antibodies against prostasomes from two different sources using both mouse and hen as hosts.", "Monoclonal antibodies against seminal prostasomes were produced by intrasplenic immunization.", "One μg of purified prostasomes was injected four times in the spleens of mice.", "Hybridomas were tested in an ELISA system, using seminal prostasomes as the coating antigen.", "Chicken polyclonal antibodies against prostasomes were produced by immunisation with seminal prostasomes and cancer prostasomes, respectively.", "3.Characterisation of Prostasome from Seminal Plasma, Prostate Tissues, Prostate Cancer Metastases and PC3 Cells The prostasome membrane is composite and 2-dimensional electrophoresis has revealed at least 80 different protein entities.", "Determination of some of the proteins associated with prostasomes has been done in an attempt to characterise or differentiate the prostasomes of the four different sources, seminal plasma, prostate tissue, prostate cancer metastases and PC3 cells.", "Results: Aminopeptidase (CD13), Dipeptidylpeptidase IV (CD26), the neuropeptides: Chromogranin A and B, Neuropeptide Y (NPY) and Vasoactiveintestinal peptide (VIP) are all present in high amounts in seminal prostasomes.", "They also appear in prostate tissue prostasomes, CD 26, Chromogranin B and VIP at equal amounts as in the seminal prostasomes, but only at about 50% of the Chromogranin A, 30% of CD13 and 4% of NPY compared to the concentrations in seminal prostasomes.", "Prostasomes derived from the prostate cancer metastases and PC3 cell line showed a somewhat different pattern with the Chromogranin A concentration being 3-5 times higher than in seminal prostasomes and NPY, VIP and CD13 less than 10% of the seminal prostasomes.", "Some differences in protein pattern between the four prostasome types could also be demonstrated by electrophoresis on SDS-PAGE.", "4.Functional Similarities Between Seminal Prostasomes and PC3 Prostasomes Prostasome-like granules are present in the PC3 prostate cancer cells.", "Since the seminal prostasomes are able to promote the forward motility of human spermatozoa, we conducted a study to determine whether PC3 prostasomes exerted similar effects to those of seminal prostasomes on buffer-washed spermatozoa from normospermic semen samples.", "Semen samples were obtained from normospermic men, according to WHO laboratory manual, during evaluation for in vitro fertilisation.", "Motile spermatozoa were obtained by a swim-up procedure.", "Prostasomes were obtained from the human prostatic carcinoma cell line PC-3 and purified according to our protocol.", "The sperm motility analysis was done in accordance with the guidelines for application of CASA technology.", "At each measurement time, at least 200 spermatozoa from each aliquot sample were analysed in order to monitor sperm movement characteristics.", "This was done with an HTM semi-automated motility analyser (Hamilton-Thorn Research.", "Inc., Danvers, Mass., USA).", "The effects of PC3 prostasomes and seminal prostasomes on the sperm cell motility over time were compared at a protein concentration of 0.1 mg/ml.", "There were no significant differences between the two types of prostasomes in their stimulatory ability.", "We conclude that PC3 prostasomes, isolated from in vitro-grown PC3 cells, bear a functional resemblance to prostasomes isolated from human seminal plasma.", "5.Anti-Prostasome Autoantibodies in Serum from Patients with Metastasis of Prostate Cancer Serum samples from 13 men with PAD verified prostate cancer with metastases were included in the study.", "As control group we used healthy blood donors, 20 men and 20 women, age 20-40 years which were all tested for low PSA values.", "An immunoassay was designed to detect anti-prostasome autoantibodies in serum.", "For enzyme-linked immunosorbent assay (ELISA), plates were coated (F96 Polysorp, Nunc) with 4 μg purified prostasomes obtained from prostate tissue (see p.1) diluted in 100 mmol/L NaHCO3, pH 9.5 (coating buffer) for 2 hours at 37° C. The plates were washed and blocked for one hour, 37° C., with the coating buffer containing 3% BSA.", "After blocking, the plates were washed 3 times with 200 μl phosphate buffered saline with 0.1% Tween (PBS-T) and then incubated with 200, patient sera (dilution 1:50 in PBS) for 2 hours, 37° C. After 3 new washes with 200 μl PBS-T, 100 μl goat-anti-human IgG-horse radish peroxidase (HRP) conjugated antibodies were added (dilution 1:1000 in PBS) and incubated for 1 hour at room temperature.", "The plates were washed 3 times with 200 μl PBS-T and incubated with substrate (Tetramethyl benzidine, Zymed Laboratories Inc, Ca, USA) for 15 minutes, room temperature and protected from light.", "The reaction was stopped by adding 50 μl sulphuric acid (1.8 mol/L).", "The absorbance was measured at 450 nm in an ELISA reader (Spectra Max 250, Molecular Devices, Ca, USA).", "The reference interval for the control group was: 0.03-0.15 (absorbancy values at 450 nm) and that of the patients was 0.23-0.34.It should be noted that all patients included in the study had an ELISA test value that was significantly elevated above the background values of the control group.", "This indicated presence of antiprostasome autoantibodies in all of the patients with PAD verified prostate cancer.", "REFERENCES 1.Nilsson B. O., Jin M. and Ronquist G. (1996) Immunolocalization of prostasomes in the human prostate.", "Upsala J Med Sci, 101:149-158.2.Renneberg H., Konrad L., Dammshäuser I., Seitz J. and Aumüller G. (1997) Immunohistochemistry of prostasomes from human semen.", "The Prostate, 30:98-106.3.Nilsson B. O., Jin M. and Ronquist G (1998) Monoclonal antibodies against human prostasomes.", "The Prostate, 35:178-184.4.Schrimpf S., Hellman U., Carlsson L., Larsson A., Ronquist G., Nilsson B. O.", "(1999) Identification of dipeptidyl peptidase IV as the antigen of a monoclonal anti-prostasome antibody.", "The Prostate, 38:35-39." ] ]	Patent_10468949
[ [ "Methods of determining west nile virus epitopes and method of using the same", "Vaccines containing one or more West Nile Virus (WNV) vaccine candidate peptides in an immunologically acceptable excipient are disclosed.", "Also provided are recombinant WNV vaccine candidate peptides, wherein the peptide is expressed from a recombinant polynucleotide such as a naked DNA vaccine.", "Additionally, methods for inducing anti-WNV immune responses in a mammalian subject are also disclosed." ], [ "1.A vaccine comprising: one or more West Nile Virus (WNV) vaccine candidate peptides selected from the group consisting of SEQ ID NO: 1-95, in an immunologically acceptable excipient.", "2.The vaccine of claim 1, wherein the peptide is between 8 amino acids and 10 amino acids in length.", "3.The vaccine of claim 1, wherein one or more of the WNV vaccine candidate peptides has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20.4.The vaccine of claim 1, wherein the peptide is complexed to a carrier protein.", "5.The vaccine of claim 1, wherein the peptide is a recombinant fusion protein.", "6.The vaccine of claim 1, wherein the excipient is an adjuvant.", "7.A recombinant WNV vaccine candidate peptide, comprising: a peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20, wherein the peptide is expressed from a recombinant polynucleotide.", "8.The recombinant peptide of claim 7, wherein the recombinant polynucleotide is a naked DNA vaccine.", "9.A method for inducing an anti-WNV immune response, comprising: administering to a mammalian subject the vaccine according to claim 1.10.The method of claim 9, wherein the induction of an anti-WNV immune response results in the raising of an anti-WNV antibody.", "11.The method of claim 9, wherein the mammalian subject is a human.", "12.The method of claim 9, wherein the vaccine is administered orally, topically, parenterally, by viral infection, or intravascularly.", "13.A method for inducing an anti-WNV immune response, comprising administering to a mammalian subject the vaccine candidate peptide according to claim 7.14.The method of claim 13, wherein the induction of an anti-WNV immune response is the raising of an anti-WNV antibody.", "15.The method of claim 13, wherein the mammalian subject is a human.", "16.The method of claim 13, wherein the vaccine is administered orally, topically, parenterally, by viral infection, and intravascularly." ], [ "<SOH> BACKGROUND <EOH>West Nile virus (WNV) is the cause of a potentially fatal form of viral encephalitis that suddenly emerged in the New York City area during 1999.The virus is a member of the flavivirus family.", "Other members of the same family include St. Louis Encephalitis, Japanese Encephalitis Virus (JEV), Hepatitis C and Dengue.", "WNV is commonly found in West Asia, Africa, and the Middle East but was not reported in the Americas until 1999.", "(Lanciotti et al., Science 286:2333-37 (1999); Wright et al., Aust.", "J. Exp.", "Biol.", "Med.", "Scie.", "61(Pt.", "6):641-53 (1983)).", "The source of the introduction of the virus to New York City is unknown.", "Introduction by an infected host (e.g.", "human or bird), by an infected vector (e.g.", "mosquito), or by bio-terrorists are potential sources of WNV listed by the United States Centers for Disease Control.", "Surveillance data reported to the CDC have indicated intensified transmission and geographic expansion of the West Nile Virus (NY99) outbreak in the northeastern United States during the last two years.", "Twelve states and the District of Columbia reported WNV epizootic activity in 2000, a significant increase over the four states reporting activity in 1999.West Nile Virus is expected to continue to spread along the East Coast of the United States in 2001 and years thereafter due to over-wintering of mosquitoes and avian migratory patterns.", "(Andersen et al., Science 286:2331-33 (1999); Rappole et al., Emerging Infectious Diseases 6(4):319-28 (2000)).", "Concern about the dissemination of WNV in the United States is supported by knowledge of current endemics and epidemics in other regions of the world.", "The largest African epidemic, with approximately 3,000 clinical cases, occurred in South Africa after heavy rains in 1974.Other outbreaks have been observed in the former Soviet Republic, Central African Republic, Kisangani in the Democratic Republic of Congo (former Zaire), Egypt, Ethiopia, India, Israel, Madagascar, Nigeria, Pakistan, Senegal, Sudan, and quite a few European countries.", "The West Nile NY99 virus that was eventually associated with the New York 1999 outbreak appears to have been circulating in Israel since 1997.Other close relatives to the West Nile NY99 virus were isolated in Italy (1998), Morocco (1996), Romania (1996), and Africa (1989, 1993, 1998).", "The epitope-driven vaccine concept is an attractive one that is being successfully pursued in a number of laboratories.", "See, e.g., Hanke et al., Vaccine 16:426 (1998); Ling-Ling et al., J. Virology 71:2292-302 (1997); Nardin et al., Immunol.", "166(1):481-89 (2001)." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The goal of this project was to demonstrate the utility of a bioinformatics/computational immunology approach for the rapid selection of epitope reagents that would permit the evaluation of cell-mediated responses in the immunopathogenesis of West Nile Virus (WNV).", "In one embodiment, the invention is concerned with the development of diagnostic reagents such as tetramers and preventive or therapeutic vaccines.", "(Altman et al., Science 274(94):6 (1996)).", "In one aspect, the invention includes a vaccine that includes one or more West Nile Virus (WNV) candidate peptides disclosed in SEQ ID NOs: 1-95 and is in an immunologically acceptable excipient.", "For example, the vaccine could contain a combination of 2, 3,4, 5, 6, etc., WNV peptides disclosed in SEQ ID NOs:1-95.In one embodiment, the invention includes a vaccine where the length of one or more WNV candidate peptides is between 8 amino acids and 10 amino acids in length.", "In another embodiment, the invention includes a vaccine where one or more WNV candidate peptides have amino acid sequences from the group disclosed in SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20.The invention also includes a vaccine containing one or more WNV candidate peptides, where the peptide is complexed to a carrier protein.", "The carrier protein may be a recombinant fusion protein.", "Additionally, the excipient may be an adjuvant.", "In another aspect, the invention includes one or more recombinant WNV candidate peptide where the peptides contain an amino acid sequence from the group disclosed in SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20 and is expressed from a recombinant polynucleotide.", "In one embodiment, the recombinant polynucleotide is a naked DNA vaccine.", "In another embodiment, the invention involves a method for inducing an anti-WNV immune response by administering a vaccine containing one or more WNV vaccine candidate peptides selected from the group consisting of SEQ ID NOs:1-95 and an immunologically acceptable excipient to a mammal.", "In a further embodiment, the induction of an anti-WNV immune response results in the raising of an anti-WNV antibody.", "In various embodiments, suitable mammals include, for example, humans, cows, pigs, horses, and dogs.", "Administration of the vaccine according to the invention may be orally, topically, parenterally, by viral infection, and/or intravascularly.", "In another embodiment, the invention involves a method for inducing an anti-WNV immune response by administering a vaccine candidate peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20, wherein the peptides are expressed from a recombinant polynucleotide.", "In a further embodiment, the induction of an anti-WNV immune response results in the raising of an anti-WNV antibody.", "In various embodiments, suitable mammals include, for example, humans, cows, pigs, horses, and dogs.", "Administration of the vaccine according to the invention may be orally, topically, parenterally, by viral infection, and/or intravascularly.", "Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.", "Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.", "All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.", "In the case of conflict, the present specification, including definitions, will control.", "In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.", "Other features and advantages of the invention will be apparent from the following detailed description and claims." ], [ "TECHNICAL FIELD OF THE INVENTION This invention relates generally to the fields of epidemiology, immnunology, and molecular biology.", "BACKGROUND West Nile virus (WNV) is the cause of a potentially fatal form of viral encephalitis that suddenly emerged in the New York City area during 1999.The virus is a member of the flavivirus family.", "Other members of the same family include St. Louis Encephalitis, Japanese Encephalitis Virus (JEV), Hepatitis C and Dengue.", "WNV is commonly found in West Asia, Africa, and the Middle East but was not reported in the Americas until 1999.", "(Lanciotti et al., Science 286:2333-37 (1999); Wright et al., Aust.", "J. Exp.", "Biol.", "Med.", "Scie.", "61(Pt.", "6):641-53 (1983)).", "The source of the introduction of the virus to New York City is unknown.", "Introduction by an infected host (e.g.", "human or bird), by an infected vector (e.g.", "mosquito), or by bio-terrorists are potential sources of WNV listed by the United States Centers for Disease Control.", "Surveillance data reported to the CDC have indicated intensified transmission and geographic expansion of the West Nile Virus (NY99) outbreak in the northeastern United States during the last two years.", "Twelve states and the District of Columbia reported WNV epizootic activity in 2000, a significant increase over the four states reporting activity in 1999.West Nile Virus is expected to continue to spread along the East Coast of the United States in 2001 and years thereafter due to over-wintering of mosquitoes and avian migratory patterns.", "(Andersen et al., Science 286:2331-33 (1999); Rappole et al., Emerging Infectious Diseases 6(4):319-28 (2000)).", "Concern about the dissemination of WNV in the United States is supported by knowledge of current endemics and epidemics in other regions of the world.", "The largest African epidemic, with approximately 3,000 clinical cases, occurred in South Africa after heavy rains in 1974.Other outbreaks have been observed in the former Soviet Republic, Central African Republic, Kisangani in the Democratic Republic of Congo (former Zaire), Egypt, Ethiopia, India, Israel, Madagascar, Nigeria, Pakistan, Senegal, Sudan, and quite a few European countries.", "The West Nile NY99 virus that was eventually associated with the New York 1999 outbreak appears to have been circulating in Israel since 1997.Other close relatives to the West Nile NY99 virus were isolated in Italy (1998), Morocco (1996), Romania (1996), and Africa (1989, 1993, 1998).", "The epitope-driven vaccine concept is an attractive one that is being successfully pursued in a number of laboratories.", "See, e.g., Hanke et al., Vaccine 16:426 (1998); Ling-Ling et al., J. Virology 71:2292-302 (1997); Nardin et al., Immunol.", "166(1):481-89 (2001).", "SUMMARY OF THE INVENTION The goal of this project was to demonstrate the utility of a bioinformatics/computational immunology approach for the rapid selection of epitope reagents that would permit the evaluation of cell-mediated responses in the immunopathogenesis of West Nile Virus (WNV).", "In one embodiment, the invention is concerned with the development of diagnostic reagents such as tetramers and preventive or therapeutic vaccines.", "(Altman et al., Science 274(94):6 (1996)).", "In one aspect, the invention includes a vaccine that includes one or more West Nile Virus (WNV) candidate peptides disclosed in SEQ ID NOs: 1-95 and is in an immunologically acceptable excipient.", "For example, the vaccine could contain a combination of 2, 3,4, 5, 6, etc., WNV peptides disclosed in SEQ ID NOs:1-95.In one embodiment, the invention includes a vaccine where the length of one or more WNV candidate peptides is between 8 amino acids and 10 amino acids in length.", "In another embodiment, the invention includes a vaccine where one or more WNV candidate peptides have amino acid sequences from the group disclosed in SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20.The invention also includes a vaccine containing one or more WNV candidate peptides, where the peptide is complexed to a carrier protein.", "The carrier protein may be a recombinant fusion protein.", "Additionally, the excipient may be an adjuvant.", "In another aspect, the invention includes one or more recombinant WNV candidate peptide where the peptides contain an amino acid sequence from the group disclosed in SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20 and is expressed from a recombinant polynucleotide.", "In one embodiment, the recombinant polynucleotide is a naked DNA vaccine.", "In another embodiment, the invention involves a method for inducing an anti-WNV immune response by administering a vaccine containing one or more WNV vaccine candidate peptides selected from the group consisting of SEQ ID NOs:1-95 and an immunologically acceptable excipient to a mammal.", "In a further embodiment, the induction of an anti-WNV immune response results in the raising of an anti-WNV antibody.", "In various embodiments, suitable mammals include, for example, humans, cows, pigs, horses, and dogs.", "Administration of the vaccine according to the invention may be orally, topically, parenterally, by viral infection, and/or intravascularly.", "In another embodiment, the invention involves a method for inducing an anti-WNV immune response by administering a vaccine candidate peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20, wherein the peptides are expressed from a recombinant polynucleotide.", "In a further embodiment, the induction of an anti-WNV immune response results in the raising of an anti-WNV antibody.", "In various embodiments, suitable mammals include, for example, humans, cows, pigs, horses, and dogs.", "Administration of the vaccine according to the invention may be orally, topically, parenterally, by viral infection, and/or intravascularly.", "Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.", "Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.", "All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.", "In the case of conflict, the present specification, including definitions, will control.", "In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.", "Other features and advantages of the invention will be apparent from the following detailed description and claims.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1a shows all 3,423 peptides obtained by parsing the West Nile Virus genome were scored using EpiMatrix and the EpiMatrix motif for HLA B07 (this matrix motif provides equivalent scores for most of the HLA B07 subtypes).", "HLA B07 is a class I major histocompatability complex (“MHC”) antigen.", "Based on previous experience, most peptides (approximately 80%) scoring above an estimated binding probability (“EBP”) of 7 for this particular matrix motif are considered likely to bind to HLA B07 in T2 B-7 assays.", "Twenty WNV peptides scoring between 20 and 50 were selected for the present study.", "FIG.", "1b shows EpiMatrix HLA B07 score distributions for a random set of 10,000 peptides, 20 peptides selected for the WNV genome, and a set of known HLA B07 ligands.", "The log of EBP for all three sets (random, known binders, WNV selection) fell in the range−5 to 5.The graph shows a frequency analysis of the data, graphing the proportion of peptides (over the total number for each set) falling within scores from −5 to 5.FIG.", "2 is a plot showing the distribution of EpiMatrix (“EMX”) scores along the length of the entire WNV genome.", "DETAILED DESCRIPTION OF THE INVENTION The NY 1999 WNV sequence was obtained from Genbank (Accession No.", "AF196835).", "(Altschul et al., Nucleic Acids Res.", "25:3389-402 (1997)).", "The WNV genome is 11,000 nucleotides long.", "The following proteins have been tentatively identified: major envelope glycoprotein E; membrane (envelope) protein M; non-structural proteins NS1, NS2A, NS3, NS4A; and RNA directed polymerase (non-structural protein 5).", "The CDC has completely sequenced the West Nile NY99 virus originally obtained from a Chilean flamingo, a West Nile NY99 equine isolate, the Italy 1998 virus, the Romania 1996 virus and the prototype Eg101, virus.", "While the latter viruses are closely related to West Nile NY99, they are neither identical to each other nor to West Nile NY99.A virus isolated in Israel, “Israel 1998”, appears to be virtually identical to the West Nile NY99.Completion of the genome sequence of the Israel 1998 West Nile virus is being pursued at the Institute Pasteur, France.", "Immune response to WNV has not been well delineated.", "CD4 T helper responses have been identified to related viruses (e.g.", "JEV, Dengue) and are felt to be essential components of protective immune response.", "Also, mobilization of dendritic cells and antigen presentation by Langerhans cells found in the dermis to T cells found in the lymphoid follicles may be involved in the development of immune responses to WNV.", "(Johnston et al., J.", "Invest.", "Dermatol.", "114(3):560-68 (2000)).", "Some CD4 T cells can be identified that respond to epitopes within JEV that are identical, or nearly identical, with sequences contained in WNV.", "For example, T cell clones have been derived from a human subject who experienced dengue illness following immunization with a live experimental dengue virus type 3 (DEN3 vaccine).", "The NS3 protein was immunodominant in the CD4+ T-cell response of this subject.", "The epitopes of four Dengue NS3-specific T-cell clones were analyzed; one of the four recognized peptide epitopes derived from WNV as well as JEV.", "These epitopes were fine-mapped.", "The smallest synthetic peptide recognized by these T cell clones was a nine amino acid peptide containing amino acids 146 to 154 of dengue-4 NS3.These results confirmed immunologic cross-reactivity between JEV and WNV.", "Other researchers have focused on the effect of WNV and other flavivirus family members on endothelial cells.", "Both ICAM-1 and MHC class I and II expression are upregulated in human endothelial cells in the first 72 hours of infection.", "Langerhans cells (LC) in the epidermis may play a role in the upregulation of immune response to the virus, processing antigen, and presenting it to T cells.", "Furthermore, researchers have postulated that LC may migrate to local lymph nodes following cutaneous infection with WNV.", "Mobilization of dendritic cells and antigen presentation by these cells to T cells in the lymphoid follicles may therefore be involved in the development of immune responses to WNV.", "In support of this hypothesis, cytotoxic T cell responses (restricted by class I MHC) and T helper responses (restricted by class II MHC) appear to be critical components of human immune response to the other members of the flavivirus family.", "(Lobigs et al., Virology 202(1):195-201 (1994); (Murali-Krishna et al., J. Gen. Virol.", "75(Pt.", "4):799-807 (1994)).", "Development of cell-mediated immunity to WNV may be a critically important barrier to infection of the central nervous system.", "Reagents that permit the evaluation of cell-mediated responses may permit researchers to better understand the immunopathogenesis of WNV, to develop diagnostic reagents such as tetramers, and to develop preventive or therapeutic vaccines.", "“MHC tetramers” are a new tool that may make field-based screening for immune responses to emerging viruses an important epidemiological tool.", "Tetramers were first developed a few years ago by Altman et al., Science 274(94):6 (1996).", "These specialized constructs bear four MHC molecules complexed with beta 2 microglobulin and a specific pathogen-derived peptide ligand.", "These novel reagents can bind directly to T cells that recognize the MHC-peptide complex.", "They can be used for direct ex vivo analysis of the frequency and phenotypes of antigen-specific T cells by flow cytometry.", "The tetramer staining assay relies only upon the interaction between the tetramer reagent and T cell receptor clusters (and possibly co-receptors) on the surface of T cells.", "The assay reduces to an absolute minimum the in vitro manipulation of the sample before detection of the antigen-specific population.", "The incubation period in humans (i.e., time from infection to onset of disease symptoms) for WNV encephalitis is usually 5 to 15 days.", "Antibodies are detectable within three to seven days.", "In contrast, based on recent tetramer-staining studies, WNV T cell responses should be detectable within two to three days of infection.", "The initial CTL response to acute infection with a virus, as measured using tetramer technology, is phenomenal.", "For example, during the acute immune response to lymphocytic choriomeningitis virus (LCMV) in BALB/c mice, 55% of all CD8+ splenocytes are stained with a LCMV specific tetramer.", "The method is extremely robust, and can detect antigen-specific populations at frequencies as low as 1:5,000 CD8+ T cells (or approximately 1:50,000 PBMC).", "As little as 2 cc of blood can be drawn (containing approximately 2 million peripheral blood mono-nuclear cells, PBMC), mixed with tetramer reagents, and analyzed by fluorescence-activated cell sorting (FACS).", "No incubation is required, and the assay does not require a priori assumptions of the class of functional responses (e.g.", "cytokine profiles), and is therefore likely to provide the most complete method for detection of the magnitude of an antigen-specific response.", "Results can be available within 60 minutes of drawing a blood sample without any additional processing (other than mixing the tetramer reagent with the blood sample).", "It is conceivable that companies holding the patent for tetramer diagnostics (Beckman Coulter, for example) will begin to develop low cost FACS machines for use in the field.", "There is no specific vaccine or anti-viral treatment available for WNV infection.", "Patients who develop symptoms of WNV encephalitis can be managed with supportive intensive care.", "Not all patients with severe neurological disease due to WNV recover.", "It is likely that a CTL response will be one critical component of immune response against WNV.", "The development of a preventive or therapeutic vaccine against this public health threat would be greatly expedited if the correlates of immune response were determined and if those correlates could be rapidly incorporated into a vaccine.", "Epitopes defined using methods such as the one described here may also be useful for evaluating immune response to WNV and developing vaccines.", "West Nile Virus is expected to continue to spread along the East Coast of the United States in the future due to over-wintering of mosquitoes and avian migratory patterns.", "Although the CDC has made recommendations that will reduce the incidence of WNV transmission in populated areas, once the virus has become established it is unlikely to be eradicated.", "Tools that will enhance epidemiological surveillance, case detection, diagnosis, research on the immunopathogenesis of WNV are likely to be of great interest to physicians, public health officials, and the lay public during vectored transmission seasons (April to November).", "A bioinformatics/computational immunology approach to epitope discovery, such as the one illustrated here, will make significant contributions to the development of new research and diagnostic reagents for West Nile Virus and other emerging infectious diseases.", "Once the epitope candidates selected using this method are confirmed in CTL assays, they may be useful for (1) screening exposed individuals, (2) investigating the immuno-pathogenesis of WNV disease in humans, (3) as components of diagnostic kits developed for the surveillance effort, and/or (4) eventually, as a tool for measuring WNV vaccine-related immune responses.", "Confirmation of T cell response to the peptides will depend on availability of peripheral blood cells from West Nile-infected patients during the next transmission season.", "Additional peptides also need to be defined and screened for binding to other HLA alleles, in order to broaden the MHC specificity of the diagnostic reagent or immunopathogenesis tools developed using this approach.", "Peptides selected by the EpiMatrix approach were not confined to any particular protein, as illustrated in FIG.", "2.The final set of peptides included four from NS-1, four from NS-2A, five from NS-3, one from NS-4A, five from NS-5, one from envelope protein E, and one from membrane protein M. A summary of WNV peptides according to the invention is given below in Table 1.Each of the 95 peptide sequences identified is shown in Column 1.Column 2 shows the sequence of each peptide.", "Column 4 shows the location of the amino acid start.", "The EBP for each peptide is shown in Column 4.Column 5 shows the “cover” value for each peptide.", "TABLE 1 SEQ ID NO.", "Dataset AA Sequence AA Start E.B.P.", "Cover 1 WNB7 001 RPSECCDTLL 2663 72.10 10.20 2 WNB7 002 GPIRFVLALL 42 59.88 19.22 3 WNB7 003 GPREFCVKVL 2703 54.68 23.89 4 WNB7 004 AVKDELNTLL 861 47.77 30.50 5 WNB7 005 APAYSFNCLG 286 47.03 31.21 6 WNB7 006 AAKKKGASLL 1337 45.30 33.00 7 WNB7 007 NPMILAAGLI 1357 36.60 42.86 8 WNB7 008 IPAGFEPEML 1680 36.02 43.64 9 WNB7 009 TPAAPSYTLK 460 35.68 44.04 10 WNB7 010 VPCRGQDELV 3259 32.57 48.01 11 WNB7 011 GPGHEEPQLV 2635 32.35 48.40 12 WNB7 012 AGMLLLSLLL 2229 31.17 50.40 13 WNB7 013 EPPEGVKYVL 2895 31.07 50.40 14 WNB7 014 MPAILIALLV 1177 30.23 51.60 15 WNB7 015 KPTGSASSLV 2842 28.79 53.59 16 WNB7 016 IPTAAGKNLC 148 25.56 58.32 17 WNB7 017 RPRWIDARVY 2098 23.99 60.64 18 WNB7 018 VPGTKIAGML 2223 23.10 62.17 19 WNB7 019 RPQRHDEKTL 1127 22.49 62.93 20 WNB7 020 SPHRVPNYNL 1777 22.40 63.31 21 WNB7 021 IPMTIAGLMF 1405 22.32 63.31 22 WNB7 022 MPRVLSLIGL 21 21.22 65.17 23 WNB7 023 RPAADGRTVM 3112 20.89 65.54 24 WNB7 024 TPGLRCLNLD 1306 20.73 65.91 25 WNB7 025 SVNMTSQVLL 2760 20.09 67.00 26 WNB7 026 EERKNFLELL 2034 19.15 68.44 27 WNB7 027 GPQYEEDVNL 2779 19.15 68.44 28 WNB7 028 APWKIWMLRM 1467 18.18 70.19 29 WNB7 029 NARDRSIALT 765 17.03 72.24 30 WNB7 030 VPISSVASLN 628 16.68 72.91 31 WNB7 031 KPWDTITNVT 2860 15.94 73.89 32 WNB7 032 LPDALQTIAL 2171 15.81 74.22 33 WNB7 033 RPRMCSREEF 2920 15.81 74.22 34 WNB7 034 TVWRNRETLM 521 15.35 75.17 35 WNB7 035 LIMKDGRTLV 3249 15.10 75.49 36 WNB7 036 KSYAQMWLLL 3289 13.64 78.23 37 WNB7 037 FVDVGVSALL 2361 13.46 78.52 38 WNB7 038 GPRTNTILED 2072 12.90 79.67 39 WNB7 039 GTRAVGKPLL 2791 12.57 80.23 40 WNB7 040 VPREHNGNEI 1753 12.51 80.23 41 WNB7 041 GAPWKIWMLR 1466 12.30 80.51 42 WNB7 042 IAGMLLLSLL 2228 12.14 81.06 43 WNB7 043 MPKVIEKMEL 2716 11.93 81.33 44 WNB7 044 EPVGKVIDLG 2600 11.88 81.33 45 WNB7 045 RWFVVLLLL 275 11.82 81.59 46 WNB7 046 AYHDARQILL 1260 11.82 81.59 47 WNB7 047 APKRLTATTE 891 11.77 81.59 48 WNB7 048 EPRSGIDTNA 481 11.67 81.86 49 WNB7 049 GGRAHRMALE 2160 11.57 82.12 50 WNB7 050 EPPFGDSYIV 666 11.22 82.64 51 WNB7 051 MIDPFQLGLL 1148 10.98 83.15 52 WNB7 052 ILRNPGYALV 251 10.88 83.40 53 WNB7 053 AAPSYTLKLG 462 10.88 83.40 54 WNB7 054 TPWAILPSVV 1485 10.74 83.65 55 WNB7 055 KPLDDRFATS 3201 10.59 83.89 56 WNB7 056 AKSYAQMWLL 3288 10.09 84.85 57 WNB7 057 AIPMTIAGLM 1404 9.91 85.08 58 WNB7 058 GPCKVPISSV 624 9.87 85.31 59 WNB7 059 KGPKVRTWLF 3172 9.87 85.31 60 WNB7 060 APELANNTFV 916 9.56 85.77 61 WNB7 061 GARFLEFEAL 3010 9.52 85.77 62 WNB7 062 HSRRSRRSLT 209 9.35 86.21 63 WNB7 063 TPADTGHGTV 604 9.35 86.21 64 WNB7 064 NVVVPLLALL 1296 9.27 86.43 65 WNB7 065 GPGKSRAVNM 7 9.06 86.65 66 WNB7 066 EPHATKQSVI 534 9.06 86.65 67 WNB7 067 GPWDEGRVEI 1057 8.98 86.86 68 WNB7 068 WPATEVMTAV 1376 8.82 87.29 69 WNB7 069 MLRKKQITVL 1688 8.74 87.29 70 WNB7 070 AAAKKKGASL 1336 8.66 87.49 71 WNB7 071 TVTVTAATLL 2382 8.62 87.49 72 WNB7 072 AGCWGQVTLT 2373 8.47 87.90 73 WNB7 073 VPNYNFLVMD 1781 8.32 88.10 74 WNB7 074 AVFLICVMTL 2258 8.24 88.30 75 WNB7 075 KPTIDVKMMN 328 8.20 88.30 76 WNB7 076 GMSWITQGLL 746 8.09 88.49 77 WNB7 077 KCRVKMEKLQ 577 8.02 88.69 78 WNB7 078 AVGGVLLFLS 777 7.95 88.88 79 WNB7 079 LAREKRPRMC 2915 7.91 88.88 80 WNB7 080 EPGKNVKNVQ 1606 7.73 89.25 81 WNB7 081 ADMIDPFQLG 1146 7.70 89.25 82 WNB7 082 RGMPRVLSLI 19 7.66 89.44 83 WNB7 083 FCSNHFTELI 3241 7.66 89.44 84 WNB7 084 VYRIMTRGLL 1527 7.52 89.62 85 WNB7 085 DPFQLGLLVV 1150 7.46 89.80 86 WNB7 086 TAIAPTRAVL 57 7.42 89.80 87 WNB7 087 LKRYEDTTLV 3419 7.39 89.97 88 WNB7 088 SMPAILIALL 1176 7.32 89.97 89 WNB7 089 LVNGVVRLLS 2850 7.32 89.97 90 WNB7 090 LVAAVIGWML 259 7.29 90.15 91 WNB7 091 LGMSNRDFLE 294 7.29 90.15 92 WNB7 092 RVKMEKLLQLK 579 7.29 90.15 93 WNB7 093 GPRSNHNRRP 1040 7.25 90.15 94 WNB7 094 AIAPTRAVLD 58 7.12 90.49 95 WNB7 095 MPNGSYISAI 1661 7.02 90.66 EBPs for the WNV peptides ranged from >20% (highly likely to bind) to <1% (very unlikely to bind).", "Peptides with EpiMatrix EBP scores in the range of 7 to 50 are more likely to bind to MHC and stimulate T cells in vitro.", "See Jin et al., AIDS Res Hum Retroviruses 16:67-76 (2000).", "Peptides with an EBP score 50 are less likely to be immunogenic.", "However, they may bind to B7 in vitro.", "See Jin et al., supra; DeGroot et al., Vaccine 2000 (in press).", "[HAS THIS PAPER PUBLISHED ALREADY?]", "Triplicate wells of peptide at 10, 20, 200 ug/ml were evaluated in each of the T2 B7 binding assays; each assay was repeated 4 times.", "Table 2 provides information on the MFI for the peptide at 200 ug/ml, the fold increase over background for the peptide at 200 ug/ml, and a summary of binding results in each of the assays.", "In the summary “binding results” column, “0/3” signifies none of the wells at any of the three concentrations gave a positive result (in one assay plate), “×4” signifies that this result was obtained in each of four assays; “×3”” signifies that this result was obtained in each of three assays, and so on.", "The “binding score” column provides the numerical sum of all the positive assays in the binding summary column.", "For example, summing the results for peptide 0005: 3/3×1, 2/3×2, 1/3×1=(3×1)+(2×2)+(1×1)=8.TABLE 2 WNV T2B7 binding assay results Avg.", "SEQ Fold Avg.", "Binding ID Inc. @ MFI1 results (total Binding NO: Peptide AA Sequence EBP 200 200 of four assays number strength 4 WNVB7 AVKDELNTLL 47.77 1.0 842.5 0/3(×4) 0 non-binder n 0004 5 WNVB7 APAYSFNCLG 47.03 1.2 743.5 3/3(×1), 8 moderate y 0005 2/3(×2), 1/3(×1) 6 WNVB7 AAKKKGASLL 45.30 1.2 708.1 1/3 × 4 4 weak y 0006 7 WNVB7 NPMILAAGLI 36.60 1.1 944.6 1/3(×2) 2 non-binder n 0007 8 WNVB7 IPAGFEPEML 36.02 1.9 1207.4 3/3 × 3, 10 strong y 0008 1/3 × 1 9 WNVB7 TPAAPSYTLK 35.68 1.5 954.5 3/3(×1), 8 moderate y 0009 2/3(×2), 1/3(×1) 10 WNVB7 VPCRGQDELV 32.57 1.0 658.6 2/3(×2) 4 weak y 0010 11 WNVB7 GPGHEEPQLV 32.35 1.0 848.9 2/3(×1) 2 non-binder n 0011 12 WNVB7 AGMLLLSLLL 31.17 — — — — — 0012 13 WNVB7 EPPEGVKYVL 31.07 1.1 681.7 3/3(×1), 6 moderate y 0013 2/3(×1), 1/3(×1) 14 WNVB7 MPAILIALLV 30.23 — — — — — 0014 15 WNVB7 KPTGSASSLV 28.79 1.7 1070.2 2/3(×2), 6 moderate y 0015 1/3(×2) 17 WNVB7 RPRWIDARVY 23.99 1.9 1714.0 3/3(×3), 11 strong y 0017 2/3(×1) 18 WNVB7 VPGTKIAGML 23.10 1.7 1088.4 3/3 × 1, 6 moderate y 0018 2/3 × 1, 1/3 × 2 19 WNVB7 RPQRHDEKTL 22.49 2.8 1644.6 3/3 × 3, 11 strong y 0019 2/3 × 1 20 WNVB7 SPHRVPNYNL 22.40 2.5 1607.9 3/3(×4) 12 strong y 0020 21 WNVB7 IPMTIAGLMF 22.32 — — — — — 0021 23 WNVB7 RPAADGRTVM 20.89 1.5 979.1 3/3 × 4 12 strong y 0023 24 WNVB7 TPGLRCLNLD 20.73 1.0 849.6 1/3(×2) 2 non-binder n 0024 25 WNVB7 SVNMTSQVLL 20.09 — — — — — 0025 96 WNVB7 PEDIDCWCTK 0.00 1.1 990.3 1/3(×1) 1 non-binder n 3399 97 WNVB7 PETPQGLAKI 0.00 1.0 588.9 0/3 × 4 0 non-binder n 3403 98 WNVB7 PFPESNSPIS 0.00 1.0 626.7 1/3(×1) 1 non-binder n 3411 99 WNVB7 PRTNTILEDN 0.00 0.9 778.6 0/3(×4) 0 non-binder n 3415 100 HIV-1 B7 GPGHKARVLA GPGH 2.2 1423.8 2/3 × 2, 8 moderate 1 1291 KARV 3/3 × 1, 2 LA 1/3 × 1 1.5 1370.9 3/3(×2), 9 moderate 4 2/3(×1), 1/3(×1) 1.6 1003.9 3/3(×2), 8 moderate 1 2/3(×1) 6 1MFI = mean fluorescence index and T2B7 binding assay results for each of the peptides.", "Twelve of the sixteen WNV peptides selected for higher likelihood of binding to B7 and tested in vitro demonstrated consistent binding in the four replicate assays.", "Of these peptides, five (0008, 0017, 0019, 0020, and 0023) stabilized HLA B7 on the surface of T2B7 cells at more than two concentrations in all four replicate assays, receiving a total binding score of 10, 11, 11, 12, and 12, respectively (Table 2).", "Five WNV peptides (0005, 0009, 0013, 0015, 0018) stabilized HLA B07 to a moderate degree (receiving binding scores of 6 to 8).", "Two WNV peptides (0006, 0010) were weak binders (binding score of 4) and three did not bind (binding score of 2).", "Table 3a shows the selected WNV peptides and their respective EpiMatrix scores.", "Table 3b shows some additional peptides that were not tested and provides reasons why such testing was not done.", "TABLES 3a and 3b Selected WNV peptides and their EpiMatrix scores SEQ peptide EBP ID number (B07 AA (EMX NO: rank) Source Sequence start score) 4 WNB7 0004 NS-1 AVKDELNTLL 861 48 5 WNB7 0005 mpM APAYSFNCLG 286 47 6 WNB7 0006 NS-2A AAKKKGASLL 1337 45 7 WNB7 0007 NS-2A NPMILAAGLI 1357 37 8 WNB7 0008 NS-3 IPAGFELEML 1680 36 9 WNB7 0009 env gp E TPAAPSYTLK 460 36 10 WNB7 0010 NS-5 VPCRGQDELV 3259 33 11 WNB7 0011 NS-5 GPGHEEPQLV 2635 32 12 WNB7 0012 NS-4A AGMLLLSLLL 2229 31 13 WNB7 0013 NS-5 EPPEGVKYVL 2895 31 14 WNB7 0014 NS-2A MPAILLALLV 1177 30 15 WNB7 0015 NS-5 KPTGSASSLV 2842 29 17 WNB7 0017 NS-3 RPRWIDARVY 2098 24 18 WNB7 0018 NS-4A VPGTKIAGML 2223 23 19 WNB7 0019 NS-1 RPQRHDEKTL 1127 22 20 WNB7 0020 NS-3 SPHRVPNYNL 1777 22 21 WNB7 0021 NS-2A IPMTIAGLMF 1405 22 23 WNB7 0023 NS-5 RPAADGRTVM 3112 21 24 WNB7 0024 NS-2A TPGLRCLNLD 1306 21 25 WNB7 0025 NS-5 SVNMTSQVLL 2760 20 96 WNB7 3399 pre-mpM PEDIDCWCTK 185 0 97 WNB7 3403 NS-1 PETPQGLAKI 827 0 101 WNB7 3407 NS-1 PRSNHNRRPG 1041 0 98 WNB7 3411 NS-3 PFPESNSPIS 1830 0 99 WNB7 3415 NS-3 PRTNTILEDN 2073 0 TABLE 3b SEQ peptide EBP ID number Reason for AA (EMX NO: (B07 rank) not testing Sequence start score) 1 WNB7 EBP > 50 RPSECCDTLL 2663 72 0001 2 WNB7 EBP > 50 GPIRFVLALL 42 60 0002 3 WNB7 EBP > 50 GPREFCVKVL 2703 55 0003 16 WNB7 not IPTAAGKNLC 148 26 0016 expressed 22 WNB7 not MPRVLSLIGL 21 21 0022 expressed 3393 additional peptides not tested (including 69 putative B07 binders and 3353 “non-binders”) The 25 peptides that were tested in vitro are shown in Table 3a.", "Twenty of the peptides were selected because they had an EpiMatrix (EMX) score (estimated binding probability, or EBP) between 7 and 50.Peptides that were not tested although they may have fit the above criteria are listed in Table 3b.", "Three peptides received EMX scores above 50 (0001, 0002, 0003); these peptides were considered to be unlikely to be epitopes based on previous experience with HLA B07 restricted HIV-1 epitopes.", "An additional two peptides were not selected, even though they were predicted to be binders (0016, 0022), because they fell in regions of the genome that were considered unlikely to be expressed, based on information provided by Genbank.", "The control peptide, WC 1291, was tested in binding assays 3 times (for each set of peptides).", "The peptide bound moderately well, receiving a total binding score of eight to nine.", "Four WNV peptides selected for low EBP (3399, 3404, 3411, and 3415, all receiving EBPs of 0.0%) did not stabilize T2B7 to a significant degree, receiving binding scores of 0 to 1, reflecting only occasional stabilization of HLA B7 over background.", "Peptides 0008 and 0019 were fairly unique, when compared to sequences of other viruses within the same flavivirus family and to sequences available in public databases.", "Peptide 0008, a strong binder, was 100% conserved in most strains of West Nile virus, and different by one amino acid from Kunjin virus (closely related).", "Peptide 0019, another strong binder, was 100% conserved in WNV and Kunjin virus and 80% conserved in JEV.", "Peptide 0017 was 100% conserved in all strains of WNV and Kunjin virus, 80% conserved in JEV and MVEV, and 90% conserved in some strains of Dengue.", "Peptide 0020, another strong binder, was conserved in West Nile and Kunjin (100%), JEV, MVEV, and Dengue (90%).", "Of the five moderately well binding peptides (0005, 0009, 0013, 0015 and 0018), 0005 was 100% conserved across WNV, Kunjin, SLE and Sindbis virus.", "Thus, it is an interesting candidate for a vaccine against all members of this family of flaviviruses.", "Peptide 0009, in contrast, was only conserved in WNV and Kunjin.", "Peptide 0013, likewise, was conserved in WNV and Kunjin but less well in JEV (80%).", "WNV 0015 was conserved in WNV, Kunjin (100%), JEV (90%), MVEV (80%), SLEV (90%) and Dengue (80%).", "WNV 0018 was conserved in WNV, JEV (90%), MVEV (80%), and SLEV (90%).", "Of the two weak binding WNV peptides (0006,0010), 0006 was 90-100% conserved in WNV, Kunjin, JEV, and MVEV.", "In contrast, weak binder 0010 was highly conserved (as high as 90%) in a very wide range of viruses (West Nile, Kunjin, Japanese encephalitis, Murray Valley encephalitis, St. Louis encephalitis, Sindbis virus, Dengue, Koutango,Alfuy, Usutu, Ntaya, Saumarez Reef, Langat, Ilheus, Meaban, Yellow fever, Cacipacore, Russian Spring-Summer encephalitis, Neudoerfl, Louping ill, Tick-borne, Kadam, Royal Farm, Kyasanur forest disease, Edge Hill, Negishi, Karshi, Omsk hemorrhagic fever, Gadgets Gully, Powassan, Tyuleniy, Stratford, Kokobera, Rocio, Sitiawan, Sokuluk).", "An additional 69 WNV peptides that might be expected to bind to HLA B07 (based on score above 7) and to stimulate T cell responses (based on score below 50 and above 7) were not tested due to funding constraints and the pilot nature of this project.", "If the reminder of the WNV B7 peptides behave as predicted, we would expect that an additional 62 ligands to be identified (for a total of 71 out of the total 94 selected).", "Based on the HIV-1 results, it is expected that approximately 56 of the peptides (60% of the original 94) would be recognized by human CTL.", "Peptides selected using the EpiMatrix HLA B07 matrix method and included in this study did not always fit the conventional, anchor based format of P in position 2 and L or F in position 9.One weak binder, AAKKKGASLL (SEQ ID NO: 6), had little in common with published HLA B07 motifs.", "Additional experiments were performed to determine whether peptides that are not selected for conformity for a given EpiMatrix scoring motif are likely to bind to that HLA molecule, in T2 binding assays.", "A set of 7 HIV-1 peptides that scored well on unrelated alleles (HLA A0201 and HLA A11) but poorly with respect to HLA B7 were tested.", "Only one of the seven peptides stabilized HLA B07 on the surface of T2B7 cells.", "Information derived from such peptides that deviate from the expected (selected ligands that do not bind, and selected non-binders that do bind) are integrated into EpiVax and TB/HIV Research Lab databases so as to improve the EpiMatrix scoring matrix model.", "HLA B07 is found in 7.7% of Blacks, 8.7 % of caucasoids, 3% of orientals.", "Clearly there is a need to map additional epitopes for other alleles.", "EpiVax currently possesses information permitting the selection of putative MHC ligands for 30 class I alleles and 90 class II alleles.", "Mapping of additional epitopes for the WNV genome is currently underway.", "Existing diagnostic tests for WNV are difficult to implement in the field, and due to the complexity of the tests and their interpretation, a great deal of inter-lab variability exists.", "Similarity between WNV and other members of the flavivirus family can also complicate the diagnosis of WNV infection.", "Patterns of antibody response are required to differentiate between WNV and related flaviviruses.", "Differentiation from Kunjin virus, counterpart or subtype of WNV found in Australia and Southeast Asia may be particularly difficult as most antibodies to Kunjin may be cross-reactive with WNV.", "Finally, the antibody tests (such as IgM capture EIA) that are available for detection of WNV infection rely on the availability of paired sera (two separate blood samples at two timepoints) to establish the diagnosis.", "Plaque tests, using vero cells, are also used to detect live virus and may be most useful to confirm mosquito infection with viable WNV.", "Plaque neutralization studies involve mixing patient antibody with the virus, plating the virus/antibody mixture on Vero cells, then counting infected Vero cells (Plaque reduction test).", "This test is highly subjective, time consuming, and results may vary from laboratory to laboratory.", "Polymerase chain reaction-based (PCR) tests can be used on cerebrospinal fluid but the sensitivity and specificity of these tests is as yet poorly defined.", "Peptide 0008 is a strong contender for a WNV-specific diagnostic assay, as it was only 80% conserved (eight out of ten amino acids) in Kunjin and was less well conserved in other members of the flavivirus family.", "Results of these studies suggest that Peptide 0008, a strong binder that scored in the range of EpiMatrix scores previously determined to be compatible with immunogenicity, would be a reasonable first candidate for the development of a tetramer-based diagnostic reagent for WNV.", "See, e.g., 3in X., et al., AIDS Res.", "Hum.", "Retroviruses, 16: 67-76 (2000).", "WNVX Nucleic Acids and Polypeptides The West Nile Virus nucleic acids and polypeptides of the invention, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as “WNVX” nucleic acid or polypeptide sequences.", "One aspect of the invention pertains to isolated nucleic acid molecules that encode WNVX polypeptides or biologically active portions thereof.", "Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify WNVX-encoding nucleic acids (e.g., WNVX mRNAs and cDNAs) and fragments for use as PCR primers for the amplification and/or mutation of WN nucleic acid molecules.", "As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.", "The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised of double-stranded DNA.", "A WNVX nucleic acid can encode a mature WNVX polypeptide.", "As used herein, a “mature” form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein.", "The naturally occurring polypeptide, precursor or proprotein includes, by way of non-limiting example, the full-length gene product, encoded by the corresponding gene.", "Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein.", "The product “mature” form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.", "Examples of such processing steps leading to a “mature” form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence.", "Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine.", "Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining.", "Further as used herein, a “mature” form of a polypeptide or protein may arise from a step of post-translational modification other than a. proteolytic cleavage event.", "Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation.", "In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.", "The term “probes”, as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use.", "Probes are used in the detection of identical, similar, or complementary nucleic acid sequences.", "Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes.", "Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.", "The term “isolated” nucleic acid molecule, as utilized herein, is one, which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.", "Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′- and 3′-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.", "For example, in various embodiments, the isolated WNVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.).", "Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.", "A nucleic acid molecule of the invention, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.", "Using all or a portion of the nucleic acid sequence of as a hybridization probe, WNVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.", "), MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.", "), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)", "A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.", "The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.", "Furthermore, oligonucleotides corresponding to WNVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.", "As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.", "A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.", "Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.", "In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides or a complement thereof.", "Oligonucleotides may be chemically synthesized and may also be used as probes.", "In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of a WNVX polypeptide).", "A nucleic acid molecule that is complementary to the nucleotide sequence is one that is sufficiently complementary to the nucleotide sequence that it can hydrogen bond with little or no mismatches to the nucleotide sequence, thereby forming a stable duplex.", "As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term “binding” means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof.", "Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.", "A physical interaction can be either direct or indirect.", "Indirect interactions may be through or due to the effects of another polypeptide or compound.", "Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.", "Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence.", "Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.", "Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution.", "Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains.", "Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.", "Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.", "Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.", "Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions.", "See e.g.", "Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.", "A “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.", "Homologous nucleotide sequences encode those sequences coding for isoforms of WNVX polypeptides.", "Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.", "Alternatively, isoforms can be encoded by different genes.", "Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.", "A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding WNVX protein.", "Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions, as well as a polypeptide possessing WNVX biological activity.", "Various biological activities of the WNVX proteins are described below.", "A WNVX polypeptide is encoded by the open reading frame (“ORF”) of a WNVX nucleic acid.", "An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide.", "A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon.", "An ORF that represents the coding sequence for a full protein begins with an ATG “start” codon and terminates with one of the three “stop” codons, namely, TAA, TAG, or TGA.", "For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both.", "For an ORF to be considered as a good candidate for coding for a bona fide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.", "The nucleotide sequences determined from the cloning of the WNVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning WNVX homologues in other cell types, e.g.", "from other tissues.", "The probe/primer typically comprises substantially purified oligonucleotide.", "The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence, or an anti-sense strand nucleotide sequence, or of a naturally occurring mutant.", "Probes based on the WNVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.", "In various embodiments, the probe further comprises a label group attached thereto, e.g.", "the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.", "Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express a WNVX protein, such as by measuring a level of a WNVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting WNVX mRNA levels or determining whether a genomic WNVX gene has been mutated or deleted.", "“A polypeptide having a biologically-active portion of a WNVX polypeptide” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.", "A nucleic acid fragment encoding a “biologically-active portion of WNVX” can be prepared by isolating a portion of a nucleic acid sequence that encodes a polypeptide having a WNVX biological activity (the biological activities of the WNVX proteins are described below), expressing the encoded portion of WNVX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of WNVX.", "WNVX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of the invention due to degeneracy of the genetic code and thus encode the same WNVX proteins as that encoded by the nucleotide sequences of the invention.", "In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:1-95.", "(See Table 1).", "In addition to the WNVX nucleotide sequences, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the WNVX polypeptides may exist within a population.", "Such genetic polymorphism in the WNVX genes may exist due to natural allelic variation.", "As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORP) encoding a WNVX protein, preferably a vertebrate WNVX protein.", "Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the WNVX genes.", "Any and all such nucleotide variations and resulting amino acid polymorphisms in the WNVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the WNVX polypeptides, are intended to be within the scope of the invention.", "Nucleic acid molecules corresponding to natural allelic variants and homologues of the WNVX cDNAs of the invention can be isolated based on their homology to the WNVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.", "Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid of the invention.", "In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length.", "In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region.", "As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.", "Homologs or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.", "As used herein, the phrase “stringent hybridization conditions” refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences.", "Stringent conditions are sequence-dependent and will be different in different circumstances.", "Longer sequences hybridize specifically at higher temperatures than shorter sequences.", "Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.", "The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.", "Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.", "Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60° C. for longer probes, primers and oligonucleotides.", "Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.", "Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.", "), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.", "A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65° C., followed by one or more washes in 0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequences of the invention corresponds to a naturally-occurring nucleic acid molecule.", "As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).", "In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of the invention or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided.", "A non-limiting example of moderate stringency hybridization conditions are hybridization in 6×SSC, 5× Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency that may be used are well-known within the art.", "See, e.g., Ausubel, et al.", "(eds.", "), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.", "In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of the invention or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.", "A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).", "See, e.g., Ausubel, et al.", "(eds.", "), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981.Proc Natl Acad Sci USA 78: 6789-6792.Conservative Mutations In addition to naturally-occurring allelic variants of WNVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequences of the encoded WNVX proteins, without altering the functional ability of said WNVX proteins.", "For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made.", "A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequences of the WNVX proteins without altering their biological activity, whereas an “essential” amino acid residue is required for such biological activity.", "For example, amino acid residues that are conserved among the WNVX proteins of the invention are predicted to be particularly non-amenable to alteration.", "Amino acids for which conservative substitutions can be made are well-known within the art.", "Another aspect of the invention pertains to nucleic acid molecules encoding WNVX proteins that contain changes in amino acid residues that are not essential for activity.", "Such WNVX proteins differ in amino acid sequence from SEQ ID NOS: 1-95 yet retain biological activity.", "In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences of SEQ ID NOS: 1-95.Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS: 1-95; more preferably at least about 70% homologous to SEQ ID NOS: 1-95; still more preferably at least about 80% homologous to SEQ ID NOS: 1-95; even more preferably at least about 90% homologous to SEQ ID NOS: 1-95; and most preferably at least about 95% homologous to SEQ ID NOS: 1-95.An isolated nucleic acid molecule encoding a WNVX protein homologous to the protein of SEQ ID NOS: 1-95 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.", "Mutations can be introduced into by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.", "Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues.", "A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.", "Families of amino acid residues having similar side chains have been defined within the art.", "These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).", "Thus, a predicted non-essential amino acid residue in the WNVX protein is replaced with another amino acid residue from the same side chain family.", "Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a WNVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for WNVX biological activity to identify mutants that retain activity.", "Following mutagenesis, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.", "The relatedness of amino acid families may also be determined based on side chain interactions.", "Substituted amino acids may be fully conserved “strong” residues or fully conserved “weak” residues.", "The “strong” group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, PYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other.", "Likewise, the “weak” group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SOND, SNDEQK, NDEQHK, NEQHRK, HEY, wherein the letters within each group represent the single letter amino acid code.", "In one embodiment, a mutant WNVX protein can be assayed for (i) the ability to form protein:protein interactions with other WNVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant WNVX protein and a WNVX ligand; or (iii) the ability of a mutant WNVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g.", "avidin proteins).", "In yet another embodiment, a mutant WNVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).", "WNVX Polynucleotides Encoding WNVX Candidate Peptides In one embodiment, the WNVX polynucleotides of the invention encode WNVX vaccine candidate peptides and express the WNVX vaccine candidate peptide in vitro in a host cell culture.", "The expressed WNVX vaccine candidate peptide immunogens, after suitable purification methods known to those of ordinary skill in the art, can then be incorporated into a pharmaceutical reagent or vaccine.", "Alternatively, a WNVX polynucleotide encoding a WNVX vaccine candidate peptide immunogen can be administered directly into a human as so-called “naked DNA” to express the peptide immunogen in vivo in a patient.", "(see, Cohen, Science 259:1691 (1993); Fynan et al., Proc.", "Nad.", "Acad.", "Sci.", "USA, 90:11478 (1993); and Wolff et al., BioTechniques 11:474 (1991)).", "As used herein, the term “naked DNA” refers to DNA stripped of accompanying proteins or modifications.", "“Naked DNA” further refers to DNA not encapsulated by a liposome or virus.", "A WNVX polynucleotide encoding a WNVX vaccine candidate peptide immunogen can be used for direct injection into the host.", "This results in expression of a WNVX vaccine candidate peptide by host cells and subsequent presentation to the immune system to induce anti-WNVX antibody formation in vivo.", "Determination of the sequences for the polynucleotide coding region that codes for the WNVX vaccine candidate peptides described herein can be performed using commercially available computer programs, such as DNA Strider and Wisconsin GCG or any other methods known to those skilled in the relevant arts.", "Due to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences can be constructed which encode the claimed peptides (see, Watson et al., Molecular Biology of the Gene, 436-437 (the Benjamin/Cummings Publishing Co. 1987)).", "Antisense Nucleic Acids Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence, or fragments, analogs or derivatives thereof.", "An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence).", "In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire WNVX coding strand, or to only a portion thereof.", "Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a WNVX protein of SEQ ID NOS: 1-95, or antisense nucleic acids complementary to a WNVX nucleic acid are additionally provided.", "In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding a WNVX protein.", "The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.", "In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding the WNVX protein.", "The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).", "Given the coding strand sequences encoding the WNVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.", "The antisense nucleic acid molecule can be complementary to the entire coding region of WNVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of WNVX mRNA.", "For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of WNVX mRNA.", "An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.", "An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.", "For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).", "Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.", "Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).", "The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a WNVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation).", "The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.", "An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.", "Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.", "For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens).", "The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.", "To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.", "In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule.", "An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other.", "See, e.g., Gaultier, et al., 1987.Nucl.", "Acids Res.", "15: 6625-6641.The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (see, e.g., Inoue, et al.", "1987.Nucl.", "Acids Res.", "15: 6131-6148) or a chimeric RNA-DNA analogue (see, e.g., Inoue, et al., 1987.FEBS Lett.", "215: 327-330.Ribozymes and PNA Moieties Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.", "These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.", "In one embodiment, an antisense nucleic acid of the invention is a ribozyme.", "Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.", "Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988.Nature 334: 585-591) can be used to catalytically cleave WNVX mRNA transcripts to thereby inhibit translation of WNVX mRNA.", "A ribozyme having specificity for a WNVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a WNVX cDNA disclosed herein.", "For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a WNVX-encoding MRNA.", "See, e.g., U.S. Pat.", "No.", "4,987,071 to Cech, et al.", "and U.S. Pat.", "No.", "5,116,742 to Cech, et al.", "WNVX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.", "See, e.g., Bartel et al., (1993) Science 261:1411-1418.Alternatively, WNVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the WNVX nucleic acid (e.g., the WNVX promoter and/or enhancers) to form triple helical structures that prevent transcription of the WNVX gene in target cells.", "See, e.g., Helene, 1991.Anticancer Drug Des.", "6: 569-84; Helene, et al.", "1992.Ann.", "N.Y. Acad.", "Sci.", "660: 27-36; Maher, 1992.Bioassays 14: 807-15.In various embodiments, the WNVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.", "For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids.", "See, e.g., Hyrup, et al., 1996.Bioorg Med Chem 4: 5-23.As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.", "The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.", "The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996.supra; Perry-O'Keefe, et al., 1996.Proc.", "Natl.", "Acad.", "Sci.", "USA 93: 14670-14675.PNAs of WNVX can be used in therapeutic and diagnostic applications.", "For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.", "PNAs of WNVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (see, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and hybridization (see, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996.supra).", "In another embodiment, PNAs of WNVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.", "For example, PNA-DNA chimeras of WNVX can be generated that may combine the advantageous properties of PNA and DNA.", "Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.", "PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996.supra).", "The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al., 1996.supra and Finn, et al., 1996.Nucl Acids Res 24: 3357-3363.For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA.", "See, e.g., Mag, et al., 1989.Nucl Acid Res 17; 5973-5988.PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment.", "See, e.g., Finn, et al., 1996.supra.", "Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.", "See, e.g., Petersen, et al., 1975.Bioorg.", "Med.", "Chem.", "Lett.", "5: 1119-11124.In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989.Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 86: 6553-6556; Lemaitre, et al., 1987.Proc.", "Natl.", "Acad.", "Sci.", "84: 648-652; PCT Publication No.", "W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.", "WO 89/10134).", "In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al., 1988.BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988.Pharm.", "Res.", "5: 539-549).", "To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.", "WNVX Polypeptides A polypeptide according to the invention includes a polypeptide including the amino acid sequence of WNVX polypeptides whose sequences are provided in SEQ ID NOS: 1-95.The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS: 1-95 while still encoding a protein that maintains its WNVX activities and physiological functions, or a functional fragment thereof.", "In general, a WNVX variant that preserves WNVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence.", "Any amino acid substitution, insertion, or deletion is encompassed by the invention.", "In favorable circumstances, the substitution is a conservative substitution as defined above.", "One aspect of the invention pertains to isolated WNVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof.", "Also provided are polypeptide fragments suitable for use as immunogens to raise anti-WNVX antibodies.", "In one embodiment, native WNVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.", "In another embodiment, WNVX proteins are produced by recombinant DNA techniques.", "Alternative to recombinant expression, a WNVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.", "An “isolated” or “purified” polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the WNVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.", "The language “substantially free of cellular material” includes preparations of WNVX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced.", "In one embodiment, the language “substantially free of cellular material” includes preparations of WNVX proteins having less than about 30% (by dry weight) of non-WNVX proteins (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-WNVX proteins, still more preferably less than about 10% of non-WNVX proteins, and most preferably less than about 5% of non-WNVX proteins.", "When the WNVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, ie., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the WNVX protein preparation.", "The language “substantially free of chemical precursors or other chemicals” includes preparations of WNVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein.", "In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of WNVX proteins having less than about 30% (by dry weight) of chemical precursors or non-WNVX chemicals, more preferably less than about 20% chemical precursors or non-WNVX chemicals, still more preferably less than about 10% chemical precursors or non-WNVX chemicals, and most preferably less than about 5% chemical precursors or non-WNVX chemicals.", "Biologically-active portions of WNVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the WNVX proteins (e.g., the amino acid sequence shown in SEQ ID NOS: 1-95) that include fewer amino acids than the full-length WNVX proteins, and exhibit at least one activity of a WNVX protein.", "Typically, biologically-active portions comprise a domain or motif with at least one activity of the WNVX protein.", "A biologically-active portion of a WNVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.", "Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native WNVX protein.", "In an embodiment, the WNVX protein has an amino acid sequence shown in SEQ ID NOS: 1-95.In other embodiments, the WNVX protein is substantially homologous to SEQ ID NOS: 1-95, and retains the functional activity of the protein of SEQ ID NOS: 1-95, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below.", "Accordingly, in another embodiment, the WNVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NOS: 1-95, and retains the functional activity of the WNVX proteins of SEQ ID NOS: 1-95.WNVX Peptides as Antigens The WNVX vaccine candidate peptides can be used as antigens for raising anti-WNVX immune responses, such as T cell responses (cytotoxic T cells or T helper cells).", "An “antigen” is a molecule or a portion of a molecule capable of stimulating an immune response, which is additionally capable of inducing an animal or human to produce antibody capable of binding to an epitope of that antigen.", "An “epitope” is that portion of any molecule capable of being recognized by and bound by a major histocompatability complex (“MHC”) molecule and recognized by a T cell or bound by an antibody.", "A typical antigen can have one or more than one epitope.", "The specific recognition indicates that the antigen will react, in a highly selective manner, with its corresponding MHC and T cell, or antibody and not with the multitude of other antibodies which can be evoked by other antigens.", "A peptide is “immunologically reactive” with a T cell or antibody when it binds to an MHC and is recognized by a T cell or binds to an antibody due to recognition (or the precise fit) of a specific epitope contained within the peptide.", "Immunological reactivity can be determined by measuring T cell response in vitro or by antibody binding, more particularly by the kinetics of antibody binding, or by competition in binding using known peptides containing an epitope against which the antibody or T cell response is directed as competitors.", "Techniques used to determine whether a peptide is immunologically reactive with a T cell or with an antibody are known in the art.", "Peptides can be screened for efficacy by in vitro and in vivo assays.", "Such assays employ immunization of an animal, e.g., a rabbit or a primate, with the peptide, and evaluation of titers antibody to WNVX or to synthetic detector peptides corresponding to variant WNVX sequences.", "Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.", "Determining Homology Between Two or More Sequences To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).", "The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.", "When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid “homology” is equivalent to amino acid or nucleic acid “identity”).", "The nucleic acid sequence homology may be determined as the degree of identity between two sequences.", "The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package.", "See, Needleman and Wunsch, 1970.J Mol Biol 48: 443-453.Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown.", "The term “sequence identity” refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.", "The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.", "The term “substantial identity” as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.", "Chimeric and Fusion Proteins The invention also provides WNVX chimeric or fusion proteins.", "As used herein, a WNVX “chimeric protein” or “fusion protein” comprises a WNVX polypeptide operatively-linked to a non-WNVX polypeptide.", "An “WNVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a WNVX protein (SEQ ID NOS: 1-95), whereas a “non-WNVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the WNVX protein, e.g., a protein that is different from the WNVX protein and that is derived from the same or a different organism.", "Within a WNVX fusion protein the WNVX polypeptide can correspond to all or a portion of a WNVX protein.", "In one embodiment, a WNVX fusion protein comprises at least one biologically-active portion of a WNVX protein.", "In another embodiment, a WNVX fusion protein comprises at least two biologically-active portions of a WNVX protein.", "In yet another embodiment, a WNVX fusion protein comprises at least three biologically-active portions of a WNVX protein.", "Within the fusion protein, the term “operatively-linked” is intended to indicate that the WNVX polypeptide and the non-WNVX polypeptide are fused in-frame with one another.", "The non-WNVX polypeptide can be fused to the N-terminus or C-terminus of the WNVX polypeptide.", "In one embodiment, the fusion protein is a GST-WNVX fusion protein in which the WNVX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences.", "Such fusion proteins can facilitate the purification of recombinant WNVX polypeptides.", "In another embodiment, the fusion protein is a WNVX protein containing a heterologous signal sequence at its N-terminus.", "In certain host cells (e.g., mammalian host cells), expression and/or secretion of WNVX can be increased through use of a heterologous signal sequence.", "In yet another embodiment, the fusion protein is a WNVX-immunoglobulin fusion protein in which the WNVX sequences are fused to sequences derived from a member of the immunoglobulin protein family.", "The WNVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a WNVX ligand and a WNVX protein on the surface of a cell, to thereby suppress WNVX-mediated signal transduction in vivo.", "The WNVX-immunoglobulin fusion proteins can be used to affect the bioavailability of a WNVX cognate ligand.", "Inhibition of the WNVX ligand/WNVX interaction may be useful therapeutically for both the treatment of flavivirus-associated disorders, as well as modulating (e.g.", "promoting or inhibiting) cell survival.", "Moreover, the WNVX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-WNVX antibodies in a subject, to purify WNVX ligands, and in screening assays to identify molecules that inhibit the interaction of WNVX with a WNVX ligand.", "A WNVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.", "For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.", "In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.", "Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al.", "(eds.)", "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).", "Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).", "A WNVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the WNVX protein.", "WNVX Agonists and Antagonists The invention also pertains to variants of the WNVX proteins that function as either WNVX agonists (i.e., mimetics) or as WNVX antagonists.", "Variants of the WNVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the WNVX protein).", "An agonist of the WNVX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the WNVX protein.", "An antagonist of the WNVX protein can inhibit one or more of the activities of the naturally occurring form of the WNVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the WNVX protein.", "Thus, specific biological effects can be elicited by treatment with a variant of limited function.", "In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the WNVX proteins.", "Variants of the WNVX proteins that function as either WNVX agonists (i.e., mimetics) or as WNVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the WNVX proteins for WNVX protein agonist or antagonist activity.", "In one embodiment, a variegated library of WNVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.", "A variegated library of WNVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential WNVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of WNVX sequences therein.", "There are a variety of methods which can be used to produce libraries of potential WNVX variants from a degenerate oligonucleotide sequence.", "Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.", "Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential WNVX sequences.", "Methods for synthesizing degenerate oligonucleotides are well-known within the art.", "See, e.g., Narang, 1983.Tetrahedron 39: 3; Itakura, et al., 1984.Annu.", "Rev.", "Biochem.", "53: 323; Itakura, et al., 1984.Science 198: 1056; Ike, et al., 1983.Nucl.", "Acids Res.", "11: 477.Polypeptide Libraries In addition, libraries of fragments of the WNVX protein coding sequences can be used to generate a variegated population of WNVX fragments for screening and subsequent selection of variants of a WNVX protein.", "In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a WNVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Si nuclease, and ligating the resulting fragment library into an expression vector.", "By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the WNVX proteins.", "Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property.", "Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of WNVX proteins.", "The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.", "Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify WNVX variants.", "See, e.g., Arkin and Yourvan, 1992.Proc.", "Natl.", "Acad.", "Sci.", "USA 89: 7811-7815; Delgrave, et al., 1993.Protein Engineering 6:327-331.Anti-WNVX Antibodies The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.", "Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab′ and F(ab′)2 fragments, and an Fab expression library.", "In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.", "Certain classes have subclasses as well, such as IgG1, IgG2, and others.", "Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.", "Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.", "An isolated WNVX protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.", "The full-length WNVX protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.", "A WNVX antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NOS:1-95, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.", "Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.", "Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.", "In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of WNVXX that is located on the surface of the protein, e.g., a hydrophilic region.", "A hydrophobicity analysis of the human WNVXX protein sequence will indicate which regions of a WNVX polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.", "As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.", "See, e.g., Hopp and Woods, 1981, Proc.", "Nat.", "Acad.", "Sci.", "USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.", "Biol.", "157: 105-142, each incorporated herein by reference in their entirety.", "Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.", "A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.", "Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a WNVX protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).", "Some of these antibodies are discussed below.", "1.Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native WNVX protein, a synthetic variant thereof, or a derivative of the foregoing.", "An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic WNVX protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.", "Furthermore, the WNVX protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.", "Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.", "The preparation can further include an adjuvant.", "Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.", "), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.", "Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).", "The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.", "Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography.", "Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.", "14, No.", "8 (Apr.", "17, 2000), pp.", "25-28).", "2.Monoclonal Antibodies The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.", "In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.", "MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.", "Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).", "In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.", "Alternatively, the lymphocytes can be immunized in vitro.", "The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.", "Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.", "The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.", "59-103].", "Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.", "Usually, rat or mouse myeloma cell lines are employed.", "The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.", "For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.", "Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.", "More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J.", "Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.", "51-63].", "The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.", "Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).", "Such techniques and assays are known in the art.", "The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal.", "Biochem., 107:220 (1980).", "It is an objective, especially important in therapeutic applications of monoclonal antibodies, to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.", "After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding,1986).", "Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.", "Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.", "The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.", "The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat.", "No.", "4,816,567.DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).", "The hybridoma cells of the invention serve as a preferred source of such DNA.", "Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.", "The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat.", "No.", "4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.", "Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.", "3.Humanized Antibodies The antibodies directed against the WNVX protein antigens of the invention can further comprise humanized antibodies or human antibodies.", "These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.", "Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.", "Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.", "(See also U.S. Pat.", "No.", "5,225,539.)", "In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.", "Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.", "In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.", "The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.", "Op.", "Struct.", "Biol., 2:593-596 (1992)).", "4.Human Antibodies Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes.", "Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.", "Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.", "77-96).", "Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.", "77-96).", "In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol.", "Biol., 227:381 (1991); Marks et al., J. Mol.", "Biol.", "222:581 (1991)).", "Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.", "Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.", "This approach is described, for example, in U.S. Pat.", "Nos.", "5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.", "(Bio/Technology 10, 779-783 (1992)); Lonberg et al.", "(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern.", "Rev.", "Immunol.", "13 65-93 (1995)).", "Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.", "(See PCT publication WO94/02602).", "The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.", "The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.", "An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.", "The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells which secrete fully human immunoglobulins.", "The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.", "Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.", "An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat.", "No.", "5,939,598.It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.", "A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat.", "No.", "5,916,771.It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.", "The hybrid cell expresses an antibody containing the heavy chain and the light chain.", "In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.5.Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat.", "No.", "4,946,778).", "In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.", "Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.", "6.Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.", "In the present case, one of the binding specificities is for an antigenic protein of the invention.", "The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.", "Methods for making bispecific antibodies are known in the art.", "Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).", "Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure.", "The purification of the correct molecule is usually accomplished by affinity chromatography steps.", "Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J.3 10:3655-3659 (1991).", "Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.", "The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.", "It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.", "DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.", "For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).", "According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.", "The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.", "In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.", "tyrosine or tryptophan).", "Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.", "alanine or threonine).", "This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.", "Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g.", "F(ab′)2 bispecific antibodies).", "Techniques for generating bispecific antibodies from antibody fragments have been described in the literature.", "For example, bispecific antibodies can be prepared using chemical linkage.", "Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments.", "These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.", "The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.", "One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.", "The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.", "Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.", "Shalaby et al., J. Exp.", "Med.", "175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule.", "Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.", "The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.", "Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.", "For example, bispecific antibodies have been produced using leucine zippers.", "Kostelny et al., J. Immunol.", "148(5):1547-1553 (1992).", "The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.", "The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.", "This method can also be utilized for the production of antibody homodimers.", "The “diabody” technology described by Hollinger et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.", "The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.", "Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.", "Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported.", "See, Gruber et al., J. Immunol.", "152:5368 (1994).", "Antibodies with more than two valencies are contemplated.", "For example, trispecific antibodies can be prepared.", "Tutt et al., J. Immunol.", "147:60 (1991).", "Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.", "Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.", "CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.", "Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.", "These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.", "Another bispecific antibody of interest binds the WNVX protein antigen described herein and further binds tissue factor (TF).", "7.Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.", "Heteroconjugate antibodies are composed of two covalently joined antibodies.", "Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat.", "No.", "4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).", "It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.", "For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.", "Examples of suitable reagents for this purpose include iminothiolate and methyl4mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.", "No.", "4,676,980.8.Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.", "For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.", "The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).", "See Caron et al., J. ExP Med., 176: 1191-1195 (1992) and Shopes, J.", "Immunol., 148: 2918-2922 (1992).", "Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.", "Cancer Research, 53: 2560-2565 (1993).", "Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.", "See Stevenson et al., Anti-Cancer Drug Design.", "3: 219-230 (1989).", "9.Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).", "Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.", "Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.", "A variety of radionuclides are available for the production of radioconjugated antibodies.", "Examples include 212Bi, 131i, 131In, 90Y, and 186Re.", "Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanedianiine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).", "For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).", "Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.", "See WO94/11026.In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.", "10.Immunoliposomes The antibodies disclosed herein can also be formulated as immunoliposomes.", "Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 82: 3688 (1985); Hwang et al., Proc.", "Natl Acad.", "Sci.", "USA 77: 4030 (1980); and U.S. Pat.", "Nos.", "4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat.", "No.", "5,013,556.Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).", "Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.", "Fab′ fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al ., J. Biol.", "Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.", "A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome.", "See Gabizon et al., J.", "National Cancer Inst., 81(19): 1484 (1989).", "11.Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention Antibodies directed against a VWNVX protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of the protein (e.g., for use in measuring levels of the protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).", "In a given embodiment, antibodies against the WNVX proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antigen binding domain, are utilized as pharmacologically-active compounds (see below).", "An antibody specific for a WNVX protein of the invention can be used to isolate the protein by standard techniques, such as immunoaffinity chromatography or immunoprecipitation.", "Such an antibody can facilitate the purification of the natural WNVX protein antigen from cells and of recombinantly produced antigen expressed in host cells.", "Moreover, such an antibody can be used to detect the antigenic protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic protein.", "Antibodies directed against the WNVX protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.", "Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.", "Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.", "Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131i, 35s or 3H.", "12.Antibody Therapeutics Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may used as therapeutic agents.", "Such agents will generally be employed to treat or prevent a disease or pathology in a subject.", "An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target.", "Such an effect may be one of two kinds, depending on the specific nature of the interaction between the given antibody molecule and the target antigen in question.", "In the first instance, administration of the antibody may abrogate or inhibit the binding of the target with an endogenous ligand to which it naturally binds.", "In this case, the antibody binds to the target and masks a binding site of the naturally occurring ligand, wherein the ligand serves as an effector molecule.", "Thus the receptor mediates a signal transduction pathway for which ligand is responsible.", "Alternatively, the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule.", "In this case the target, a receptor having an endogenous ligand which may be absent or defective in the disease or pathology, binds the antibody as a surrogate effector ligand, initiating a receptor-based signal transduction event by the receptor.", "A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective.", "As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response.", "The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered.", "Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight.", "Common dosing frequencies may range, for example, from twice daily to once a week.", "13.Pharmaceutical Compositions of Antibodies Antibodies specifically binding a WNVX protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions.", "Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed.", "(Alfonso R. Gennaro, et al., editors) Mack Pub.", "Co., Easton, Pa: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol.", "4), 1991, M. Dekker, New York.", "If the antigenic WNVX protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred.", "However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells.", "Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.", "For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence.", "Such peptides can be synthesized chemically and/or produced by recombinant DNA technology.", "See, e.g., Marasco et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 90: 7889-7893 (1993).", "The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.", "Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.", "Such molecules are suitably present in combination in amounts that are effective for the purpose intended.", "The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.", "The formulations to be used for in vivo administration must be sterile.", "This is readily accomplished by filtration through sterile filtration membranes.", "Sustained-release preparations can be prepared.", "Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.", "Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat.", "No.", "3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.", "While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.", "14.ELISA Assay An agent for detecting an analyte protein is an antibody capable of binding to an analyte protein, preferably an antibody with a detectable label.", "Antibodies can be polyclonal, or more preferably, monoclonal.", "An intact antibody, or a fragment thereof (e.g., Fab or F(ab)2) can be used.", "The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.", "Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.", "The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.", "Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph.", "That is, the detection method of the invention can be used to detect an analyte MRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.", "For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations.", "In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.", "In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations.", "Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol.", "42, J. R. Crowther (Ed.)", "Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and “Practice and Thory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-an analyte protein antibody.", "For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.", "WNVX Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a WNVX protein, or derivatives, fragments, analogs or homologs thereof.", "As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.", "One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.", "Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.", "Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).", "Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.", "Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked.", "Such vectors are referred to herein as “expression vectors”.", "In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.", "In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.", "However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.", "The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.", "Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).", "The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals).", "Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).", "Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).", "It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.", "The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., WNVX proteins, mutant forms of WNVX proteins, fusion proteins, etc.).", "The recombinant expression vectors of the invention can be designed for expression of WNVX proteins in prokaryotic or eukaryotic cells.", "For example, WNVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells.", "Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).", "Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.", "Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins.", "Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.", "Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.", "Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.", "Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.", "Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.)", "and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.", "Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).", "One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein.", "See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128.Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al., 1992.Nucl.", "Acids Res.", "20: 2111-2118).", "Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.", "In another embodiment, the WNVX expression vector is a yeast expression vector.", "Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987.EMBO J.", "6: 229-234), pMFa (Kurjan and Herskowitz, 1982.Cell 30: 933-943), pJRY88 (Schultz et al., 1987.Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).", "Alternatively, WNVX can be expressed in insect cells using baculovirus expression vectors.", "Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983.Mol.", "Cell.", "Biol.", "3: 2156-2165) and the pVL series (Lucklow and Summers, 1989.Virology 170: 31-39).", "In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.", "Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987.EMBO J.", "6: 187-195).", "When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements.", "For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL.", "2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).", "Tissue-specific regulatory elements are known in the art.", "Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987.Genes Dev.", "1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988.Adv.", "Immunol.", "43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989.EMBO J.", "8: 729-733) and immunoglobulins (Banetji, et al., 1983.Cell 33: 729-740; Queen and Baltimore, 1983.Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989.Proc.", "Natl.", "Acad.", "Sci.", "USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat.", "No.", "4,873,316 and European Application Publication No.", "264,166).", "Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989.Genes Dev.", "3: 537-546).", "The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation.", "That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to WNVX mRNA.", "Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.", "The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.", "For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., “Antisense RNA as a molecular tool for genetic analysis,” Reviews-Trends in Genetics, Vol.", "1(1) 1986.Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced.", "The terms “host cell” and “recombinant host cell” are used interchangeably herein.", "It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell.", "Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.", "A host cell can be any prokaryotic or eukaryotic cell.", "For example, WNVX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).", "These hosts can be used in connection with poxvirus vectors, such as vaccinia or swinepox.", "Suitable non-pathogenic viruses, which can be engineered to carry the synthetic gene into the cells of the host include poxviruses, such as vaccinia, adenovirus, retroviruses and the like.", "A number of such non-pathogenic viruses are commonly used for human gene therapy, and as carrier for other vaccine agents, and are known and selectable by one of skill in the art.", "Another preferred system includes the baculovirus expression system and vectors.", "Other suitable host cells are known to those skilled in the art.", "Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.", "As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.", "Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al.", "(MOLECULAR CLONING: A LABORATORY MANUAL.", "2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.", "For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome.", "In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.", "Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.", "Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding WNVX or can be introduced on a separate vector.", "Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).", "A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) WNVX protein.", "Accordingly, the invention further provides methods for producing WNVX protein using the host cells of the invention.", "In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding WNVX protein has been introduced) in a suitable medium such that WNVX protein is produced.", "In another embodiment, the method further comprises isolating WNVX protein from the medium or the host cell.", "Transgenic WNVX Animals The host cells of the invention can also be used to produce non-human transgenic animals.", "For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which WNVX protein-coding sequences have been introduced.", "Such host cells can then be used to create non-human transgenic animals in which exogenous WNVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous WNVX sequences have been altered.", "Such animals are useful for studying the function and/or activity of WNVX protein and for identifying and/or evaluating modulators of WNVX protein activity.", "As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.", "Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.", "A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.", "As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous WNVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.", "A transgenic animal of the invention can be created by introducing WNVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal.", "The WNVX cDNA sequences of the invention can be introduced as a transgene into the genome of a non-human animal.", "Alternatively, a non-human homologue of the WNVX gene, such as a mouse WNVX gene, can be isolated based on hybridization to the WNVX cDNA (described further supra) and used as a transgene.", "Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.", "A tissue-specific regulatory sequence(s) can be operably-linked to the WNVX transgene to direct expression of WNVX protein to particular cells.", "Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat.", "Nos.", "4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986.In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.", "Similar methods are used for production of other transgenic animals.", "A transgenic founder animal can be identified based upon the presence of the WNVX transgene in its genome and/or expression of WNVX mRNA in tissues or cells of the animals.", "A transgenic founder animal can then be used to breed additional animals carrying the transgene.", "Moreover, transgenic animals carrying a transgene-encoding WNVX protein can further be bred to other transgenic animals carrying other transgenes.", "To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a WNYX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the WNVX gene.", "In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous WNVX gene is functionally disrupted (ie., no longer encodes a functional protein; also referred to as a “knock out” vector).", "Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous WNVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous WNVX protein).", "In the homologous recombination vector, the altered portion of the WNVX gene is flanked at its 5′- and 3′-termini by additional nucleic acid of the WNVX gene to allow for homologous recombination to occur between the exogenous WNVX gene carried by the vector and an endogenous WNVX gene in an embryonic stem cell.", "The additional flanking WNVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.", "Typically, several kilobases of flanking DNA (both at the 5′- and 3′-termini) are included in the vector.", "See, e.g., Thomas, et al., 1987.Cell 51: 503 for a description of homologous recombination vectors.", "The vector is then introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced WNVX gene has homologously-recombined with the endogenous WNVX gene are selected.", "See, e.g., Li, et al., 1992.Cell 69: 915.The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras.", "See, e.g., Bradley, 1987.In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed.", "IRL, Oxford, pp.", "113-152.A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.", "Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene.", "Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991.Curr.", "Opin.", "Biotechnol.", "2: 823-829; PCT International Publication Nos.", ": WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene.", "One example of such a system is the cre/loxP recombinase system of bacteriophage P1.For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992.Proc.", "Natl.", "Acad.", "Sci.", "USA 89: 6232-6236.Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae.", "See, O'Gorman, et al., 1991.Science 251:1351-1355.If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.", "Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.", "Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al., 1997.Nature 385: 810-813.In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase.", "The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.", "The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.", "The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.", "Pharmaceutical Compositions The WNVX nucleic acid molecules, WNVX proteins, and anti-WNVX antibodies (also referred to herein as “active compounds”) of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration.", "Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.", "As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.", "Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.", "Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin.", "Liposomes and non-aqueous vehicles such as fixed oils may also be used.", "The use of such media and agents for pharmaceutically active substances is well known in the art.", "Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.", "Supplementary active compounds can also be incorporated into the compositions.", "A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.", "Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.", "Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.", "The pH can-be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.", "The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.", "Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.", "For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).", "In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists.", "It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.", "The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.", "The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.", "Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.", "In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.", "Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.", "Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a WNVX protein or anti-WNVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.", "Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.", "In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.", "Oral compositions generally include an inert diluent or an edible carrier.", "They can be enclosed in gelatin capsules or compressed into tablets.", "For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.", "In one embodiment, the active compounds may be incorporated into lypoamino acid conjugates as described in Toth, et al., J.", "Drug Target, 2:217-239 (1994).", "Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.", "Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.", "The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.", "For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.", "Systemic administration can also be by transmucosal or transdermal means.", "For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.", "Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.", "Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.", "For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.", "The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.", "In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.", "Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.", "Methods for preparation of such formulations will be apparent to those skilled in the art.", "The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.", "These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat.", "No.", "4,522,811.It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.", "Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.", "The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.", "The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.", "Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat.", "No.", "5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994.Proc.", "Natl.", "Acad.", "Sci.", "USA 91: 3054-3057).", "The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.", "Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.", "The vaccine composition can include as the active agents, one of the following components: (a) an WNVX vaccine candidate peptide immunogen, which can be in the form of recombinant proteins or, alternatively, can be in the form of a mixture of carrier protein conjugates; (b) a polynucleotide encoding a WNVX vaccine candidate; (c) a recombinant virus carrying the synthetic gene or molecule; and (d) a bacteria carrying the WNVX vaccine candidate.", "The selected active component may be present in a pharmaceutically acceptable carrier, and the vaccine composition can also contain additional ingredients.", "Formulations containing the WNVX vaccine candidate peptide may also contain other active agents, including, but not limited to, such as adjuvants and immunostimulatory cytokines, such as IL-12 and other well-known cytokines, for the vaccine compositions.", "WNVX vaccine candidate peptide immunogens may be linked to a suitable carrier in order to improve the efficacy of antigen presentation to the immune system.", "Such carriers can be, for instance, organic polymers.", "A carrier protein can enhance the immunogenicity of the peptide immunogen.", "Such a carrier can be a larger molecule, which has an adjuvant effect.", "Exemplary conventional protein carriers include, keyhole limpet hemocyan, E. coli DnaK protein, galactokinase (galK, which catalyzes the first step of galactose metabolism in bacteria), ubiquitin, α-mating factor, β-galactosidase, and influenza NS-1 protein.", "Toxoids (i.e., nucleic acid sequences which encode the naturally occurring toxin, with sufficient modifications to eliminate its toxic activity) such as diphtheria toxoid and tetanus toxoid can also be employed as carriers.", "Similarly, a variety of bacterial heat shock proteins, e.g., mycobacterial hsp-70, can be used.", "Glutathione reductase (GST) is another useful carrier.", "One of skill in the art can readily determine and select an appropriate carrier.", "Viruses can be modified by recombinant DNA technology such as, e.g.", "rhinovirus, poliovirus, vaccinia, or influenzavirus, etc.", "The peptide can be linked to a modified, i.e., attenuated or recombinant virus such as modified influenza virus or modified hepatitis B virus or to parts of a virus, e.g., to a viral glycoprotein such as, e.g., hemagglutinin of influenza virus or surface antigen of hepatitis B virus, in order to increase the immunological response against WNVX-infected cells.", "Other antigen carrier systems may also be used to enhance immunogenicity.", "In one embodiment, the immunogens of the invention are incorporated into a multi-peptide conjugate (MPC) system, where the immunogens are synthesized and coupled to a core peptide template using known methods of peptide synthesis and solution chemistry.", "See e.g., Boykins, et al., Peptides 21:9-17 (2000).", "In another embodiment, the immunogens are incorporated into a Multiple Antigen Peptide System (MAPS) as described by Tam in U.S. Pat.", "No.", "5,229,490.Where the polynucleotides of the invention are naked DNA vaccines, suitable vehicles for direct DNA, plasmid polynucleotide, or recombinant vector administration include, without limitation, saline, sucrose, protamine, polybrene, polylysine, polycations, proteins, calcium phosphate, or spermidine.", "See e.g, PCT International patent application WO 94/01139.As with the immunogenic compositions, the amounts of components in the DNA and vector compositions and the mode of administration, e.g., injection or intranasal, can be selected and modified by one of skill in the art.", "Generally, each dose will comprise between about 50 μg to about 1 mg of immunogen-encoding DNA per ml of a sterile solution.", "For recombinant viruses containing the coding polynucleotide, the doses can range from about 20 to about 50 ml of saline solution containing concentrations of from about 1×107 to 1×1010 pfu/ml recombinant virus of the invention.", "One suitable human dosage is about 20 ml saline solution at the above concentrations.", "However, it is understood that one of skill in the art can alter such dosages depending upon the identity of the recombinant virus and the make-up of the immunogen that it is being delivered to the host.", "The amounts of the commensal bacteria carrying the synthetic gene or molecules to be delivered to the patient will generally range between about 103 to about 1012 cells/kg.", "These dosages, will of course, be altered by one of skill in the art depending upon the bacterium being used and the particular composition containing immunogens being delivered by the live bacterium.", "The pharmaceutical and vaccine compositions of the invention can be included in a container, pack, or dispenser together with instructions for administration.", "Screening and Detection Methods The isolated nucleic acid molecules of the invention can be used to express WNVX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect WNVX mRNA (e.g., in a biological sample) or a genetic lesion in a WNVX gene, and to modulate WNVX activity, as described further, below.", "In addition, the WNVX proteins can be used to screen drugs or compounds that modulate the WNVX protein activity or expression.", "In addition, the anti-WNVX antibodies of the invention can be used to detect and isolate WNVX proteins and modulate WNVX activity.", "The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.", "Screening Assays The invention provides a method (also referred to herein as a “screening assay”) for identifying modulators, ie., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to WNVX proteins or have a stimulatory or inhibitory effect on, e.g., WNVX protein expression or WNVX protein activity.", "The invention also includes compounds identified in the screening assays described herein.", "In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a WNVX protein or polypeptide or biologically-active portion thereof.", "The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.", "The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds.", "See, e.g., Lam, 1997.Anticancer Drug Design 12: 145.A “small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.", "Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.", "Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.", "Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993.Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 90: 6909; Erb, et al., 1994.Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 91: 11422; Zuckermann, et al., 1994.J.", "Med.", "Chem.", "37: 2678; Cho, et al., 1993.Science 261: 1303; Carrell, et al., 1994.Angew.", "Chem.", "Int.", "Ed.", "Engl.", "33: 2059; Carell, et al., 1994.Angew.", "Chem.", "Int.", "Ed.", "Engl.", "33: 2061; and Gallop, et al., 1994.J.", "Med.", "Chem.", "37:1233.Libraries of compounds may be presented in solution (e.g., Houghten, 1992.Biotechniques 13: 412-421), or on beads (Lam, 1991.Nature 354: 82-84), on chips (Fodor, 1993.Nature 364: 555-556), bacteria (Ladner, U.S. Pat.", "No.", "5,223,409), spores (Ladner, U.S. Pat.", "No.", "5,233,409), plasmids (Cull, et al., 1992.Proc.", "Natl.", "Acad.", "Sci.", "USA 89: 1865-1869) or on phage (Scott and Smith, 1990.Science 249: 386-390; Devlin, 1990.Science 249: 404-406;.Cwirla, et al., 1990.Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 87: 6378-6382; Felici, 1991.J.", "Mol.", "Biol.", "222: 301-310; Ladner, U.S. Pat.", "No.", "5,233,409.).", "In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of WNVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a WNVX protein determined.", "The cell, for example, can of mammalian origin or a yeast cell.", "Determining the ability of the test compound to bind to the WNVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the WNVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex.", "For example, test compounds can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.", "Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.", "In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of WNVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds WNVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a WNVX protein, wherein determining the ability of the test compound to interact with a WNVX protein comprises determining the ability of the test compound to preferentially bind to WNVX protein or a biologically-active portion thereof as compared to the known compound.", "In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of WNVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the WNVX protein or biologically-active portion thereof.", "Determining the ability of the test compound to modulate the activity of WNVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the WNVX protein to bind to or interact with a WNVX target molecule.", "As used herein, a “target molecule” is a molecule with which a WNVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a WNVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.", "A WNVX target molecule can be a non-WNVX molecule or a WNVX protein or polypeptide of the invention.", "In one embodiment, a WNVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extraceliular signal (e.g.", "a signal generated by binding of a compound to a membrane-bound WNVX molecule) through the cell membrane and into the cell.", "The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with WNVX.", "Determining the ability of the WNVX protein to bind to or interact with a WNVX target molecule can be accomplished by one of the methods described above for determining direct binding.", "In one embodiment, determining the ability of the WNVX protein to bind to or interact with a WNVX target molecule can be accomplished by determining the activity of the target molecule.", "For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (ie.", "intracellular Ca2+, diacylglycerol, IP3, etc.", "), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a WNVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.", "In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting a WNVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the WNVX protein or biologically-active portion thereof Binding of the test compound to the WNVX protein can be determined either directly or indirectly as described above.", "In one such embodiment, the assay comprises contacting the WNVX protein or biologically-active portion thereof with a known compound which binds WNVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a WNVX protein, wherein determining the ability of the test compound to interact with a WNVX protein comprises determining the ability of the test compound to preferentially bind to WNVX or biologically-active portion thereof as compared to the known compound.", "In still another embodiment, an assay is a cell-free assay comprising contacting WNVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g.", "stimulate or inhibit) the activity of the WNVX protein or biologically-active portion thereof.", "Determining the ability of the test compound to modulate the activity of WNVX can be accomplished, for example, by determining the ability of the WNVX protein to bind to a WNVX target molecule by one of the methods described above for determining direct binding.", "In an alternative embodiment, determining the ability of the test compound to modulate the activity of WNVX protein can be accomplished by determining the ability of the WNVX protein further modulate a WNVX target molecule.", "For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra.", "In yet another embodiment, the cell-free assay comprises contacting the WNVX protein or biologically-active portion thereof with a known compound which binds WNVX protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a WNVX protein, wherein determining the ability of the test compound to interact with a WNVX protein comprises determining the ability of the WNVX protein to preferentially bind to or modulate the activity of a WNVX target molecule.", "The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of WNVX protein.", "In the case of cell-free assays comprising the membrane-bound form of WNVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of WNVX protein is maintained in solution.", "Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).", "In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either WNVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.", "Binding of a test compound to WNVX protein, or interaction of WNVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants.", "Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.", "In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix.", "For example, GST-WNVX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)", "or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or WNVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).", "Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra.", "Alternatively, the complexes can be dissociated from the matrix, and the level of WNVX protein binding or activity determined using standard techniques.", "Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention.", "For example, either the WNVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.", "Biotinylated WNVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).", "Alternatively, antibodies reactive with WNVX protein or target molecules, but which do not interfere with binding of the WNVX protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or WNVX protein trapped in the wells by antibody conjugation.", "Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the WNVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the WNVX protein or target molecule.", "In another embodiment, modulators of WNVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of WNVX mRNA or protein in the cell is determined.", "The level of expression of WNVX mRNA or protein in the presence of the candidate compound is compared to the level of expression of WNVX mRNA or protein in the absence of the candidate compound.", "The candidate compound can then be identified as a modulator of WNVX MRNA or protein expression based upon this comparison.", "For example, when expression of WNVX mRNA or protein is greater (Le., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of WNVX MRNA or protein expression.", "Alternatively, when expression of WNVX mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of WNVX mRNA or protein expression.", "The level of WNVX mRNA or protein expression in the cells can be determined by methods described herein for detecting WNVX MRNA or protein.", "In yet another aspect of the invention, the WNVX proteins can be used as “bait proteins” in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat.", "No.5,283,317; Zervos, et al., 1993.Cell 72: 223-232; Madura, et al., 1993.J.", "Biol.", "Chem.", "268: 12046-12054; Bartel, et al., 1993.Biotechniques 14: 920-924; Iwabuchi, et al., 1993.Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with WNVX (“WNVX-binding proteins” or “WNVX-bp”) and modulate WNVX activity.", "Such WNVX-binding proteins are also likely to be involved in the propagation of signals by the WNVX proteins as, for example, upstream or downstream elements of the WNVX pathway.", "The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.", "Briefly, the assay utilizes two different DNA constructs.", "In one construct, the gene that codes for WNVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).", "In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor.", "If the “bait” and the “prey” proteins are able to interact, in vivo, forming a WNVX-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity.", "This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor.", "Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with WNVX.", "The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.", "Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents.", "By way of example, and not of limitation, these sequences can be used to: (i) locate gene regions associated with viral susceptibility; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample.", "Some of these applications are described in the subsections, below.", "Predictive Medicine The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.", "Accordingly, one aspect of the invention relates to diagnostic assays for determining WNVX protein and/or nucleic acid expression as well as WNVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with WNVX infection.", "The disorders include flavivirus disorders such as St. Louis Encephalitis, Japanese Encephalitis, Hepatitis C, and Dengue.", "The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with WNVX protein, nucleic acid expression or activity.", "For example, mutations in a WNVX gene can be assayed in a biological sample.", "Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with WNVX protein, nucleic acid expression, or biological activity.", "Another aspect of the invention provides methods for determining WNVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as “pharmacogenomics”).", "Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.)", "Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the activity of WNVX in clinical trials.", "These and other agents are described in further detail in the following sections.", "Diagnostic Assays An exemplary method for detecting the presence or absence of WNVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting WNVX protein or nucleic acid (e.g., MRNA, genomic DNA) that encodes WNVX protein such that the presence of WNVX is detected in the biological sample.", "An agent for detecting WNVX MRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to WNVX mRNA or genomic DNA.", "The nucleic acid probe can be, for example, a fill-length WNVX nucleic acid, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to WNVX mRNA or genomic DNA.", "Other suitable probes for use in the diagnostic assays of the invention are described herein.", "An agent for detecting WNVX protein is an antibody capable of binding to WNVX protein, preferably an antibody with a detectable label.", "Antibodies can be polyclonal, or more preferably, monoclonal.", "An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used.", "The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (ie., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.", "Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.", "The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.", "That is, the detection method of the invention can be used to detect WNVX “mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.", "For example, in vitro techniques for detection of WNVX mRNA include Northern hybridizations and in situ hybridizations.", "In vitro techniques for detection of WNVX protein include enzyme linked immunosorbent assays (EUSAs), Western blots, immunoprecipitations, and immunofluorescence.", "In vitro techniques for detection of WNVX genomic DNA include Southern hybridizations.", "Furthermore, in vivo techniques for detection of WNVX protein include introducing into a subject a labeled anti-WNVX antibody.", "For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.", "In one embodiment, the biological sample contains protein molecules from the test subject.", "Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.", "A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.", "In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting WNVX protein, mRNA, or genomic DNA, such that the presence of WNVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of WNVX protein, mRNA or genomic DNA in the control sample with the presence of WNVX protein, mRNA or genomic DNA in the test sample.", "The invention also encompasses kits for detecting the presence of WNVX in a biological sample.", "For example, the kit can comprise: a labeled compound or agent capable of detecting WNVX protein or MRNA in a biological sample; means for determining the amount of WNVX in the sample; and means for comparing the amount of WNVX in the sample with a standard.", "The compound or agent can be packaged in a suitable container.", "The kit can further comprise instructions for using the kit to detect WNVX protein or nucleic acid.", "Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects susceptible to WNVX infection.", "For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with WNVX protein, nucleic acid expression or activity.", "Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder.", "Thus, the invention provides a method for identifying a disease or disorder associated with WNVX infection in which a test sample is obtained from a subject and WNVX protein or nucleic acid (e.g., MRNA, genomic DNA) is detected, wherein the presence of WNVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with WNVX.", "As used herein, a “test sample” refers to a biological sample obtained from a subject of interest.", "For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.", "Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with WNVX infection.", "For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder.", "Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with WNVX infection in which a test sample is obtained and WNVX protein or nucleic acid is detected (e.g., wherein the presence of WNVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with WNVX infection).", "The methods of the invention can also be used to detect genetic lesions in a WNVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation.", "In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a WNVX-protein, or the misexpression of the WNVX gene.", "For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from a WNVX gene; (ii) an addition of one or more nucleotides to a WNVX gene; (iii) a substitution of one or more nucleotides of a WNVX gene, (iv) a chromosomal rearrangement of a WNVX gene; (v) an alteration in the level of a messenger RNA transcript of a WNVX gene, (vi) aberrant modification of a WNVX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of a WNVX gene, (viii) a non-wild-type level of a WNVX protein, (ix) allelic loss of a WNVX gene, and (x) inappropriate post-translational modification of a WNVX protein.", "As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a WNVX gene.", "A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.", "However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.", "In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.", "Nos.", "4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al., 1988.Science 241: 1077-1080; and Nakazawa, et al., 1994.Proc.", "Natl.", "Acad.", "Sci.", "USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the WNVX-gene (see, Abravaya, et al., 1995.Nucl.", "Acids Res.", "23: 675-682).", "This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, MRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to a WNVX gene under conditions such that hybridization and amplification of the WNVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.", "It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.", "Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990.Proc.", "Natl.", "Acad.", "Sci.", "USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al., 1989.Proc.", "Natl.", "Acad.", "Sci.", "USA 86: 1173-1177); Qβ Replicase (see, Lizardi, et al, 1988.BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art.", "These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.", "In an alternative embodiment, mutations in a WNVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.", "For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared.", "Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.", "Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.", "No.", "5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.", "In other embodiments, genetic mutations in WNVX nucleic acids can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes.", "See, e.g., Cronin, et al., 1996.Human Mutation 7: 244-255; Kozal, et al., 1996.Nat.", "Med.", "2: 753-759.For example, genetic mutations in WNVX nucleic acids can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al., supra.", "Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes.", "This step allows the identification of point mutations.", "This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.", "Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.", "In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the WNVX gene and detect mutations by comparing the sequence of the sample WNVX with the corresponding wild-type (control) sequence.", "Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977.Proc.", "Natl.", "Acad.", "Sci.", "USA 74: 560 or Sanger, 1977.Proc.", "Natl.", "Acad.", "Sci.", "USA 74: 5463.It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al., 1995.Diotechniques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No.", "WO 94/16101; Cohen, et al., 1996.Adv.", "Chromatography 36: 127-162; and Griffin, et al., 1993.Appl.", "Biochem.", "Biotechnol.", "38: 147-159).", "Other methods for detecting mutations in the WNVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes.", "See, e.g., Myers, et al., 1985.Science 230: 1242.In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type WNVX sequence with potentially mutant RNA or DNA obtained from a tissue sample.", "The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.", "For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions.", "In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions.", "After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation.", "See, e.g., Cotton, et al., 1988.Proc.", "Natl.", "Acad.", "Sci.", "USA 85: 4397; Saleeba, et al., 1992.Methods Enzymol.", "217: 286-295.In an embodiment, the control DNA or RNA can be labeled for detection.", "In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in WNVX cDNAs obtained from samples of cells.", "For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches.", "See, e.g., Hsu, et al., 1994.Carcinogenesis 15: 1657-1662.According to an exemplary embodiment, a probe based on a WNVX sequence, e.g., a wild-type WNVX sequence, is hybridized to a cDNA or other DNA product from a test cell(s).", "The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like.", "See, e.g., U.S. Pat.", "No.", "5,459,039.In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in WNVX genes.", "For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids.", "See, e.g., Orita, et al., 1989.Proc.", "Nat].", "Acad.", "Sci.", "USA: 86: 2766; Cotton, 1993.Mutat.", "Res.", "285: 125-144; Hayashi, 1992.Genet.", "Anal.", "Tech.", "Appl.", "9: 73-79.Single-stranded DNA fragments of sample and control WNVX nucleic acids will be denatured and allowed to renature.", "The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.", "The DNA fragments may be labeled or detected with labeled probes.", "The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.", "In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility.", "See, e.g., Keen, et al., 1991.Trends Genet.", "7: 5.In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE).", "See, e.g., Myers, et al., 1985.Nature 313: 495.When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.", "In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA.", "See, e.g., Rosenbaum and Reissner, 1987.Biophys.", "Chem.", "265: 12753.Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension.", "For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found.", "See, e.g., Saild, et al., 1986.Nature 324: 163; Saiki, et al., 1989.Proc.", "Natl.", "Acad.", "Sci.", "USA 86: 6230.Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.", "Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention.", "Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al., 1989.Nucl.", "Acids Res.", "17: 2437-2448) or at the extreme 3′-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993.Tibtech.", "11: 238).", "In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection.", "See, e.g., Gasparini, et al., 1992.Mol.", "Cell Probes 6: 1.It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification.", "See, e.g., Barany, 1991.Proc.", "Natl.", "Acad.", "Sci.", "USA 88: 189.In such cases, ligation will occur only if there is a perfect match at the 3′-terminus of the 5′ sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.", "The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a WNVX gene.", "Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which WNVX is expressed may be utilized in the prognostic assays described herein.", "However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.", "Pharmacogenomics Agents, or modulators that have a stimulatory or inhibitory effect on WNVX activity (e.g., WNVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include flavivirus associated disorders.", "In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered.", "Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.", "Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype.", "Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens.", "Accordingly, the activity of WNVX protein, expression of WNVX nucleic acid, or mutation content of WNVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.", "Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons.", "See e.g., Eichelbaum, 1996.Clin.", "Exp.", "Pharmacol.", "Physiol., 23: 983-985; Linder, 1997.Clin.", "Chem., 43: 254-266.In general, two types of pharmacogenetic conditions can be differentiated.", "Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism).", "These pharmacogenetic conditions can occur either as rare defects or as polymorphisms.", "For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.", "As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.", "The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug.", "These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM).", "The prevalence of PM is different among different populations.", "For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6.Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses.", "If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine.", "At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses.", "Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.", "Thus, the activity of VWNVX protein, expression of WNVX nucleic acid, or mutation content of WNVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.", "In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype.", "This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a WNVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.", "Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of WNVX (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials.", "For example, the effectiveness of an agent determined by a screening assay as described herein to increase WNVX gene expression, protein levels, or upregulate WNVX activity, can be monitored in clinical trails of subjects exhibiting decreased WNVX gene expression, protein levels, or downregulated WNVX activity.", "Alternatively, the effectiveness of an agent determined by a screening assay to decrease WNVX gene expression, protein levels, or downregulate WNVX activity, can be monitored in clinical trails of subjects exhibiting increased WNVX gene expression, protein levels, or upregulated WNVX activity.", "In such clinical trials, the expression or activity of WNVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a “read out” or markers of the immune responsiveness of a particular cell.", "By way of example, and not of limitation, genes, including WNVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates WNVX activity (e.g., identified in a screening assay as described herein) can be identified.", "Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of WNVX and other genes implicated in the disorder.", "The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of WNVX or other genes.", "In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent.", "Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.", "In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a WNVX protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the WNVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the WNVX protein, MRNA, or genomic DNA in the pre-administration sample with the WNVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.", "For example, increased administration of the agent may be desirable to increase the expression or activity of WNVX to higher levels than detected, i.e., to increase the effectiveness of the agent.", "Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of WNVX to lower levels than detected, i.e., to decrease the effectiveness of the agent.", "Methods of Treatment The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant WNVX expression or activity.", "The disorders include flavivirus-related disorders.", "These methods of treatment will be discussed more fully, below.", "Disease and Disorders Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity.", "Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner.", "Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to “knockout” endoggenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989.Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.", "Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (Le., are agonists to) activity.", "Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner.", "Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.", "Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide).", "Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.)", "and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).", "Prophylactic Methods In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant WNVX expression or activity, by administering to the subject an agent that modulates WNVX expression or at least one WNVX activity.", "Subjects at risk for a disease that is caused or contributed to by aberrant WNVX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.", "Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the WNVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.", "Depending upon the type of WNVX aberrancy, for example, a WNVX agonist or WNVX antagonist agent can be used for treating the subject.", "The appropriate agent can be determined based on screening assays described herein.", "The prophylactic methods of the invention are further discussed in the following subsections.", "Therapeutic Methods Another aspect of the invention pertains to methods of modulating WNVX expression or activity for therapeutic purposes.", "The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of WNVX protein activity associated with the cell.", "An agent that modulates WNVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a WNVX protein, a peptide, a WNVX peptidomimetic, or other small molecule.", "In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) WNVX expression or activity.", "In another embodiment, the method involves administering a WNVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant WNVX expression or activity.", "In another embodiment, the agent inhibits one or more WNVX protein activity.", "Examples of such inhibitory agents include antisense WNVX nucleic acid molecules and anti-WNVX antibodies.", "These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).", "As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a WNVX protein or nucleic acid molecule.", "The method for reducing the levels of WNVX involves exposing a human to a WNVX vaccine candidate peptides, actively inducing antibodies that react with WNVX, and impairing the multiplication of WNVX in vivo.", "This method is appropriate for an WNVX infected subject with a competent immune system, or an uninfected or recently infected subject.", "The method induces antibodies, which react with WNVX, which reduces multiplication during any initial acute infection with WNVX.", "The terms “treating,” “treatment,” and the like are used herein to mean obtaining a desired pharmacologic or physiologic effect.", "The effect can be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, or can be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.", "“Treating” as used herein covers any treatment and includes: (a) preventing a disorder from occurring in a subject that can be predisposed to a disorder, but has not yet been diagnosed as having it; (b) inhibiting the disorder, i.e., arresting its development; or (c) relieving or ameliorating the disorder.", "An “effective amount” or “therapeutically effective amount” is the amount sufficient to obtain the desired physiological effect.", "An effective amount of the WNVX vaccine candidate peptide or vector expressing WNVX vaccine candidate peptides is generally determined by the physician in each case on the basis of factors normally considered by one skilled in the art to determine appropriate dosages, including the age, sex, and weight of the subject to be treated, the condition being treated, and the severity of the medical condition being treated.", "Among such patients suitable for treatment with this method are WNVX infected patients.", "Method of Administration WNVX vaccine candidate peptides can be administered in a variety of ways, orally, topically, parenterally e.g.", "subcutaneously, intraperitoneally, by viral infection, intravascularly, etc.", "Depending upon the manner of introduction, the WNVX vaccine candidate peptides can be formulated in a variety of ways.", "The concentration of WNVX vaccine candidate peptides in the formulation can vary from about 0.1-100 wt.", "%.", "The amount of the WNVX vaccine candidate peptide or polynucleotides of the invention present in each vaccine dose is selected with regard to consideration of the patient's age, weight, sex, general physical condition and the like.", "The amount of WNVX vaccine candidate peptide required to induce an immune response, preferably a protective response, or produce an exogenous effect in the patient without significant adverse side effects varies depending upon the pharmaceutical composition employed and the optional presence of an adjuvant.", "Generally, for the compositions containing WNVX vaccine candidate peptide, each dose will comprise between about 50 Ag to about 1 mg of the WNVX vaccine candidate peptide immunogens/ml of a sterile solution.", "A more preferred dosage can be about 200 μg of WNVX vaccine candidate peptide immunogen.", "Other dosage ranges can also be contemplated by one of skill in the art Initial doses can be optionally followed by repeated boosts, where desirable.", "The method can involve chronically administering the WNVX vaccine candidate peptide composition.", "For therapeutic use or prophylactic use, repeated dosages of the immunizing compositions can be desirable, such as a yearly booster or a booster at other intervals.", "The dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.", "Usually a daily dosage of active ingredient can be about 0.01 to 100 mg/kg of body weight.", "Ordinarily 1.0 to 5, and preferably 1 to 10 mg/kg/day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.", "The WNVX vaccine candidate peptide can be employed in chronic treatments for subjects at risk of acute infection.", "A dosage frequency for such “acute” infections may range from daily dosages to once or twice a week intravenously or intramuscularly, for a duration of about 6 weeks.", "The peptides can also be employed in chronic treatments for infected patients.", "In infected patients, the frequency of chronic administration can range from daily dosages to once or twice a week i.v.", "or i.m., and may depend upon the half-life of the immunogen (e.g., about 7-21 days).", "However, the duration of chronic treatment for such infected patients is anticipated to be an indefinite, but prolonged period.", "For such therapeutic uses, the WNVX vaccine candidate peptide formulations and modes of administration are substantially identical to those described specifically above and can be administered concurrently or simultaneously with other conventional therapeutics.", "In another embodiment, mulitple WNVX nucleic acids and proteins are used simultaneously as a combination vaccine.", "See e.g., Rennels, et al., Pediatrics, 96:576-79 (1995); Eskola, et al., Vaccine, 8(2):107-10 (1990).", "In an alternative embodiment, mulitple WNVX nucleic acids and proteins are utilized in multi-stage vaccine regimes, such as the prime-boost method described in Ramshaw, et al., Immunology Today, 21:4:163-65 (2000).", "Determination of the Biological Effect of the Therapeutic In various embodiments of the invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.", "In various specific embodiments, in vitro assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s).", "Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects.", "Similarly, for in vivo testing, any of the animal model system known in the art may be used prior to administration to human subjects.", "Prophylactic and Therapeutic Uses of the Compositions of the Invention The WNVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of flavivirus disorders.", "As an example, a cDNA encoding the WNVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof.", "By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from a flaviviral disorder.", "Both the novel nucleic acid encoding the WNVX protein, and the WNVX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.", "A further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties).", "These materials are further useful in the generation of antibodies which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.", "The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.", "EXAMPLES Example 1 Obtaining the WNV Sequence The NY 1999 WNV sequence was obtained from the Genbank (Genbank accession number AF196835).", "The 3,433 amino acids contained in the Genbank translation were parsed into 3,424 10-amino acid long frames, each 10 amino acid-long peptide sequence overlapping the previous peptide sequence by one amino acid (see Table 1 for an illustration).", "The sequences of these 3,424 10 mers were stored in a database (WNV peptide database).", "Table 2 provides an example of the analysis performed to select candidate B07 ligands from the WNV genome.", "The sequence of WNV was parsed into overlapping peptides, 10 amino acids in length, overlapping by one amino acid, starting with the first amino acid to be translated from the WNV sequence (AA 0001).", "The resulting 3,423 10 mers were then compared to the EpiMatrix B07 matrix and evaluated for match to the matrix pattern.", "Peptides that best matched the matrix received the highest EBP which is the value that EpiMatrix uses to describe the probability that the peptide will bind to B07 in vitro and in vivo.", "Of the 6 overlapping peptides in this particular region of the WNV sequence shown here, WNVB7 0019 received the best EMX score (22.49), and could be considered therefore the most likely candidate for in vitro studies (of this set of 6 peptides).", "The 25 peptides tested in vitro are shown in Table 3a.", "Twenty of the peptides were selected because they had an EpiMatrix (EMX) score (estimated binding probability, or EBP) between 7 and 50.Peptides that were not tested even though they may fit the above criteria are listed in Table 3b.", "Three peptides received EMX scores above 50 (0001, 0002, 0003).", "These peptides were considered to be unlikely to be epitopes based on previous experience with HLA B07 restricted HIV-1 epitopes (TB/HIV Research Lab, unpublished data).", "Additionally, two peptides were not selected, even though they were predicted to be binders (0016, 0022), because they fell in regions of the genome that were considered unlikely to be expressed, based on information provided by Genbank.", "Example 2 EpiMatrix Analysis EpiMatrix is a matrix-based algorithm that ranks 10 amino acid long segments, overlapping by 9 amino acids, from any protein sequence by estimated probability of binding to a selected MHC molecule.", "(De Groot et al., AIDS Research and Human Retroviruses 13:539-41 (1997)).", "The procedure for developing matrix motifs was published by Schafer et al, 16 Vaccine 1998 (1998).", "We have constructed matrix motifs for 32 HLA class I alleles, one murine allele (H-2 Kd) and several human class II alleles.", "Putative MHC ligands are selected by scoring each 10-mer frame in a protein sequence.", "This score, or estimated binding probability (EBP), is derived by comparing the sequence of the 10-mer to the matrix of 10 amino acid sequences known to bind to each MHC allele.", "Retrospective studies have demonstrated that EpiMatrix accurately predicts published MHC ligands (Jesdale et al., in Vaccines '97 (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1997)).", "An additional feature of EpiMatrix is that it can measure the MHC binding potential of each 10 amino acid long snapshot to a number of human HLA, and therefore can be used to identify regions of MHC binding potential clustering.", "Other laboratories have confirmed cross-presentation of peptides within HLA “superfamilies” (A11, A3, A31, A33 and A68) (Jesdale et al., in Vaccines '97 (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1997)).", "Presumably, vaccines containing such “clustered” or promiscuous epitopes will have an advantage over vaccines composed of epitopes that are not “clustered.", "In work performed in the TB/HIV Research Lab, we have confirmed cross-MHC binding that was predicted by EpiMatrix.", "Each of the peptides was then evaluated using EpiMatrix, a matrix-based algorithm that ranks 10 amino acid peptides by estimated probability of binding to a selected MHC molecule as follows.", "The peptides are scored by estimating the relative promotion or inhibition of binding for each amino acid, compared to known MHC binders for that allele.", "This information is summed across the peptide and a summary score (EMX score) is assigned to the entire peptide.", "After comparing the EMX score to the scores of known MHC ligands, EpiMatrix arrives at an “estimated binding probability” (abbreviated as EBP, but not strictly a probability).", "The EBP describes the proportion of peptides with EpiMatrix scores as high or higher that will bind to a given MHC molecule.", "EBPs range from 100% (highly likely to bind) to less than 1% (very unlikely to bind).", "The EpiMatrix approach was used to screen the West Nile Virus NY99 genome for putative HLA B07 restricted epitopes.", "Ninety-four of 3,433 WNV peptides scored above a predetermined cutoff suggesting that they would be likely to bind to HLA B07.Sixteen of the 94 candidate B07 ligands and four peptides that were not expected to be ligands based on their EpiMatrix score were synthesized.", "Twelve of the sixteen putative HLA B07 ligands (75%) were shown stabilize HLA B07 molecules on the surface of T2B7 cells.", "Five of these peptides, IPAGFEPEML (WNV B07 0008) (SEQ ID NO: 8); RPRWIDARVY (WNV B07 0017) (SEQ ID NO: 17); RPQRHDEKTL (WNV Be 07 0019) (SEQ ID NO: 19); SPHRVPNYNL (WNV B07 0020) (SEQ ID NO:20); and RPAADGRTVM (WNV B07 0023) (SEQ D NO 23) bound with much greater affinity to B07 in vitro than another, previously published B07 epitope GPGHKARVLA (from HIV-1) (SEQ ID NO: 96).", "None of four selected “non-binders” stabilized HLA B07 to a significant degree.", "MHC ligands identified using this method may be used to screen T cells derived from WNV-exposed individuals for cell-mediated response to WNV or to develop diagnostic reagents such as tetramers for epidemiological surveillance.", "Using the EpiMatrix approach, five excellent B07-restricted T cell epitope candidates for an emerging infectious disease were rapidly identified.", "Overall, twelve of 16 (75%) peptides selected for this study bound in T2B7 binding assays.", "Sixteen WNV peptides were screened.", "Twelve epitope candidates were identified over the course of 20 working days.", "Five of these candidates exhibited excellent binding to HLA B07 in vitro, suggesting that they might be excellent reagents for developing tetramer assays.", "The largest source of delay in the process was peptide synthesis (four weeks from placement of order to receipt of the first set of peptides.", "This entire process could be accelerated, if more rapid access to MHC ligands were necessary.", "The binding studies described here are a first step along the path to confirming immunogenicity, however, in cases (such as WNV) where access to T cells from infected individuals is limited, both the bioinformatics step and the binding assays can be executed without clinical specimens.", "Once the epitope candidates selected using this method are confirmed in CTL assays, they may be useful for (1) screening exposed individuals, (2) investigating the immuno-pathogenesis of WNV disease in humans, (3) as components of diagnostic kits developed for the surveillance effort, and/or (4) eventually, as a tool for measuring WNV vaccine-related immune responses.", "Confirmation of T cell response to the peptides will depend on availability of peripheral blood cells from West Nile-infected patients during the next transmission season.", "Additional peptides would also need to be defined and screened for binding to other HLA alleles, in order to broaden the MHC specificity of the diagnostic reagent or immunopathogenesis tools developed using this approach.", "Example 3 Analysis of Overall Scores, Compared to a Random Set and a Set of Known HLA B07 Binders The 3,424 10 mers derived from WNV were compared to the EpiMatrix B07 matrix and evaluated for match to the matrix pattern.", "The majority of decamers scored for the entire WNV genome (using the HLA B07 scoring matrix) fell below 1% EBP score (FIG.", "1a).", "This is also generally true for other proteins we have analyzed (Xia Jin et al., 1999, K. Bond, J. McNicholl, manuscript in preparation, and unpublished data from the TB/HIV Research Lab).", "FIG.", "1b shows the distribution of HLA B07 scores of a set of 10,000 random peptides (plotted as their natural logs, so as to better distribute EBP scores falling below 1), compared to scores for a set of more than 300 known binders (compiled and maintained at EpiVax) and to the scores of the set of WNV peptides selected for this study.", "As can be seen in the figure, the set of peptides selected for this study fell well within the EBP range for a set of more than 300 known HLA B07 ligands.", "Table 4 provides an illustration of the analysis performed to select candidate B07 ligands from the WNV genome.", "Of the 6 overlapping peptides in this particular region of the WNV sequence shown here, WNVB7 0019 received the best EMX score (22.49), and could be considered therefore the most likely candidate for in vitro studies (of this set of 6 peptides).", "TABLE 4 Scoring overlapping peptides using EpiMatrix motif HLA B07 SEQ ID AA peptide number NO: Start (B07 rank) Sequence EBP 102 1123 WNB7 3119 GMEIRPQRHD 0.04 103 1124 WNB7 2818 MEIRPQRHDE 0.08 104 1125 WNB7 0591 EIRPQRHDEK 1.12 105 1126 WNB7 2660 IRPQRHDEKT 0.1 106 1127 WNB7 0019 RPQRHDEKTL 22.49 107 1128 WNB7 2661 PQRHDEKTLV 0.1 This table provides an example of the analysis performed to select candidate B07 ligands from the WNV genome.", "The sequence of WNV was parsed into overlapping peptides, 10 amino acids in length, (“10 mers”) overlapping by one amino acid, starting with the first amino acid to be translated from the WNV sequence (AA 0001).", "The resulting 3,423 “10 mers” were then compared to the EpiMatrix B07 matrix and evaluated for match to the matrix pattern.", "Peptides that best matched the matrix received the highest estimated binding probability (“EBP”) which is the value that EpiMatrix uses to describe the probability that the peptide will bind to B07 in vitro and in vivo.", "Of the 6 overlapping peptides in this particular region of the WNV sequence shown here, WNVB7 0019 received the best EMX score (22.49), and could be considered therefore the most likely candidate for in vitro studies (of this set of 6 peptides).", "Peptides scoring above an EBP of 20% and below 50% (FIG.", "1a) were selected for screening in vitro.", "Ninety four of the 3,424 10 mers scored above 7%.", "Of these, 20 were selected, scoring between an EBP of 50 and an EBP of 20.There were 3 peptides scoring above 50 (001, 002, 003, Table 2b); these were not synthesized nor screened for this study because scores in this range are less likely to be ligands and epitopes (TB/HIV Research Lab and EpiVax unpublished results).", "Two peptides scoring between 50 and 20 (0016 and 0022) were also not tested because they did not fall within a region of the WNV genome belonging to a mature WNV protein, based on information available in the Genbank database (Table 2b).", "Four peptides could not be synthesized to sufficient purity for this study (0012, 0014, 0021, 0025) and, in addition, the amino acid sequence of peptide 0012 was found to overlap to a significant degree with the human genome (0012) and for that reason was eliminated.", "Four “non-binder” peptides and a known binder (1291) were also synthesized.", "A total of 21 peptides were available for testing in vitro.", "Of the 3,424 peptides scored in this analysis, 3,330 peptides scored below an EBP of 7% (3,424-94).", "These were considered unlikely to bind to HLA B07.Thus, using the EpiMatrix approach, 3330 WNV peptides were set aside as unlikely candidates for HLA B07 binding studies.", "This represents a 97% reduction in the complete set of peptides that could have been tested for the WNV NY99 genome (3,330/3,424).", "Rather than testing every possible peptide in the search for epitopes, some researchers have adopted a standard “overlapping” approach (constructing a set of 10 amino acid long peptides overlapping by 4 amino acids covering the entire genome, for example).", "This strategy (10/4 OL set) would still require the synthesis of a total of 685 10 mer peptides, approximately 30 times more peptides than were synthesized and tested using the EpiMatrix approach.", "Example 4 Selection of Peptides Based on previous analyses, higher EpiMatrix scores suggest greater MHC binding potential.", "Therefore the top scoring WNV peptides were considered for further evaluation in vitro.", "Twenty peptides scoring below an EBP of 50 and above an EBP of 20 (Table 3a) were selected for screening in vitro.", "Peptides scoring with an EBP above 50 were not selected because peptides with this score are less likely to be immunogenic, even though they may bind to B7 in vitro (See, e.g., Jin X, et al., AIDS Res Hum Retroviruses 2000; 16:67-76; De Groot AS, et al., Vaccine.", "(In press 2001.)).", "Peptides scoring below an EBP of 20 and above an EBP of 7 are considered likely to bind, however they were not synthesized for the study because of the pilot project (e.g.", "limited funds) nature of this study.", "Four peptides of the lowest scoring WNV peptides (EBP=0.00%, Table 2b) were synthesized to test the hypothesis that low scoring peptides derived from WNV would not bind to HLA B07 in vitro (predicted non-binders).", "One well-defined, previously published B07-restricted epitope (derived from HIV) was also synthesized to serve as a positive control for the assays.", "Example 5 Cross-Reactivity Analyses Following the binding analysis, using the proprietary tool Conservatrix, these sequences were aligned and compared for other related flaviviruses and identified (and tagged these peptides in the database) all highly unique sequences in WNV.", "In an intermediate step designed to avoid selecting epitopes that have any cross-reactivity with ‘self’, each of the highly selected epitopes was passed through the Blast engine at NCBI, using our proprietary tool BlastiMer.", "Any sequence that was associated with (i.e.", "is over 80% identical to the 10 amino acid WNV NY99 sequence) a peptide component of equivalent length contained in the human genome (accessible and published to date) was set aside.", "This only occurred for one of the peptides, peptide 0012, which was found to be highly conserved in the public human genome database at 8 out of the 10 amino acids in the sequence (peptide 0012 was not tested in vitro).", "Example 6 Peptide Synthesis Peptides corresponding to the epitope selections were prepared by Fmoc synthesis on an automated Rainen Symphony/Protein Technologies synthesizer.", "(Synpep, Dublin, Calif.) The peptides are delivered 95% pure as ascertained by HPLC, Mass Spec, and UV scan (insuring purity, mass and spectrum respectively).", "The peptides were shipped as a lyophilized powder.", "This powder was diluted in a minimal volume of DMSO and then brought up to stock concentration (1 mg/mi) in RPMI 1640 (Sigma, St Louis, Mo.)).", "Four peptides could not be purified to specifications, therefore these peptides (0012, 0014, 0021, and 0025) were not evaluated in vitro.", "Example 7 MHC Binding Studies The T2B7 binding assay method is well described in the literature and operational in the laboratory.", "This assay relies on the ability of exogenously added peptides to stabilize the class I MHC/beta 2 microglobulin structure on the surface of transporters associated with antigen processing (TAP)-deficient cell lines.", "Briefly, HLA B07 T2 cell lines were prepared by incubating overnight (16 hours) at 26° C. Just prior to the binding assay these cells were washed twice in serum-free media.", "Solutions of the test peptides at three concentrations (final concentration of 10, 20, and 200 ug/ml in RPMI 1640 (Sigma, St. Louis, Mo.))", "were plated in triplicate wells of a 96 well, round-bottom assay plate (Beckton Dickinson, Lincoln Park, N.J.).", "Sixteen wells containing cells without peptide were included in each plate, serving as the no-peptide control (background control) for the assay.", "100,000 cells were added to each well, and the plates were then incubated for four hours at 37° C., 5% CO2 The plates were then spun at 110×g for 10 minutes at 4° C., supernatant is discarded and the remaining cells are re-suspended.", "One hundred uL of diluted primary antibody-containing hybridoma supernatant (1:10 dilution of ME1 supernatant produced by HB-119 cell line from ATCC) in staining buffer (PBS, 5% FBS, 0.1% sodium azide) was added to all of the wells except 8 wells per assay plate (each containing 200 ug/ml of one study peptide, these wells served as controls for non-specific binding of secondary antibody).", "Primary antibody was incubated with the peptide-pulsed cells for 30 minutes at 4° C. After washing three times, the cells were re-suspended, and 100 uL of a 1:250 dilution of FITC-labeled secondary antibody (FITC labeled Goat F(ab′)2 Anti-mouse IgG (H+L) from Caltag) in staining buffer were added to all wells.", "The plates were incubated for 30 minutes at 4° C., and subsequently washed three times.", "The contents of each well were then resuspended in 200 uL of fixing buffer (PBS, 1% paraformaldehyde), and aliquoted into FACS tubes.", "The 16 negative control wells in each plate contained no peptide but did contain cells, primary antibody and secondary antibody.", "An additional set of triplicate wells was plated with peptide at the highest concentration (200 ug/ml), but no primary antibody was added to the wells to control for non-specific secondary antibody binding.", "One positive control peptide (the known B07 binder) was tested at three concentrations (in triplicate wells) in each assay plate.", "Following fixing, the presence of fluorescent secondary antibody on the surface of T2 cells (gated to the appropriate cell size) was measured at 488 nm on a FACScan flow cytometer (Becton-Dickinson, New Jersey).", "The mean linear fluorescence (MIS) of 10,000 events was measured and compared to the background fluorescence of cells plated in (no peptide) control wells.", "A positive response was defined as a 10% increase over baseline MLF and p<0.05, in more than one concentration in each of four assays.", "The assays are repeated 4 times for each concentration of peptide.", "Each peptide was tested in a total of 36 wells (triplicate wells, three concentrations, four assays).", "The B07 molecule was considered to be stabilized on the surface of the T2B7 cells if the average of the mean linear fluorescence for the triplicate wells at each concentration of peptide was >10% higher than the average of the 16 negative control wells (and p<0.05 in two-way comparison by ANOVA).", "Binding was rated as strong, moderate, weak, or none, based on the number of significantly positive wells by pair-wise ANOVA.", "See Table 2, supra.", "Other Embodiments Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow.", "In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.", "The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein.", "Other aspects, advantages, and modifications considered to be within the scope of the following claims." ] ]	Patent_10468950
[ [ "Cloning of transgenic animals comprising artificial chromosomes", "The invention is directed in part to totipotent cells that have one or more artificial chromosomes; processes for producing such cells; processes for using such cells (e.g., nuclear transfer); transgenic embryos and transgenic animals cloned from such cells; and processes for producing such embryos and animals." ], [ "1.A method of producing a transgenic ungulate embryo by nuclear transfer of a nuclear donor cell into an enucleated recipient cell, the method comprising: (a) fusing a nuclear donor cell, or a nucleus thereof, with an enucleated recipient cell of the same species as the nuclear donor cell to form a nuclear transfer embryo, wherein a heterologous DNA molecule of greater than 100 kilobase pairs is injected into said oocyte prior to or following fusion, whereby said nuclear transfer embryo comprises said heterologous DNA molecule; and (b) activating the nuclear transfer embryo to provide said transgenic ungulate embryo.", "2.A method according to claim 1, wherein said transgenic ungulate embryo is cultured, and wherein said culturing step comprises selection for one or more markers of said heterologous DNA molecule.", "3.A method according to claim 1, wherein the transgenic ungulate is selected from the group consisting of a bovine, an ovine, a caprine, and a porcine.", "4.A method according to claim 1, wherein the heterologous DNA molecule comprises one or more telomeres, one or more centromeres, and one or more origins of replication.", "5.A method according to claim 1, wherein the heterologous DNA molecule is contained within the cells of the transgenic ungulate embryo on a replication unit that comprises essentially no homologous DNA.", "6.A method according to claim 1, wherein said nuclear transfer embryo is cultured to at least the two cell stage, wherein at least 50% of the cells of the transgenic ungulate embryo comprise the heterologous DNA molecule.", "7.A method according to claim 1, wherein the nuclear donor cell is selected from the group consisting of a somatic cell, a primordial germ cell, an embryonic germ cell, and an embryonic stem cell.", "8.A method according to claim 1, wherein the heterologous DNA comprises a plurality of copies of at least one transgene.", "9.A method according to claim 1, wherein said heterologous DNA molecule is between 100 kilobase pairs and 500 megabase pairs.", "10.A method according to claim 1, wherein said heterologous DNA molecule is an artificial chromosome.", "11.A method of producing a transgenic ungulate from a transgenic ungulate embryo of claim 1, the method comprising: transferring said transgenic ungulate embryo into a maternal host so as to produce a fetus of full fetal development and parturition to generate said transgenic ungulate.", "12.A method according to claim 11, wherein prior to said transferring step, said transgenic ungulate embryo is cultured to at least the two cell stage.", "13.A method according to claim 11, wherein at least 50% of the cells of the transgenic ungulate comprise the heterologous DNA molecule.", "14.A method of producing a transgenic ungulate from a transgenic ungulate embryo of claim 1, the method comprising: (a) transferring said transgenic ungulate embryo into a maternal host so as to produce a fetus; (b) obtaining a nuclear donor cell from said fetus, wherein said cell comprises said heterologous DNA molecule; (c) fusing said nuclear donor cell, or a nucleus thereof, with an enucleated recipient cell of the same species as the said cell to form a second nuclear transfer embryo, wherein said second nuclear transfer embryo comprises said heterologous DNA molecule; (d) activating said second nuclear transfer embryo to provide a second transgenic ungulate embryo; and (e) transferring said second transgenic ungulate embryo into a maternal host so as to produce a fetus of full fetal development and parturition to generate said transgenic ungulate.", "15.A method according to claim 14, wherein prior to said transferring step, said transgenic ungulate embryo and/or said second transgenic ungulate embryo is cultured to at least the two cell stage.", "16.A method according to claim 14, wherein at least 50% of the cells of the transgenic ungulate comprise the heterologous DNA molecule.", "17.A method of producing a transgenic ungulate from a transgenic ungulate embryo of claim 1, the method comprising: (a) transferring said transgenic ungulate embryo into a maternal host so as to produce a fetus; (b) obtaining one or more cells from said fetus, wherein one or more of said cells comprise said heterologous DNA molecule, and culturing said one or more cells to obtain a cell culture; (c) fusing a nuclear donor cell obtained from said cell culture, or a nucleus thereof, with an enucleated recipient cell of the same species as said nuclear donor cell to form a second nuclear transfer embryo, wherein said second nuclear transfer embryo comprises said heterologous DNA molecule; (d) activating said second nuclear transfer embryo to provide a second transgenic ungulate embryo; and (e) transferring said second transgenic ungulate embryo into a maternal host so as to produce a fetus of full fetal development and parturition to generate said transgenic ungulate.", "18.A method according to claim 17, wherein prior to said transferring step, said transgenic ungulate embryo and/or said second transgenic ungulate embryo is cultured to at least the two cell stage.", "19.A method according to claim 17, wherein at least 50% of the cells of the transgenic ungulate comprise the heterologous DNA molecule.", "20.A method according to claim 17, wherein said culturing step comprises selection for one or more markers of said heterologous DNA molecule, whereby at least 90% of cells in said cell culture comprise said heterologous DNA molecule.", "21.A transgenic ungulate embryo comprising one or more cells, wherein at least 50% of said one or more cells comprise an artificial chromosome.", "22.A transgenic ungulate, wherein at least 50% of the cells making up said ungulate comprise an artificial chromosome." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.", "Researchers have been developing methods for cloning mammalian animals over the past two decades.", "These reported methods typically include the steps of (1) isolating a pluripotent or totipotent cell; (2) inserting the cell or nucleus isolated from the cell into an enucleated oocyte (i.e., the oocyte's nucleus was previously extracted), and (3) allowing the embryo to mature in vivo.", "The first successful nuclear transfer experiment using mammalian cells was reported in 1983, when pronuclei isolated from a murine (mouse) zygote were inserted into an enucleated oocyte and resulted in like offspring(s).", "See, e.g., McGrath & Solter, 1983 , Science 220:1300-1302.Subsequently, other workers described the production of chimeric murine embryos (e.g., embryos that contain a subset of cells having significantly different nuclear DNA from other cells in the embryo) using murine primordial germ cells (PGCs).", "These cells are and can give rise to pluripotent cells (e.g., cells that can differentiate into other types of cells, and which may, but are not required to, differentiate into a grown animal).", "See, e.g., Matsui et al., 1992 , Cell 70:841-847 and Resnick et al., 1992, Nature 359:550; Kato et al., 1994, Journal of Reproduction and Fertility Abstract Series, Society For the Study of Fertility, Annual Conference, Southampton, 13:38.Progress has also been reported in the field of cloning ovine (sheep) animals (see, e.g., Willadsen, 1986, Nature 320:63-65; Campbell et al., 1996, Nature 380:64-66; PCT Publication WO 95/20042; Wilmut et al., 1997, Nature 385:810-813; PCT Publication WO 96/07732; PCT Publication WO 97/07668; and PCT publication WO 97/07669; and McCreath et al., 2000, Nature, 405:1066-1069), and bovine animals, (see, e.g., U.S. Pat.", "Nos.", "4,994,384 and 5,057,420; Sims & First, 1993, Theriogenology 39:313; Keefer et al., 1994, Mol.", "Reprod.", "Dev.", "38:264-268; Delhaise et al., 1995, Reprod.", "Fert.", "Develop.", "7:1217-1219; Lavoir 1994, J. Reprod.", "Dev.", "37:413-424; Stice et al., 1996, Biol.", "Reprod.", "54: 100-110; and PCT application WO 95/10599 entitled “Embryonic Stem Cell-Like Cells”).", "Researchers have also disclosed methods that resulted in cloned bovine animals (cattle).", "Bovines have been cloned using an embryonic cell derived from a 2-64 cell embryo as a nuclear donor.", "This bovine animal was reportedly cloned by utilizing nuclear transfer techniques set forth in U.S. Pat.", "Nos.", "4,994,384 and 5,057,420.Others reported that cloned bovine embryos were formed where an inner cell mass cell of a blastocyst stage embryo was utilized as a nuclear donor in a nuclear transfer procedure (Sims & First, 1993, Theriogenology 39:313; Keefer et al., 1994, Mol.", "Reprod.", "Dev.", "38:264-268; and U.S. Pat.", "No.", "6,107,543); a PGC isolated from fetal tissue as a nuclear donor (Delhaise et al., 1995, Reprod.", "Fert.", "Develop.", "7:1217-1219; Lavoir 1994, J. Reprod.", "Dev.", "37:413-424; and PCT application WO 95/10599 entitled “Embryonic Stem Cell-Like Cells”); a proliferating somatic cell (U.S. Pat.", "No.", "5,945,577); and a reprogrammed nonembryonic cell (U.S. Pat.", "No.", "6,011,197) Additionally, researchers have reported methods for obtaining cloned porcine animals and porcine chimeric animals, specifically, where a nuclear donor obtained from a 4-cell embryo is placed inside an enucleated zygote.", "See, e.g., Prather et al., 1989, Biology of Reproduction 41: 414-418; Piedrahita et al., 1998, Biology of Reproduction 58: 1321-1329; and WO 94/26884, “Embryonic Stem Cells for Making Chimeric and Transgenic Ungulates,” Wheeler, published Nov. 24, 1994.Also, researchers have reported nuclear transfer experiments using porcine nuclear donors and porcine oocytes.", "See., e.g., Nagashima et al., 1997, Mol.", "Reprod.", "Dev.", "48: 339-343; Nagashima et al., 1992, J. Reprod.", "Dev.", "38: 73-78; Prather et al., 1989, Biol.", "Reprod.", "41: 414-419; Prather et al., 1990, Exp.", "Zool.", "255: 355-358; Saito et al., 1992, Assis.", "Reprod.", "Tech.", "Andro.", "259: 257-266; Terlouw et al., 1992, Theriogenology 37: 309, Pokajaeva et al., Nature 407, 86-90 (2000); Onishi et al., Science 289 1188-1190 (2000); and Betthauser et al., Nature Biotechnology 18: 1055-1059 (2000).", "Researchers have also developed methods for generating transgenic cells, which may be applicable to the production of transgenic animals.", "Although several viral vectors, non-viral vectors, and other delivery systems have been developed for establishing transgenic cells, many of these technologies are constrained by multiple limitations.", "Specifically, these limitations include (1) the size of inserted DNA is limited to approximately 10 kilobases (kb); (2) integration of the DNA of interest cannot be specifically targeted into the cell's nuclear DNA; and (3) expression of a recombinant product from the DNA of interest cannot be well controlled.", "See, e.g., Mitani et al., 1993, Trends Biotech, 11: 162-166; U.S. Pat.", "No.", "5,633,067, “Method of Producing a Transgenic Bovine or Transgenic Bovine Embryo,” DeBoer et al, issued May 27, 1997; U.S. Pat.", "No.", "5,612,205, “Homologous Recombination in Mammalian Cells,” Kay et al., issued Mar.", "18, 1997; and PCT publication WO 93/22432, “Method for Identifying Transgenic Pre-Implantation Embryos,” all of which are incorporated by reference herein in their entirety, including all figures, drawings, and tables.", "Artificial chromosome technology is not constrained by the above-defined limitations.", "Moreover, researchers have discovered that artificial chromosomes can be replicated de novo.", "See, e.g., Kereso et al., 1996, Chromosome Research 4: 226-239, Holló et al., 1996, Chromosome Research 4: 240-247, U.S. Pat.", "No.", "6,025,155, and U.S. Pat.", "No.", "6,077,697.Each reference used to provide background information in this section is hereby incorporated by reference in its entirety, including ant tables, figures, and claims.", "Despite progress towards cloning mammals and establishing transgenic cells, there remains a great need in the art for materials and methods that enhance the efficiency for cloning transgenic animals.", "In particular, there remains a great need in the art to provide pluripotent and totipotent transgenic cells that can be utilized as nuclear donors.", "Furthermore, there remains a long felt need in the art for providing cell lines that are karyotypically stable and transgenic, which can be utilized in processes for cloning transgenic animals." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention relates in part to transgenic, totipotent, mammalian cells comprising one or more large, heterologous DNA constructs of 100 kbp or more.", "Preferably, the large DNA construct(s) are artificial chromosomes.", "Mammalian cells containing the large heterologous DNA construct(s) may be used for producing transgenic embryos and transgenic animals cloned from such cells.", "The invention is also directed in part to processes for producing totipotent cells that comprise one or more large, heterologous DNA constructs; processes for utilizing such cells; and processes for producing transgenic embryos and transgenic animals cloned from such cells.", "Thus, in a first aspect, the invention features a method for producing transgenic cells by inserting a large, heterologous DNA construct of 100 kbp or more into cells.", "Such cells may preferably be used as nuclear donor cells in methods to produce transgenic animals, most preferably ungulates.", "Preferably, a large, heterologous DNA construct is at least 200 Kbp, at least 300 Kbp, at least 400 Kbp, at least 500 Kbp, at least 750 Kbp, at least 1 Mbp, at least 5 Mbp, at least 10 Mbp, at least 20 Mbp, at least 50 Mbp, at least 100 Mbp, at least 500 Mbp, or at least 1000 Mbp.", "Particularly useful are artificial chromosomes of between 100 Kbp and 500 Mbp; between 500 Kbp and 500 Mbp; and between 1 Mbp and 500 Mbp.", "In certain embodiments, the large, heterologous DNA construct(s) of this aspect are artificial chromosomes.", "Advantages of using artificial chromosomes include: (1) target DNA greater than 10 kb can be inserted into cells; (2) the location of target DNA of interest can be controlled; (3) transgenic animals and embryos containing large foreign genes, or a large copy number of one or more foreign genes, in a majority of cells can be obtained; and (4) the expression levels of a recombinant product from the DNA of interest can be manipulated in vitro.", "Specifically, expression levels can be manipulated by controlling the copy number of target DNA and/or its regulation by promoters, enhancers, etc., in an artificial chromosome, as defined in greater detail hereafter.", "The term “artificial chromosome” as used herein refers to nucleic acid molecules that are generated by the manipulation of DNA, contain a centromere, and are capable of stable, autonomous replication in cells.", "An artificial chromosome (1) can replicate with naturally occurring chromosomes in the nucleus of target cell; (2) can be large in size (ranging in size from 100 kilobase pairs (Kbp) to 1000 megabase pairs (Mbp) in length, or more); (3) typically comprises a centromere, origins of replication, and telomeres; and (4) can comprise neutral DNA.", "Neutral DNA does not encode products that significantly alter the functions of a cell in which the artificial chromosome is located.", "For example, neutral DNA may encode ribosomal RNA.", "It is not typical that increasing levels of ribosomal RNA significantly alters cell functions.", "Neutral DNA can also be referred to as “satellite DNA.” Preferably, an artificial chromosome is at least 200 Kbp, at least 300 Kbp, at least 400 Kbp, at least 500 Kbp, at least 750 Kbp, at least 1 Mbp, at least 5 Mbp, at least 10 Mbp, at least 20 Mbp, at least 50 Mbp, at least 100 Mbp, at least 500 Mbp, or at least 1000 Mbp.", "Particularly useful are artificial chromosomes of between 100 Kbp and 500 Mbp; between 500 Kbp and 500 Mbp; and between 1 Mbp and 500 Mbp.", "Materials and methods for producing, identifying, and characterizing artificial chromosomes are well known in the art.", "See, e.g., Kereso et al., 1996, Chromosome Research 4: 226-239, Holló et al., 1996, Chromosome Research 4: 240-247, International publication nos.", "WO00/18941, WO98/08964, WO97/16533 and WO97/40183, and U.S. Pat.", "Nos.", "5,721,118, 6,025,155, 6,077,697, and 6,133,503, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.", "These publications also describe shuttle vectors useful for incorporating target DNA into artificial chromosomes.", "Artificial chromosomes can arise from a portion of a natural chromosome by manipulation.", "Artificial chromosomes can be detected in cells by using chromosome identification techniques well known in the art.", "An example of such a technique is chromosome karyotype analysis.", "Mammalian artificial chromosomes (MACs) can be generated by cellular mediated chromosome assembly from transfected alphoid, telomeric and marker DNAs (Harrington J. J. et al.", "Nature Genetics, 15, 345-355, 1997; Ikeno, M. et al, Nature Biotechnology 16, 431-439, 1998; Henning, K. A. et al, PNAS USA 96, 592-597, 1999) and even from non-alphoid DNA (du Sart D, et al, Nature Genetics 16, 144-153, 1997).", "Minichromosomes may be generated by fragmenting natural human chromosomes using telomere-directed breakage (Shen M H, et al, Human Molecular Genetics 6, 1375-1382, 1997; Shen M H et al, Current Biology 10, 31-34, 1999).", "It is possible to transfer human-murine minichromosome chimeras (Shen M H et al, Current Biology 10, 31-34, 1999), fragmented human minichromosomes Tomizuka K et al, Nature Genetics 16, 133-143, 1997; Tomizuka K et al, PNAS USA 97, 722-797, 2000), and human small accessory chromosomes (SACs; Vermeesch J R et al, Human Genetics 105, 611-618, 1999) via microcell-mediated chromosome transfer (MMCT) to recipient cells.", "The term “target DNA” as used herein refers to DNA that is intended to be or has been incorporated into a large heterologous DNA construct, preferably an artificial chromosome.", "The term “heterologous” is defined below.", "Target DNA can encode multiple types of recombinant products, as defined hereafter, and may exist in multiple copies when introduced into an artificial chromosome.", "One advantage of artificial chromosome technology is that target DNA copy number can be controlled and monitored in an artificial chromosome in vitro before the artificial chromosome comprising the target DNA is introduced into a cell.", "In addition, depending on the promoter used, expression can also be monitored in vitro.", "This advantage is contrasted with many existing techniques for creating transgenic cells, which cause random insertion of target DNA into a cell nuclear DNA.", "Materials and methods for introducing target DNA into an artificial chromosome and materials and methods for introducing the resulting artificial chromosome into cells are defined hereafter.", "The term “heterologous nucleic acid” refers to nucleic acids having (1) a nucleic acid sequence that differs from the nucleic acid sequences present in cell's naturally occurring nuclear DNA; (2) a subset of nucleic acid having a nucleotide sequence that is present in cell nuclear DNA, but that exists in different proportions in the heterologous nucleic acid than in cell nuclear DNA; (3) a nucleic acid sequence originating from a species other than the species from which cell nuclear DNA originates; and (4) a nucleic acid sequence that differs from the DNA sequences present in cell's naturally occurring mitochondrial DNA.", "Artificial chromosomes, such as mammalian artificial chromosomes [MACs], can be generated and isolated by the methods described in the publications above.", "In particularly preferred embodiments, two types of artificial chromosomes are used, both of which function in cells as stable, functional chromsomes.", "One type, herein referred to as ACEs (“Artificial Chromosome Expression systems” based on satellite DNA) is a stable heterochromatic chromosome, and the other type is a de novo-formed minichromosomes based on amplification of euchromatin.", "Artificial chromosomes, and, in particular the two preferred types discussed above, provide an extra-genomic locus for targeted integration of up to multi-megabase pair size DNA fragments that contain single or multiple genes, including multiple copies of a single gene operatively linked to one promoter or each copy or several copies linked to separate promoters.", "Thus, methods provided can be used to introduce genes via MACs into cells and tissues of ungulate mammals.", "The artificial chromosomes with integrated heterologous DNA may be used in methods of production of gene products, particularly products that require expression of multigenic biosynthetic pathways, and also are intended for delivery into the nuclei of cells, such as nuclear donor cells used in nuclear transfer procedures, for production of transgenic ungulate mammals.", "Additionally, such artificial chromosomes provide extra-genomic specific integration sites for introduction of genes encoding proteins of interest and permit up to multi-megabase size DNA integration so that, for example, genes encoding an entire metabolic pathway, a very large gene such as the cystic fibrosis transmembrane conductance regulator gene (approximately 250 kb genomic DNA gene), or several genes, such as multiple genes encoding a series of antigens for preparation of a multivalent vaccine, can be stably introduced into a cell.", "The artificial chromosomes described herein, including ACEs and euchromatin-based minichromosomes, can be generated by introducing heterologous DNA, preferably including DNA encoding one or multiple selectable marker(s), into cells, preferably a stable cell line, growing the cells under selective conditions, and identifying from among the resulting cell clones those that include chromosomes with more than one centromere, fragments thereof, and/or heterochromatic structures.", "Amplification that produces the additional centromere(s) occurs in cells that contain chromosomes in which heterologous DNA has integrated near the centromere in the pericentric region of the chromosome.", "Selected cells comprising intermediates in the formation of such artificial chromosomes can then be used to generate complete artificial chromosomes.", "For example, continued culture of cells containing a formerly dicentric chromosome under conditions that destabilize chromosomes (such as BrdU treatment) and/or under selective conditions can yield ACEs.", "Similarly, artificial chromosomes can be generated by culturing cells with multi-centric (typically dicentric) chromosomes under conditions whereby the chromosome breaks to form a minichromosome and a formerly dicentric chromosome.", "Among the MACs provided herein can be ACEs, which are predominantly heterochromatic (i.e., contain more heterochromatin than euchromatin, and preferably contain about 70% heterochromatin), and can comprise repeating units of short satellite DNA, so that without insertion of heterologous or foreign DNA, the chromosomes preferably contain no genetic information.", "They can thus be used as “safe” vectors for delivery of DNA to mammalian hosts because they do not contain any potentially harmful genes.", "ACEs are generated, not from the minichromosome fragment as, for example, in U.S. Pat.", "No.", "5,288,625 (which is incorporated herein by reference in its entirety including all figures, tables, and drawings), but from the fragment of the formerly dicentric chromosome.", "In addition, euchromatic minichromosomes can be generated.", "Methods for generating one type of MAC, the minichromosome, is described in U.S. Pat.", "No.", "5,288,625 (which is incorporated herein by reference in its entirety including all figures, tables, and drawings), along with its use for the expression of heterologous DNA are provided.", "In preferred embodiments, (1) the artificial chromosome is an ACEs comprising one or more markers; (2) a marker is an antibiotic resistance gene selected from the group consisting of neomycin resistance gene, hygromycin resistance gene, and puromycin resistance gene; (3) the artificial chromosome comprises a DNA sequence that encodes one or more recombinant products; (4) a recombinant product is a ribozyme; (5) a recombinant product is antisense RNA; (6) a recombinant product is a peptide; (7) a recombinant product is a polypeptide; (8) a recombinant product is a protein; (9) a recombinant product is an enzyme; (10) a recombinant product is expressed in a biological fluid; (11) a recombinant product is expressed in a tissue; (12) a recombinant product confers resistance to one or more parasites and/or diseases; (13) an artificial chromosome comprises one or more regulatory elements; (14) a regulatory element is a promoter element; (15) a promoter element is selected from the group consisting of milk protein promoter, urine protein promoter, blood protein promoter, tear duct protein promoter, synovial protein promoter, mandibular gland protein promoter, casein promoter, β-casein promoter, melanocortin promoter, milk serum protein promoter, α-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, and α-actin promoter; (17) a regulatory element is a repressor element; (18) a regulatory element is an insulator element; and (19) a regulatory element is an enhancer element.", "The term “marker” as used herein refers to any DNA sequence that distinguishes a cell comprising an artificial chromosome, or a precursor thereof, from a cell that does not comprise the artificial chromosome or precursor.", "For example, a marker can be used in the initial steps of generating ACEs, whereby the marker distinguishes a cell containing a foreign nucleic acid from a cell that does not contain the foreign nucleic acid.", "Multiple types of markers, such as genes encoding green fluorescent protein, antibiotic resistance, β-galactosidase, glutamine synthetase, thymidine kinase, cytosine deaminase, and dihydrofolate reductase are well known in the art.", "Preferred as markers are DNA sequences that encode a molecule which directly or indirectly inactivates a drug that retards the growth of cells not expressing such a molecule.", "Examples of these latter described markers are blasticidin-S, neomycin, hygromycin, and puromycin resistance genes.", "These examples are not meant to be limiting and the invention relates in part to any marker known in the art.", "The term “ribozyme” as used herein refers to ribonucleic acid molecules that can cleave other RNA molecules in specific regions.", "Ribozymes can bind to discrete regions on a RNA molecule, and then specifically cleave a region within that binding region or adjacent to the binding region.", "Ribozyme techniques can thereby decrease the amount of polypeptide translated from formerly intact message RNA molecules.", "For specific descriptions of ribozymes, see U.S. Pat.", "No.", "5,354,855, entitled “RNA Ribozyme which Cleaves Substrate RNA without Formation of a Covalent Bond,” Cech et al., issued on Oct. 11, 1994, and U.S. Pat.", "No.", "5,591,610, entitled “RNA Ribozyme Polymerases, Dephosphorylases, Restriction Endoribonucleases and Methods,” Cech et al., issued on Jan. 7, 1997, both of which are incorporated by reference in their entireties including all figures, tables, and drawings.", "The term “antisense RNA” as used herein refers to any RNA that binds to mRNA with enough affinity to decrease the amount of protein translated from the mRNA.", "The amount of protein translated from the mRNA is preferably decreased by more than 20%; more preferably decreased by more than 50%, 70%, and 80%; and most preferably decreased by more than 90%.", "Antisense RNA materials and methods are well known in the art.", "The terms “biological fluid” and the term “tissue” as used herein refer to any fluid or tissue in or from a biological organism.", "The fluids may include, but are not limited to, tears, saliva, milk, urine, amniotic fluid, semen, plasma, oviductal fluid, and synovial fluid.", "The tissues may include, but are not limited to, lung, heart, blood, liver, muscle, brain, pancreas, skin, and others.", "The term “confers resistance” as used herein refers to the ability of a recombinant product to completely abrogate or partially alleviate the symptoms of a disease or parasitic condition.", "Hence, if a disease is related to inflammation, for example, a recombinant product can confer resistance to that inflammation if the inflammation decreases upon expression of the recombinant product.", "A recombinant product may confer resistance or partially confer resistance to a disease or parasitic condition, for example, if the recombinant product is an antisense RNA molecule that specifically binds to an mRNA molecule encoding a polypeptide responsible for the inflammation.", "In preferred embodiments, the DNA with the selectable marker that is introduced into cells to generate artificial chromosomes includes sequences that target it to the pericentric region of the chromosome.", "Integration of the DNA into existing chromosomes in the cells can induce amplification that results in generation of additional centromeres.", "Transgenic Cells Large heterologous nucleic acid constructs, such as artificial chromosomes, can then be introduced into cells to produce stable transformed cell lines and cells.", "Introduction is effected by any suitable method or combination of methods including, but not limited to microinjection, cell fusion, microcell fusion, electroporation, sonoporation, electrofusion, projectile bombardment, calcium phosphate precipitation, lipid-mediated transfer systems, ligand/receptor systems and other such methods well known to the skilled artisan.", "ACEs in particular can be readily isolated and used for gene delivery.", "These artificial chromosomes can also be used in gene product production systems, production of humanized genetically transformed animal organs, and, most preferably, the generation of transgenic ungulates.", "In certain embodiments, the invention relates to transgenic, totipotent, mammalian cells comprising at least one artificial chromosome, but the invention relates in part to any number of artificial chromosomes in a totipotent mammalian cell.", "A totipotent mammalian cell preferably comprises ten or fewer artificial chromosomes; more preferably comprises six or fewer artificial chromosomes, four or fewer artificial chromosomes, or two or fewer artificial chromosomes; and most preferably comprises one artificial chromosome.", "If a totipotent mammalian cell of the invention comprises more than one artificial chromosome, the artificial chromosomes may be identical or may differ from one another.", "The term “transgenic” as used herein in reference to cells refers to a cell that comprises heterologous nucleic acid, preferably deoxyribonucleic acid (DNA).", "In preferred embodiments, a transgenic cell comprises one or more heterologous DNA sequences.", "In other preferred embodiments, a transgenic cell is a cell in which one or more endogenous genes have been deleted, duplicated, activated, or modified.", "In particularly preferred embodiments, a transgenic cell comprises both one or more heterologous DNA sequences, and one or more endogenous genes that have been deleted, duplicated, activated, or modified.", "An artificial chromosome present in a transgenic cell can comprise heterologous DNA.", "Heterologous DNA can encode multiple types of recombinant products, as defined hereafter.", "The term “transgene” as used herein refers to a single gene that is partially or entirely transgenic in origin.", "In certain embodiments, greater than 50% of the transgene consists of heterologous DNA.", "In preferred embodiments, greater than 75% of the transgene consists of heterologous DNA, greater than 80% of the transgene consists of heterologous DNA, greater than 90% of the transgene consists of heterologous DNA, greater than 95% of the transgene consists of heterologous DNA, greater than 98% of the transgene consists of heterologous DNA, and 100% of the transgene consists of heterologous DNA.", "The term “different nucleic acid sequence” as used herein refers to nucleic acid sequences that are not substantially similar.", "The term “substantially similar” as used herein in reference to nucleic acid sequences refers to two nucleic acid sequences having preferably 80% or more nucleic acid identity, more preferably 90% or more nucleic acid identity or most preferably 95% or more nucleic acid identity.", "Nucleic acid identity is a property of nucleic acid sequences that measures their similarity or relationship when aligned by means known to one skilled in the art.", "Identity is measured by dividing the number of identical bases in the two sequences by the total number of bases and multiplying the product by 100.Thus, two copies of exactly the same sequence have 100% identity, while sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity.", "Those skilled in the art will recognize that several computer programs are available for determining sequence identity and similarity using standard parameters, for example Gapped BLAST or PSI-BLAST (Altschul, et al.", "(1997) Nucleic Acids Res.", "25:3389-3402), BLAST (Altschul, et al.", "(1990) J. Mol.", "Biol.", "215:403-410), and Smith-Waterman (Smith, et al.", "(1981) J. Mol.", "Biol.", "147:195-197).", "Preferably, the default settings of these programs will be employed, but those skilled in the art recognize whether these settings need to be changed and know how to make the changes.", "The term “substantially similar” as used herein in reference to amino acid sequences refers to two amino acid sequences having preferably 50% or more amino acid identity, more preferably 70% or more amino acid identity or most preferably 90% or more amino acid identity.", "Amino acid identity is a property of amino acid sequence that measures their similarity or relationship.", "Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100.Thus, two copies of exactly the same sequence have 100% identity, while sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity.", "“Similarity” in protein sequences is measured by dividing the number of identical residues plus the number of conservatively substituted residues (see Bowie, et al.", "Science, (1999) 247, 1306-1310, which is incorporated herein by reference in its entirety, including any drawings, figures, or tables) by the total number of residues and gaps and multiplying the product by 100.“Similarity” in nucleic acid sequences is measured by dividing the number of identical bases by the total number of residues and gaps and multiplying the product by 100.The term “recombinant product” as used herein refers to the product produced from a target DNA sequence.", "A recombinant product can be a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a polypeptide that binds to a regulatory element (a term described hereafter), a structural protein, an RNA molecule, and/or a ribozyme, for example.", "These products are well defined in the art.", "This list of products is for illustrative purposes only and the invention relates to other types of recombinant products.", "In preferred embodiments, (1) the mammalian cell is an ungulate cell; (2) the ungulate is selected from the group consisting of bovids, ovids, cervids, suids, equids and camelids; (3) the ungulate is bovine; (4) the mammalian cell is a nonembryonic cell; (5) the mammalian cell is a fetal cell; and (6) the mammalian cell is an adult cell.", "The term “mammalian” as used herein refers to any animal of the class Mammalia.", "Preferably, a mammalian cell or cell line is a placental, a monotreme and a marsupial.", "Most preferably, a mammalian cell or cell line is a canid, felid, murid, leporid, ursid, mustelid, ungulate, ovid, suid, equid, bovid, caprid, cervid, and a human or non-human primate.", "A mammalian cell or cell line can be isolated from any source of mammalian cells including, but not limited to, a mammalian embryo, a mammalian fetus, and a mammalian animal.", "The term “canid” as used herein refers to any animal of the family Canidae.", "Preferably, a canid cell or cell line is isolated from a wolf, a jackal, a fox, and a domestic dog.", "The term “felid” as used herein refers to any animal of the family Felidae.", "Preferably, a felid cell or cell line is isolated from a lion, a tiger, a leopard, a cheetah, a cougar, and a domestic cat.", "The term “murid” as used herein refers to any animal of the family Muridae.", "Preferably, a murid cell or cell line is isolated from a mouse and a rat.", "The term “leporid” as used herein refers to any animal of the family Leporidae.", "Preferably, a leporid cell or cell line is isolated from a rabbit.", "The term “ursid” as used herein refers to any animal of the family Ursidae.", "Preferably, a ursid cell or cell line is isolated from a bear.", "The term “mustelid” as used herein refers to any animal of the family Mustelidae.", "Preferably, a mustelid cell or cell line is isolated from a weasel, a ferret, an otter, a mink, and a skunk.", "The term “primate” as used herein refers to any animal of the Primate order.", "Preferably, a primate cell or cell line is isolated from an ape, a monkey, a chimpanzee, and a lemur.", "The term “ungulate” as used herein refers to any animal of the polyphyletic group formerly known as the taxon Ungulata.", "Preferably, an ungulate cell or cell line is isolated from a camel, a hippopotamus, a horse, a tapir, and an elephant.", "Most preferably, an ungulate cell or cell line is isolated from a sheep, a cow, a goat, and a pig.", "Especially preferred in the bovine species are Bos taurus, Bos indicus , and Bos buffaloes cows or bulls.", "The term “ovid” as used herein refers to any animal of the family Ovidae.", "Preferably, an ovid cell or cell line is isolated from a sheep.", "The term “suid” as used herein refers to any animal of the family Suidae.", "Preferably, a suid cell or cell line is isolated from a pig or a boar.", "The term “equid” as used herein refers to any animal of the family Equidae.", "Preferably, an equid cell or cell line is isolated from a zebra or an ass.", "Most preferably, an equid cell or cell line is isolated from a horse.", "The term “bovid” as used herein refers to any animal of the family Bovidae.", "Preferably, an bovid cell or cell line is isolated from an antelope, an oxen, a cow, and a bison.", "The term “caprid” as used herein refers to any animal of the family Caprinae.", "Preferably, a caprid cell or cell line is isolated from a goat.", "The term “cervid” as used herein refers to any animal of the family Cervidae.", "Preferably, a cervid cell or cell line is isolated from a deer.", "The term “immortalized” or “permanent” as used herein in reference to cells refers to cells that have exceeded the Hayflick limit.", "The Hayflick limit can be defined as the number of cell divisions that occur before a cell line becomes senescent.", "Hayflick set this limit to approximately 60 divisions for most non-immortalized cells.", "See, e.g., Hayflick and Moorhead, 1961, Exp.", "Cell.", "Res.", "25: 585-621; and Hayflick, 1965, Exp.", "Cell Research 37: 614-636, incorporated herein by reference in their entireties including all figures, tables, and drawings.", "Therefore, an immortalized cell line can be distinguished from non-immortalized cell lines if the cells in the cell line are able to undergo more than 60 divisions.", "If the cells of a cell line are able to undergo more than 60 cell divisions, the cell line is an immortalized or permanent cell line.", "The immortalized cells of the invention are preferably able to undergo more than 70 divisions, are more preferably able to undergo more than 80 divisions, and are most preferably able to undergo more than 90 cell divisions.", "The terms “primary culture” and “primary cell” refer to cells taken from a tissue source, and their progeny, grown in culture before subdivision and transfer to a subculture.", "The terms “plated” or “plating” as used herein in reference to cells refer to establishing cell cultures in vitro.", "For example, cells can be diluted in cell culture media and then added to a cell culture plate or cell culture dish.", "Cell culture plates are commonly known to a person of ordinary skill in the art.", "Cells may be plated at a variety of concentrations and/or cell densities.", "In preferred embodiments, plated cells may grow to confluence.", "The meaning of the term “cell plating” can also extend to the term “cell passaging.” Cells of the invention can be passaged using cell culture techniques well known to those skilled in the art.", "The term “cell passaging” refers to such techniques which typically involve the steps of (1) releasing cells from a solid support and disassociation of these cells, and (2) diluting the cells in fresh media suitable for cell proliferation.", "Immortalized cells can be successfully grown by plating the cells in conditions where they lack cell to cell contact.", "Cell passaging may also refer to removing a portion of liquid medium bathing cultured cells and adding liquid medium from another source to the cell culture to dilute the cell concentration.", "The term “proliferation” as used herein in reference to cells refers to a group of cells that can increase in size and/or can increase in numbers over a period of time.", "The term “confluence” as used herein refers to a group of cells where a large percentage of the cells are physically contacted with at least one other cell in that group.", "Confluence may also be defined as a group of cells that grow to a maximum cell density in the conditions provided.", "For example, if a group of cells can proliferate in a monolayer and they are placed in a culture vessel in a suitable growth medium, they are confluent when the monolayer has spread across a significant surface area of the culture vessel.", "The surface area covered by the cells preferably represents about 50% of the total surface area, more preferably represents about 70% of the total surface area, and most preferably represents about 90% of the total surface area.", "The cells and cell lines of the instant invention may be cultured.", "The term “cultured” as used herein in reference to cells refers to one or more cells that are undergoing cell division or not undergoing cell division in an in vitro environment.", "An in vitro environment can be any medium known in the art that is suitable for maintaining cells in vitro, such as suitable liquid media or agar, for example.", "Specific examples of suitable in vitro environments for cell cultures are described in Culture of Animal Cells: a manual of basic techniques (3 rd edition), 1994, R. I. Freshney (ed.", "), Wiley-Liss, Inc.; Cells : a laboratory manual (vol.", "1), 1998; D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.", "), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J. Morgan John Wiley and Sons, Ltd., each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.", "Cells may be cultured in suspension and/or in monolayers with one or more substantially similar cells.", "Cells may be cultured in suspension and/or in monolayers with a heterogeneous population of cells.", "The term “heterogeneous” as utilized in the previous sentence can relate to any cell characteristics, such as cell type and cell cycle stage, for example.", "Cells may be cultured in suspension, cultured as monolayers attached to a solid support, and/or cultured on a layer of feeder cells, for example.", "The term “feeder cells” is defined hereafter.", "Furthermore, cells may be successfully cultured by plating the cells in conditions where they lack cell to cell contact.", "Preferably, cultured cells undergo cell division and are cultured for at least 5 days, more preferably for at least 10 days or 20 days, and most preferably for at least 30 days.", "Preferably, a significant number of cultured cells do not terminate while in culture.", "The terms “terminate” and “significant number” are defined hereafter.", "Nearly any type of cell can be placed in cell culture conditions.", "Cultured cells can be utilized to establish a cell line.", "In particularly preferred embodiments, a cell may be “clonally propagated.” In these embodiments, cells are diluted to an extent such that, statistically, some or all of the culture vessels into which the diluted cells are placed will contain only a single cell.", "Thus, the culture that grows within these culture vessels will be derived from a single cell.", "Materials and methods for clonally propagating cells are described in U.S. patent application Ser.", "No.", "09/753,323 (attorney docket number 030653.0026.CIP1, filed Dec. 28, 2000), which is hereby incorporated in its entirety.", "The term “terminating” and “terminate” as used herein with regard to cultured cells may refer to cells that undergo cell death, which can be measured using multiple techniques known to those skilled in the art (e.g., CytoTox96® Cytotoxicity Assay, Promega, Inc. catalog no.", "G1780; Celltiter96® Aqueous Cell Proliferation Assay Kit, Promega, Inc. catalog no.", "G3580; and Trypan Blue solution for cytotoxicity assays, Sigma catalog no.", "T6146).", "Termination may also be a result of apoptosis, which can be measured using multiple techniques known to persons skilled in the art (e.g., Dead End™ Apoptosis Detection Kit, Promega, Inc. catalog no.", "G7130).", "Terminated cells may be identified as those that have undergone cell death and/or apoptosis and have released from a solid surface in culture.", "In addition, terminated cells may lack intact membranes which can be identified by procedures described above.", "Also, terminated cells may exhibit decreased metabolic activity, which may be caused in part by decreased mitochondrial activity that can be identified by rhodamine 1,2,3, for example.", "Furthermore, termination can be refer to cell cultures where a significant number of cultured cells terminate.", "The term “significant number” in the preceding sentence refers to about 80% of the cells in culture, preferably about 90% of the cells in culture, more preferably about 100% of the cells in culture, and most preferably 100% of the cells in culture.", "The term “suspension” as used herein refers to cell culture conditions in which the cells are not attached to a solid support.", "Cells proliferating in suspension can be stirred while proliferating using apparatus well known to those skilled in the art.", "The term “monolayer” as used herein refers to cells that are attached to a solid support while proliferating in suitable culture conditions.", "A small portion of the cells proliferating in the monolayer under suitable growth conditions may be attached to cells in the monolayer but not to the solid support.", "Preferably less than 15% of these cells are not attached to the solid support, more preferably less than 10% of these cells are not attached to the solid support, and most preferably less than 5% of these cells are not attached to the solid support.", "The term “substantially similar” as used herein in reference to mammalian cells refers to cells from the same organism and the same tissue.", "Substantially similar can also refer to cell populations that have not significantly differentiated.", "For example, preferably less than 15% of the cells in a population of cells have differentiated, more preferably less than 10% of the cell population have differentiated, and most preferably less than 5% of the cell population have differentiated.", "The term “cell line” as used herein refers to cultured cells that can be passaged one or more times.", "The invention preferably relates to cell lines that can be passaged more than 2, 5, 10, 15, 20, 30, 50, 80, 100, and 200 times.", "The concept of cell passaging is defined previously.", "In preferred embodiments, (1) the mammalian cell is subject to manipulation; (2) the manipulation comprises the step of nuclear transfer; (3) the nuclear transfer comprises the step of inserting the totipotent mammalian cell into a recipient oocyte; (4) the manipulation comprises a step of cryopreservation of the mammalian cell; (5) the manipulation comprises a step of thawing of the mammalian cell; (6) the manipulation comprises a step of culturing the mammalian cell; (7) the manipulation comprises a step of passaging the mammalian cell; (8) the manipulation comprises a step of synchronizing the mammalian cell; (9) the manipulation comprises a step of introducing the mammalian cell to feeder cells; and (10) the manipulation comprises a step of dissociating the mammalian cell from other cells.", "The term “manipulation” as used herein refers to the common usage of the term, which is the management or handling directed towards some object.", "Examples of manipulations are described herein.", "The term “thawing” as used herein refers to the process of increasing the temperature of a cryopreserved cell, embryo, or portions of animals.", "Methods of thawing cryopreserved materials such that they are active after the thawing process are well-known to those of ordinary skill in the art.", "The term “dissociating” as used herein refers to the materials and methods useful for pulling a cell away from another cell.", "For example, a blastomere (i.e., a cellular member of a morula or blastocyst stage embryo) can be pulled away from the rest of the developing cell mass by techniques and apparatus well known to a person of ordinary skill in the art.", "See, e.g., U.S. Pat.", "No.", "4,994,384, entitled “Multiplying Bovine Embryos,” issued on Feb. 19, 1991, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "Alternatively, cells proliferating in culture can be separated from one another to facilitate such processes as cell passaging, which is described previously.", "In addition, dissociation of a cultured cell from a group of cultured cells can be useful as a first step in the process of nuclear transfer, as described hereafter.", "When a cell is dissociated from an embryo, the dissociation manipulation can be useful for such processes as re-cloning, a process described herein, as well as a step for multiplying the number of embryos.", "The term “non-embryonic cell” as used herein refers to a cell that is not isolated from an embryo.", "Non-embryonic cells can be differentiated or non-differentiated.", "Non-embryonic cells refers to nearly any somatic cell, such as cells isolated from an ex utero animal.", "These examples are not meant to be limiting.", "The term “fetus” as used herein refers to a developing cell mass that has implanted into the uterine membrane of a maternal host.", "A fetus can include such defining features as a genital ridge, for example.", "A genital ridge is a feature easily identified by a person of ordinary skill in the art, and is a recognizable feature in fetuses of most animal species.", "The term “fetal cell” as used herein refers to any cell isolated from and/or has arisen from a fetus or derived from a fetus.", "The term “non-fetal cell” is a cell that is not derived or isolated from a fetus.", "When cells are isolated from a fetus, such cells are preferably isolated from fetuses where the fetus is between 20 days and parturition, between 30 days and 100 days, more preferably between 35 days and 70 days and between 40 days and 60 days, and most preferably about a 55 day fetus.", "An age of a fetus can be determined from the time that an embryo, which develops into the fetus, is established.", "The term “about” with respect to fetuses refers to plus or minus five days.", "The term “parturition” as used herein refers to a time that a fetus is delivered from female recipient.", "A fetus can be delivered from a female recipient by abortion, c-section, or birth.", "In preferred embodiments, the cells and cell lines of the instant invention are primary cells, embryonic cells, non-embryonic cells, fetal cells, genital ridge cells, primordial germ cells, embryonic germ cells, embryonic stem cells, somatic cells, adult cells, fibroblasts, differentiated cells, undifferentiated cells, amniotic cells, ovarian follicular cells, and cumulus cells.", "Preferably, such cells grow to confluent monolayers in culture.", "The term “primordial germ cell” as used herein refers to a diploid somatic cell capable of becoming a germ cell.", "Primordial germ cells can be isolated from the genital ridge of a developing cell mass.", "The genital ridge is a section of a developing cell mass that is well-known to a person of ordinary skill in the art.", "See, e.g., Strelchenko, 1996, Theriogenology 45: 130-141 and Lavoir 1994, J. Reprod.", "Dev.", "37: 413-424.Such cells, when cultured, are referred to by the skilled artisan as “embryonic germ cells.” The term “embryonic stem cell” as used herein refers to pluripotent cells isolated from an embryo that are maintained in in vitro cell culture.", "Embryonic stem cells may be cultured with or without feeder cells.", "Embryonic stem cells can be established from embryonic cells isolated from embryos at any stage of development, including blastocyst stage embryos and pre-blastocyst stage embryos.", "Embryonic stem cells are well known to a person of ordinary skill in the art.", "See, e.g., WO 97/37009, entitled “Cultured Inner Cell Mass Cell-Lines Derived from Ungulate Embryos,” Stice & Golueke, published Oct. 9, 1997, and Yang & Anderson, 1992, Theriogenology 38: 315-335, both of which are incorporated herein by reference in their entireties, including all figures, tables, and drawings.", "The term “differentiated cell” as used herein refers to a cell that has developed from an unspecialized phenotype to that of a specialized phenotype.", "For example, embryonic cells can differentiate into an epithelial cell lining the intestine.", "It is highly unlikely that differentiated cells revert into their precursor cells in vivo or in vitro.", "However, materials and methods of the invention can reprogram differentiated cells into immortalized, totipotent cells.", "Differentiated cells can be isolated from a fetus or a live born animal, for example.", "The term “undifferentiated cell” as used herein refers to a cell that has an unspecialized phenotype and is capable of differentiating.", "An example of an undifferentiated cell is a stem cell.", "The term “asynchronous population” as used herein refers to cells that are not arrested at any one stage of the cell cycle.", "Many cells can progress through the cell cycle and do not arrest at any one stage, while some cells can become arrested at one stage of the cell cycle for a period of time.", "Some known stages of the cell cycle are G 0 , G 1 , S, G 2 , and M. An asynchronous population of cells is not manipulated to synchronize into any one or predominantly into any one of these phases.", "Cells can be arrested in the G 0 stage of the cell cycle, for example, by utilizing multiple techniques known in the art, such as by serum deprivation.", "Examples of methods for arresting non-immortalized cells in one part of the cell cycle are discussed in WO 97/07669, entitled “Quiescent Cell Populations for Nuclear Transfer,” hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "The terms “synchronous population” and “synchronizing” as used herein refer to a fraction of cells in a population that are arrested (i.e., the cells are not dividing) in a discrete stage of the cell cycle.", "Preferably, about 50% of the cells in a population of cells are arrested in one stage of the cell cycle, more preferably about 70% of the cells in a population of cells are arrested in one stage of the cell cycle, and most preferably about 90% of the cells in a population of cells are arrested in one stage of the cell cycle.", "Cell cycle stage can be distinguished by relative cell size as well as by a variety of cell markers well known to a person of ordinary skill in the art.", "For example, cells can be distinguished by such markers by using flow cytometry techniques well known to a person of ordinary skill in the art.", "Alternatively, cells can be distinguished by size utilizing techniques well known to a person of ordinary skill in the art, such as by the utilization of a light microscope and a micrometer, for example.", "The term “adult cell” as used herein refers to a cell from a live-born animal.", "The term “amniotic cell” as used herein refers to any cultured or non-cultured cell isolated from amniotic fluid.", "Examples of methods for isolating and culturing amniotic cells are discussed in Bellow et al., 1996, Theriogenology 45: 225; Garcia & Salaheddine, 1997, Theriogenology 47: 1003-1008; Leibo & Rail, 1990, Theriogenology 33: 531-552; and Vos et al., 1990, Vet.", "Rec.", "127: 502-504, each of which is incorporated herein by reference in its entirety, including all figures tables and drawings.", "Particularly preferred are cultured amniotic cells that are rounded (e.g., cultured amniotic cells that do not display a fibroblast-like morphology).", "Also preferred amniotic cells are fetal fibroblast cells.", "The terms “fibroblast,” fibroblast-like,” “fetal,” and “fetal fibroblast” are defined hereafter.", "The term “fibroblast” as used herein refers to cultured cells having a flattened and elongated morphology that are able to grow in monolayers.", "Preferably, fibroblasts grow to confluent monolayers in culture.", "While fibroblasts characteristically have a flattened appearance when cultured on culture media plates, fetal fibroblast cells can also have a spindle-like morphology.", "Fetal fibroblasts may require density limitation for growth, may generate type I collagen, and may have a finite life span in culture of approximately fifty generations.", "Preferably, fetal fibroblast cells rigidly maintain a diploid chromosomal content.", "For a description of fibroblast cells, see, e.g., Culture of Animal Cells: a manual of basic techniques (3 rd edition), 1994, R. I. Freshney (ed), Wiley-Liss, Inc., incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "The term “uterine cell” as used herein refers to any cell isolated from a uterus.", "Preferably, a uterine cell is a cell deriving from a pregnant adult animal.", "In preferred embodiments, uterine cells are cells obtained from fluid that fills the uterine cavity.", "Such cells can be obtained by numerous methods well known in the art such as amniocentesis.", "The term “ovarian follicular cell” as used herein refers to a cultured or non-cultured cell obtained from an ovarian follicle, other than an oocyte.", "Follicular cells may be isolated from ovarian follicles at any stage of development, including primordial follicles, primary follicles, secondary follicles, growing follicles, vesicular follicles, maturing follicles, mature follicles, and graafian follicles.", "Furthermore, follicular cells may be isolated when an oocyte in an ovarian follicle is immature (i.e., an oocyte that has not progressed to metaphase II) or when an oocyte in an ovarian follicle is mature (i.e., an oocyte that has progressed to metaphase II or a later stage of development).", "Preferred follicular cells include, but are not limited to, pregranulosa cells, granulosa cells, theca cells, columnar cells, stroma cells, theca interna cells, theca externa cells, mural granulosa cells, luteal cells, and corona radiata cells.", "Particularly preferred follicular cells are cumulus cells.", "Various types of follicular cells are known and can be readily distinguished by those skilled in the art.", "See, e.g., Laboratory Production of Cattle Embryos, 1994, Ian Gordon, CAB International; Anatomy and Physiology of Farm Animals (5th ed.", "), 1992, R. D. Frandson and T. L. Spurgeon, Lea & Febiger, each of which is incorporated herein by reference in its entirety including all figures, drawings, and tables.", "Individual types of follicular cells may be cultured separately, or a mixture of types may be cultured together.", "The term “cumulus cell” as used herein refers to any cultured or non-cultured cell isolated from cells and/or tissue surrounding an oocyte.", "Persons skilled in the art can readily identify cumulus cells.", "Examples of methods for isolating and/or culturing cumulus cells are discussed in Damiani et al., 1996, Mol.", "Reprod.", "Dev.", "45: 521-534; Long et al., 1994, J: Reprod.", "Fert.", "102: 361-369; and Wakayama et al., 1998, Nature 394: 369-373, each of which is incorporated herein by reference in its entireties, including all figures, tables, and drawings.", "Cumulus cells may be isolated from ovarian follicles at any stage of development, including primordial follicles, primary follicles, secondary follicles, growing follicles, vesicular follicles, maturing follicles, mature follicles, and graafian follicles.", "Cumulus cells may be isolated from oocytes in a number of manners well known to a person of ordinary skill in the art.", "For example, cumulus cells can be separated from oocytes by pipeting the cumulus cell/oocyte complex through a small bore pipette, by exposure to hyaluronidase, or by mechanically disrupting (e.g.", "vortexing) the cumulus cell/oocyte complex.", "Additionally, exposure to Ca ++ /Mg ++ free media can remove cumulus from immature oocytes.", "Also, cumulus cell cultures can be established by placing matured oocytes in cell culture media.", "Once cumulus cells are removed from media containing increased LH/FSH concentrations, they can to attach to the culture plate.", "In a preferred embodiment, the culturing process can comprise the step of selecting totipotent mammalian cells comprising at least one artificial chromosome.", "The term “selection” as used herein refers to a process for identifying cells that comprise a large heterologous nucleic acid construct, such as an artificial chromosome.", "Selection can be effected by identifying a marker region incorporated in an artificial chromosome.", "The term “marker” is defined previously.", "Preferably, from 50% to 100% of cells in cell cultures that have undergone selection comprise an artificial chromosome.", "In particularly preferred embodiments, greater than or equal to 50% of cells in cell cultures that have undergone selection comprise an artificial chromosome.", "More preferably, greater than or equal to 75% of cells in cell cultures that have undergone selection comprise an artificial chromosome.", "Most preferably, greater than or equal to 90% of cells in cell cultures that have undergone selection comprise an artificial chromosome.", "The term “feeder cells” as used herein refers to cells grown in co-culture with target cells.", "Target cells can be precursor cells and totipotent cells, for example.", "Feeder cells can provide, for example, peptides, polypeptides, electrical signals, organic molecules (e.g., steroids), nucleic acid molecules, growth factors (e.g., bFGF), other factors (e.g., cytokines such as LIF and steel factor), and metabolic nutrients to target cells.", "Certain cells, such as immortalized, totipotent cells may not require feeder cells for healthy growth.", "Feeder cells preferably grow in a mono-layer.", "Feeder cells can be established from multiple cell types.", "Examples of these cell types are fetal cells, mouse cells, Buffalo rat liver cells, and oviductal cells.", "These examples are not meant to be limiting.", "Tissue samples can be broken down to establish a feeder cell line by methods well known in the art (e.g., by using a blender).", "Feeder cells may originate from the same or different animal species as the precursor cells.", "In an example of feeder cells established from fetal cells, ungulate fetuses and preferably bovine fetuses may be utilized to establish a feeder cell line where one or more cell types have been removed from the fetus (e.g., primordial germ cells, cells in the head region, and cells in the body cavity region).", "When an entire fetus is utilized to establish a fetal feeder cell line, feeder cells (e.g., fibroblast cells) and precursor cells (e.g., primordial germ cells) can arise from the same source (e.g., one fetus).", "The term “drug” as used herein refers to any type of molecule that retards the normal growth rate of a cell.", "A normal cell growth rate is measured in the absence of drug.", "A drug may also lyse cells.", "In another aspect, the invention features a method for producing transgenic ungulates by introducing a large heterologous nucleic acid construct into a nuclear donor cell, then fusing this nuclear donor cell into an enucleated recipient cell to form a nuclear transfer embryo, activating this embryo, and finally transferring this embryo into a maternal host to produce a transgenic animal.", "In particularly preferred embodiments, the transgenic animal is an ungulate.", "Nuclear Transfer Most preferably, transgenic animals are prepared by introducing a heterologous nucleic acid molecule, preferably an artificial chromosome, into a nuclear donor cell, then fusing this nuclear donor cell into an enucleated recipient cell, most preferably an enucleated oocyte, to form a nuclear transfer embryo, activating this embryo, and finally transferring this embryo into a maternal host to produce a transgenic animal.", "In preferred embodiments, the artificial chromosome(s) is introduced into the cybrid by introduction into the nuclear donor cell prior to the fusion with the enucleated recipient cell or enucleated oocyte.", "In other preferred embodiments, the artificial chromosome(s) is introduced into the cybrid formed by fusion of the nuclear donor cell with the enucleated recipient cell or enucleated oocyte.", "In yet other preferred embodiments, the artificial chromosome(s) is introduced into the cybrid simultaneously with the fusion of the nuclear donor cell with the enucleated recipient cell or enucleated oocyte The terms “nuclear transfer” and “nuclear transfer procedure” as used herein refer to introducing a full complement of nuclear DNA from one cell to an enucleated cell.", "Nuclear transfer methods are well known to a person of ordinary skill in the art.", "See, U.S. Pat.", "No.", "4,994,384 to Prather et al., entitled “Multiplying Bovine Embryos,” issued on Feb. 19, 1991; U.S. Pat.", "No.", "5,057,420 to Massey, entitled “Bovine Nuclear Transplantation,” issued on Oct. 15, 1991; U.S. Pat.", "No.", "5,994,619, issued on Nov. 30, 1999 to Stice et al., entitled “Production of Chimeric Bovine or Porcine Animals Using Cultured Inner Cell Mass Cells; U.K.", "Patents Nos.", "GB 2,318,578 GB 2,331,751, issued on Jan. 19, 2000 to Campbell et al.", "and Wilmut et al., respectively, entitled “Quiescent Cell Populations For Nuclear Transfer”; U.S. Pat.", "No.", "6,011,197 to Strelchenko et al., entitled “Method of Cloning Bovines Using Reprogrammed Non-Embryonic Bovine Cells,” issued on Jan. 4, 2000; and in U.S. patent application Ser.", "No.", "09/753,323 entitled “Method of Cloning Porcine Animals (attorney docket number 030653.0026.CIP1, filed Dec. 28, 2000), each of which are hereby incorporated by reference in its entirety including all figures, tables and drawings.", "Nuclear transfer may be accomplished by using oocytes that are not surrounded by a zona pellucida.", "In a nuclear transfer procedure, a nuclear donor cell, or the nucleus thereof, is introduced into a recipient cell.", "A recipient cell is preferably an oocyte and is preferably enucleated.", "However, the invention relates in part to nuclear transfer, where a nucleus of an oocyte is not physically extracted from the oocyte.", "It is possible to establish a nuclear transfer embryo where nuclear DNA from the donor cell is replicated during cellular divisions.", "See, e.g., Wagoner et al., 1996, “Functional enucleation of bovine oocytes: effects of centrifugation and ultraviolet light,” Theriogenology 46: 279-284.In addition, nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte.", "Also, nuclear transfer may be accomplished by combining one nuclear donor, one or more enucleated oocytes, and the cytoplasm of one or more enucleated oocytes.", "The resulting combination of a nuclear donor cell and a recipient cell can be referred to variously as a “nuclear transfer embryo,” a “hybrid cell,” or a “cybrid.” Furthermore, a nuclear donor may arise from an animal of the same species from which a nuclear recipient is isolated.", "Alternatively, a nuclear donor may arise from an animal of a different specie from which a nuclear recipient is isolated.", "For example, a differentiated cell isolated from an ear punch of a water buffalo may be utilized as a nuclear donor and an oocyte isolated from a bovine animal may be utilized as a nuclear acceptor.", "Thus, xenospecific nuclear transfer is contemplated by the instant invention.", "The term “nuclear donor” as used herein refers to any cell, or nucleus thereof, having nuclear DNA that can be translocated into an oocyte.", "A nuclear donor may be a nucleus that has been isolated from a cell.", "Multiple techniques are available to a person of ordinary skill in the art for isolating a nucleus from a cell and then utilizing the nucleus as a nuclear donor.", "See, e.g., U.S. Pat.", "Nos.", "4,664,097, 6,011,197, and 6,107,543, each of which is hereby incorporated by reference in its entirety including all figures, tables and drawings.", "Any type of cell can serve as a nuclear donor.", "Examples of nuclear donor cells include, but are not limited to, cultured and non-cultured cells isolated from an embryo arising from the union of two gametes in vitro or in vivo; embryonic stem cells (ES cells) arising from cultured embryonic cells (e.g., pre-blastocyst cells and inner cell mass cells); cultured and non-cultured cells arising from inner cell mass cells isolated from embryos; cultured and non-cultured pre-blastocyst cells; cultured and non-cultured fetal cells; cultured and non-cultured adult cells; cultured and non-cultured primordial germ cells; cultured and non-cultured germ cells (e.g., embryonic germ cells); cultured and non-cultured somatic cells isolated from an animal; cultured and non-cultured cumulus cells; cultured and non-cultured amniotic cells; cultured and non-cultured fetal fibroblast cells; cultured and non-cultured genital ridge cells; cultured and non-cultured differentiated cells; cultured and non-cultured cells in a synchronous population; cultured and non-cultured cells in an asynchronous population; cultured and non-cultured serum-starved cells; cultured and non-cultured permanent cells; and cultured and non-cultured totipotent cells.", "See, e.g., Piedrahita et al., 1998, Biol.", "Reprod.", "58: 1321-1329; Shim et al., 1997, Biol.", "Reprod.", "57: 1089-1095; Tsung et al., 1995, Shih Yen Sheng Wu Hsueh Pao 28: 173-189; and Wheeler, 1994, Reprod.", "Fertil.", "Dev.", "6: 563-568, each of which is incorporated herein by reference in its entirety including all figures, drawings, and tables.", "In addition, a nuclear donor may be a cell that was previously frozen or cryopreserved.", "The term “activation” refers to any materials and methods useful for stimulating a cell to divide before, during, and after a nuclear transfer step.", "Cybrids may require stimulation in order to divide after a nuclear transfer has occurred.", "The invention pertains to any activation materials and methods known to a person of ordinary skill in the art.", "Although electrical pulses are sometimes sufficient for stimulating activation of cybrids, other means are sometimes useful or necessary for proper activation of the cybrid.", "Chemical materials and methods useful for activating embryos are described below in other preferred embodiments of the invention.", "Examples of non-electrical means for activation include agents such as ethanol; inositol trisphosphate (IP 3 ); Ca ++ ionophores (e.g., ionomycin) and protein kinase inhibitors (e.g., 6-dimethylaminopurine (MAP)); temperature change; protein synthesis inhibitors (e.g., cyclohexamide); phorbol esters such as phorbol 12-myristate 13-acetate (PMA); mechanical techniques; and thapsigargin.", "The invention includes any activation techniques known in the art.", "See, e.g., U.S. Pat.", "No.", "5,496,720, entitled “Parthenogenic Oocyte Activation” to Susko-Parrish et al., issued on Mar.", "5, 1996; and U.S. Pat.", "No.", "6,077,710, issued on Jun.", "20, 2000, each of which is incorporated by reference herein in its entirety, including all figures, tables, and drawings.", "The term “fusion” as used herein in reference to nuclear transfer refers to the combination of portions of lipid membranes corresponding to the nuclear donor and the recipient oocyte.", "Lipid membranes can correspond to the plasma membranes of cells or nuclear membranes, for example.", "The fusion can occur between the nuclear donor and recipient oocyte when they are placed adjacent to one another, or when the nuclear donor is placed in the perivitelline space of the recipient oocyte, for example.", "Specific examples for translocation of the totipotent mammalian cell into the oocyte are described hereafter in other preferred embodiments.", "These techniques for translocation are fully described in the references cited previously herein in reference to nuclear transfer.", "The term “electrical pulses” as used herein refers to subjecting the nuclear donor and recipient oocyte to electric current.", "For nuclear transfer, the nuclear donor and recipient oocyte can be aligned between electrodes and subjected to electrical current.", "The electrical current can be alternating current or direct current.", "The electrical current can be delivered to cells for a variety of different times as one pulse or as multiple pulses.", "The cells are typically cultured in a suitable medium for the delivery of electrical pulses.", "Examples of electrical pulse conditions utilized for nuclear transfer are described in the references and patents previously cited herein in reference to nuclear transfer.", "The term “fusion agent” as used herein in reference to nuclear transfer refers to any compound or biological organism that can increase the probability that portions of plasma membranes from different cells will fuse when a totipotent mammalian cell nuclear donor is placed adjacent to the recipient oocyte.", "In preferred embodiments fusion agents are selected from the group consisting of polyethylene glycol (PEG), trypsin, dimethylsulfoxide (DMSO), lectins, agglutinin, viruses, and Sendai virus.", "These examples are not meant to be limiting and other fusion agents known in the art are applicable and included herein.", "The term “suitable concentration” as used herein in reference to fusion agents, refers to any concentration of a fusion agent that affords a measurable amount of fusion.", "Fusion can be measured by multiple techniques well known to a person of ordinary skill in the art, such as by utilizing a light microscope, dyes, and fluorescent lipids, for example.", "The term “totipotent” as used herein refers to a cell, embryo, or fetus capable of giving rise to a live born animal.", "The term “totipotent” can also refer to a cell that gives rise to all of the cells in a particular animal.", "A totipotent cell can give rise to all of the cells of an animal when it is utilized in a procedure for developing an embryo from one or more nuclear transfer steps.", "Totipotent cells, embryos, and fetuses may also be used to generate incomplete animals such as those useful for organ harvesting, e.g., having genetic modifications to eliminate growth of an organ or appendage by manipulation of a homeotic gene.", "The term “live born” as used herein preferably refers to an animal that exists ex utero.", "A “live born” animal may be an animal that is alive for at least one second from the time it exits the maternal host.", "A “live born” animal may not require the circulatory system of an in utero environment for survival.", "A “live born” animal may be an ambulatory animal.", "Such animals can include pre- and post-pubertal animals.", "As discussed previously, a live born animal may lack a portion of what exists in a normal animal of its kind.", "In preferred embodiments, (1) totipotent cells arise from at least one precursor cell; (2) a precursor cell is isolated from and/or arises from any region of a ungulate animal; (3) a precursor cell is isolated from and/or arises from any cell in culture; (4) a precursor cell is selected from the group consisting of a primary cell, a non-embryonic cell, a non-fetal cell, a differentiated cell, an undifferentiated cell, a somatic cell, an embryonic cell, a fetal cell, an embryonic stem cell, a primordial germ cell, a genital ridge cell, a cumulus cell, an amniotic cell, a fetal fibroblast cell, a uterine cell, an ovarian follicular cell, a cumulus cell, an hepatocyte, an embryonic germ cell, an adult cell, a cell isolated from an asynchronous population of cells, and a cell isolated from a synchronized population of cells where the synchronous population is not arrested in the G 0 stage of the cell cycle; (5) totipotent cells have a morphology of an embryonic germ cell.", "The terms “precursor cell” or “precursor cells” as used herein refer to a cell or cells used to create a cell line of totipotent cells.", "Precursor cells can be isolated from, any animal, preferably from a mammal, and more preferably from an ungulate.", "The precursor cell or cells may be isolated from nearly any cellular entity.", "For example, a precursor cell or cells may be isolated from blastocysts, embryos, fetuses, and cell lines (e.g., cell lines established from embryonic cells), preferably isolated from fetuses and/or cell lines established from fetal cells, and more preferably isolated from ex utero animals and/or cell cultures and/or cell lines established from such ex utero animals.", "An ex utero animal may exist as a newborn animal, adolescent animal, yearling animal, and adult animal.", "The ex utero animals may be alive or post mortem.", "Examples of precursor cells include, but are not limited to, non-embryonic cells; non-fetal cells; differentiated cells; adult cells; somatic cells; embryonic cells; fetal cells; embryonic stem cells; primordial germ cells; genital ridge cells; uterine cells; amniotic cells; ovarian follicular cells; cumulus cells; cells isolated from an asynchronous population of cells; and cells isolated from a synchronized population of cells where the synchronous population is not arrested in the G 0 stage of the cell cycle; and any of the forgoing that are cultured, cultured as cell lines and/or totipotent.", "The term “arises from” as used herein refers to the conversion of one or more cells into one or more cells having at least one differing characteristic.", "For example, (1) a non-totipotent precursor cell can be converted into a totipotent cell by utilizing features of the invention described hereafter; (2) a precursor cell can develop a cell morphology of an embryonic germ cell; (3) a precursor cell can give rise to a cultured cell; (4) a precursor cell can give rise to a cultured cell line; and (5) a precursor cell can give rise to a cultured permanent cell line.", "A conversion process can be referred to as a reprogramming step.", "In addition, the term “arises from” refers to establishing totipotent embryos from totipotent cells of the invention by using a nuclear transfer process, as described hereafter.", "The terms “reprogramming” or “reprogrammed” as used herein refer to materials and methods that can convert a non-totipotent cell into a totipotent cell.", "Distinguishing features between totipotent and non-totipotent cells are described previously.", "An example of materials and methods for converting non-totipotent cells into totipotent cells is to incubate precursor cells with a receptor ligand cocktail.", "Receptor ligand cocktails are described hereafter.", "In preferred embodiments, culturing of a cell is a sufficient stimulus to render a cell totipotent.", "The term “reprogramming” or “reprogrammed” as used herein can also refer to materials and methods that can convert a cell into another cell having at least one differing characteristic.", "Also, such materials and methods may reprogram or convert a cell into another cell type that is not typically expressed during the life cycle of the former cell.", "For example, (1) a non-totipotent cell can be reprogrammed into an totipotent cell; (2) a precursor cell can be reprogrammed into a cell having a morphology of an EG cell; and (3) a precursor cell can be reprogrammed into a totipotent cell.", "An example of materials and methods for converting a precursor cell into a totipotent cell having EG cell morphology is described hereafter.", "The term “isolated” as used herein in reference to cells refers to a cell that is mechanically separated from another group of cells.", "Examples of a group of cells are a developing cell mass, a cell culture, a cell line, and an animal.", "These examples are not meant to be limiting and the invention relates to any group of cells.", "Methods for isolating one or more cells from another group of cells are well known in the art.", "See, e.g., Culture of Animal Cells: a manual of basic techniques (3 rd edition), 1994, R. I. Freshney (ed.", "), Wiley-Liss, Inc.; Cells: a laboratory manual (vol.", "1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.", "), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J. Morgan, John Wiley and Sons, Ltd.", "The terms “cryopreservation” or “cryopreserved” as used herein refer to freezing a cell, embryo, or animal of the invention.", "The cells, embryos, or portions of animals of the invention are frozen at temperatures lower than 0° C., preferably lower than −80° C., more preferably at temperatures lower than −140° C., and most preferably at temperatures lower than −196° C. Cells and embryos in the invention can be cryopreserved for an indefinite amount of time.", "It is known that biological materials can be cryopreserved for more than fifty years.", "For example, semen that is cryopreserved for more than fifty years can be utilized to artificially inseminate a female bovine animal.", "Methods and tools for cryopreservation are well-known to those skilled in the art.", "See, e.g., U.S. Pat.", "No.", "5,160,312, entitled “Cryopreservation Process for Direct Transfer of Embryos,” issued to Voelkel on Nov. 3, 1992, hereby incorporated by reference herein in its entirety, including all figures, tables, and drawings.", "For the purposes of the present invention, the terms “embryo” or “embryonic” as used herein refer to a developing cell mass that has not implanted into the uterine membrane of a maternal host.", "Hence, the term “embryo” as used herein can refer to a fertilized oocyte, a cybrid (defined herein), a pre-blastocyst stage developing cell mass, and/or any other developing cell mass that is at a stage of development prior to implantation into the uterine membrane of a maternal host.", "Embryos of the invention may not display a genital ridge.", "Hence, an “embryonic cell” is isolated from and/or has arisen from an embryo.", "An embryo can represent multiple stages of cell development.", "For example, a one cell embryo can be referred to as a zygote, a solid spherical mass of cells resulting from a cleaved embryo can be referred to as a morula, and an embryo having a blastocoel can be referred to as a blastocyst.", "The terms “enucleated oocyte” or “enucleated recipient cell” as used herein refer to an oocyte which has had its nucleus removed.", "Typically, a needle can be placed into an oocyte and the nucleus can be aspirated into the inner space of the needle.", "The needle can be removed from the oocyte without rupturing the plasma membrane.", "This enucleation technique is well known to a person of ordinary skill in the art.", "See, U.S. Pat.", "No.", "4,994,384; U.S. Pat.", "No.", "5,057,420; and Willadsen, 1986, Nature 320:63-65.An enucleated oocyte can be prepared from a young or an aged oocyte.", "An enucleated oocyte is preferably prepared from an oocyte that has been matured, in vitro or in vivo, for some period of time.", "This time can vary, depending on the source species for the oocyte.", "For example, bovine oocytes are preferably matured for between 10 hours and 40 hours, more preferably for between 16 hours and 36 hours, and most preferably between 20 hours and 32 hours.", "In contrast, porcine oocytes are preferably matured for greater than 24 hours, and more preferably matured for greater than 36 hours.", "In particularly preferred embodiments, a porcine oocyte is matured for more than 40 hours, up to about 96 hours, more preferably from 42-54 hours, and even more preferably from 42 to 48 hours.", "The terms “maturation” and “matured” as used herein refer to process in which an oocyte is incubated in a medium in vitro.", "Oocytes can be incubated with multiple media well known to a person of ordinary skill in the art.", "See, e.g., Saito et al., 1992, Roux 's Arch.", "Dev.", "Biol.", "201: 134-141 for bovine organisms and Wells et al., 1997, Biol.", "Repr.", "57: 385-393 for ovine organisms and also Mattioli et al., 1989, Theriogenology 31: 1201-1207; Jolliff & Prather, 1997, Biol.", "Reprod.", "56: 544-548; Funahashi & Day, 1993, J. Reprod.", "Fert.", "98: 179-185; Nagashima et al., 1997, Mol.", "Reprod.", "Dev.", "38: 339-343; Abeydeera et al., 1998, Biol.", "Reprod.", "58: 213-218; Funahashi et al., 1997, Biol.", "Reprod.", "57: 49-53; and Sawai et al., 1997, Biol.", "Reprod.", "57: 1-6, each of which are incorporated herein by reference in their entireties including all figures, tables, and drawings.", "Maturation media can comprise multiple types of components, including microtubule inhibitors (e.g.", "cytochalasin B), hormones and growth factors.", "Other examples of components that can be incorporated into maturation media are discussed in WO 97/07668, entitled “Unactivated Oocytes as Cytoplast Recipients for Nuclear Transfer,” Campbell & Wilmut, published on Mar.", "6, 1997, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "The time of maturation can be determined from the time that an oocyte is placed in a maturation medium to the time that the oocyte is subject to a manipulation (e.g., enucleation, nuclear transfer, fusion, and/or activation).", "Oocytes can be matured for any period of time: an oocyte can be matured for greater than 10 hours, greater than 20 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours, greater than 60 hours, greater than 72 hours, and greater than 90 hours.", "The term “about” with respect to oocyte maturation refers to plus or minus 3 hours.", "An oocyte can also be matured in vivo.", "Time of maturation may be the time that an oocyte receives an appropriate stimulus to resume meiosis to the time that the oocyte is manipulated.", "Similar maturation periods described above for in vitro matured oocytes apply to in vivo matured oocytes.", "Nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte.", "In addition, nuclear transfer may be accomplished by combining one nuclear donor, one or more enucleated oocytes, and the cytoplasm of one or more enucleated oocytes.", "The term “young oocyte” as used herein refers to an oocyte that has been matured in vitro for a time less than or equal to the length of time between the onset of estrus and ovulation in vivo.", "For example, the onset of estrus is signaled by a surge in leutenizing hormone.", "A cow typically ovulates about 26 hours following the onset of estrus.", "Thus, a young oocyte is an oocyte matured for about 26 hours or less, preferably 16 to 17 hours.", "Methods for measuring the length of time between the onset of estrus and ovulation are well known to the skilled artisan.", "See, e.g., P. T. Cupps, “Reproduction in Domestic Animals,” Fourth Edition, Academic Press, San Diego, Calif., USA, 1991.For horses, ovulation occurs about 33 hours after onset of estrus; for pigs, about 40 hours; for sheep and goats, about 24-36 hours; for dogs, about 40-50 hours; and for cats, about 24-36 hours.", "The term “young oocyte” may also refer to an oocyte that has been matured and ovulated in vivo and that is collected at about the time of ovulation.", "The term about in this context refers to +/−1 hour.", "Oocytes can be isolated from live animals using methods well known to a person of ordinary skill in the art.", "See, e.g., Pieterse et al., 1988, “Aspiration of bovine oocytes during transvaginal ultrasound scanning of the ovaries,” Theriogenology 30: 751-762.Oocytes can be isolated from ovaries or oviducts of deceased or live born animals.", "Suitable media for in vitro culture of oocytes are well known to a person of ordinary skill in the art.", "See, e.g., U.S. Pat.", "No.", "5,057,420, which is incorporated by reference herein.", "Some young oocytes can be identified by the appearance of their ooplasm.", "Because certain cellular material (e.g., lipids) have not yet dispersed within the ooplasm.", "Young oocytes can have a pycnotic appearance.", "A pycnotic appearance can be characterized as clumping of cytoplasmic material.", "For example, in bovines, a “pycnotic” appearance is to be contrasted with the appearance of oocytes that are older than 28 hours, which have a more homogenous appearing ooplasm.", "The term “aged oocyte” as used herein refers to an oocyte that has been matured in vitro for a time greater than the length of time between the onset of estrus and ovulation in vivo.", "The term “aged oocyte” may also refer to an oocyte that has been matured and ovulated in vivo and that is collected later than about 1 hour after the time of ovulation.", "An aged oocyte can be identified by its characteristically homogenous ooplasm.", "This appearance is to be contrasted with the pycnotic appearance of young oocytes as described previously herein.", "The age of the oocyte can be defined by the time that has elapsed between the time that the oocyte is placed in a suitable maturation medium and the time that the oocyte is activated.", "The age of the oocyte can dramatically enhance the efficiency of nuclear transfer.", "For example, an aged oocyte can be more susceptible to activation stimuli than a young oocyte.", "The term “ovulated in vivo” as used herein refers to an oocyte that is isolated from an animal a certain number of hours after the animal exhibits characteristics that it is in estrus.", "The characteristics of an animal in estrus are well known to a person of ordinary skill in the art, as described in references disclosed herein.", "The terms “maternal recipient” and “recipient female” as used herein refer to a female animal which is implanted with an embryo for development of the embryo.", "A maternal recipient may be either homospecific or xenospecific to the implanted embryo.", "For example it has been shown in the art that bovine embryos can develop in the oviducts of sheep.", "Stice & Keefer, 1993, “Multiple generational bovine embryo cloning,” Biology of Reproduction 48: 715-719.Implanting techniques are well known to a person of ordinary skill in the art.", "See, e.g., Polge & Day, 1982, “Embryo transplantation and preservation,” Control of Pig Reproduction , D J A Cole and G R Foxcroft, eds., London, UK, Butterworths, pp.", "227-291; Gordon, 1997, “Embryo transfer and associated techniques in pigs,” Controlled reproduction in pigs (Gordon, ed), CAB International, Wallingford UK, pp 164-182; and Kojima, 1998, “Embryo transfer,” Manual of pig embryo transfer procedures , National Livestock Breeding Center, Japanese Society for Development of Swine Technology, pp 76-79, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "The term “replication unit” as used herein refers to that portion of a chromosome or other DNA molecule capable of being replicated that is copied from a given origin of replication.", "A chromosome in eukaryotes has many replication units.", "The term “origin of replication” refers to the location in a DNA molecule where its replication begins.", "The term “essentially no homologous DNA” means that the DNA molecule in question comprises almost entirely heterologous DNA.", "Preferably, a molecule which contains essentially no homologous DNA comprises at least 98%, 99%, 99.5%, or 99.9% heterologous DNA when the number of base pairs of heterologous DNA in the molecule is divided by the overall number of base pairs in the molecule.", "The term “homologous DNA” as used herein refers to DNA having the same nucleic acid sequence as DNA sequences present in cell nuclear DNA.", "The term “germ line” refers to those cells which give rise to the reproductive cells of an organism.", "These cells contain the complete haploid genome of an organism and will pass these DNA molecules to the descendants of the organism in question.", "The term “somatic cell” refers to those cells of an organism which are not involved in the production of gametes, e.g., they are not involved in passing the genome to the next generation of the organism in question.", "Transgenic Embryos, Fetuses, and Animals In yet another aspect, the instant invention relates in part to any embryos, fetuses, and animals emanating from totipotent mammalian cells of the invention, where one or more cells in these developing cell masses comprise at least one large heterologous nucleic acid construct, most preferably an artificial chromosome.", "In preferred embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells of the embryos, fetuses, and animals emanating from totipotent mammalian cells of the invention comprise at least one large heterologous nucleic acid construct.", "Most preferably, between 90% and all of the cells of the embryos, fetuses, and animals emanating from totipotent mammalian cells of the invention comprise at least one large heterologous nucleic acid construct.", "Such embryos, fetuses, and animals are known in the art as being “transgenic.” In certain embodiments, the large heterologous nucleic acid construct is an artificial chromosome, most preferably an ACEs or a euchromatin-based minichromosome.", "The cells of the embryos, fetuses, and animals that comprise at least one artificial chromosome preferably comprise ten or fewer artificial chromosomes; more preferably comprise six or fewer artificial chromosomes, four or fewer artificial chromosomes, or two or fewer artificial chromosomes; and most preferably comprise one artificial chromosome.", "If the cells of the embryos, fetuses, and animals of the invention comprise more than one artificial chromosome, the artificial chromosomes may be identical or may differ from one another.", "The term “transgenic” as used herein in reference to embryos, fetuses and animals refers to an embryo, fetus or animal comprising one or more cells that contain heterologous nucleic acids.", "In preferred embodiments, a transgenic embryo, fetus, or animal comprises one or more transgenic cells.", "While germ line transmission is not a requirement of transgenic embryos, fetuses, or animals as that term is used herein, in particularly preferred embodiments a transgenic embryo, fetus, or animal can pass its transgenic characteristic(s) through the germ line.", "In certain embodiments, a transgenic embryo, fetus or animal expresses one or more transgenes as transgenic RNA and protein molecules.", "Most preferably, a transgenic embryo, fetus or animal results from a nuclear transfer procedure using a transgenic nuclear donor cell.", "Transgenic totipotent mammalian embryos can be established from cultured cybrids emanating from one or more nuclear transfer procedures, where one of the nuclear transfer procedures utilizes a totipotent mammalian cell harboring at least one artificial chromosome as a nuclear donor.", "A transgenic totipotent fetus can be established, for example, from a transgenic totipotent embryo that has been implanted into the uterus of a suitable female host.", "Cloned transgenic mammalian animals of the invention can be established from totipotent mammalian cells, totipotent mammalian embryos, and totipotent mammalian fetuses of the invention.", "In certain embodiments, a transgenic animal embryo is produced by nuclear transfer of a nuclear donor cell into an enucleated recipient cell according to the following method: (a) a heterologous DNA molecule of greater than 100 kilobase pairs is introduced into one or more ungulate cells by microcell fusion; (b) the one or more cells are cultured to provide a cell culture; (c) a nuclear donor cell obtained from the cell culture is fused with an enucleated recipient cell to form a nuclear transfer embryo comprising the heterologous DNA molecule; and (d) the nuclear transfer embryo is activated to provide the transgenic ungulate embryo.", "In particularly preferred embodiments, method further comprises one or more of the following: the culturing step comprises selection for one or more markers of said heterologous DNA molecule, whereby at least 90% of cells in said cell culture comprise the heterologous DNA molecule; the transgenic animal is an ungulate selected from the group consisting of a bovine, an ovine, a caprine, and a porcine; the heterologous DNA molecule comprises one or more telomeres, one or more centromeres, and one or more origins of replication; the heterologous DNA molecule is contained within the cells of the transgenic ungulate embryo on a replication unit that comprises essentially no homologous DNA; the activated nuclear transfer embryo is cultured to at least the two cell stage, wherein at least 50% of the cells of the transgenic ungulate embryo comprise the heterologous DNA molecule; the nuclear donor cell is selected from the group consisting of a somatic cell, a primordial germ cell, an embryonic germ cell, and an embryonic stem cell; the heterologous DNA molecule comprises a plurality of copies of at least one transgene; the heterologous DNA molecule is between 100 kilobase pairs and 500 megabase pairs in size; and the heterologous DNA molecule is an artificial chromosome.", "In yet another aspect, the invention features a method of using a cloned transgenic fetus or animal, where one or more cells of the fetus or animal comprise one or more large heterologous nucleic acid constructs.", "The method of using a cloned transgenic fetus or animal comprises the step of isolating at least one component from the fetus or animal.", "The term “component” as used herein can relate to any portion of a fetus or animal.", "A component can be selected from the group consisting of fluid, biological fluid, cell, tissue, organ, gamete, embryo, and fetus.", "The term “gamete” as used herein refers to any cell participating, directly or indirectly, in the reproductive system of an animal.", "Examples of gametes are spermatocytes, spermatogonia, oocytes, and oogonia.", "Gametes can be present in fluids, tissues, and organs collected from animals (e.g., sperm is present in semen).", "For example, methods of collecting semen for the purposes of artificial insemination are well known to a person of ordinary skill in the art.", "See, e.g., Physiology of Reproduction and Artificial Insemination of Cattle (2nd edition), Salisbury et al., copyright 1961, 1978, WH Freeman & Co., San Francisco.", "However, the invention relates to the collection of any type of gamete from an animal.", "The term “tissue” is defined previously.", "The term “organ” relates to any organ isolated from a fetus or animal, or any portion of an organ.", "Examples of organs and tissues are neuronal tissue, brain tissue, spleen, heart, lung, gallbladder, pancreas, testis, ovary, intestine, skin, and kidney.", "These examples are not limiting and the invention relates to any organ and any tissue isolated from a cloned animal of the invention.", "In preferred embodiments, (1) fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses can be subject to manipulation; (2) the manipulation comprises isolating at least one component from an animal or fetus; (3) the manipulation comprises the step of cryopreserving the components; (4) the manipulation comprises the step of thawing components; (5) the manipulation comprises the step of separating the semen into X-chromosome bearing semen and Y-chromosome bearing semen; (6) the manipulation comprises methods of preparing the semen for artificial insemination; (7) the manipulation comprises the step of purification of desired polypeptide(s) from the component; (8) the manipulation comprises concentration of the components; and (9) the manipulation comprises the step of transferring one or more cloned cells, cloned tissues, cloned organs, and/or portions of cloned organs to a recipient organism (e.g., the recipient organism may be of a different species than the donor source).", "The term “separating” as used herein in reference to separating semen refers to methods well known to a person skilled in the art for fractionating a semen sample into sex-specific fractions.", "This type of separation can be accomplished by using flow cytometers that are commercially available.", "Methods of utilizing flow cytometers from separating sperm by genetic content are well known in the art.", "In addition, semen can be separated by its sex-associated characteristics by other methods well known to a person of ordinary skill in the art.", "See, U.S. Pat.", "Nos.", "5,439,362, 5,346,990, and 5,021,244, entitled “Sex-Associated Membrane Proteins and Methods for Increasing the Probability that Offspring Will Be of a Desired Sex,” Spaulding, issued on Aug. 8, 1995, Sep. 13, 1994, and Jun.", "4, 1991 respectively, all of which are incorporated herein by reference in their entireties including all figures, tables, and drawings.", "Semen preparation methods are well known to someone of ordinary skill in the art.", "Examples of these preparative steps are described in Physiology of Reproduction and Artificial Insemination of Cattle (2nd.", "edition), Salisbury et al., copyright 1961, 1978, W.H.", "Freeman & Co., San Francisco.", "The term “purification” as used herein refers to increasing the specific activity of a particular polypeptide or polypeptides in a sample.", "Specific activity can be expressed as the ratio between the activity of the target polypeptide and the concentration of total polypeptide in the sample.", "Activity can be catalytic activity and/or binding activity, for example.", "Alternatively, specific activity can be expressed as the ratio between the concentration of the target polypeptide and the concentration of total polypeptide.", "Purification methods include dialysis, centrifugation, and column chromatography techniques, which are well-known procedures to a person of ordinary skill in the art.", "See, e.g., Young et al., 1997, “Production of biopharmaceutical proteins in the milk of transgenic dairy animals,” BioPharm 10(6): 34-38.The term “transferring” as used herein can relate to shifting cells, tissues, organs, and/or portions of organs to an animal.", "The cells, tissues, organs, and/or portions of organs can be, for example, (a) developed in vitro and then transferred to an animal, (b) removed from an animal and transferred to another animal of a different specie, (c) removed from an animal and transferred to another animal of the same specie, (d) removed from one portion of an animal (e.g., the leg of an animal) and then transferred to another portion of the same animal (e.g., the brain of the animal), and/or (e) any combination of the foregoing.", "The term “transferring” can relate to adding cells, tissues, and/or organs to an animal and can also relate to removing cells, tissues, and/or organs from an animal and replacing them with cells, tissues, and/or organs from another source.", "The term “transferring” as used herein can also refer to implanting one or more cells, tissues, organs, and/or portions of organs from the cloned mammalian animal into another organism.", "For example, neuronal tissue from a cloned mammalian organism can be grafted into an appropriate area in the human nervous system to treat neurological diseases such as Alzheimer's disease.", "Alternatively, cloned cells, tissues, and/or organs originating from a porcine organism may be transferred to a human recipient.", "Surgical methods for accomplishing this preferred aspect of the invention are well known to a person of ordinary skill in the art.", "Transferring procedures may include the step of removing or deleting cells, tissues, or organs from a recipient organism before a transfer step.", "Of particular interest are transgenic animals that express genes that confer resistance or reduce susceptibility to disease.", "Since multiple genes can be introduced on an ACEs, a series of genes encoding an antigen can be introduced, which upon expression will serve to immunize [in a manner similar to a multivalent vaccine] the host animal against the diseases for which exposure to the antigens provide immunity or some protection.", "Also of interest are transgenic animals that serve as models of certain diseases and disorders for use in studying the disease and developing therapeutic treatments and cures thereof.", "Such animal models of disease express genes [typically carrying a disease-associated mutation], which are introduced into the animal on a MAC, preferably an ACEs, and which induce the disease or disorder in the animal.", "Similarly, MACs carrying genes encoding antisense RNA may be introduced into animal cells to generate conditional “knock-out” transgenic animals.", "In such animals, expression of the antisense RNA results in decreased or complete elimination of the products of genes corresponding to the antisense RNA.", "Of further interest are transgenic mammals that harbor MAC-carried genes encoding therapeutic proteins that are expressed in the animal's milk.", "Transgenic animals for use in xenotransplantation, which express MAC-carried genes that serve to humanize the animal's organs, are also of interest.", "Genes that might be used in humanizing animal organs include those encoding human surface antigens.", "The invention relates in part to any disease or parasitic condition known in the art.", "See, e.g., Hagan & Bruners Infectious Diesases of Domestic Animals (7th edition), Gillespie & Timoney, copyright 1981, Cornell University Press, Ithaca N.Y.", "Examples of parasites include, but are not limited to, worms, insects, invertebrate, bacterial, viral, and eukaryotic parasites.", "These parasites can lead to diseased states that can be controlled by the materials and methods of the invention.", "The term “regulatory element” as used herein refers to a DNA or RNA sequence that can increase or decrease the amount of product produced from another DNA or RNA sequence.", "The regulatory element can cause the constitutive production of the product (e.g., the product can be expressed constantly).", "Alternatively, the regulatory element can enhance or diminish the production of a recombinant product in an inducible fashion (e.g., the product can be expressed in response to a specific signal).", "The regulatory element can be controlled, for example, by nutrition, by light, or by adding a substance to the transgenic organism's system.", "Examples of regulatory elements well-known to those of ordinary skill in the art are promoters, enhancers, insulators, and repressors.", "See, e.g., Transgenic Animals, Generation and Use, 1997, Edited by L. M. Houdebine, Hardwood Academic Publishers, Australia, hereby incorporated herein by reference in its entirety including all figures, tables, and drawings.", "The terms “promoters,” “promoter,” or “promoter elements” as used herein refer to a DNA sequence that is located adjacent to a DNA sequence that encodes a recombinant product.", "A promoter is preferably operatively linked to the adjacent DNA sequence.", "A promoter typically increases the amount of recombinant product expressed from a DNA sequence as compared to the amount of the expressed recombinant product when no promoter exists.", "A promoter from one organism can be utilized to enhance recombinant product expression from a DNA sequence that originates from another organism.", "In addition, one promoter element can increase an amount of recombinant products expressed for multiple DNA sequences attached in tandem.", "Hence, one promoter element can enhance the expression of one or more recombinant products.", "Multiple promoter elements are well-known to persons of ordinary skill in the art.", "Examples of promoter elements are described hereafter.", "The terms “enhancers,” “enhancer” or “enhancer elements” as used herein refer to a DNA sequence that is located adjacent to the DNA sequence that encodes a recombinant product.", "Enhancer elements are typically located upstream of a promoter element or can be located downstream of the coding DNA sequence (e.g., the DNA sequence transcribed or translated into a recombinant product or products).", "Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream of the DNA sequence that encodes the recombinant product.", "Enhancer elements can increase the amount of recombinant product expressed from a DNA sequence above the increased expression afforded by a promoter element.", "Multiple enhancer elements are readily available to persons of ordinary skill in the art.", "The terms “insulators,” “insulator,” or “insulator elements” as used herein refer to DNA sequences that flank the DNA sequence encoding the recombinant product.", "Insulator elements can direct the recombinant product expression to specific tissues in an organism.", "Multiple insulator elements are well known to persons of ordinary skill in the art.", "See, e.g., Geyer, 1997, Curr.", "Opin.", "Genet.", "Dev.", "7: 242-248, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "The terms “repressor” or “repressor element” as used herein refer to a DNA sequence located in proximity to the DNA sequence that encodes the recombinant product, where the repressor sequence can decrease the amount of recombinant product expressed from that DNA sequence.", "Repressor elements can be controlled by the binding of a specific molecule or specific molecules to the repressor element DNA sequence.", "These molecules can either activate or deactivate the repressor element.", "Multiple repressor elements are available to a person of ordinary skill in the art.", "The terms “milk protein promoter,” “urine protein promoter,” “blood protein promoter,” “tear duct protein promoter,” “synovial protein promoter,” “spermatogenesis protein promoter,” and “mandibular gland protein promoter” refer to promoter elements that regulate the specific expression of proteins within the specified fluid or gland or cell type in an animal.", "For example, a milk protein promoter is a regulatory element that can control the expression of a protein that is expressed in the milk of an animal.", "Other promoters, such as β-casein promoter, melanocortin promoter, milk serum protein promoter, casein promoter, α-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, and α-actin promoter, for example, are well known to a person of ordinary skill in the art.", "The terms “insertion” and “introduction” as used herein in reference to artificial chromosomes or other large heterologous nucleic acid constructs refer to translocating one or more such artificial chromosomes or constructs from the outside of a cell to the inside of a cell.", "Insertion can be effected in at least two manners: by mechanical delivery and non-mechanical delivery.", "The term “mechanical delivery” as used herein refers to processes that utilize an apparatus that directly or indirectly introduces DNA (e.g., one or more artificial chromosomes) into one or more cells.", "Examples of mechanical delivery of DNA into cells include, but are not limited to, microinjection, particle bombardment, sonoporation, and electroporation.", "The term “non-mechanical delivery” as used herein refers to non-mechanical processes such as diffusive processes, for example.", "For instance, non-mechanical delivery may be effected by introducing DNA (e.g., an artificial chromosome) and one or more reagents to a medium bathing cell surfaces, where the reagents increase the probability that the DNA enters the cells.", "Such reagents are well known in the art, such as liposomes, acyl moieties, peptide moieties, saccharide moieties, and/or polyethylene glycol (PEG), for example.", "Such reagents may be complexed with the target molecule and the reagents may be introduced to cells in vivo and/or ex vivo.", "These examples are not meant to be limiting and the invention relates in part to any non-mechanical form of insertion.", "The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments, as well as from the claims.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "INTRODUCTION The invention relates in part to the cloning of animals that comprise heterologous DNA molecules.", "Such transgenic animals preferably contain at least about 100 kilobase pairs of exogenous DNA in structures known to the skilled artisan as “artificial chromosomes.” BACKGROUND OF THE INVENTION The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.", "Researchers have been developing methods for cloning mammalian animals over the past two decades.", "These reported methods typically include the steps of (1) isolating a pluripotent or totipotent cell; (2) inserting the cell or nucleus isolated from the cell into an enucleated oocyte (i.e., the oocyte's nucleus was previously extracted), and (3) allowing the embryo to mature in vivo.", "The first successful nuclear transfer experiment using mammalian cells was reported in 1983, when pronuclei isolated from a murine (mouse) zygote were inserted into an enucleated oocyte and resulted in like offspring(s).", "See, e.g., McGrath & Solter, 1983, Science 220:1300-1302.Subsequently, other workers described the production of chimeric murine embryos (e.g., embryos that contain a subset of cells having significantly different nuclear DNA from other cells in the embryo) using murine primordial germ cells (PGCs).", "These cells are and can give rise to pluripotent cells (e.g., cells that can differentiate into other types of cells, and which may, but are not required to, differentiate into a grown animal).", "See, e.g., Matsui et al., 1992, Cell 70:841-847 and Resnick et al., 1992, Nature 359:550; Kato et al., 1994, Journal of Reproduction and Fertility Abstract Series, Society For the Study of Fertility, Annual Conference, Southampton, 13:38.Progress has also been reported in the field of cloning ovine (sheep) animals (see, e.g., Willadsen, 1986, Nature 320:63-65; Campbell et al., 1996, Nature 380:64-66; PCT Publication WO 95/20042; Wilmut et al., 1997, Nature 385:810-813; PCT Publication WO 96/07732; PCT Publication WO 97/07668; and PCT publication WO 97/07669; and McCreath et al., 2000, Nature, 405:1066-1069), and bovine animals, (see, e.g., U.S. Pat.", "Nos.", "4,994,384 and 5,057,420; Sims & First, 1993, Theriogenology 39:313; Keefer et al., 1994, Mol.", "Reprod.", "Dev.", "38:264-268; Delhaise et al., 1995, Reprod.", "Fert.", "Develop.", "7:1217-1219; Lavoir 1994, J. Reprod.", "Dev.", "37:413-424; Stice et al., 1996, Biol.", "Reprod.", "54: 100-110; and PCT application WO 95/10599 entitled “Embryonic Stem Cell-Like Cells”).", "Researchers have also disclosed methods that resulted in cloned bovine animals (cattle).", "Bovines have been cloned using an embryonic cell derived from a 2-64 cell embryo as a nuclear donor.", "This bovine animal was reportedly cloned by utilizing nuclear transfer techniques set forth in U.S. Pat.", "Nos.", "4,994,384 and 5,057,420.Others reported that cloned bovine embryos were formed where an inner cell mass cell of a blastocyst stage embryo was utilized as a nuclear donor in a nuclear transfer procedure (Sims & First, 1993, Theriogenology 39:313; Keefer et al., 1994, Mol.", "Reprod.", "Dev.", "38:264-268; and U.S. Pat.", "No.", "6,107,543); a PGC isolated from fetal tissue as a nuclear donor (Delhaise et al., 1995, Reprod.", "Fert.", "Develop.", "7:1217-1219; Lavoir 1994, J. Reprod.", "Dev.", "37:413-424; and PCT application WO 95/10599 entitled “Embryonic Stem Cell-Like Cells”); a proliferating somatic cell (U.S. Pat.", "No.", "5,945,577); and a reprogrammed nonembryonic cell (U.S. Pat.", "No.", "6,011,197) Additionally, researchers have reported methods for obtaining cloned porcine animals and porcine chimeric animals, specifically, where a nuclear donor obtained from a 4-cell embryo is placed inside an enucleated zygote.", "See, e.g., Prather et al., 1989, Biology of Reproduction 41: 414-418; Piedrahita et al., 1998, Biology of Reproduction 58: 1321-1329; and WO 94/26884, “Embryonic Stem Cells for Making Chimeric and Transgenic Ungulates,” Wheeler, published Nov. 24, 1994.Also, researchers have reported nuclear transfer experiments using porcine nuclear donors and porcine oocytes.", "See., e.g., Nagashima et al., 1997, Mol.", "Reprod.", "Dev.", "48: 339-343; Nagashima et al., 1992, J. Reprod.", "Dev.", "38: 73-78; Prather et al., 1989, Biol.", "Reprod.", "41: 414-419; Prather et al., 1990, Exp.", "Zool.", "255: 355-358; Saito et al., 1992, Assis.", "Reprod.", "Tech.", "Andro.", "259: 257-266; Terlouw et al., 1992, Theriogenology 37: 309, Pokajaeva et al., Nature 407, 86-90 (2000); Onishi et al., Science 289 1188-1190 (2000); and Betthauser et al., Nature Biotechnology 18: 1055-1059 (2000).", "Researchers have also developed methods for generating transgenic cells, which may be applicable to the production of transgenic animals.", "Although several viral vectors, non-viral vectors, and other delivery systems have been developed for establishing transgenic cells, many of these technologies are constrained by multiple limitations.", "Specifically, these limitations include (1) the size of inserted DNA is limited to approximately 10 kilobases (kb); (2) integration of the DNA of interest cannot be specifically targeted into the cell's nuclear DNA; and (3) expression of a recombinant product from the DNA of interest cannot be well controlled.", "See, e.g., Mitani et al., 1993, Trends Biotech, 11: 162-166; U.S. Pat.", "No.", "5,633,067, “Method of Producing a Transgenic Bovine or Transgenic Bovine Embryo,” DeBoer et al, issued May 27, 1997; U.S. Pat.", "No.", "5,612,205, “Homologous Recombination in Mammalian Cells,” Kay et al., issued Mar.", "18, 1997; and PCT publication WO 93/22432, “Method for Identifying Transgenic Pre-Implantation Embryos,” all of which are incorporated by reference herein in their entirety, including all figures, drawings, and tables.", "Artificial chromosome technology is not constrained by the above-defined limitations.", "Moreover, researchers have discovered that artificial chromosomes can be replicated de novo.", "See, e.g., Kereso et al., 1996, Chromosome Research 4: 226-239, Holló et al., 1996, Chromosome Research 4: 240-247, U.S. Pat.", "No.", "6,025,155, and U.S. Pat.", "No.", "6,077,697.Each reference used to provide background information in this section is hereby incorporated by reference in its entirety, including ant tables, figures, and claims.", "Despite progress towards cloning mammals and establishing transgenic cells, there remains a great need in the art for materials and methods that enhance the efficiency for cloning transgenic animals.", "In particular, there remains a great need in the art to provide pluripotent and totipotent transgenic cells that can be utilized as nuclear donors.", "Furthermore, there remains a long felt need in the art for providing cell lines that are karyotypically stable and transgenic, which can be utilized in processes for cloning transgenic animals.", "SUMMARY OF THE INVENTION The invention relates in part to transgenic, totipotent, mammalian cells comprising one or more large, heterologous DNA constructs of 100 kbp or more.", "Preferably, the large DNA construct(s) are artificial chromosomes.", "Mammalian cells containing the large heterologous DNA construct(s) may be used for producing transgenic embryos and transgenic animals cloned from such cells.", "The invention is also directed in part to processes for producing totipotent cells that comprise one or more large, heterologous DNA constructs; processes for utilizing such cells; and processes for producing transgenic embryos and transgenic animals cloned from such cells.", "Thus, in a first aspect, the invention features a method for producing transgenic cells by inserting a large, heterologous DNA construct of 100 kbp or more into cells.", "Such cells may preferably be used as nuclear donor cells in methods to produce transgenic animals, most preferably ungulates.", "Preferably, a large, heterologous DNA construct is at least 200 Kbp, at least 300 Kbp, at least 400 Kbp, at least 500 Kbp, at least 750 Kbp, at least 1 Mbp, at least 5 Mbp, at least 10 Mbp, at least 20 Mbp, at least 50 Mbp, at least 100 Mbp, at least 500 Mbp, or at least 1000 Mbp.", "Particularly useful are artificial chromosomes of between 100 Kbp and 500 Mbp; between 500 Kbp and 500 Mbp; and between 1 Mbp and 500 Mbp.", "In certain embodiments, the large, heterologous DNA construct(s) of this aspect are artificial chromosomes.", "Advantages of using artificial chromosomes include: (1) target DNA greater than 10 kb can be inserted into cells; (2) the location of target DNA of interest can be controlled; (3) transgenic animals and embryos containing large foreign genes, or a large copy number of one or more foreign genes, in a majority of cells can be obtained; and (4) the expression levels of a recombinant product from the DNA of interest can be manipulated in vitro.", "Specifically, expression levels can be manipulated by controlling the copy number of target DNA and/or its regulation by promoters, enhancers, etc., in an artificial chromosome, as defined in greater detail hereafter.", "The term “artificial chromosome” as used herein refers to nucleic acid molecules that are generated by the manipulation of DNA, contain a centromere, and are capable of stable, autonomous replication in cells.", "An artificial chromosome (1) can replicate with naturally occurring chromosomes in the nucleus of target cell; (2) can be large in size (ranging in size from 100 kilobase pairs (Kbp) to 1000 megabase pairs (Mbp) in length, or more); (3) typically comprises a centromere, origins of replication, and telomeres; and (4) can comprise neutral DNA.", "Neutral DNA does not encode products that significantly alter the functions of a cell in which the artificial chromosome is located.", "For example, neutral DNA may encode ribosomal RNA.", "It is not typical that increasing levels of ribosomal RNA significantly alters cell functions.", "Neutral DNA can also be referred to as “satellite DNA.” Preferably, an artificial chromosome is at least 200 Kbp, at least 300 Kbp, at least 400 Kbp, at least 500 Kbp, at least 750 Kbp, at least 1 Mbp, at least 5 Mbp, at least 10 Mbp, at least 20 Mbp, at least 50 Mbp, at least 100 Mbp, at least 500 Mbp, or at least 1000 Mbp.", "Particularly useful are artificial chromosomes of between 100 Kbp and 500 Mbp; between 500 Kbp and 500 Mbp; and between 1 Mbp and 500 Mbp.", "Materials and methods for producing, identifying, and characterizing artificial chromosomes are well known in the art.", "See, e.g., Kereso et al., 1996, Chromosome Research 4: 226-239, Holló et al., 1996, Chromosome Research 4: 240-247, International publication nos.", "WO00/18941, WO98/08964, WO97/16533 and WO97/40183, and U.S. Pat.", "Nos.", "5,721,118, 6,025,155, 6,077,697, and 6,133,503, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.", "These publications also describe shuttle vectors useful for incorporating target DNA into artificial chromosomes.", "Artificial chromosomes can arise from a portion of a natural chromosome by manipulation.", "Artificial chromosomes can be detected in cells by using chromosome identification techniques well known in the art.", "An example of such a technique is chromosome karyotype analysis.", "Mammalian artificial chromosomes (MACs) can be generated by cellular mediated chromosome assembly from transfected alphoid, telomeric and marker DNAs (Harrington J. J. et al.", "Nature Genetics, 15, 345-355, 1997; Ikeno, M. et al, Nature Biotechnology 16, 431-439, 1998; Henning, K. A. et al, PNAS USA 96, 592-597, 1999) and even from non-alphoid DNA (du Sart D, et al, Nature Genetics 16, 144-153, 1997).", "Minichromosomes may be generated by fragmenting natural human chromosomes using telomere-directed breakage (Shen M H, et al, Human Molecular Genetics 6, 1375-1382, 1997; Shen M H et al, Current Biology 10, 31-34, 1999).", "It is possible to transfer human-murine minichromosome chimeras (Shen M H et al, Current Biology 10, 31-34, 1999), fragmented human minichromosomes Tomizuka K et al, Nature Genetics 16, 133-143, 1997; Tomizuka K et al, PNAS USA 97, 722-797, 2000), and human small accessory chromosomes (SACs; Vermeesch J R et al, Human Genetics 105, 611-618, 1999) via microcell-mediated chromosome transfer (MMCT) to recipient cells.", "The term “target DNA” as used herein refers to DNA that is intended to be or has been incorporated into a large heterologous DNA construct, preferably an artificial chromosome.", "The term “heterologous” is defined below.", "Target DNA can encode multiple types of recombinant products, as defined hereafter, and may exist in multiple copies when introduced into an artificial chromosome.", "One advantage of artificial chromosome technology is that target DNA copy number can be controlled and monitored in an artificial chromosome in vitro before the artificial chromosome comprising the target DNA is introduced into a cell.", "In addition, depending on the promoter used, expression can also be monitored in vitro.", "This advantage is contrasted with many existing techniques for creating transgenic cells, which cause random insertion of target DNA into a cell nuclear DNA.", "Materials and methods for introducing target DNA into an artificial chromosome and materials and methods for introducing the resulting artificial chromosome into cells are defined hereafter.", "The term “heterologous nucleic acid” refers to nucleic acids having (1) a nucleic acid sequence that differs from the nucleic acid sequences present in cell's naturally occurring nuclear DNA; (2) a subset of nucleic acid having a nucleotide sequence that is present in cell nuclear DNA, but that exists in different proportions in the heterologous nucleic acid than in cell nuclear DNA; (3) a nucleic acid sequence originating from a species other than the species from which cell nuclear DNA originates; and (4) a nucleic acid sequence that differs from the DNA sequences present in cell's naturally occurring mitochondrial DNA.", "Artificial chromosomes, such as mammalian artificial chromosomes [MACs], can be generated and isolated by the methods described in the publications above.", "In particularly preferred embodiments, two types of artificial chromosomes are used, both of which function in cells as stable, functional chromsomes.", "One type, herein referred to as ACEs (“Artificial Chromosome Expression systems” based on satellite DNA) is a stable heterochromatic chromosome, and the other type is a de novo-formed minichromosomes based on amplification of euchromatin.", "Artificial chromosomes, and, in particular the two preferred types discussed above, provide an extra-genomic locus for targeted integration of up to multi-megabase pair size DNA fragments that contain single or multiple genes, including multiple copies of a single gene operatively linked to one promoter or each copy or several copies linked to separate promoters.", "Thus, methods provided can be used to introduce genes via MACs into cells and tissues of ungulate mammals.", "The artificial chromosomes with integrated heterologous DNA may be used in methods of production of gene products, particularly products that require expression of multigenic biosynthetic pathways, and also are intended for delivery into the nuclei of cells, such as nuclear donor cells used in nuclear transfer procedures, for production of transgenic ungulate mammals.", "Additionally, such artificial chromosomes provide extra-genomic specific integration sites for introduction of genes encoding proteins of interest and permit up to multi-megabase size DNA integration so that, for example, genes encoding an entire metabolic pathway, a very large gene such as the cystic fibrosis transmembrane conductance regulator gene (approximately 250 kb genomic DNA gene), or several genes, such as multiple genes encoding a series of antigens for preparation of a multivalent vaccine, can be stably introduced into a cell.", "The artificial chromosomes described herein, including ACEs and euchromatin-based minichromosomes, can be generated by introducing heterologous DNA, preferably including DNA encoding one or multiple selectable marker(s), into cells, preferably a stable cell line, growing the cells under selective conditions, and identifying from among the resulting cell clones those that include chromosomes with more than one centromere, fragments thereof, and/or heterochromatic structures.", "Amplification that produces the additional centromere(s) occurs in cells that contain chromosomes in which heterologous DNA has integrated near the centromere in the pericentric region of the chromosome.", "Selected cells comprising intermediates in the formation of such artificial chromosomes can then be used to generate complete artificial chromosomes.", "For example, continued culture of cells containing a formerly dicentric chromosome under conditions that destabilize chromosomes (such as BrdU treatment) and/or under selective conditions can yield ACEs.", "Similarly, artificial chromosomes can be generated by culturing cells with multi-centric (typically dicentric) chromosomes under conditions whereby the chromosome breaks to form a minichromosome and a formerly dicentric chromosome.", "Among the MACs provided herein can be ACEs, which are predominantly heterochromatic (i.e., contain more heterochromatin than euchromatin, and preferably contain about 70% heterochromatin), and can comprise repeating units of short satellite DNA, so that without insertion of heterologous or foreign DNA, the chromosomes preferably contain no genetic information.", "They can thus be used as “safe” vectors for delivery of DNA to mammalian hosts because they do not contain any potentially harmful genes.", "ACEs are generated, not from the minichromosome fragment as, for example, in U.S. Pat.", "No.", "5,288,625 (which is incorporated herein by reference in its entirety including all figures, tables, and drawings), but from the fragment of the formerly dicentric chromosome.", "In addition, euchromatic minichromosomes can be generated.", "Methods for generating one type of MAC, the minichromosome, is described in U.S. Pat.", "No.", "5,288,625 (which is incorporated herein by reference in its entirety including all figures, tables, and drawings), along with its use for the expression of heterologous DNA are provided.", "In preferred embodiments, (1) the artificial chromosome is an ACEs comprising one or more markers; (2) a marker is an antibiotic resistance gene selected from the group consisting of neomycin resistance gene, hygromycin resistance gene, and puromycin resistance gene; (3) the artificial chromosome comprises a DNA sequence that encodes one or more recombinant products; (4) a recombinant product is a ribozyme; (5) a recombinant product is antisense RNA; (6) a recombinant product is a peptide; (7) a recombinant product is a polypeptide; (8) a recombinant product is a protein; (9) a recombinant product is an enzyme; (10) a recombinant product is expressed in a biological fluid; (11) a recombinant product is expressed in a tissue; (12) a recombinant product confers resistance to one or more parasites and/or diseases; (13) an artificial chromosome comprises one or more regulatory elements; (14) a regulatory element is a promoter element; (15) a promoter element is selected from the group consisting of milk protein promoter, urine protein promoter, blood protein promoter, tear duct protein promoter, synovial protein promoter, mandibular gland protein promoter, casein promoter, β-casein promoter, melanocortin promoter, milk serum protein promoter, α-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, and α-actin promoter; (17) a regulatory element is a repressor element; (18) a regulatory element is an insulator element; and (19) a regulatory element is an enhancer element.", "The term “marker” as used herein refers to any DNA sequence that distinguishes a cell comprising an artificial chromosome, or a precursor thereof, from a cell that does not comprise the artificial chromosome or precursor.", "For example, a marker can be used in the initial steps of generating ACEs, whereby the marker distinguishes a cell containing a foreign nucleic acid from a cell that does not contain the foreign nucleic acid.", "Multiple types of markers, such as genes encoding green fluorescent protein, antibiotic resistance, β-galactosidase, glutamine synthetase, thymidine kinase, cytosine deaminase, and dihydrofolate reductase are well known in the art.", "Preferred as markers are DNA sequences that encode a molecule which directly or indirectly inactivates a drug that retards the growth of cells not expressing such a molecule.", "Examples of these latter described markers are blasticidin-S, neomycin, hygromycin, and puromycin resistance genes.", "These examples are not meant to be limiting and the invention relates in part to any marker known in the art.", "The term “ribozyme” as used herein refers to ribonucleic acid molecules that can cleave other RNA molecules in specific regions.", "Ribozymes can bind to discrete regions on a RNA molecule, and then specifically cleave a region within that binding region or adjacent to the binding region.", "Ribozyme techniques can thereby decrease the amount of polypeptide translated from formerly intact message RNA molecules.", "For specific descriptions of ribozymes, see U.S. Pat.", "No.", "5,354,855, entitled “RNA Ribozyme which Cleaves Substrate RNA without Formation of a Covalent Bond,” Cech et al., issued on Oct. 11, 1994, and U.S. Pat.", "No.", "5,591,610, entitled “RNA Ribozyme Polymerases, Dephosphorylases, Restriction Endoribonucleases and Methods,” Cech et al., issued on Jan. 7, 1997, both of which are incorporated by reference in their entireties including all figures, tables, and drawings.", "The term “antisense RNA” as used herein refers to any RNA that binds to mRNA with enough affinity to decrease the amount of protein translated from the mRNA.", "The amount of protein translated from the mRNA is preferably decreased by more than 20%; more preferably decreased by more than 50%, 70%, and 80%; and most preferably decreased by more than 90%.", "Antisense RNA materials and methods are well known in the art.", "The terms “biological fluid” and the term “tissue” as used herein refer to any fluid or tissue in or from a biological organism.", "The fluids may include, but are not limited to, tears, saliva, milk, urine, amniotic fluid, semen, plasma, oviductal fluid, and synovial fluid.", "The tissues may include, but are not limited to, lung, heart, blood, liver, muscle, brain, pancreas, skin, and others.", "The term “confers resistance” as used herein refers to the ability of a recombinant product to completely abrogate or partially alleviate the symptoms of a disease or parasitic condition.", "Hence, if a disease is related to inflammation, for example, a recombinant product can confer resistance to that inflammation if the inflammation decreases upon expression of the recombinant product.", "A recombinant product may confer resistance or partially confer resistance to a disease or parasitic condition, for example, if the recombinant product is an antisense RNA molecule that specifically binds to an mRNA molecule encoding a polypeptide responsible for the inflammation.", "In preferred embodiments, the DNA with the selectable marker that is introduced into cells to generate artificial chromosomes includes sequences that target it to the pericentric region of the chromosome.", "Integration of the DNA into existing chromosomes in the cells can induce amplification that results in generation of additional centromeres.", "Transgenic Cells Large heterologous nucleic acid constructs, such as artificial chromosomes, can then be introduced into cells to produce stable transformed cell lines and cells.", "Introduction is effected by any suitable method or combination of methods including, but not limited to microinjection, cell fusion, microcell fusion, electroporation, sonoporation, electrofusion, projectile bombardment, calcium phosphate precipitation, lipid-mediated transfer systems, ligand/receptor systems and other such methods well known to the skilled artisan.", "ACEs in particular can be readily isolated and used for gene delivery.", "These artificial chromosomes can also be used in gene product production systems, production of humanized genetically transformed animal organs, and, most preferably, the generation of transgenic ungulates.", "In certain embodiments, the invention relates to transgenic, totipotent, mammalian cells comprising at least one artificial chromosome, but the invention relates in part to any number of artificial chromosomes in a totipotent mammalian cell.", "A totipotent mammalian cell preferably comprises ten or fewer artificial chromosomes; more preferably comprises six or fewer artificial chromosomes, four or fewer artificial chromosomes, or two or fewer artificial chromosomes; and most preferably comprises one artificial chromosome.", "If a totipotent mammalian cell of the invention comprises more than one artificial chromosome, the artificial chromosomes may be identical or may differ from one another.", "The term “transgenic” as used herein in reference to cells refers to a cell that comprises heterologous nucleic acid, preferably deoxyribonucleic acid (DNA).", "In preferred embodiments, a transgenic cell comprises one or more heterologous DNA sequences.", "In other preferred embodiments, a transgenic cell is a cell in which one or more endogenous genes have been deleted, duplicated, activated, or modified.", "In particularly preferred embodiments, a transgenic cell comprises both one or more heterologous DNA sequences, and one or more endogenous genes that have been deleted, duplicated, activated, or modified.", "An artificial chromosome present in a transgenic cell can comprise heterologous DNA.", "Heterologous DNA can encode multiple types of recombinant products, as defined hereafter.", "The term “transgene” as used herein refers to a single gene that is partially or entirely transgenic in origin.", "In certain embodiments, greater than 50% of the transgene consists of heterologous DNA.", "In preferred embodiments, greater than 75% of the transgene consists of heterologous DNA, greater than 80% of the transgene consists of heterologous DNA, greater than 90% of the transgene consists of heterologous DNA, greater than 95% of the transgene consists of heterologous DNA, greater than 98% of the transgene consists of heterologous DNA, and 100% of the transgene consists of heterologous DNA.", "The term “different nucleic acid sequence” as used herein refers to nucleic acid sequences that are not substantially similar.", "The term “substantially similar” as used herein in reference to nucleic acid sequences refers to two nucleic acid sequences having preferably 80% or more nucleic acid identity, more preferably 90% or more nucleic acid identity or most preferably 95% or more nucleic acid identity.", "Nucleic acid identity is a property of nucleic acid sequences that measures their similarity or relationship when aligned by means known to one skilled in the art.", "Identity is measured by dividing the number of identical bases in the two sequences by the total number of bases and multiplying the product by 100.Thus, two copies of exactly the same sequence have 100% identity, while sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity.", "Those skilled in the art will recognize that several computer programs are available for determining sequence identity and similarity using standard parameters, for example Gapped BLAST or PSI-BLAST (Altschul, et al.", "(1997) Nucleic Acids Res.", "25:3389-3402), BLAST (Altschul, et al.", "(1990) J. Mol.", "Biol.", "215:403-410), and Smith-Waterman (Smith, et al.", "(1981) J. Mol.", "Biol.", "147:195-197).", "Preferably, the default settings of these programs will be employed, but those skilled in the art recognize whether these settings need to be changed and know how to make the changes.", "The term “substantially similar” as used herein in reference to amino acid sequences refers to two amino acid sequences having preferably 50% or more amino acid identity, more preferably 70% or more amino acid identity or most preferably 90% or more amino acid identity.", "Amino acid identity is a property of amino acid sequence that measures their similarity or relationship.", "Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100.Thus, two copies of exactly the same sequence have 100% identity, while sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity.", "“Similarity” in protein sequences is measured by dividing the number of identical residues plus the number of conservatively substituted residues (see Bowie, et al.", "Science, (1999) 247, 1306-1310, which is incorporated herein by reference in its entirety, including any drawings, figures, or tables) by the total number of residues and gaps and multiplying the product by 100.“Similarity” in nucleic acid sequences is measured by dividing the number of identical bases by the total number of residues and gaps and multiplying the product by 100.The term “recombinant product” as used herein refers to the product produced from a target DNA sequence.", "A recombinant product can be a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a polypeptide that binds to a regulatory element (a term described hereafter), a structural protein, an RNA molecule, and/or a ribozyme, for example.", "These products are well defined in the art.", "This list of products is for illustrative purposes only and the invention relates to other types of recombinant products.", "In preferred embodiments, (1) the mammalian cell is an ungulate cell; (2) the ungulate is selected from the group consisting of bovids, ovids, cervids, suids, equids and camelids; (3) the ungulate is bovine; (4) the mammalian cell is a nonembryonic cell; (5) the mammalian cell is a fetal cell; and (6) the mammalian cell is an adult cell.", "The term “mammalian” as used herein refers to any animal of the class Mammalia.", "Preferably, a mammalian cell or cell line is a placental, a monotreme and a marsupial.", "Most preferably, a mammalian cell or cell line is a canid, felid, murid, leporid, ursid, mustelid, ungulate, ovid, suid, equid, bovid, caprid, cervid, and a human or non-human primate.", "A mammalian cell or cell line can be isolated from any source of mammalian cells including, but not limited to, a mammalian embryo, a mammalian fetus, and a mammalian animal.", "The term “canid” as used herein refers to any animal of the family Canidae.", "Preferably, a canid cell or cell line is isolated from a wolf, a jackal, a fox, and a domestic dog.", "The term “felid” as used herein refers to any animal of the family Felidae.", "Preferably, a felid cell or cell line is isolated from a lion, a tiger, a leopard, a cheetah, a cougar, and a domestic cat.", "The term “murid” as used herein refers to any animal of the family Muridae.", "Preferably, a murid cell or cell line is isolated from a mouse and a rat.", "The term “leporid” as used herein refers to any animal of the family Leporidae.", "Preferably, a leporid cell or cell line is isolated from a rabbit.", "The term “ursid” as used herein refers to any animal of the family Ursidae.", "Preferably, a ursid cell or cell line is isolated from a bear.", "The term “mustelid” as used herein refers to any animal of the family Mustelidae.", "Preferably, a mustelid cell or cell line is isolated from a weasel, a ferret, an otter, a mink, and a skunk.", "The term “primate” as used herein refers to any animal of the Primate order.", "Preferably, a primate cell or cell line is isolated from an ape, a monkey, a chimpanzee, and a lemur.", "The term “ungulate” as used herein refers to any animal of the polyphyletic group formerly known as the taxon Ungulata.", "Preferably, an ungulate cell or cell line is isolated from a camel, a hippopotamus, a horse, a tapir, and an elephant.", "Most preferably, an ungulate cell or cell line is isolated from a sheep, a cow, a goat, and a pig.", "Especially preferred in the bovine species are Bos taurus, Bos indicus, and Bos buffaloes cows or bulls.", "The term “ovid” as used herein refers to any animal of the family Ovidae.", "Preferably, an ovid cell or cell line is isolated from a sheep.", "The term “suid” as used herein refers to any animal of the family Suidae.", "Preferably, a suid cell or cell line is isolated from a pig or a boar.", "The term “equid” as used herein refers to any animal of the family Equidae.", "Preferably, an equid cell or cell line is isolated from a zebra or an ass.", "Most preferably, an equid cell or cell line is isolated from a horse.", "The term “bovid” as used herein refers to any animal of the family Bovidae.", "Preferably, an bovid cell or cell line is isolated from an antelope, an oxen, a cow, and a bison.", "The term “caprid” as used herein refers to any animal of the family Caprinae.", "Preferably, a caprid cell or cell line is isolated from a goat.", "The term “cervid” as used herein refers to any animal of the family Cervidae.", "Preferably, a cervid cell or cell line is isolated from a deer.", "The term “immortalized” or “permanent” as used herein in reference to cells refers to cells that have exceeded the Hayflick limit.", "The Hayflick limit can be defined as the number of cell divisions that occur before a cell line becomes senescent.", "Hayflick set this limit to approximately 60 divisions for most non-immortalized cells.", "See, e.g., Hayflick and Moorhead, 1961, Exp.", "Cell.", "Res.", "25: 585-621; and Hayflick, 1965, Exp.", "Cell Research 37: 614-636, incorporated herein by reference in their entireties including all figures, tables, and drawings.", "Therefore, an immortalized cell line can be distinguished from non-immortalized cell lines if the cells in the cell line are able to undergo more than 60 divisions.", "If the cells of a cell line are able to undergo more than 60 cell divisions, the cell line is an immortalized or permanent cell line.", "The immortalized cells of the invention are preferably able to undergo more than 70 divisions, are more preferably able to undergo more than 80 divisions, and are most preferably able to undergo more than 90 cell divisions.", "The terms “primary culture” and “primary cell” refer to cells taken from a tissue source, and their progeny, grown in culture before subdivision and transfer to a subculture.", "The terms “plated” or “plating” as used herein in reference to cells refer to establishing cell cultures in vitro.", "For example, cells can be diluted in cell culture media and then added to a cell culture plate or cell culture dish.", "Cell culture plates are commonly known to a person of ordinary skill in the art.", "Cells may be plated at a variety of concentrations and/or cell densities.", "In preferred embodiments, plated cells may grow to confluence.", "The meaning of the term “cell plating” can also extend to the term “cell passaging.” Cells of the invention can be passaged using cell culture techniques well known to those skilled in the art.", "The term “cell passaging” refers to such techniques which typically involve the steps of (1) releasing cells from a solid support and disassociation of these cells, and (2) diluting the cells in fresh media suitable for cell proliferation.", "Immortalized cells can be successfully grown by plating the cells in conditions where they lack cell to cell contact.", "Cell passaging may also refer to removing a portion of liquid medium bathing cultured cells and adding liquid medium from another source to the cell culture to dilute the cell concentration.", "The term “proliferation” as used herein in reference to cells refers to a group of cells that can increase in size and/or can increase in numbers over a period of time.", "The term “confluence” as used herein refers to a group of cells where a large percentage of the cells are physically contacted with at least one other cell in that group.", "Confluence may also be defined as a group of cells that grow to a maximum cell density in the conditions provided.", "For example, if a group of cells can proliferate in a monolayer and they are placed in a culture vessel in a suitable growth medium, they are confluent when the monolayer has spread across a significant surface area of the culture vessel.", "The surface area covered by the cells preferably represents about 50% of the total surface area, more preferably represents about 70% of the total surface area, and most preferably represents about 90% of the total surface area.", "The cells and cell lines of the instant invention may be cultured.", "The term “cultured” as used herein in reference to cells refers to one or more cells that are undergoing cell division or not undergoing cell division in an in vitro environment.", "An in vitro environment can be any medium known in the art that is suitable for maintaining cells in vitro, such as suitable liquid media or agar, for example.", "Specific examples of suitable in vitro environments for cell cultures are described in Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed.", "), Wiley-Liss, Inc.; Cells: a laboratory manual (vol.", "1), 1998; D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.", "), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J. Morgan John Wiley and Sons, Ltd., each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.", "Cells may be cultured in suspension and/or in monolayers with one or more substantially similar cells.", "Cells may be cultured in suspension and/or in monolayers with a heterogeneous population of cells.", "The term “heterogeneous” as utilized in the previous sentence can relate to any cell characteristics, such as cell type and cell cycle stage, for example.", "Cells may be cultured in suspension, cultured as monolayers attached to a solid support, and/or cultured on a layer of feeder cells, for example.", "The term “feeder cells” is defined hereafter.", "Furthermore, cells may be successfully cultured by plating the cells in conditions where they lack cell to cell contact.", "Preferably, cultured cells undergo cell division and are cultured for at least 5 days, more preferably for at least 10 days or 20 days, and most preferably for at least 30 days.", "Preferably, a significant number of cultured cells do not terminate while in culture.", "The terms “terminate” and “significant number” are defined hereafter.", "Nearly any type of cell can be placed in cell culture conditions.", "Cultured cells can be utilized to establish a cell line.", "In particularly preferred embodiments, a cell may be “clonally propagated.” In these embodiments, cells are diluted to an extent such that, statistically, some or all of the culture vessels into which the diluted cells are placed will contain only a single cell.", "Thus, the culture that grows within these culture vessels will be derived from a single cell.", "Materials and methods for clonally propagating cells are described in U.S. patent application Ser.", "No.", "09/753,323 (attorney docket number 030653.0026.CIP1, filed Dec. 28, 2000), which is hereby incorporated in its entirety.", "The term “terminating” and “terminate” as used herein with regard to cultured cells may refer to cells that undergo cell death, which can be measured using multiple techniques known to those skilled in the art (e.g., CytoTox96® Cytotoxicity Assay, Promega, Inc. catalog no.", "G1780; Celltiter96® Aqueous Cell Proliferation Assay Kit, Promega, Inc. catalog no.", "G3580; and Trypan Blue solution for cytotoxicity assays, Sigma catalog no.", "T6146).", "Termination may also be a result of apoptosis, which can be measured using multiple techniques known to persons skilled in the art (e.g., Dead End™ Apoptosis Detection Kit, Promega, Inc. catalog no.", "G7130).", "Terminated cells may be identified as those that have undergone cell death and/or apoptosis and have released from a solid surface in culture.", "In addition, terminated cells may lack intact membranes which can be identified by procedures described above.", "Also, terminated cells may exhibit decreased metabolic activity, which may be caused in part by decreased mitochondrial activity that can be identified by rhodamine 1,2,3, for example.", "Furthermore, termination can be refer to cell cultures where a significant number of cultured cells terminate.", "The term “significant number” in the preceding sentence refers to about 80% of the cells in culture, preferably about 90% of the cells in culture, more preferably about 100% of the cells in culture, and most preferably 100% of the cells in culture.", "The term “suspension” as used herein refers to cell culture conditions in which the cells are not attached to a solid support.", "Cells proliferating in suspension can be stirred while proliferating using apparatus well known to those skilled in the art.", "The term “monolayer” as used herein refers to cells that are attached to a solid support while proliferating in suitable culture conditions.", "A small portion of the cells proliferating in the monolayer under suitable growth conditions may be attached to cells in the monolayer but not to the solid support.", "Preferably less than 15% of these cells are not attached to the solid support, more preferably less than 10% of these cells are not attached to the solid support, and most preferably less than 5% of these cells are not attached to the solid support.", "The term “substantially similar” as used herein in reference to mammalian cells refers to cells from the same organism and the same tissue.", "Substantially similar can also refer to cell populations that have not significantly differentiated.", "For example, preferably less than 15% of the cells in a population of cells have differentiated, more preferably less than 10% of the cell population have differentiated, and most preferably less than 5% of the cell population have differentiated.", "The term “cell line” as used herein refers to cultured cells that can be passaged one or more times.", "The invention preferably relates to cell lines that can be passaged more than 2, 5, 10, 15, 20, 30, 50, 80, 100, and 200 times.", "The concept of cell passaging is defined previously.", "In preferred embodiments, (1) the mammalian cell is subject to manipulation; (2) the manipulation comprises the step of nuclear transfer; (3) the nuclear transfer comprises the step of inserting the totipotent mammalian cell into a recipient oocyte; (4) the manipulation comprises a step of cryopreservation of the mammalian cell; (5) the manipulation comprises a step of thawing of the mammalian cell; (6) the manipulation comprises a step of culturing the mammalian cell; (7) the manipulation comprises a step of passaging the mammalian cell; (8) the manipulation comprises a step of synchronizing the mammalian cell; (9) the manipulation comprises a step of introducing the mammalian cell to feeder cells; and (10) the manipulation comprises a step of dissociating the mammalian cell from other cells.", "The term “manipulation” as used herein refers to the common usage of the term, which is the management or handling directed towards some object.", "Examples of manipulations are described herein.", "The term “thawing” as used herein refers to the process of increasing the temperature of a cryopreserved cell, embryo, or portions of animals.", "Methods of thawing cryopreserved materials such that they are active after the thawing process are well-known to those of ordinary skill in the art.", "The term “dissociating” as used herein refers to the materials and methods useful for pulling a cell away from another cell.", "For example, a blastomere (i.e., a cellular member of a morula or blastocyst stage embryo) can be pulled away from the rest of the developing cell mass by techniques and apparatus well known to a person of ordinary skill in the art.", "See, e.g., U.S. Pat.", "No.", "4,994,384, entitled “Multiplying Bovine Embryos,” issued on Feb. 19, 1991, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "Alternatively, cells proliferating in culture can be separated from one another to facilitate such processes as cell passaging, which is described previously.", "In addition, dissociation of a cultured cell from a group of cultured cells can be useful as a first step in the process of nuclear transfer, as described hereafter.", "When a cell is dissociated from an embryo, the dissociation manipulation can be useful for such processes as re-cloning, a process described herein, as well as a step for multiplying the number of embryos.", "The term “non-embryonic cell” as used herein refers to a cell that is not isolated from an embryo.", "Non-embryonic cells can be differentiated or non-differentiated.", "Non-embryonic cells refers to nearly any somatic cell, such as cells isolated from an ex utero animal.", "These examples are not meant to be limiting.", "The term “fetus” as used herein refers to a developing cell mass that has implanted into the uterine membrane of a maternal host.", "A fetus can include such defining features as a genital ridge, for example.", "A genital ridge is a feature easily identified by a person of ordinary skill in the art, and is a recognizable feature in fetuses of most animal species.", "The term “fetal cell” as used herein refers to any cell isolated from and/or has arisen from a fetus or derived from a fetus.", "The term “non-fetal cell” is a cell that is not derived or isolated from a fetus.", "When cells are isolated from a fetus, such cells are preferably isolated from fetuses where the fetus is between 20 days and parturition, between 30 days and 100 days, more preferably between 35 days and 70 days and between 40 days and 60 days, and most preferably about a 55 day fetus.", "An age of a fetus can be determined from the time that an embryo, which develops into the fetus, is established.", "The term “about” with respect to fetuses refers to plus or minus five days.", "The term “parturition” as used herein refers to a time that a fetus is delivered from female recipient.", "A fetus can be delivered from a female recipient by abortion, c-section, or birth.", "In preferred embodiments, the cells and cell lines of the instant invention are primary cells, embryonic cells, non-embryonic cells, fetal cells, genital ridge cells, primordial germ cells, embryonic germ cells, embryonic stem cells, somatic cells, adult cells, fibroblasts, differentiated cells, undifferentiated cells, amniotic cells, ovarian follicular cells, and cumulus cells.", "Preferably, such cells grow to confluent monolayers in culture.", "The term “primordial germ cell” as used herein refers to a diploid somatic cell capable of becoming a germ cell.", "Primordial germ cells can be isolated from the genital ridge of a developing cell mass.", "The genital ridge is a section of a developing cell mass that is well-known to a person of ordinary skill in the art.", "See, e.g., Strelchenko, 1996, Theriogenology 45: 130-141 and Lavoir 1994, J. Reprod.", "Dev.", "37: 413-424.Such cells, when cultured, are referred to by the skilled artisan as “embryonic germ cells.” The term “embryonic stem cell” as used herein refers to pluripotent cells isolated from an embryo that are maintained in in vitro cell culture.", "Embryonic stem cells may be cultured with or without feeder cells.", "Embryonic stem cells can be established from embryonic cells isolated from embryos at any stage of development, including blastocyst stage embryos and pre-blastocyst stage embryos.", "Embryonic stem cells are well known to a person of ordinary skill in the art.", "See, e.g., WO 97/37009, entitled “Cultured Inner Cell Mass Cell-Lines Derived from Ungulate Embryos,” Stice & Golueke, published Oct. 9, 1997, and Yang & Anderson, 1992, Theriogenology 38: 315-335, both of which are incorporated herein by reference in their entireties, including all figures, tables, and drawings.", "The term “differentiated cell” as used herein refers to a cell that has developed from an unspecialized phenotype to that of a specialized phenotype.", "For example, embryonic cells can differentiate into an epithelial cell lining the intestine.", "It is highly unlikely that differentiated cells revert into their precursor cells in vivo or in vitro.", "However, materials and methods of the invention can reprogram differentiated cells into immortalized, totipotent cells.", "Differentiated cells can be isolated from a fetus or a live born animal, for example.", "The term “undifferentiated cell” as used herein refers to a cell that has an unspecialized phenotype and is capable of differentiating.", "An example of an undifferentiated cell is a stem cell.", "The term “asynchronous population” as used herein refers to cells that are not arrested at any one stage of the cell cycle.", "Many cells can progress through the cell cycle and do not arrest at any one stage, while some cells can become arrested at one stage of the cell cycle for a period of time.", "Some known stages of the cell cycle are G0, G1, S, G2, and M. An asynchronous population of cells is not manipulated to synchronize into any one or predominantly into any one of these phases.", "Cells can be arrested in the G0 stage of the cell cycle, for example, by utilizing multiple techniques known in the art, such as by serum deprivation.", "Examples of methods for arresting non-immortalized cells in one part of the cell cycle are discussed in WO 97/07669, entitled “Quiescent Cell Populations for Nuclear Transfer,” hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "The terms “synchronous population” and “synchronizing” as used herein refer to a fraction of cells in a population that are arrested (i.e., the cells are not dividing) in a discrete stage of the cell cycle.", "Preferably, about 50% of the cells in a population of cells are arrested in one stage of the cell cycle, more preferably about 70% of the cells in a population of cells are arrested in one stage of the cell cycle, and most preferably about 90% of the cells in a population of cells are arrested in one stage of the cell cycle.", "Cell cycle stage can be distinguished by relative cell size as well as by a variety of cell markers well known to a person of ordinary skill in the art.", "For example, cells can be distinguished by such markers by using flow cytometry techniques well known to a person of ordinary skill in the art.", "Alternatively, cells can be distinguished by size utilizing techniques well known to a person of ordinary skill in the art, such as by the utilization of a light microscope and a micrometer, for example.", "The term “adult cell” as used herein refers to a cell from a live-born animal.", "The term “amniotic cell” as used herein refers to any cultured or non-cultured cell isolated from amniotic fluid.", "Examples of methods for isolating and culturing amniotic cells are discussed in Bellow et al., 1996, Theriogenology 45: 225; Garcia & Salaheddine, 1997, Theriogenology 47: 1003-1008; Leibo & Rail, 1990, Theriogenology 33: 531-552; and Vos et al., 1990, Vet.", "Rec.", "127: 502-504, each of which is incorporated herein by reference in its entirety, including all figures tables and drawings.", "Particularly preferred are cultured amniotic cells that are rounded (e.g., cultured amniotic cells that do not display a fibroblast-like morphology).", "Also preferred amniotic cells are fetal fibroblast cells.", "The terms “fibroblast,” fibroblast-like,” “fetal,” and “fetal fibroblast” are defined hereafter.", "The term “fibroblast” as used herein refers to cultured cells having a flattened and elongated morphology that are able to grow in monolayers.", "Preferably, fibroblasts grow to confluent monolayers in culture.", "While fibroblasts characteristically have a flattened appearance when cultured on culture media plates, fetal fibroblast cells can also have a spindle-like morphology.", "Fetal fibroblasts may require density limitation for growth, may generate type I collagen, and may have a finite life span in culture of approximately fifty generations.", "Preferably, fetal fibroblast cells rigidly maintain a diploid chromosomal content.", "For a description of fibroblast cells, see, e.g., Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed), Wiley-Liss, Inc., incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "The term “uterine cell” as used herein refers to any cell isolated from a uterus.", "Preferably, a uterine cell is a cell deriving from a pregnant adult animal.", "In preferred embodiments, uterine cells are cells obtained from fluid that fills the uterine cavity.", "Such cells can be obtained by numerous methods well known in the art such as amniocentesis.", "The term “ovarian follicular cell” as used herein refers to a cultured or non-cultured cell obtained from an ovarian follicle, other than an oocyte.", "Follicular cells may be isolated from ovarian follicles at any stage of development, including primordial follicles, primary follicles, secondary follicles, growing follicles, vesicular follicles, maturing follicles, mature follicles, and graafian follicles.", "Furthermore, follicular cells may be isolated when an oocyte in an ovarian follicle is immature (i.e., an oocyte that has not progressed to metaphase II) or when an oocyte in an ovarian follicle is mature (i.e., an oocyte that has progressed to metaphase II or a later stage of development).", "Preferred follicular cells include, but are not limited to, pregranulosa cells, granulosa cells, theca cells, columnar cells, stroma cells, theca interna cells, theca externa cells, mural granulosa cells, luteal cells, and corona radiata cells.", "Particularly preferred follicular cells are cumulus cells.", "Various types of follicular cells are known and can be readily distinguished by those skilled in the art.", "See, e.g., Laboratory Production of Cattle Embryos, 1994, Ian Gordon, CAB International; Anatomy and Physiology of Farm Animals (5th ed.", "), 1992, R. D. Frandson and T. L. Spurgeon, Lea & Febiger, each of which is incorporated herein by reference in its entirety including all figures, drawings, and tables.", "Individual types of follicular cells may be cultured separately, or a mixture of types may be cultured together.", "The term “cumulus cell” as used herein refers to any cultured or non-cultured cell isolated from cells and/or tissue surrounding an oocyte.", "Persons skilled in the art can readily identify cumulus cells.", "Examples of methods for isolating and/or culturing cumulus cells are discussed in Damiani et al., 1996, Mol.", "Reprod.", "Dev.", "45: 521-534; Long et al., 1994, J: Reprod.", "Fert.", "102: 361-369; and Wakayama et al., 1998, Nature 394: 369-373, each of which is incorporated herein by reference in its entireties, including all figures, tables, and drawings.", "Cumulus cells may be isolated from ovarian follicles at any stage of development, including primordial follicles, primary follicles, secondary follicles, growing follicles, vesicular follicles, maturing follicles, mature follicles, and graafian follicles.", "Cumulus cells may be isolated from oocytes in a number of manners well known to a person of ordinary skill in the art.", "For example, cumulus cells can be separated from oocytes by pipeting the cumulus cell/oocyte complex through a small bore pipette, by exposure to hyaluronidase, or by mechanically disrupting (e.g.", "vortexing) the cumulus cell/oocyte complex.", "Additionally, exposure to Ca++/Mg++ free media can remove cumulus from immature oocytes.", "Also, cumulus cell cultures can be established by placing matured oocytes in cell culture media.", "Once cumulus cells are removed from media containing increased LH/FSH concentrations, they can to attach to the culture plate.", "In a preferred embodiment, the culturing process can comprise the step of selecting totipotent mammalian cells comprising at least one artificial chromosome.", "The term “selection” as used herein refers to a process for identifying cells that comprise a large heterologous nucleic acid construct, such as an artificial chromosome.", "Selection can be effected by identifying a marker region incorporated in an artificial chromosome.", "The term “marker” is defined previously.", "Preferably, from 50% to 100% of cells in cell cultures that have undergone selection comprise an artificial chromosome.", "In particularly preferred embodiments, greater than or equal to 50% of cells in cell cultures that have undergone selection comprise an artificial chromosome.", "More preferably, greater than or equal to 75% of cells in cell cultures that have undergone selection comprise an artificial chromosome.", "Most preferably, greater than or equal to 90% of cells in cell cultures that have undergone selection comprise an artificial chromosome.", "The term “feeder cells” as used herein refers to cells grown in co-culture with target cells.", "Target cells can be precursor cells and totipotent cells, for example.", "Feeder cells can provide, for example, peptides, polypeptides, electrical signals, organic molecules (e.g., steroids), nucleic acid molecules, growth factors (e.g., bFGF), other factors (e.g., cytokines such as LIF and steel factor), and metabolic nutrients to target cells.", "Certain cells, such as immortalized, totipotent cells may not require feeder cells for healthy growth.", "Feeder cells preferably grow in a mono-layer.", "Feeder cells can be established from multiple cell types.", "Examples of these cell types are fetal cells, mouse cells, Buffalo rat liver cells, and oviductal cells.", "These examples are not meant to be limiting.", "Tissue samples can be broken down to establish a feeder cell line by methods well known in the art (e.g., by using a blender).", "Feeder cells may originate from the same or different animal species as the precursor cells.", "In an example of feeder cells established from fetal cells, ungulate fetuses and preferably bovine fetuses may be utilized to establish a feeder cell line where one or more cell types have been removed from the fetus (e.g., primordial germ cells, cells in the head region, and cells in the body cavity region).", "When an entire fetus is utilized to establish a fetal feeder cell line, feeder cells (e.g., fibroblast cells) and precursor cells (e.g., primordial germ cells) can arise from the same source (e.g., one fetus).", "The term “drug” as used herein refers to any type of molecule that retards the normal growth rate of a cell.", "A normal cell growth rate is measured in the absence of drug.", "A drug may also lyse cells.", "In another aspect, the invention features a method for producing transgenic ungulates by introducing a large heterologous nucleic acid construct into a nuclear donor cell, then fusing this nuclear donor cell into an enucleated recipient cell to form a nuclear transfer embryo, activating this embryo, and finally transferring this embryo into a maternal host to produce a transgenic animal.", "In particularly preferred embodiments, the transgenic animal is an ungulate.", "Nuclear Transfer Most preferably, transgenic animals are prepared by introducing a heterologous nucleic acid molecule, preferably an artificial chromosome, into a nuclear donor cell, then fusing this nuclear donor cell into an enucleated recipient cell, most preferably an enucleated oocyte, to form a nuclear transfer embryo, activating this embryo, and finally transferring this embryo into a maternal host to produce a transgenic animal.", "In preferred embodiments, the artificial chromosome(s) is introduced into the cybrid by introduction into the nuclear donor cell prior to the fusion with the enucleated recipient cell or enucleated oocyte.", "In other preferred embodiments, the artificial chromosome(s) is introduced into the cybrid formed by fusion of the nuclear donor cell with the enucleated recipient cell or enucleated oocyte.", "In yet other preferred embodiments, the artificial chromosome(s) is introduced into the cybrid simultaneously with the fusion of the nuclear donor cell with the enucleated recipient cell or enucleated oocyte The terms “nuclear transfer” and “nuclear transfer procedure” as used herein refer to introducing a full complement of nuclear DNA from one cell to an enucleated cell.", "Nuclear transfer methods are well known to a person of ordinary skill in the art.", "See, U.S. Pat.", "No.", "4,994,384 to Prather et al., entitled “Multiplying Bovine Embryos,” issued on Feb. 19, 1991; U.S. Pat.", "No.", "5,057,420 to Massey, entitled “Bovine Nuclear Transplantation,” issued on Oct. 15, 1991; U.S. Pat.", "No.", "5,994,619, issued on Nov. 30, 1999 to Stice et al., entitled “Production of Chimeric Bovine or Porcine Animals Using Cultured Inner Cell Mass Cells; U.K.", "Patents Nos.", "GB 2,318,578 GB 2,331,751, issued on Jan. 19, 2000 to Campbell et al.", "and Wilmut et al., respectively, entitled “Quiescent Cell Populations For Nuclear Transfer”; U.S. Pat.", "No.", "6,011,197 to Strelchenko et al., entitled “Method of Cloning Bovines Using Reprogrammed Non-Embryonic Bovine Cells,” issued on Jan. 4, 2000; and in U.S. patent application Ser.", "No.", "09/753,323 entitled “Method of Cloning Porcine Animals (attorney docket number 030653.0026.CIP1, filed Dec. 28, 2000), each of which are hereby incorporated by reference in its entirety including all figures, tables and drawings.", "Nuclear transfer may be accomplished by using oocytes that are not surrounded by a zona pellucida.", "In a nuclear transfer procedure, a nuclear donor cell, or the nucleus thereof, is introduced into a recipient cell.", "A recipient cell is preferably an oocyte and is preferably enucleated.", "However, the invention relates in part to nuclear transfer, where a nucleus of an oocyte is not physically extracted from the oocyte.", "It is possible to establish a nuclear transfer embryo where nuclear DNA from the donor cell is replicated during cellular divisions.", "See, e.g., Wagoner et al., 1996, “Functional enucleation of bovine oocytes: effects of centrifugation and ultraviolet light,” Theriogenology 46: 279-284.In addition, nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte.", "Also, nuclear transfer may be accomplished by combining one nuclear donor, one or more enucleated oocytes, and the cytoplasm of one or more enucleated oocytes.", "The resulting combination of a nuclear donor cell and a recipient cell can be referred to variously as a “nuclear transfer embryo,” a “hybrid cell,” or a “cybrid.” Furthermore, a nuclear donor may arise from an animal of the same species from which a nuclear recipient is isolated.", "Alternatively, a nuclear donor may arise from an animal of a different specie from which a nuclear recipient is isolated.", "For example, a differentiated cell isolated from an ear punch of a water buffalo may be utilized as a nuclear donor and an oocyte isolated from a bovine animal may be utilized as a nuclear acceptor.", "Thus, xenospecific nuclear transfer is contemplated by the instant invention.", "The term “nuclear donor” as used herein refers to any cell, or nucleus thereof, having nuclear DNA that can be translocated into an oocyte.", "A nuclear donor may be a nucleus that has been isolated from a cell.", "Multiple techniques are available to a person of ordinary skill in the art for isolating a nucleus from a cell and then utilizing the nucleus as a nuclear donor.", "See, e.g., U.S. Pat.", "Nos.", "4,664,097, 6,011,197, and 6,107,543, each of which is hereby incorporated by reference in its entirety including all figures, tables and drawings.", "Any type of cell can serve as a nuclear donor.", "Examples of nuclear donor cells include, but are not limited to, cultured and non-cultured cells isolated from an embryo arising from the union of two gametes in vitro or in vivo; embryonic stem cells (ES cells) arising from cultured embryonic cells (e.g., pre-blastocyst cells and inner cell mass cells); cultured and non-cultured cells arising from inner cell mass cells isolated from embryos; cultured and non-cultured pre-blastocyst cells; cultured and non-cultured fetal cells; cultured and non-cultured adult cells; cultured and non-cultured primordial germ cells; cultured and non-cultured germ cells (e.g., embryonic germ cells); cultured and non-cultured somatic cells isolated from an animal; cultured and non-cultured cumulus cells; cultured and non-cultured amniotic cells; cultured and non-cultured fetal fibroblast cells; cultured and non-cultured genital ridge cells; cultured and non-cultured differentiated cells; cultured and non-cultured cells in a synchronous population; cultured and non-cultured cells in an asynchronous population; cultured and non-cultured serum-starved cells; cultured and non-cultured permanent cells; and cultured and non-cultured totipotent cells.", "See, e.g., Piedrahita et al., 1998, Biol.", "Reprod.", "58: 1321-1329; Shim et al., 1997, Biol.", "Reprod.", "57: 1089-1095; Tsung et al., 1995, Shih Yen Sheng Wu Hsueh Pao 28: 173-189; and Wheeler, 1994, Reprod.", "Fertil.", "Dev.", "6: 563-568, each of which is incorporated herein by reference in its entirety including all figures, drawings, and tables.", "In addition, a nuclear donor may be a cell that was previously frozen or cryopreserved.", "The term “activation” refers to any materials and methods useful for stimulating a cell to divide before, during, and after a nuclear transfer step.", "Cybrids may require stimulation in order to divide after a nuclear transfer has occurred.", "The invention pertains to any activation materials and methods known to a person of ordinary skill in the art.", "Although electrical pulses are sometimes sufficient for stimulating activation of cybrids, other means are sometimes useful or necessary for proper activation of the cybrid.", "Chemical materials and methods useful for activating embryos are described below in other preferred embodiments of the invention.", "Examples of non-electrical means for activation include agents such as ethanol; inositol trisphosphate (IP3); Ca++ ionophores (e.g., ionomycin) and protein kinase inhibitors (e.g., 6-dimethylaminopurine (MAP)); temperature change; protein synthesis inhibitors (e.g., cyclohexamide); phorbol esters such as phorbol 12-myristate 13-acetate (PMA); mechanical techniques; and thapsigargin.", "The invention includes any activation techniques known in the art.", "See, e.g., U.S. Pat.", "No.", "5,496,720, entitled “Parthenogenic Oocyte Activation” to Susko-Parrish et al., issued on Mar.", "5, 1996; and U.S. Pat.", "No.", "6,077,710, issued on Jun.", "20, 2000, each of which is incorporated by reference herein in its entirety, including all figures, tables, and drawings.", "The term “fusion” as used herein in reference to nuclear transfer refers to the combination of portions of lipid membranes corresponding to the nuclear donor and the recipient oocyte.", "Lipid membranes can correspond to the plasma membranes of cells or nuclear membranes, for example.", "The fusion can occur between the nuclear donor and recipient oocyte when they are placed adjacent to one another, or when the nuclear donor is placed in the perivitelline space of the recipient oocyte, for example.", "Specific examples for translocation of the totipotent mammalian cell into the oocyte are described hereafter in other preferred embodiments.", "These techniques for translocation are fully described in the references cited previously herein in reference to nuclear transfer.", "The term “electrical pulses” as used herein refers to subjecting the nuclear donor and recipient oocyte to electric current.", "For nuclear transfer, the nuclear donor and recipient oocyte can be aligned between electrodes and subjected to electrical current.", "The electrical current can be alternating current or direct current.", "The electrical current can be delivered to cells for a variety of different times as one pulse or as multiple pulses.", "The cells are typically cultured in a suitable medium for the delivery of electrical pulses.", "Examples of electrical pulse conditions utilized for nuclear transfer are described in the references and patents previously cited herein in reference to nuclear transfer.", "The term “fusion agent” as used herein in reference to nuclear transfer refers to any compound or biological organism that can increase the probability that portions of plasma membranes from different cells will fuse when a totipotent mammalian cell nuclear donor is placed adjacent to the recipient oocyte.", "In preferred embodiments fusion agents are selected from the group consisting of polyethylene glycol (PEG), trypsin, dimethylsulfoxide (DMSO), lectins, agglutinin, viruses, and Sendai virus.", "These examples are not meant to be limiting and other fusion agents known in the art are applicable and included herein.", "The term “suitable concentration” as used herein in reference to fusion agents, refers to any concentration of a fusion agent that affords a measurable amount of fusion.", "Fusion can be measured by multiple techniques well known to a person of ordinary skill in the art, such as by utilizing a light microscope, dyes, and fluorescent lipids, for example.", "The term “totipotent” as used herein refers to a cell, embryo, or fetus capable of giving rise to a live born animal.", "The term “totipotent” can also refer to a cell that gives rise to all of the cells in a particular animal.", "A totipotent cell can give rise to all of the cells of an animal when it is utilized in a procedure for developing an embryo from one or more nuclear transfer steps.", "Totipotent cells, embryos, and fetuses may also be used to generate incomplete animals such as those useful for organ harvesting, e.g., having genetic modifications to eliminate growth of an organ or appendage by manipulation of a homeotic gene.", "The term “live born” as used herein preferably refers to an animal that exists ex utero.", "A “live born” animal may be an animal that is alive for at least one second from the time it exits the maternal host.", "A “live born” animal may not require the circulatory system of an in utero environment for survival.", "A “live born” animal may be an ambulatory animal.", "Such animals can include pre- and post-pubertal animals.", "As discussed previously, a live born animal may lack a portion of what exists in a normal animal of its kind.", "In preferred embodiments, (1) totipotent cells arise from at least one precursor cell; (2) a precursor cell is isolated from and/or arises from any region of a ungulate animal; (3) a precursor cell is isolated from and/or arises from any cell in culture; (4) a precursor cell is selected from the group consisting of a primary cell, a non-embryonic cell, a non-fetal cell, a differentiated cell, an undifferentiated cell, a somatic cell, an embryonic cell, a fetal cell, an embryonic stem cell, a primordial germ cell, a genital ridge cell, a cumulus cell, an amniotic cell, a fetal fibroblast cell, a uterine cell, an ovarian follicular cell, a cumulus cell, an hepatocyte, an embryonic germ cell, an adult cell, a cell isolated from an asynchronous population of cells, and a cell isolated from a synchronized population of cells where the synchronous population is not arrested in the G0 stage of the cell cycle; (5) totipotent cells have a morphology of an embryonic germ cell.", "The terms “precursor cell” or “precursor cells” as used herein refer to a cell or cells used to create a cell line of totipotent cells.", "Precursor cells can be isolated from, any animal, preferably from a mammal, and more preferably from an ungulate.", "The precursor cell or cells may be isolated from nearly any cellular entity.", "For example, a precursor cell or cells may be isolated from blastocysts, embryos, fetuses, and cell lines (e.g., cell lines established from embryonic cells), preferably isolated from fetuses and/or cell lines established from fetal cells, and more preferably isolated from ex utero animals and/or cell cultures and/or cell lines established from such ex utero animals.", "An ex utero animal may exist as a newborn animal, adolescent animal, yearling animal, and adult animal.", "The ex utero animals may be alive or post mortem.", "Examples of precursor cells include, but are not limited to, non-embryonic cells; non-fetal cells; differentiated cells; adult cells; somatic cells; embryonic cells; fetal cells; embryonic stem cells; primordial germ cells; genital ridge cells; uterine cells; amniotic cells; ovarian follicular cells; cumulus cells; cells isolated from an asynchronous population of cells; and cells isolated from a synchronized population of cells where the synchronous population is not arrested in the G0 stage of the cell cycle; and any of the forgoing that are cultured, cultured as cell lines and/or totipotent.", "The term “arises from” as used herein refers to the conversion of one or more cells into one or more cells having at least one differing characteristic.", "For example, (1) a non-totipotent precursor cell can be converted into a totipotent cell by utilizing features of the invention described hereafter; (2) a precursor cell can develop a cell morphology of an embryonic germ cell; (3) a precursor cell can give rise to a cultured cell; (4) a precursor cell can give rise to a cultured cell line; and (5) a precursor cell can give rise to a cultured permanent cell line.", "A conversion process can be referred to as a reprogramming step.", "In addition, the term “arises from” refers to establishing totipotent embryos from totipotent cells of the invention by using a nuclear transfer process, as described hereafter.", "The terms “reprogramming” or “reprogrammed” as used herein refer to materials and methods that can convert a non-totipotent cell into a totipotent cell.", "Distinguishing features between totipotent and non-totipotent cells are described previously.", "An example of materials and methods for converting non-totipotent cells into totipotent cells is to incubate precursor cells with a receptor ligand cocktail.", "Receptor ligand cocktails are described hereafter.", "In preferred embodiments, culturing of a cell is a sufficient stimulus to render a cell totipotent.", "The term “reprogramming” or “reprogrammed” as used herein can also refer to materials and methods that can convert a cell into another cell having at least one differing characteristic.", "Also, such materials and methods may reprogram or convert a cell into another cell type that is not typically expressed during the life cycle of the former cell.", "For example, (1) a non-totipotent cell can be reprogrammed into an totipotent cell; (2) a precursor cell can be reprogrammed into a cell having a morphology of an EG cell; and (3) a precursor cell can be reprogrammed into a totipotent cell.", "An example of materials and methods for converting a precursor cell into a totipotent cell having EG cell morphology is described hereafter.", "The term “isolated” as used herein in reference to cells refers to a cell that is mechanically separated from another group of cells.", "Examples of a group of cells are a developing cell mass, a cell culture, a cell line, and an animal.", "These examples are not meant to be limiting and the invention relates to any group of cells.", "Methods for isolating one or more cells from another group of cells are well known in the art.", "See, e.g., Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed.", "), Wiley-Liss, Inc.; Cells:a laboratory manual (vol.", "1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.", "), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J. Morgan, John Wiley and Sons, Ltd.", "The terms “cryopreservation” or “cryopreserved” as used herein refer to freezing a cell, embryo, or animal of the invention.", "The cells, embryos, or portions of animals of the invention are frozen at temperatures lower than 0° C., preferably lower than −80° C., more preferably at temperatures lower than −140° C., and most preferably at temperatures lower than −196° C. Cells and embryos in the invention can be cryopreserved for an indefinite amount of time.", "It is known that biological materials can be cryopreserved for more than fifty years.", "For example, semen that is cryopreserved for more than fifty years can be utilized to artificially inseminate a female bovine animal.", "Methods and tools for cryopreservation are well-known to those skilled in the art.", "See, e.g., U.S. Pat.", "No.", "5,160,312, entitled “Cryopreservation Process for Direct Transfer of Embryos,” issued to Voelkel on Nov. 3, 1992, hereby incorporated by reference herein in its entirety, including all figures, tables, and drawings.", "For the purposes of the present invention, the terms “embryo” or “embryonic” as used herein refer to a developing cell mass that has not implanted into the uterine membrane of a maternal host.", "Hence, the term “embryo” as used herein can refer to a fertilized oocyte, a cybrid (defined herein), a pre-blastocyst stage developing cell mass, and/or any other developing cell mass that is at a stage of development prior to implantation into the uterine membrane of a maternal host.", "Embryos of the invention may not display a genital ridge.", "Hence, an “embryonic cell” is isolated from and/or has arisen from an embryo.", "An embryo can represent multiple stages of cell development.", "For example, a one cell embryo can be referred to as a zygote, a solid spherical mass of cells resulting from a cleaved embryo can be referred to as a morula, and an embryo having a blastocoel can be referred to as a blastocyst.", "The terms “enucleated oocyte” or “enucleated recipient cell” as used herein refer to an oocyte which has had its nucleus removed.", "Typically, a needle can be placed into an oocyte and the nucleus can be aspirated into the inner space of the needle.", "The needle can be removed from the oocyte without rupturing the plasma membrane.", "This enucleation technique is well known to a person of ordinary skill in the art.", "See, U.S. Pat.", "No.", "4,994,384; U.S. Pat.", "No.", "5,057,420; and Willadsen, 1986, Nature 320:63-65.An enucleated oocyte can be prepared from a young or an aged oocyte.", "An enucleated oocyte is preferably prepared from an oocyte that has been matured, in vitro or in vivo, for some period of time.", "This time can vary, depending on the source species for the oocyte.", "For example, bovine oocytes are preferably matured for between 10 hours and 40 hours, more preferably for between 16 hours and 36 hours, and most preferably between 20 hours and 32 hours.", "In contrast, porcine oocytes are preferably matured for greater than 24 hours, and more preferably matured for greater than 36 hours.", "In particularly preferred embodiments, a porcine oocyte is matured for more than 40 hours, up to about 96 hours, more preferably from 42-54 hours, and even more preferably from 42 to 48 hours.", "The terms “maturation” and “matured” as used herein refer to process in which an oocyte is incubated in a medium in vitro.", "Oocytes can be incubated with multiple media well known to a person of ordinary skill in the art.", "See, e.g., Saito et al., 1992, Roux 's Arch.", "Dev.", "Biol.", "201: 134-141 for bovine organisms and Wells et al., 1997, Biol.", "Repr.", "57: 385-393 for ovine organisms and also Mattioli et al., 1989, Theriogenology 31: 1201-1207; Jolliff & Prather, 1997, Biol.", "Reprod.", "56: 544-548; Funahashi & Day, 1993, J. Reprod.", "Fert.", "98: 179-185; Nagashima et al., 1997, Mol.", "Reprod.", "Dev.", "38: 339-343; Abeydeera et al., 1998, Biol.", "Reprod.", "58: 213-218; Funahashi et al., 1997, Biol.", "Reprod.", "57: 49-53; and Sawai et al., 1997, Biol.", "Reprod.", "57: 1-6, each of which are incorporated herein by reference in their entireties including all figures, tables, and drawings.", "Maturation media can comprise multiple types of components, including microtubule inhibitors (e.g.", "cytochalasin B), hormones and growth factors.", "Other examples of components that can be incorporated into maturation media are discussed in WO 97/07668, entitled “Unactivated Oocytes as Cytoplast Recipients for Nuclear Transfer,” Campbell & Wilmut, published on Mar.", "6, 1997, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "The time of maturation can be determined from the time that an oocyte is placed in a maturation medium to the time that the oocyte is subject to a manipulation (e.g., enucleation, nuclear transfer, fusion, and/or activation).", "Oocytes can be matured for any period of time: an oocyte can be matured for greater than 10 hours, greater than 20 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours, greater than 60 hours, greater than 72 hours, and greater than 90 hours.", "The term “about” with respect to oocyte maturation refers to plus or minus 3 hours.", "An oocyte can also be matured in vivo.", "Time of maturation may be the time that an oocyte receives an appropriate stimulus to resume meiosis to the time that the oocyte is manipulated.", "Similar maturation periods described above for in vitro matured oocytes apply to in vivo matured oocytes.", "Nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte.", "In addition, nuclear transfer may be accomplished by combining one nuclear donor, one or more enucleated oocytes, and the cytoplasm of one or more enucleated oocytes.", "The term “young oocyte” as used herein refers to an oocyte that has been matured in vitro for a time less than or equal to the length of time between the onset of estrus and ovulation in vivo.", "For example, the onset of estrus is signaled by a surge in leutenizing hormone.", "A cow typically ovulates about 26 hours following the onset of estrus.", "Thus, a young oocyte is an oocyte matured for about 26 hours or less, preferably 16 to 17 hours.", "Methods for measuring the length of time between the onset of estrus and ovulation are well known to the skilled artisan.", "See, e.g., P. T. Cupps, “Reproduction in Domestic Animals,” Fourth Edition, Academic Press, San Diego, Calif., USA, 1991.For horses, ovulation occurs about 33 hours after onset of estrus; for pigs, about 40 hours; for sheep and goats, about 24-36 hours; for dogs, about 40-50 hours; and for cats, about 24-36 hours.", "The term “young oocyte” may also refer to an oocyte that has been matured and ovulated in vivo and that is collected at about the time of ovulation.", "The term about in this context refers to +/−1 hour.", "Oocytes can be isolated from live animals using methods well known to a person of ordinary skill in the art.", "See, e.g., Pieterse et al., 1988, “Aspiration of bovine oocytes during transvaginal ultrasound scanning of the ovaries,” Theriogenology 30: 751-762.Oocytes can be isolated from ovaries or oviducts of deceased or live born animals.", "Suitable media for in vitro culture of oocytes are well known to a person of ordinary skill in the art.", "See, e.g., U.S. Pat.", "No.", "5,057,420, which is incorporated by reference herein.", "Some young oocytes can be identified by the appearance of their ooplasm.", "Because certain cellular material (e.g., lipids) have not yet dispersed within the ooplasm.", "Young oocytes can have a pycnotic appearance.", "A pycnotic appearance can be characterized as clumping of cytoplasmic material.", "For example, in bovines, a “pycnotic” appearance is to be contrasted with the appearance of oocytes that are older than 28 hours, which have a more homogenous appearing ooplasm.", "The term “aged oocyte” as used herein refers to an oocyte that has been matured in vitro for a time greater than the length of time between the onset of estrus and ovulation in vivo.", "The term “aged oocyte” may also refer to an oocyte that has been matured and ovulated in vivo and that is collected later than about 1 hour after the time of ovulation.", "An aged oocyte can be identified by its characteristically homogenous ooplasm.", "This appearance is to be contrasted with the pycnotic appearance of young oocytes as described previously herein.", "The age of the oocyte can be defined by the time that has elapsed between the time that the oocyte is placed in a suitable maturation medium and the time that the oocyte is activated.", "The age of the oocyte can dramatically enhance the efficiency of nuclear transfer.", "For example, an aged oocyte can be more susceptible to activation stimuli than a young oocyte.", "The term “ovulated in vivo” as used herein refers to an oocyte that is isolated from an animal a certain number of hours after the animal exhibits characteristics that it is in estrus.", "The characteristics of an animal in estrus are well known to a person of ordinary skill in the art, as described in references disclosed herein.", "The terms “maternal recipient” and “recipient female” as used herein refer to a female animal which is implanted with an embryo for development of the embryo.", "A maternal recipient may be either homospecific or xenospecific to the implanted embryo.", "For example it has been shown in the art that bovine embryos can develop in the oviducts of sheep.", "Stice & Keefer, 1993, “Multiple generational bovine embryo cloning,” Biology of Reproduction 48: 715-719.Implanting techniques are well known to a person of ordinary skill in the art.", "See, e.g., Polge & Day, 1982, “Embryo transplantation and preservation,” Control of Pig Reproduction, D J A Cole and G R Foxcroft, eds., London, UK, Butterworths, pp.", "227-291; Gordon, 1997, “Embryo transfer and associated techniques in pigs,” Controlled reproduction in pigs (Gordon, ed), CAB International, Wallingford UK, pp 164-182; and Kojima, 1998, “Embryo transfer,” Manual of pig embryo transfer procedures, National Livestock Breeding Center, Japanese Society for Development of Swine Technology, pp 76-79, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "The term “replication unit” as used herein refers to that portion of a chromosome or other DNA molecule capable of being replicated that is copied from a given origin of replication.", "A chromosome in eukaryotes has many replication units.", "The term “origin of replication” refers to the location in a DNA molecule where its replication begins.", "The term “essentially no homologous DNA” means that the DNA molecule in question comprises almost entirely heterologous DNA.", "Preferably, a molecule which contains essentially no homologous DNA comprises at least 98%, 99%, 99.5%, or 99.9% heterologous DNA when the number of base pairs of heterologous DNA in the molecule is divided by the overall number of base pairs in the molecule.", "The term “homologous DNA” as used herein refers to DNA having the same nucleic acid sequence as DNA sequences present in cell nuclear DNA.", "The term “germ line” refers to those cells which give rise to the reproductive cells of an organism.", "These cells contain the complete haploid genome of an organism and will pass these DNA molecules to the descendants of the organism in question.", "The term “somatic cell” refers to those cells of an organism which are not involved in the production of gametes, e.g., they are not involved in passing the genome to the next generation of the organism in question.", "Transgenic Embryos, Fetuses, and Animals In yet another aspect, the instant invention relates in part to any embryos, fetuses, and animals emanating from totipotent mammalian cells of the invention, where one or more cells in these developing cell masses comprise at least one large heterologous nucleic acid construct, most preferably an artificial chromosome.", "In preferred embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells of the embryos, fetuses, and animals emanating from totipotent mammalian cells of the invention comprise at least one large heterologous nucleic acid construct.", "Most preferably, between 90% and all of the cells of the embryos, fetuses, and animals emanating from totipotent mammalian cells of the invention comprise at least one large heterologous nucleic acid construct.", "Such embryos, fetuses, and animals are known in the art as being “transgenic.” In certain embodiments, the large heterologous nucleic acid construct is an artificial chromosome, most preferably an ACEs or a euchromatin-based minichromosome.", "The cells of the embryos, fetuses, and animals that comprise at least one artificial chromosome preferably comprise ten or fewer artificial chromosomes; more preferably comprise six or fewer artificial chromosomes, four or fewer artificial chromosomes, or two or fewer artificial chromosomes; and most preferably comprise one artificial chromosome.", "If the cells of the embryos, fetuses, and animals of the invention comprise more than one artificial chromosome, the artificial chromosomes may be identical or may differ from one another.", "The term “transgenic” as used herein in reference to embryos, fetuses and animals refers to an embryo, fetus or animal comprising one or more cells that contain heterologous nucleic acids.", "In preferred embodiments, a transgenic embryo, fetus, or animal comprises one or more transgenic cells.", "While germ line transmission is not a requirement of transgenic embryos, fetuses, or animals as that term is used herein, in particularly preferred embodiments a transgenic embryo, fetus, or animal can pass its transgenic characteristic(s) through the germ line.", "In certain embodiments, a transgenic embryo, fetus or animal expresses one or more transgenes as transgenic RNA and protein molecules.", "Most preferably, a transgenic embryo, fetus or animal results from a nuclear transfer procedure using a transgenic nuclear donor cell.", "Transgenic totipotent mammalian embryos can be established from cultured cybrids emanating from one or more nuclear transfer procedures, where one of the nuclear transfer procedures utilizes a totipotent mammalian cell harboring at least one artificial chromosome as a nuclear donor.", "A transgenic totipotent fetus can be established, for example, from a transgenic totipotent embryo that has been implanted into the uterus of a suitable female host.", "Cloned transgenic mammalian animals of the invention can be established from totipotent mammalian cells, totipotent mammalian embryos, and totipotent mammalian fetuses of the invention.", "In certain embodiments, a transgenic animal embryo is produced by nuclear transfer of a nuclear donor cell into an enucleated recipient cell according to the following method: (a) a heterologous DNA molecule of greater than 100 kilobase pairs is introduced into one or more ungulate cells by microcell fusion; (b) the one or more cells are cultured to provide a cell culture; (c) a nuclear donor cell obtained from the cell culture is fused with an enucleated recipient cell to form a nuclear transfer embryo comprising the heterologous DNA molecule; and (d) the nuclear transfer embryo is activated to provide the transgenic ungulate embryo.", "In particularly preferred embodiments, method further comprises one or more of the following: the culturing step comprises selection for one or more markers of said heterologous DNA molecule, whereby at least 90% of cells in said cell culture comprise the heterologous DNA molecule; the transgenic animal is an ungulate selected from the group consisting of a bovine, an ovine, a caprine, and a porcine; the heterologous DNA molecule comprises one or more telomeres, one or more centromeres, and one or more origins of replication; the heterologous DNA molecule is contained within the cells of the transgenic ungulate embryo on a replication unit that comprises essentially no homologous DNA; the activated nuclear transfer embryo is cultured to at least the two cell stage, wherein at least 50% of the cells of the transgenic ungulate embryo comprise the heterologous DNA molecule; the nuclear donor cell is selected from the group consisting of a somatic cell, a primordial germ cell, an embryonic germ cell, and an embryonic stem cell; the heterologous DNA molecule comprises a plurality of copies of at least one transgene; the heterologous DNA molecule is between 100 kilobase pairs and 500 megabase pairs in size; and the heterologous DNA molecule is an artificial chromosome.", "In yet another aspect, the invention features a method of using a cloned transgenic fetus or animal, where one or more cells of the fetus or animal comprise one or more large heterologous nucleic acid constructs.", "The method of using a cloned transgenic fetus or animal comprises the step of isolating at least one component from the fetus or animal.", "The term “component” as used herein can relate to any portion of a fetus or animal.", "A component can be selected from the group consisting of fluid, biological fluid, cell, tissue, organ, gamete, embryo, and fetus.", "The term “gamete” as used herein refers to any cell participating, directly or indirectly, in the reproductive system of an animal.", "Examples of gametes are spermatocytes, spermatogonia, oocytes, and oogonia.", "Gametes can be present in fluids, tissues, and organs collected from animals (e.g., sperm is present in semen).", "For example, methods of collecting semen for the purposes of artificial insemination are well known to a person of ordinary skill in the art.", "See, e.g., Physiology of Reproduction and Artificial Insemination of Cattle (2nd edition), Salisbury et al., copyright 1961, 1978, WH Freeman & Co., San Francisco.", "However, the invention relates to the collection of any type of gamete from an animal.", "The term “tissue” is defined previously.", "The term “organ” relates to any organ isolated from a fetus or animal, or any portion of an organ.", "Examples of organs and tissues are neuronal tissue, brain tissue, spleen, heart, lung, gallbladder, pancreas, testis, ovary, intestine, skin, and kidney.", "These examples are not limiting and the invention relates to any organ and any tissue isolated from a cloned animal of the invention.", "In preferred embodiments, (1) fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses can be subject to manipulation; (2) the manipulation comprises isolating at least one component from an animal or fetus; (3) the manipulation comprises the step of cryopreserving the components; (4) the manipulation comprises the step of thawing components; (5) the manipulation comprises the step of separating the semen into X-chromosome bearing semen and Y-chromosome bearing semen; (6) the manipulation comprises methods of preparing the semen for artificial insemination; (7) the manipulation comprises the step of purification of desired polypeptide(s) from the component; (8) the manipulation comprises concentration of the components; and (9) the manipulation comprises the step of transferring one or more cloned cells, cloned tissues, cloned organs, and/or portions of cloned organs to a recipient organism (e.g., the recipient organism may be of a different species than the donor source).", "The term “separating” as used herein in reference to separating semen refers to methods well known to a person skilled in the art for fractionating a semen sample into sex-specific fractions.", "This type of separation can be accomplished by using flow cytometers that are commercially available.", "Methods of utilizing flow cytometers from separating sperm by genetic content are well known in the art.", "In addition, semen can be separated by its sex-associated characteristics by other methods well known to a person of ordinary skill in the art.", "See, U.S. Pat.", "Nos.", "5,439,362, 5,346,990, and 5,021,244, entitled “Sex-Associated Membrane Proteins and Methods for Increasing the Probability that Offspring Will Be of a Desired Sex,” Spaulding, issued on Aug. 8, 1995, Sep. 13, 1994, and Jun.", "4, 1991 respectively, all of which are incorporated herein by reference in their entireties including all figures, tables, and drawings.", "Semen preparation methods are well known to someone of ordinary skill in the art.", "Examples of these preparative steps are described in Physiology of Reproduction and Artificial Insemination of Cattle (2nd.", "edition), Salisbury et al., copyright 1961, 1978, W.H.", "Freeman & Co., San Francisco.", "The term “purification” as used herein refers to increasing the specific activity of a particular polypeptide or polypeptides in a sample.", "Specific activity can be expressed as the ratio between the activity of the target polypeptide and the concentration of total polypeptide in the sample.", "Activity can be catalytic activity and/or binding activity, for example.", "Alternatively, specific activity can be expressed as the ratio between the concentration of the target polypeptide and the concentration of total polypeptide.", "Purification methods include dialysis, centrifugation, and column chromatography techniques, which are well-known procedures to a person of ordinary skill in the art.", "See, e.g., Young et al., 1997, “Production of biopharmaceutical proteins in the milk of transgenic dairy animals,” BioPharm 10(6): 34-38.The term “transferring” as used herein can relate to shifting cells, tissues, organs, and/or portions of organs to an animal.", "The cells, tissues, organs, and/or portions of organs can be, for example, (a) developed in vitro and then transferred to an animal, (b) removed from an animal and transferred to another animal of a different specie, (c) removed from an animal and transferred to another animal of the same specie, (d) removed from one portion of an animal (e.g., the leg of an animal) and then transferred to another portion of the same animal (e.g., the brain of the animal), and/or (e) any combination of the foregoing.", "The term “transferring” can relate to adding cells, tissues, and/or organs to an animal and can also relate to removing cells, tissues, and/or organs from an animal and replacing them with cells, tissues, and/or organs from another source.", "The term “transferring” as used herein can also refer to implanting one or more cells, tissues, organs, and/or portions of organs from the cloned mammalian animal into another organism.", "For example, neuronal tissue from a cloned mammalian organism can be grafted into an appropriate area in the human nervous system to treat neurological diseases such as Alzheimer's disease.", "Alternatively, cloned cells, tissues, and/or organs originating from a porcine organism may be transferred to a human recipient.", "Surgical methods for accomplishing this preferred aspect of the invention are well known to a person of ordinary skill in the art.", "Transferring procedures may include the step of removing or deleting cells, tissues, or organs from a recipient organism before a transfer step.", "Of particular interest are transgenic animals that express genes that confer resistance or reduce susceptibility to disease.", "Since multiple genes can be introduced on an ACEs, a series of genes encoding an antigen can be introduced, which upon expression will serve to immunize [in a manner similar to a multivalent vaccine] the host animal against the diseases for which exposure to the antigens provide immunity or some protection.", "Also of interest are transgenic animals that serve as models of certain diseases and disorders for use in studying the disease and developing therapeutic treatments and cures thereof.", "Such animal models of disease express genes [typically carrying a disease-associated mutation], which are introduced into the animal on a MAC, preferably an ACEs, and which induce the disease or disorder in the animal.", "Similarly, MACs carrying genes encoding antisense RNA may be introduced into animal cells to generate conditional “knock-out” transgenic animals.", "In such animals, expression of the antisense RNA results in decreased or complete elimination of the products of genes corresponding to the antisense RNA.", "Of further interest are transgenic mammals that harbor MAC-carried genes encoding therapeutic proteins that are expressed in the animal's milk.", "Transgenic animals for use in xenotransplantation, which express MAC-carried genes that serve to humanize the animal's organs, are also of interest.", "Genes that might be used in humanizing animal organs include those encoding human surface antigens.", "The invention relates in part to any disease or parasitic condition known in the art.", "See, e.g., Hagan &Bruners Infectious Diesases of Domestic Animals (7th edition), Gillespie & Timoney, copyright 1981, Cornell University Press, Ithaca N.Y.", "Examples of parasites include, but are not limited to, worms, insects, invertebrate, bacterial, viral, and eukaryotic parasites.", "These parasites can lead to diseased states that can be controlled by the materials and methods of the invention.", "The term “regulatory element” as used herein refers to a DNA or RNA sequence that can increase or decrease the amount of product produced from another DNA or RNA sequence.", "The regulatory element can cause the constitutive production of the product (e.g., the product can be expressed constantly).", "Alternatively, the regulatory element can enhance or diminish the production of a recombinant product in an inducible fashion (e.g., the product can be expressed in response to a specific signal).", "The regulatory element can be controlled, for example, by nutrition, by light, or by adding a substance to the transgenic organism's system.", "Examples of regulatory elements well-known to those of ordinary skill in the art are promoters, enhancers, insulators, and repressors.", "See, e.g., Transgenic Animals, Generation and Use, 1997, Edited by L. M. Houdebine, Hardwood Academic Publishers, Australia, hereby incorporated herein by reference in its entirety including all figures, tables, and drawings.", "The terms “promoters,” “promoter,” or “promoter elements” as used herein refer to a DNA sequence that is located adjacent to a DNA sequence that encodes a recombinant product.", "A promoter is preferably operatively linked to the adjacent DNA sequence.", "A promoter typically increases the amount of recombinant product expressed from a DNA sequence as compared to the amount of the expressed recombinant product when no promoter exists.", "A promoter from one organism can be utilized to enhance recombinant product expression from a DNA sequence that originates from another organism.", "In addition, one promoter element can increase an amount of recombinant products expressed for multiple DNA sequences attached in tandem.", "Hence, one promoter element can enhance the expression of one or more recombinant products.", "Multiple promoter elements are well-known to persons of ordinary skill in the art.", "Examples of promoter elements are described hereafter.", "The terms “enhancers,” “enhancer” or “enhancer elements” as used herein refer to a DNA sequence that is located adjacent to the DNA sequence that encodes a recombinant product.", "Enhancer elements are typically located upstream of a promoter element or can be located downstream of the coding DNA sequence (e.g., the DNA sequence transcribed or translated into a recombinant product or products).", "Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream of the DNA sequence that encodes the recombinant product.", "Enhancer elements can increase the amount of recombinant product expressed from a DNA sequence above the increased expression afforded by a promoter element.", "Multiple enhancer elements are readily available to persons of ordinary skill in the art.", "The terms “insulators,” “insulator,” or “insulator elements” as used herein refer to DNA sequences that flank the DNA sequence encoding the recombinant product.", "Insulator elements can direct the recombinant product expression to specific tissues in an organism.", "Multiple insulator elements are well known to persons of ordinary skill in the art.", "See, e.g., Geyer, 1997, Curr.", "Opin.", "Genet.", "Dev.", "7: 242-248, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.", "The terms “repressor” or “repressor element” as used herein refer to a DNA sequence located in proximity to the DNA sequence that encodes the recombinant product, where the repressor sequence can decrease the amount of recombinant product expressed from that DNA sequence.", "Repressor elements can be controlled by the binding of a specific molecule or specific molecules to the repressor element DNA sequence.", "These molecules can either activate or deactivate the repressor element.", "Multiple repressor elements are available to a person of ordinary skill in the art.", "The terms “milk protein promoter,” “urine protein promoter,” “blood protein promoter,” “tear duct protein promoter,” “synovial protein promoter,” “spermatogenesis protein promoter,” and “mandibular gland protein promoter” refer to promoter elements that regulate the specific expression of proteins within the specified fluid or gland or cell type in an animal.", "For example, a milk protein promoter is a regulatory element that can control the expression of a protein that is expressed in the milk of an animal.", "Other promoters, such as β-casein promoter, melanocortin promoter, milk serum protein promoter, casein promoter, α-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, and α-actin promoter, for example, are well known to a person of ordinary skill in the art.", "The terms “insertion” and “introduction” as used herein in reference to artificial chromosomes or other large heterologous nucleic acid constructs refer to translocating one or more such artificial chromosomes or constructs from the outside of a cell to the inside of a cell.", "Insertion can be effected in at least two manners: by mechanical delivery and non-mechanical delivery.", "The term “mechanical delivery” as used herein refers to processes that utilize an apparatus that directly or indirectly introduces DNA (e.g., one or more artificial chromosomes) into one or more cells.", "Examples of mechanical delivery of DNA into cells include, but are not limited to, microinjection, particle bombardment, sonoporation, and electroporation.", "The term “non-mechanical delivery” as used herein refers to non-mechanical processes such as diffusive processes, for example.", "For instance, non-mechanical delivery may be effected by introducing DNA (e.g., an artificial chromosome) and one or more reagents to a medium bathing cell surfaces, where the reagents increase the probability that the DNA enters the cells.", "Such reagents are well known in the art, such as liposomes, acyl moieties, peptide moieties, saccharide moieties, and/or polyethylene glycol (PEG), for example.", "Such reagents may be complexed with the target molecule and the reagents may be introduced to cells in vivo and/or ex vivo.", "These examples are not meant to be limiting and the invention relates in part to any non-mechanical form of insertion.", "The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments, as well as from the claims.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention relates in part to totipotent cells comprising at least one artificial chromosome.", "These cells can be utilized in nuclear transfer processes for establishing cloned transgenic embryos, fetuses, and animals.", "These cells and other materials and methods of the invention represent an improvement towards establishing cloned transgenic animals.", "One improvement towards establishing cloned transgenic animals is that large units of target DNA (e.g., heterologous DNA) may be introduced to cells.", "Specifically, target DNA larger than 10 kb can be introduced into artificial chromosomes and these artificial chromosomes can be inserted into cells.", "Hence, genes and regulatory sequences larger than 10 kb in length can be inserted into one artificial chromosome and then introduced to cells; multiple types of regulatory sequences and multiple types of genes can be inserted into an artificial chromosome and then introduced to cells; and multiple copies of a given target DNA sequence can be incorporated into an artificial chromosome and then incorporated into a cell.", "These are examples of improvements and the invention also relates to other improvements.", "In another improvement, the invention provides materials and methods that can provide in vitro control of in vivo recombinant product expression levels.", "Specifically, artificial chromosomes comprising a known copy number of a target DNA can be selected before the artificial chromosome comprising the target DNA is inserted into a cell of interest.", "The number of copies can be quantified by a variety of techniques known in the art, such as Southern blot and FISH procedures, for example.", "Hence, in contrast to techniques currently applied in the art for establishing cloned transgenic animals, expression levels of one or more recombinant products can be controlled by the number of copies of target DNA present in an artificial chromosome that encode these recombinant products.", "Another improvement towards establishing cloned transgenic animals is that the location of target DNA within a transgenic cell can be controlled.", "Artificial chromosomes remain distinct and separate from endogenous genomic DNA (i.e., they exist as “extra-genomic” elements in the nucleus of the transgenic cell), thus providing a controlled and known locations of target DNA.", "This advantage is contrasted with many existing techniques for creating transgenic cells, which cause random insertion of target DNA into cell nuclear DNA.", "Because random insertion decreases efficiencies for establishing transgenic cells due to unproductive insertions that must be quantified in vivo, the materials and methods defined herein are improvements over the technology currently utilized in the art to clone transgenic animals.", "The following descriptions define processes for establishing totipotent cells comprising one or more artificial chromosomes, introducing one or more artificial chromosomes into cells, and processes for utilizing totipotent cells that comprise one or more artificial chromosomes.", "Establishing Totipotent Cells The invention relates in part to establishing totipotent mammalian cells comprising one or more artificial chromosomes.", "These cells can be useful as nuclear donors in nuclear transfer procedures.", "Utilizing these cells in nuclear transfer procedures can improve the efficiency of processes for establishing cloned and transgenic embryos, fetuses, and animals.", "Totipotent mammalian cells can be established from nearly any type of precursor cell.", "Examples of precursor cells are non-embryonic cells; non-fetal cells; differentiated cells; somatic cells; embryonic cells; fetal cells; embryonic stem cells; primordial germ cells; genital ridge cells; cells isolated from an asynchronous population of cells; and cells isolated from a synchronized population of cells where the synchronous population is not arrested in the G0 stage of the cell cycle; and any of the forgoing that are cultured, cultured as cell lines, immortalized, and/or totipotent.", "These examples are not meant to be limiting and the invention relates to any precursor cell known in the art.", "Processes for establishing totipotent cells having at least one artificial chromosome comprise one or more of the following procedures: (1) inserting at least one artificial chromosome into a cell; (2) introducing a stimulus to a cell; (3) conducting one or more nuclear transfer procedures, which may optionally include fusion and/or activation steps; and (4) selecting cells that comprise at least one artificial chromosome.", "These procedures can be conducted in any order and each procedure may be repeated more than once.", "Materials and methods for introducing at least one artificial chromosome into a cell and selecting for cells that comprise an artificial chromosome are defined hereafter.", "Materials and methods for (1) culturing cells; (2) passaging and plating cells; (3) preparing and administering a stimulus to cells; (4) preparing feeder cells; (5) and (6) establishing totipotent cells, are defined in PCT application number WO 98/39416 entitled “Method of Cloning Animals,” Strelchenko et al., filed Mar.", "5, 1998, which is hereby incorporated herein by reference in its entirety including all figures, tables, and drawings.", "Preparing Artificial Chromosomes Artificial chromosome expression systems (ACes) containing multiple copies of several transgenes were incorporated into bovine embryos produced using nuclear transfer technologies.", "In the first approach, ACes were incorporated into bovine embryos by direct injection of ACes in enucleated oocytes just prior to their electrofusion with a nuclear donor cell to form a cybrid.", "Direct injection of ACes yield blastocysts in which up to 25% of the cells contained ACes.", "In another approach, nuclear donor cells that contained ACes were enriched using hygromycin B and then used to generate nuclear transfer blastocysts that contain an artificial chromosome in 90% or more of cells for the purpose of producing cloned animals with specific traits or that express commercially relevant proteins in the mammary glands or other tissues.", "The transfection of nuclear donor cells used in cloning has the distinct advantages of permitting the pre-selection of transgenic cells prior to nuclear transfer and providing for transgene incorporation in the majority of nucleated cells in the organism in the first generation.", "Because of the advantages of using transgenic nuclear donor cells, cloning has become the method of choice to generate transgenic animals.", "However, there are several limitations to current methods to produce transgenic nuclear donor cells.", "One limitation is that if the transgenes insert randomly into the genome, the expression of the transgene cannot be predicted.", "Position effects on transgene expression occur after all forms of gene delivery-microinjection, transfection, electroporation, infection, etc.", "In addition, random transgene integration is likely to cause mutations of critical genomic loci and generate a wide variety of abnormal phenotypes in cells and animals (Constantini et al., 1989; Rossant, 1991; Favor and Morawetz, 1992; Rijkers et al., 1994; Kurth, 1998; Woychik and Alagramam, 1998).", "If targeting to specific loci via homologous recombination is used, the size and copy number of the transgenes are limited.", "In addition, introducing several different transgenes into the same locus simultaneously would likely prove difficult.", "An approach to overcome the limitations of both random transgene insertion and transgene targeting is to introduce into nuclear transfer embryos an entirely new chromosome, ACes.", "The advantage of ACes is that instead of genes being inserted at random into the existing chromosomes of a cell or embryo, genes are engineered onto a separate chromosome with its own structure for extra-genomic maintenance and replication.", "ACes with specific transgenes of interest have been transmitted through the germline of mice following their injection into the pronuclei of zygotes (Coet al.", "2000).", "In addition, these artificial chromosomes have been transferred into cells in vitro via microcell mediated chromosome transfer (Telenius et al.", "1999) and other non-viral methods.", "Artificial chromosomes are well known in the art.", "In particular, materials and methods for (1) preparing artificial chromosomes de novo, and (2) preparing recombinant vectors suitable for inserting heterologous DNA (e.g., heterologous with respect to artificial chromosome DNA and/or heterologous with respect to cell nuclear DNA) into an artificial chromosome are well known in the art.", "See, e.g., Kereso et al., 1996, Chromosome Research 4: 226-239, Holló et al., 1996, Chromosome Research 4: 240-247, U.S. Pat.", "No.", "6,025,155, and U.S. Pat.", "No.", "6,077,697.Artificial chromosomes can comprise multiple elements, including (1) one or more repressor elements; (2) one or more insulator elements; (3) one or more promoter elements; (4) one or more enhancer elements; (5) one or more units of target DNA; (6) one or more units of neutral DNA; (7) a centromere; (8) one or more origins of replication; and (9) one or more telomeres.", "These elements are defined previously and are well known in the art.", "These elements are examples and the invention relates in part to other DNA elements known in the art.", "These elements can be repeated in any number in an artificial chromosome, and can be located in any order in an artificial chromosome.", "Repeated elements can be contiguous and/or non-contiguous.", "Related but different elements can also exist in an artificial chromosome.", "For example, an artificial chromosome may comprise one or more copies of target DNA that encodes one type of recombinant product and one or more copies of another type of target DNA that encodes another type of recombinant product.", "Target DNA can encode recombinant products including, but not limited to, ribozymes; antisense RNA; peptides; polypeptides; proteins; structural proteins; antibodies; enzymes; and portions, fragments, mutants, deletions, and fusions of any of the foregoing.", "Examples of recombinant products encoded by target DNA include hormones, enzymes, growth factors, clotting factors, apolipoproteins, receptors, drugs, pharmaceuticals, bioceuticals, nutraceuticals, oncogenes, tumor antigens, tumor suppressors, cytokines, viral antigens, parasitic antigens, bacterial antigens and chemically synthesized polymers and polymers biosynthesized and/or modified by chemical, cellular and/or enzymatic processes.", "Specific examples of recombinant products include proinsulin, insulin, growth hormone, androgen receptors, casein, milk proteins, muscle proteins, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding proteins, epidermal growth factor, TGF-α, TGF-β, platelet-derived growth factor (PDGF), angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor, and angiogenin), matrix proteins (Type I collagen, Type IV collagen, Type VII collagen, laminin), oncogenes (ras, fos, myc, erb, src, sis, jun), E6 or E7 transforming sequence, p53 protein, cytokine receptor, IL-1, IL-6, IL-8, IL-2, α, β, or γIFN, GMCSF, GCSF, viral capsid protein, and proteins from viral, bacterial and parasitic organisms.", "Other specific proteins or polypeptides which can be expressed include: phenylalanine hydroxylase, α-1-antitrypsin, cholesterol-7α-hydroxylase, truncated apolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoprotein A1, LDL receptor, scavenger receptor for oxidized lipoproteins, molecular variants of each, VEGF, and combinations thereof.", "Other examples are clotting factors, fibrinogen, factor VIII, Von Willebrands Factor, α-glucosidase, apolipoproteins, drugs, tumor antigens, viral antigens, parasitic antigens, monoclonal antibodies, and bacterial antigens.", "One skilled in the art readily appreciates that these proteins belong to a wide variety of classes of proteins, and that other proteins within these classes can also be used.", "These are only examples and are not meant to be limiting in any way.", "It should also be noted that target DNA includes (1) nucleic acid sequences not normally found in the cells; (2) nucleic acid molecules which are normally found in the cells but not expressed at physiological significant levels; (3) nucleic acid sequences normally found in the cells and normally expressed at physiological desired levels; (4) other nucleic acid sequences which can be modified for expression in cells; and (5) any combination of the above.", "Examples of promoter elements include, but are not limited to, milk protein promoter, urine protein promoter, blood protein promoter, tear duct protein promoter, synovial protein promoter, mandibular gland protein promoter, casein promoter, β-casein promoter, melanocortin promoter, milk serum protein promoter, α-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, and α-actin promoter.", "Materials and methods for manipulating DNA of mammalian cells are well-known to a person of ordinary skill in the art.", "See, e.g., Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press, hereby incorporated by reference in its entirety including all figures, tables, and drawings.", "Introducing Artificial Chromosomes into Cells Artificial chromosomes comprise DNA molecules that can be introduced into cells.", "Materials and methods for introducing DNA molecules into cells are well known in the art.", "The invention relates to introducing one or more artificial chromosomes into any type of cell.", "Examples of cell types are defined herein.", "Furthermore, artificial chromosomes can be introduced into cells when the cells are incorporated within fluids, tissues, organs, and animals.", "DNA molecules can be introduced into cells by utilizing at least two types of processes.", "First, DNA can be inserted into cells by using mechanical processes, where DNA is physically inserted into a cell.", "Examples of mechanical processes are microinjection, sonoporation, electroporation, and particle bombardment.", "Second, DNA can be introduced into cells by using non-mechanical processes.", "Examples of diffusive processes known in the art are viral processes, non-viral processes, liposome-mediated cell fusion processes, peptide-mediated cell fusion processes, ligand/receptor processes, and diffusion processes that insert an entire complement of nuclear DNA into cells.", "The above-identified examples are not meant to be limiting and the invention relates to any combinations of materials and methods for introducing DNA into cells that are known in the art.", "Materials and methods for DNA introduction processes are well known in the art.", "These methods include, but are not limited to, direct DNA transfer techniques, cell microinjection methods, micro particle bombardment processes, electroporation methods, cell fusion techniques, microcell fusion processes, lipid mediated transfer methods, lipofection processes, liposome mediated methods, protoplast regeneration processes, protoplast fusion methods, and polycation mediated techniques.", "See, e.g., Hogan et al., 1994, Manipulating the Mouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (see especially pages 255-264 and Appendix 3); Liszewski, 1998, “Non-viral Strategies for Gene Therapy,” Genetic Engineering News (Jan. 1, 1998): 13, 28, 32; Keown et al., 1990, Methods in Enzymology 185: 527-537; Mansour et al., 1988, Nature 336: 348-352; U.S. Pat.", "No.", "5,491,075; U.S. Pat.", "No.", "5,482,928; U.S. Pat.", "No.", "5,424,409; U.S. Pat.", "No.", "5,470,708; Kuo and Saltzman, 1996, “Novel Systems for Controlled Delivery of Macromolecules,” Critical Reviews in Eukaryotic Gene Expression 6(1): 59-73; Monsigny et al., 1994, “Glycoconjugates as carriers for specific delivery of therapeutic drugs and genes,” Advanced Drug Delivery Reviews 14: 1-24; Nicolau and Cudd, 1989, “Liposomes as Carriers of DNA,” Critical Review in Therapeutic Drug Carrier Systems 6: 239-271; Papisov, 1995, “Modeling in vivo transfer of long-circulating polymers (two classes of long circulating polymers and factors affecting their transfer in vivo),” Advanced Drug Delivery Reviews 16: 127-139; Szoka and Papahadjopouls, 1980, “Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes),” Annual Reviews of Biophysics and Bioengineering 9: 467-508; Gottschalk et al., 1996, “A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells,” Gene Therapy 3: 448-457; Nicolau et al., 1987, “Liposomes as Carriers for in vivo Gene Transfer and Expression,” Methods in Enzymology 149: 157-176; Simons et al., 1988, “Gene Transfer Into Sheep,” Bio/Technology 6: 179-183; Yang, 1992, “Gene Transfer into Mammalian Somatic Cells in vivo,” Critical Reviews in Biotechnology 12: 335-356; Behr, 1993, “Synthetic Gene-Transfer Vectors,” Acc.", "Chem.", "Res.", "26: 274-278; Davis et al., 1993, “Direct Gene Transfer into Skeletal Muscle in vivo: Factors Affecting Efficiency of Transfer and Stability of Expression,” Human Gene Therapy 4: 151-159; Gao and Huang, 1995, “Cationic liposome-mediated gene transfer,” Gene Therapy 2: 710-722; Rhodes et al., WO 93/14778 dated Aug. 5, 1993, PCT/US93/00492 dated Jan. 21, 1993, entitled “Ex Vivo Gene Transfer”; Wigler et al., 1978, “Biochemiical Transfer of Single-Copy Eucaryotic Genes Using Total Cellular DNA as Donor,” Cell 14: 725-731; Yang et al., 1990, “In vivo and in vitro Gene Transfer to Mammalian Somatic Cells by Particle Bombardment,” Proc.", "Natl.", "Acad.", "Sci.", "USA 87: 9568-9572; and Wagner et al., 1991, “DNA-Binding Transferrin Conjugates as Functional Gene-Delivery Agents: Synthesis by Linkage of Polylysine or Ethidium Homodimer to the Transferrin Carbohydrate Moiety,” Bioconjugate Chem.", "2: 226-231; Wigler et al., 1979, Proc.", "Natl.", "Acad.", "Sci.", "USA 76:1373-1376; Strauss, 1996, Meth.", "Mol.", "Biol.", "54:307-327; Lambert, 1991, Proc.", "Natl.", "Acad.", "Sci.", "USA 88:5907-5911; U.S. Pat.", "No.", "5,396,767, Sawford et al., Somatic Cell Mol.", "Genet.", "13:279-284; Dhar et al., 1984, Somatic Cell Mol.", "Genet.", "10:547-559; McNeill-Killary et al., 1995, Meth.", "Enzymol.", "254:133-152; Teifel et al., 1995, Biotechniques 19:79-80; Albrecht et al., 1996, Ann.", "Hematol.", "72:73-79; Holmen et al., 1995, In Vitro Cell Dev.", "Biol.", "Anim.", "31:347-351; Remy et al., 1994, Bioconjug.", "Chem.", "5:647-654; Le Bolch et al., 1995, Tetrahedron Lett.", "36:6681-6684; Loeffler et al., 1993, Methl.", "Enzynol.", "217:599-618; Teifel et al., 1995, Biotechniques 19:79-80; Albrecht et al., 1996, Ann.", "Hematol.", "72:73-79; Holman et al., 1995, In Vitro Cell Dev.", "Biol.", "Anim.", "31:347-351; Remy et al., 1994, Bioconjug.", "Chem.", "5:647-654; Le Bolch et al., 1995, Tetrahedron Lett.", "36:6681-6684; Loeffler et al., 1993, Meth.", "Enzymol.", "217:599-618; Strauss, 1996, Meth.", "Mol.", "Biol.", "54:307-327; Brazolot et al.", "1991, Mol.", "Repro.", "Dev.", "30:304-312; U.S. Pat.", "Nos.", "4,955,378, 4,923,814, 4,476,004, 4,906,576 and 4,441,972; International PCT application publication No.", "WO 91/00358; U.S. Pat.", "Nos.", "4,784,737, 5,501,967, 5,501,662, 5,019,034, 5,503,999; Fromm et al., 1985, Proc.", "Natl.", "Acad.", "Sci.", "USA 82:5824-5828; Aq Biotechnology News 7:3 and 17 September/October 1990; U.S. Pat.", "Nos.", "5,240,840, 4,806,476, 5,298,429, 5,396,767; Fournier, 1981, Proc.", "Nat.", "Acad.", "Sci.", "USA 78:6349-6353; Lambert et al., 1991, Proc.", "Natl.", "Acad.", "Sci.", "USA 88:5907-59; Dieken et al., 1996, Natura Genet.", "12:174-182, all of which are incorporated by reference herein in their entirety, including all figures, tables, and drawings.", "For a cell that is to be used as a nuclear donor, one or more artificial chromosomes can be introduced into the cell prior to placing the cell into the perivitelline space of the recipient oocyte, or after placing the cell into the perivitelline space and just prior to fusion.", "In other embodiments, one or more artificial chromosomes can be introduced into the recipient oocyte prior to fusion with the nuclear donor cell.", "In yet other embodiments, one or more artificial chromosomes can be introduced into a pre-formed cybrid prepared by fusion of a nuclear donor cell with a recipient oocyte.", "Additionally, in certain preferred embodiments, one or more artificial chromosomes are not introduced into the cell, but rather allowed to associate with the exterior of either the nuclear donor cell or the recipient oocyte.", "In these embodiments, the artificial chromosome can be carried into the cybrid at the time of fusion.", "Cells comprising one or more artificial chromosomes can be selected against cells that do not comprise an artificial chromosome by identifying the artificial chromosome directly and by detecting a marker present in an artificial chromosome.", "Methods for identifying an artificial chromosome directly are well known in the art.", "Examples of such methods include FISH (fluorescent in situ hybridization) and chromosome karyotyping, where the artificial chromosome can be detected in one cell by simply counting the number of chromosomes in the cell.", "An artificial chromosome is present in a cell if the cell has one or more chromosomes in addition to the number of chromosomes normally present in such a cell.", "One cell that contains one or more artificial chromosomes can be utilized to establish a cell culture of such cells.", "Markers are well known in the art.", "Examples of markers are drug resistance genes, such as genes that render a cell resistant to such drugs as neomycin, hygromycin, blasticidin S, and puromycin.", "These examples are not meant to be limiting.", "Other examples of markers are genes that express enzymes that directly or indirectly modify substrates, such as a gene which encodes β-galactosidase and a gene that encodes luciferase.", "If an artificial chromosome harbors more than one marker, multiple markers can be identified.", "Similarly, if a cell comprises more than one artificial chromosome, where each artificial chromosome harbors a distinct marker, multiple markers may be identified.", "Materials and methods for conducting such selection processes are well known in the art.", "Examples of processes for detecting markers in cells are polymerase chain reaction and FISH procedures by utilizing appropriate DNA probes.", "Hence, identifying cells comprising one or more artificial chromosomes can be accomplished by utilizing materials and methods well known in the art.", "Processes that Utilize Totipotent Cells Comprising Artificial Chromosomes The invention relates in part to processes that utilize totipotent cells comprising artificial chromosomes.", "These cells are preferably utilized as nuclear donors in nuclear transfer processes, where the nuclear donor is inserted into a recipient oocyte.", "Nuclear transfer procedures typically include a translocation step, where the nuclear donor is inserted into a recipient oocyte.", "Nuclear transfer procedures can optionally include a fusion step (e.g., effected by one or more electric pulses and/or one or more fusion agents) and can optionally include an activation step (e.g., electrostimulation and/or ionomycin coupled with DMAP).", "Nuclear transfer processes may include one or more nuclear transfer cycles and the various steps in each cycle can be executed in any order and may be repeated more than once in any cycle.", "Nuclear transfer processes can give rise to cloned embryos, where the embryonic cells comprise one or more artificial chromosomes.", "One or more nuclear transfer cycles may be utilized to establish cloned embryos, fetuses, and animals of the invention.", "These cloned embryos can develop into a cloned fetus where the fetal cells comprise one or more artificial chromosomes.", "Cloned fetuses may be established, for example, when cloned transgenic embryos are implanted into an uterus of a suitable recipient female.", "Cloned transgenic animals may be established when cloned transgenic fetuses are allowed to develop into an animal.", "Cells isolated from cloned embryos, fetuses, and animals can be subjected to selection processes defined previously to determine whether the cells comprise one or more artificial chromosomes.", "In addition, entire embryos and fetuses may be subjected to these selection processes.", "Cells obtained from fetuses, embryos, and animals produced by the nuclear transfer procedures described herein can be used in a second nuclear transfer, or recloning, procedure.", "For example, blastomeres from a first nuclear transfer embryo can be used as nuclear donors, or used to establish a cell line, which cells are used as nuclear donors.", "Alternatively, a fetus can be harvested from a maternal host, and one or more cells used directly as nuclear donors, or used to establish a cell line, which cells are used as nuclear donors.", "Such cells can undergo selection for the presence of an artificial chromosome, as described herein.", "In addition, cells obtained from a transgenic animal can be used directly as nuclear donors, or used to establish a nuclear donor cell line.", "Materials and methods for (1) conducting one or more nuclear transfer cycles; (2) conducting nuclear donor insertion processes; (3) conducting fusion processes; (4) conducting activation processes; (5) preparing oocytes as nuclear recipients; (6) preparing totipotent cells as nuclear donors; (7) culturing embryos; (8) manipulating embryos; (9) implanting one or more embryos into an uterus of an appropriate animal; (10) manipulating fetuses; and (11) using cloned animals, are defined in PCT application entitled “Method of Cloning Animals,” Strelchenko et al., filed Mar.", "5, 1998.EXAMPLES The examples below are non-limiting and are merely representative of various aspects and features of the present invention.", "Example 1 Incorporating Artificial Chromosomes Into Cells The present invention describes methods to incorporate artificial chromosomes into the nuclei of bovine embryos.", "One of these methods, using microcell mediated chromosome transfer into nuclear transfer donor cells, has yielded bovine blastocysts where greater than 90% of the cells contain one ACes/cell.", "Another method where ACes were injected into enucleated oocytes prior to fusion with a nuclear transfer donor cell has produced blastocysts where up to 25% of the cells contain one ACes/cell.", "The potential advantage of injecting ACes into enucleated oocytes is that transgenic embryos could be generated more quickly since the microcell fusion and selection processes would be avoided.", "The combination of ACes and nuclear transfer technologies, as described herein, makes possible the generation of transgenic embryos containing one or more artificial chromosomes in a majority of cells.", "ACes are ideal chromosome vectors, they can be engineered with large payloads (Mb), isolated, delivered, and maintained as discrete non-integrating chomosomes for long-term stability in animals, and they eliminate the concerns of insertional mutagenesis.", "Another useful feature is that transgene expression from ACes can be evaluated in cell lines and mice prior to generating transgenic animals.", "Generation of β-ACes.", "The formation of the satellite DNA-based artificial chromosome has been described previously (Kereso, et al.", "1996).", "The murine ACes described therein contained approximately 60 million base pairs (Mb) of DNA and included centromere, telomeres, blocks of murine satellite repeats, and two regions of heterologous DNA, including five copies of the lacZ gene encoding β-galactosidase and six copies of the hygromycin phosphotransferase gene which conferred hygromycin resistance (“β-ACes”).", "Generation of Embryonic Germ (EG) Cells.", "Genital ridge cells from bovine fetuses of age 50-60 days were isolated as follows.", "The genital ridges from a fetus were minced in 2 ml of TL-HEPES (Bio Whittaker #04-616F) containing 3 mg/ml protease (Sigma #p6911) using sterile surgical blades.", "The minced genital ridges were incubated at 37° C. for 40-50 minutes, and then disaggregated by tituration with a 2 ml syringe and 25 gauge needle.", "The disaggregated genital ridge cell suspension was combined with 10 ml of TL-HEPES in a 15 ml sterile tube and centrifuged at 300×g for 10 min.", "After aspiration of the supernatant, the disaggregated cells were resuspended in 10 ml of α-MEM medium (Gibco #32571-037) plus 10% FBS (Hyclone #A-111-D), and 0.1 mM β-mercaptoethanol.", "The resuspended cells were divided evenly between ten culture flasks (75 cm2) containing mitotically inactivated fetal mouse feeder cells and culture with α-MEM medium (10 ml) supplemented with 25 ng/ml each of bovine basic fibroblast growth factor (bFGF) and human recombinant leukemia inhibitory factor (hrLIF).", "After 7-10 days in culture, the culture flasks had become confluent with small, densely packed cells referred to as embryonic germ (“EG”) cells.", "The EG cells were passaged into 25 culture dishes (5×106 cells/10 cm dish) containing 7 ml of α-MEM medium for the microcell fusion procedure.", "Microcell Transfer of β-ACes to EG Cells.", "Microcells carrying β-ACes were produced from the rodent/hybrid cell line mM2C1 and transferred to bovine EG cells as described before (Telenius et al., 1999) with minor modifications.", "142×106 metaphase cells were loaded on five 50% Percoll (Pharmacia) cushions, generating 660×106 microcells.", "540×106 microcells were fused to 1.25×108 recipient EG cells.", "Fusion was performed with 37% PEG 1450 (Sigma) for 3 minutes at 37° C., followed by drug-selection in 0.125 mg/ml hygromycin-B (Calbiochem) beginning 18-24 hours post-fusion.", "The duration of drug selection was 10-14 days.", "Hygromycin resistant cells were used for nuclear transfer 14-30 days following initiation of drug selection.", "Example 2 Nuclear Transfer Nuclear transfer of hygromycin-resistant EG cells was performed as described above (and in Strelchenko et al.", "2000).", "Enucleated oocyte-donor cell complexes were fused by electroporation, activated chemically, and cultured for 7-10 days.", "Isolation of ACes β-ACes were isolated and purified by flow cytometry according to de Jong et al.", "(1999) except for the modification of the sheath buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 100 mM NaCl, 30 μM spermine, and 70 μM spermidine) (J-buffer).", "Just prior to use, the β-ACes were concentrated by centrifugation at 2500×g for 15 min at 4° C. in a swinging bucket rotor.", "All but 50 μl of the supernatant was removed and the pellet was resuspended by gently tapping the tube.", "Microinjection of β-ACes into Cybrids Prior to Fusion Enucleated oocytes with the donor cell in position under the zona pellucida (enucleated oocyte-donor cell complexes) were placed in a 25 μl drop of TL-HEPES medium on a 10 cm petri dish.", "10-20 μl of concentrated β-ACes were placed in a separate drop of J-Buffer (25 μl).", "The drops were then covered with mineral oil.", "A single ACes was drawn into a microinjection needle (Humagen # MIC-10,7 μm O.D.)", "and the needle was pressed against the oocyte plasma membrane adjacent to the nuclear donor cell.", "With controlled negative pressure, the membrane was drawn into the needle until the oocyte membrane ruptured.", "The cytoplasm drawn into the needle by this process was gently expelled back into the oocyte with the β-ACes such that the single β-ACes were placed adjacent to the nuclear donor cell.", "The ACes-containing oocyte was then fused with the donor cell by electroporation using standard procedures (Strelchenko et al., 2000).", "The resulting embryos were activated and cultured for 7-10 days as described previously (Strelchenko et al., 2000).", "Microinjection of β-ACes into Cybrids Subsequent to Fusion After fusion of a nuclear donor cell with an enucleated oocyte, as described herein, the resulting cybrid is placed in a drop of TL-HEPES under mineral oil on a 10 cm petri dish.", "10 to 20 μL of concentrated β-ACes are placed in a separate drop of J-Buffer.", "A single β-ACes is drawn into a microinjection needle (Humagen # MIC-10,7 μm O.D.)", "and the needle is pressed against the cybrid plasma membrane, preferably near the donor cell nucleus.", "With controlled negative pressure, the cybrid membrane is drawn into the needle until the oocyte membrane ruptures.", "The cytoplasm drawn into the pipet by this process is gently expelled back into the oocyte with the β-ACes such that the single β-ACes is placed adjacent to the donor cell nucleus.", "Fluorescent In Situ Hybridization (FISH) Metaphase preps of 7 to 10 day old expanded blastocysts were prepared by growing them for 12 hours in medium containing 1 μg/ml colchicine.", "The blastocyst was placed on a slide in 30 μl of 2:1 dH2O:medium for 5 minutes.", "As much liquid as possible was removed and 30 μl of 0.01 M HCl/0.1% Tween-20 was added to the embryo until the zona pellucida dissolved and the cells started to disaggregate.", "A drop of cold fixative (3:1 methanol:acetic acid) was dropped onto the cells.", "The slide was allowed to dry and age at least 24 hours before proceeding with FISH.", "Extended embryo cultures were generated by culturing blastocysts in medium in 25 cm2 culture flasks and were prepared for FISH in the same manner as the blastocysts with the exception that a portion of the attached cultures were dislodged from the tissue flask using an 18 gauge needle and a 1 ml pipet.", "All general DNA manipulations were performed by standard procedures (See, e.g., Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press).", "Genomic DNA was prepared from bovine EG cells using the Wizard genomic kit (Promega).", "Primers 5′-ATCCAGACAGACAAGACAAGACAT-3′ and 5′-TTCCAGCGAGCGGCAAGGAC-3′ were used to amplify a 1.9 Kb fragment from bovine satellite 1.709 (Accession No.", "X00979; Skowronski et al., 1984).", "PCR amplification conditions were as follows: 25 cycles at 90° C. for 30 sec., 50° C. for 30 sec., 72° C. for 120 sec.", "The PCR product was purified and digested with EcoRI and subcloned into the plasmid vector Bluescript SK (Stratagene), generating plasmid pBSAT-1.709.A biotinylated bovine satellite 1.709 DNA probe was prepared from pBSAT-1.709 DNA using the Biotin-Nick Translation Mix (Boehringer Mannheim).", "Digoxigenin labeled mouse major satellite DNA probe was prepared from plasmid pSAT-2 (Wong and Rattner, 1988) using the DIG-Nick Translation Mix Boehringer Mannheim).", "FISH was performed as previously described (Pinkel et al., 1986).", "Bovine satellite probe does not cross hybridize to murine or hamster DNA sequences; the mouse major satellite probe does not cross hybridize with bovine DNA sequences.", "Microcell Transfer Two microcell transfer experiments resulted in successful generation of hygromycin-B resistant (hygR) EG cell colonies (>65).", "HygR-EG cells were used as nuclear donor cells in nuclear transfer and 33 blastocysts were generated from 136 cybrids (24%).", "Expanded blastocysts (21) were placed in extended embryo culture to generate a cell line.", "Two of these embryo cell lines were proliferated to a point where FISH analysis could be performed.", "One embryo cell line comprised 1 β-ACes/cell in greater than 90% of the cells while the other cell line comprised 2 β-ACes/cell in greater than 90% of the cells.", "The method of introducing β-ACes via microcell fusion has the advantage of producing blastocysts with little or no mosaicism.", "Since transgenic EG cells which have survived a similar selection regime as those described herein has produced live calves (Strelchenko et al., 2000), β-ACes-containing EG cells can be expected to be successful as nuclear donors for cloning bovines.", "Injection of ACes into Enucleated Oocyte-Donor Cell Complexes β-ACes were injected into 140 enucleated oocyte-donor cell complexes and the resulting enucleated oocyte-donor cell complexes were fused, activated and cultured as described elsewhere.", "β-ACes-injected cybrids developed at similar frequencies (39%) as the noninjected cybrids.", "Twenty-eight blastocysts were analyzed by FISH, of which 12 (43%) blastocysts contained β-ACes.", "Mosaicism in the transgenic blastocysts ranged from 1-25% (1 at 25%, 1 at 10%, 4 at 2%, and 6 at 1%).", "While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention.", "One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein.", "The cell lines, embryos, animals, and processes and methods for producing them are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.", "Modifications therein and other uses will occur to those skilled in the art.", "These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.", "It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.", "All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains.", "All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.", "The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.", "Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms.", "The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.", "Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.", "In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.", "For example, if X is described as selected from the group consisting of neomycin, hygromycin, and puromycin, claims for X being neomycin and claims for X being hygromycin and puromycin are fully described.", "Other embodiments are set forth within the following claims." ] ]	Patent_10468951
[ [ "Method and vehicle for pavement surface dressing", "According to the invention, the surface is dressed by synchronous spreading of binder and gravel on the pavement as follows: a) a first layer of hot bitumen (b1) having a temperature greater than 100° C. is spread on the pavement (S); b) a film of hot water (e) is spread on top of said first layer, c) a second layer of hot bitumen (b2) having a temperature greater than 100° C. is spread on top of said film of hot water; d) a layer of gravel e) is spread on top of all of the above.", "Pavement dressing." ], [ "1.Method for applying a surface coating by the synchronous spreading of bonding agent and chippings onto a roadway, according to which; a) a first layer of hot bitumen (b1), at a temperature above 100° C., is spread onto the roadway (S); b) onto this first layer is deposited a film of hot water (e), c) onto this film of hot water is spread a second layer of hot bitumen (b2), at a temperature above 100° C.; d) over the whole thing is spread a layer of chippings (c).", "2.Method according to claim 1, characterised in that bitumen taken to a temperature close to 130° C. and water taken to a temperature close to 100° C. are used.", "3.Method according to claim 1 or 2, characterised in that the chippings are spread less than two seconds and, preferably, less than one second after the second layer of bitumen is spread.", "4.Method according to one of claims 1 to 3, characterised in that the total spreading time is less than about four seconds.", "5.Vehicle for applying a surface coating by synchronous spreading of bonding agent and chippings onto a roadway, with the chassis (41) bearing a hot bitumen tank (6) and a tipper (7) containing chippings (c), this vehicle being fitted with a first boom (1) for dispensing hot bitumen (b1), which in placed in front—considering the forward operational movement (F) of the vehicle (4)—of a chipping spreader device (70), characterised in that the chassis (41) additionally bears a hot water tank (5), the vehicle being fitted with a second boom (3) for dispensing hot bitumen (b2), also placed in front of said chipping spreader device (70), and with a hot water (e) spray boom (2) placed between the two bitumen dispensing booms (1; 3)." ], [ "The present invention relates to a method and a machine for applying a surface coating by the synchronous spreading of bonding agent and chippings onto a roadway.", "By “synchronous spreading” will be understood in the present description, as well as in the claims which follow, a single action spreading, with the chippings being spread immediately, as quickly as possible, after the spreading of the bitumen, with the result that the chippings are laid into a hot bonding agent, the temperature of which is practically the same as that at which it is kept on the spreading machine.", "To spread synchronously, a lorry type vehicle is traditionally used with a chassis bearing a hot bitumen tank and a tipper containing the chippings, this vehicle being fitted with a boom for dispensing bitumen onto the roadway which is placed in front—considering the vehicle's forward operational movement—of a chipping spreader device.", "A vehicle of this kind, usually called a “bi-spreader”, is for example, marketed by the applicant company under the commercial name “CHIPSEALER”.", "The objective of the present invention is to propose a method for applying the surface coating which, while being simple to implement, makes it possible to obtain, on the one hand a good adhesion of the coating on the road surface, on the other hand improved cohesion of the coating, and in particular a particularly effective anchoring of the chippings within the layer of bonding agent.", "This objective is reached, in accordance with the invention, by operating in the following way a) a first layer of hot bitumen with a temperature above 100° C. in spread onto the roadway; b) onto this first layer is deposited a film of hot water; c) onto this film of hot water is spread a second layer of hot bitumen, with a temperature above 100° C.; d) over the whole thing is spread a layer or chippings.", "Furthermore according to a certain number of additional characteristics, which do not restrict the invention: bitumen taken to a temperature close to 130° C. and water taken to a temperature close to 100° C. are used; the chippings are spread less than two seconds and, preferably, leer than one second after the second layer of bitumen is spread; the total spreading time is less than about four seconds.", "The vehicle for applying a surface coating by synchronous spreading of bonding agent onto a roadway, which is also the subject of the present invention, comprises a chassis bearing a hot bitumen tank and a tipper containing chippings, this vehicle being fitted with a first boom for dispensing hot bitumen onto the roadway, which is placed in front—considering the vehicle's forward operational movement—of a chipping spreader device.", "This vehicle is remarkable in that the chassis additionally bears a hot water tank, the vehicle being fitted additionally with a second boom for dispensing hot bitumen, also placed in front of said chipping spreader device, and with a hot water spray boom placed between the two bitumen dispensing booms.", "Other characteristics and advantages of the invention will emerge from the description and from the appended drawings, in which FIGS.", "1 to 3 are diagrams illustrating the method of the invention, while FIG.", "4 is a diagrammatic side view of the vehicle used to implement the process.", "In FIG.", "1 has been shown diagrammatically the method of coating a roadway S, by synchronous spreading of bonding agent and chippings.", "In this figure two bonding agent dispensing booms have been denoted by the reference numbers 1 and 3.These two booms extend parallel to each other, transversally relative to the roadway.", "In a known way, each of the booms is composed of a plurality of spray nozzles located side-by-side in the transverse direction, each able to emit a conical jet, each jet covering the neighbouring jet in such a way as to ensure a homogeneous distribution of the bonding agent sprayed onto the roadway.", "The direction of forward movement of the spreading system is denoted by the arrow F. Between the two booms 1 and 3 is inserted a device for spraying hot water e on the roadway, with the reference number 2, and symbolised by a watering can.", "This device may also be constituted in practice by a set of juxtaposed nozzles which spray the water in the form of fine droplets according to a homogeneous distribution.", "To the rear of the spreading assembly 1-2-3, the chippings c are deposited by gravity, with one of them being shown as it falls.", "Booms 1 and 3 are adapted to spray onto the roadway hot bitumen b1 and b2 respectively; the temperature of the bitumen is preferably about 130° C. The temperature of the hot water is close to 100° C. The volume of water e deposited is very markedly smaller than the volumes of bitumen b, and b2.The quantities of bitumen b1 and b2 supplied by each of the booms 1 and 3 are advantageously the same or close to each other.", "These quantities are naturally dependent on the thickness of the coating layer it is desired to apply, a thickness to which the particle size distribution of the chippings used is also adapted.", "The bitumen b1 forms a first layer, the main function of which is to anchor the new layer to the surface S being treated; the bitumen b, will particularly fill in cracks or other irregularities in the surface of the roadway.", "The film of water e is covered, immediately after it has been sprayed onto the first layer, by the equally hot bitumen b2 of the second layer.", "Since the water is also hot, close to its boiling point, micro-bubbles of foaming water vapour can be instantly seen to form, causing a significant and quasi-instantaneous expansion of the mix.", "The layer of chippings c is therefore embedded into a foamy and expanded bonding agent, FIG.", "2A is a diagrammatic view from above showing a layer of chippings that has just been deposited into a standard bonding agent L. FIG.", "2B is a similar view showing the layer of chippings deposited in the expanded bitumen B applied in accordance with the invention.", "A comparison or these two figures reveals that the chippings are much more fully embedded in the second case.", "In practice, the expansion of the bitumen in the upper layer occurs very fast and continues to occur after the chippings c have been deposited, as is shown in FIGS.", "3A and 3B.", "In FIG.", "3A the position is shown just at the moment the chippings are deposited into the expanding bitumen, denoted b1; the height of the bitumen layer is denoted h1.The water caught in the two layers of bitumen is converted very rapidly into steam, continuing to expand and more exactly to expand the upper layer in the intervals separating the different chippings to reach a height h2 substantially greater than h1, such that the entire lower portion as well as the central part of each chipping is coated within the bonding agent.", "After the water has evaporated this height can be seen to go down; however the bitumen retains an extensive contact with the chippings by forming a meniscus helping them to get fully anchored into the layer of bitumen, even after this has cooled and the water which it contained has evaporated.", "A remarkable adhesion of the layer is thus obtained relative to the road surface S together with a firm anchoring of the chippings within this layer.", "The two bitumens b1 and b2 are not necessarily identical in composition or quality.", "To implement the method it is important for the different subsequent spreading operations, namely the spreading of bitumen b1, the spreading of the water and the spreading of the bitumen b2 and the spreading of the chippings c to be carried out one after the other in quick succession.", "Thus, the chippings are spread to advantage lose than 2 seconds after the spreading of the second bitumen layer and preferably less than one second after this spreading.", "The total spreading time, between the spreading of the first layer b1 and the spreading of the chippings c is preferably less than about 4 seconds; it is for example about 2 seconds.", "The vehicle 4 diagrammatically shown in FIG.", "4 allows the method that has just been described, to be implemented.", "It is a vehicle of the bi-spreader type, including a driver's cabin 40 and a chassis 41, the latter bearing in the front part a bitumen tank 6 and in the rear part an articulated tipper 7.The tank 6 is heat-insulated and contains a bitumen which is for example at a temperature of about 130° C. The tipper 7 contains chippings or loose stones.", "It is articulated on the chassis at the base of its rear part, and comprises chipping dispensing means 70, spreading being carried out in a conventional way, by using a flap 71 which allows the chippings to be distributed homogeneously as they fall onto the roadway S. Devices of this type are described for example in the documents FR-B-2 528 085, FR-A-1 200 883 and FR-A-2 538 014.The rear of the tipper is also fitted with a walkway 72, which, in a known way, is adapted to receive an operator.", "In accordance with the invention, the chassis 41 additionally supports a tank 5 intended to contain hot water.", "In the example shown, this tank is located between the cabin 40 and the bitumen tank 6.The tank 5 is also heat-insulated and allows the water it contains to be kept at a temperature close to 100° C. It is considered that this vehicle is adapted to work in the direction of its forward movement symbolised by the arrow F. Just in front of the flap 71 is placed an assembly of three booms 1, 2, 3.Booms 1 and 3 are adapted to dispense bitumen b1, b2 from the tank 6.This dispensing is carried out by appropriate means of known type.", "In FIG.", "4 has been shown a stop valve 60 located at the outlet of the tank 6, a dispensing pipe 61, a feed regulation and distribution valve 62, and a pair of secondary pipes 63, 64, which feed each of the booms 1, 3 respectively.", "By means of the feed regulation valve 62, the flow rates of the jets emitted by each of the booms 1, 3 can be varied if necessary.", "The hot water dispensing boom 2 is located between the two booms 1 and 3.It is fed from the tank 5 via a conduit 51, upstream of which is located a stop and feed regulation valve 50.Thus, depending on working conditions it is possible to adjust the flow rate of each valve individually.", "The loose stone flow rate may also be adjusted by appropriate means of known type with which the dispensing device 70 is fitted.", "It goes without saying that in the possible event of two bitumens of different qualities being used, the vehicle is fitted with two tanks 6 each containing one bitumen.", "In the event of a vehicle being used which operates in reverse motion, the booms 1-2-3 are placed to the rear of the chipping dispenser means 70, and their arrangement is reversed (boom 1 to the rear of booms 2 and 3).", "The normal working speed of such a vehicle is about 1 metre/second.", "By way of example, the bitumen is spread at a proportion of between 0.5 and 2.5 kg/m2; the water is proportioned at between 5 and 300 g/m2.The particle size distribution of the chippings has a value of between {fraction (2/4)} mm and {fraction (10/14)} mm." ] ]	Patent_10468960
[ [ "Electrohydraulic brake system and method for operating the same", "The invention relates to an electrohydraulic brake system in which, in a normal operating mode, a pedal cylinder (1) which is coupled to a brake pedal (16) can be acted on either by a high-pressure reservoir (12) or a low-pressure reservoir (13) via an electrovalve (4).", "Likewise, a brake cylinder (2) which is coupled to a brake device (17) can be coupled either to the high-pressure reservoir (12) or the low-pressure reservoir (13) via an electrovalve (7).", "If there is a fault, by switching over electrovalves (3, 5, 6), the system is changed over into a safety mode in which the pedal cylinder (1) and the brake cylinder (2) are coupled directly hydraulically.", "In order to ensure a defined volume of hydraulic fluid in the hydraulic path (13) here, an additional cylinder (8) is provided whose spring-loaded side which expands in the safety mode can carry off excess hydraulic fluid from the brake cylinder (2).", "In addition, hydraulic fluid can be removed from the low-pressure reservoir (13) via a nonreturn valve (11)." ], [ "1.An electrohydraulic brake system, containing a) a pedal cylinder (1) which is coupled to a brake pedal (16), b) a brake cylinder (2) which is coupled to brake devices (17), c) at least one valve (3, 6) which is preferably electrically activated and which, in a safety mode, can bring about a hydraulic path (13) with a direct connection between the pedal cylinder (1) and brake cylinder (2), wherein the brake system has a balancing device (8, 9, 10, 11) with which, in the safety mode, a defined volume of hydraulic fluid can be brought about in the hydraulic path (13).", "2.The electrohydraulic brake system as claimed in claim 1, wherein the balancing device contains an additional cylinder (8) with a spring-loaded piston, the working volume, which expands under spring effect, of the cylinder being capable of being coupled to the hydraulic path (13) in the safety mode by means of a preferably electrically activated valve (9).", "3.The brake system as claimed in claim 2, wherein, outside the safety mode, the working volume, which is compressed under spring effect, of the additional cylinder (8) can be coupled to a high-pressure reservoir (12) via a preferably electrically activated valve (10).", "4.The brake system as claimed in one of claims 1 to 3, wherein the hydraulic path (13) is coupled to a low-pressure reservoir (13) via a nonreturn valve (11).", "5.The brake system as claimed in one of claims 1 to 4, wherein the latter has a low-pressure reservoir (13) to which, in the safety mode, the respective second sides of the pedal cylinder (1), brake cylinder (2) and/or additional cylinder (8) are coupled.", "6.The brake system as claimed in one of claims 1 to 5, wherein the pedal cylinder (1) and/or the brake cylinder (2) is/are coupled to a preferably electrically activated valve (4, 7) via which, outside the safety mode, the cylinder volumes can be connected optionally to a low-pressure reservoir (13) or to a high-pressure reservoir (12).", "7.A method for operating an electrohydraulic brake system, in particular a brake system as claimed in one of claims 1 to 6, containing a) a pedal cylinder (1) which is coupled to a brake pedal (16), b) a brake cylinder (2) which is coupled to brake devices (17), in which case, at the changeover into a safety mode, a hydraulic path (13) is brought about with a direct connection between the pedal cylinder (1) and brake cylinder (2), wherein, in the safety mode, a defined volume of hydraulic fluid is brought about in the hydraulic path (13).", "8.The method as claimed in claim 7, wherein, at the changeover into the safety mode, hydraulic fluid is sucked out of the brake cylinder (2) until its piston has reached its home position.", "9.The method as claimed in claim 7 or 8, wherein hydraulic fluid is fed to the hydraulic path (13) if the pressure there drops below a predefined pressure.", "10.The method as claimed in one of claims 7 to 9, wherein, outside the safety mode, high pressure or low pressure is applied to the pedal cylinder (1) and/or brake cylinder (2) under the control of electrovalves (4, 7)." ], [ "The invention relates to an electrohydraulic brake system containing a pedal cylinder which is coupled to a brake pedal, a brake cylinder which is coupled to brake devices and at least one valve which, in a safety mode, can bring about a hydraulic path with a direct connection between the pedal cylinder and brake cylinder.", "In addition, the invention relates to a method for operating such a brake system.", "Electrohydraulic brake systems are known, for example, from WO 00/68053.They have a high-pressure reservoir and a low-pressure reservoir for a hydraulic fluid as well as electromagnetic valves (“servovalves”), the activation of which can cause the aforesaid reservoirs to be selectively coupled to a brake cylinder in order to generate a brake pressure in the brake devices (disk brakes, drum brakes or the like).", "The braking force can thus be set independently, of the pedal position of the brake pedal and does not have to be generated by means of the physical force of the driver.", "However, in order to give the driver a customary feedback sensation when the brake pedal is activated, systems have been developed with electrohydraulic activation of the pedal travel.", "In these systems, the brake pedal is coupled to a pedal cylinder which may be of one-sided or two-sided design and whose working volume or volumes can be coupled either to a high-pressure reservoir or a low-pressure reservoir by a controller via a further electromagnetic valve.", "Furthermore, it is known to equip electrohydraulic brake systems with a safety system which, when there is a fault—such as in particular failure of the pressure in the high-pressure reservoir—permits the brake to be activated.", "For this purpose, suitable switching of electromagnetic valves ensures that a hydraulic path containing two corresponding working volumes of pedal cylinder and brake cylinder is brought about so that a direct hydraulic active connection is produced between the brake pedal and the brake devices.", "However, the problem with these systems is that the changeover into the safety mode can take place at a time at which the pistons in the pedal cylinder and brake cylinder are in positions where they do not correspond so that there is too little or too much hydraulic fluid in the hydraulic path for normal hydraulic operation.", "If, for example, there is too much hydraulic fluid in the hydraulic path, the brake cylinder piston cannot assume its zero position.", "The safety system thus indeed guarantees braking at any time, but not the release of the brakes.", "Against this background, the object of the present invention has been to make available an electrohydraulic brake system and a method for operating it which, in the safety mode, ensures the widest possible functional range of the brake system in each case.", "This object is achieved by means of a brake system having the features of claim 1 and by means of a method having the features of claim 7.Advantageous refinements are contained in the subclaims.", "The electrohydraulic brake system according to the invention contains a “pedal cylinder” which is coupled to a brake pedal, and a “brake cylinder” which is coupled to brake devices.", "In addition, it contains at least one preferably electrically activated valve which can be activated in such a way that, in a safety mode, it can bring about a hydraulic path with a direct hydraulic connection between the pedal cylinder and brake cylinder.", "According to the definition, the hydraulic path includes the connecting line and the coupled working volumes of the pedal cylinder and brake cylinder.", "In the hydraulic path it is possible, in particular, for the working volume, which is compressed when the brake pedal is activated, of the pedal cylinder to be coupled to the working volume, which expands when the brake is applied, of the brake cylinder.", "The brake system is defined by the fact that it has a balancing device with which, in the safety mode, a defined volume of hydraulic fluid can be brought about in the hydraulic path.", "The fact that a defined volume of hydraulic fluid is made certain by the balancing device ensures that a reliable function with a broad working range is achieved between the brake pedal and brake devices when there is direct hydraulic coupling.", "In particular permanent blocking of the brakes cannot occur as a result of an excess of hydraulic fluid.", "According to one preferred refinement of the brake system, it has an additional cylinder with a spring-loaded piston, that working volume of the additional cylinder which expands under the effect of the spring being capable of being coupled to the hydraulic path by means of preferably electrically activated valve.", "Such coupling may take place, in particular, at the changeover into safety mode, after which the working volume, which expands under the effect of the spring, of the additional cylinder can take off excess hydraulic fluid from the hydraulic path.", "It is advantageous here that this withdrawal of hydraulic fluid is brought about as a result of the expansion of a spring and is thus independent of the hydraulic pressure which may have dropped during a changeover into the safety mode.", "According to one development of the refinement mentioned above, the working volume, which is compressed under the effect of the spring, of the additional cylinder can be coupled to a high-pressure reservoir via a preferably electrically activated valve.", "This coupling may be brought about here in particular outside the safety mode so that high pressure then prevails in the aforesaid working volume and causes the piston of the additional cylinder to be displaced with compression of the spring.", "In this way it is ensured that during a possible changeover into the safety mode the spring is in the compressed state and can therefore ensure the desired expansion of the spring-end volume.", "In one preferred refinement of the brake system, the hydraulic path is coupled via a nonreturn valve to a low-pressure reservoir with hydraulic fluid.", "In this way it is possible to bring about a situation in which not less than a predefined quantity of hydraulic fluid is located in the hydraulic path.", "Should this in fact be the case at the beginning, as a result at some time a situation is produced in which a lower pressure prevails in the hydraulic path than in the low-pressure reservoir.", "However, at this time, the nonreturn valve opens and thus brings about an inflow of hydraulic fluid to the required extent.", "Furthermore, the brake system may contain a low-pressure reservoir for hydraulic fluid, to which reservoir, in the safety mode, the respectively second sides of a two-sided pedal cylinder are coupled to a two-sided brake cylinder and/or a two-sided additional cylinder.", "Such coupling brings about defined force relationships and a system-wide equalization of volume of the hydraulic fluid.", "In addition, the pedal cylinder and/or the brake cylinder can be coupled to a (further) preferably electrically activated valve via which, outside the safety mode, the cylinder working volumes can either be connected to a low-pressure reservoir or a high-pressure reservoir.", "In this way, the position of the piston and movement of the piston of the cylinders can be controlled electrically in a desired way.", "The invention also relates to a method for operating an electrohydraulic brake system, the brake system being capable of is being configured in particular in the way explained above.", "The brake system contains a pedal cylinder which is coupled to a brake pedal and a brake cylinder which is coupled to brake devices, a hydraulic path with a direct connection between the pedal cylinder and brake cylinder being brought about at the changeover into a safety mode.", "The method is defined by the fact that in the safety mode a defined volume of hydraulic fluid is brought about in the hydraulic path of the pedal cylinder and brake cylinder.", "The result of this is that the brake system also has the widest possible working range in the safety mode.", "In particular in the method, hydraulic fluid can be sucked out of the brake cylinder at the changeover into the safety mode until the piston of said brake cylinder has reached its home position.", "This home position of the piston constitutes a defined state which is to be assumed when the brake devices are completely released.", "In addition, hydraulic fluid can be fed to the hydraulic path if the pressure in the hydraulic path drops below a predefined pressure.", "This provides an equalization of volume if there should be too little hydraulic fluid in the hydraulic path at the start of the safety mode as a result of the position of the pistons of the pedal cylinder and brake cylinder which happens to be present.", "Furthermore, it is preferred if, outside the safety mode, high pressure or low pressure is applied to the pedal cylinder and/or the brake cylinder controlled via preferably electrically activated valves.", "In this way, servofunctions of the brake system can be generated.", "The invention is explained in more detail below by way of example using the figures, in which: FIG.", "1 shows a brake system according to the invention, and FIGS.", "2a-d show various initial states of the pedal cylinder and brake cylinder at the changeover into a safety mode.", "FIG.", "1 is a schematic view of the circuit diagram of an electrohydraulic brake system according to the invention, the illustrated position of the valves corresponding to the normal operating mode in the “regulated mode”.", "The brake system contains a pedal cylinder 1 whose piston is coupled to a brake pedal 16.A spring force which forces the piston into a home position (inactivated brake pedal) acts on the piston.", "The pedal cylinder 1 is embodied as a double-acting cylinder whose two reciprocal working volumes can be coupled either to a high-pressure reservoir 12 or low-pressure reservoir 13 for hydraulic fluid via an associated electromagnetic servovalve or proportional valve 4 in accordance with the predefined values of a controller (not illustrated).", "In this way, a force can be exerted selectively on the piston of the pedal cylinder 1 and thus on the brake pedal 16 by activating the servovalve 4.Furthermore, the brake system contains a brake cylinder 2 which is coupled to a brake device 17 in order to generate a braking force at the wheels.", "The brake cylinder 2 is also of double-acting design with two reciprocal working volumes in the example illustrated, the one working volume being capable of being connected either to the high-pressure reservoir 12 or the low-pressure reservoir 13 via a servovalve 7 in accordance with the predefined values of a controller.", "The other working volume of the brake cylinder 2 is connected directly to the low-pressure reservoir 13 in the example illustrated.", "With a brake system of the design described above, it is possible to implement a normal electrohydraulic braking operating mode in which electrohydraulic activation of the pedal travel can take place by means of the pedal cylinder 1, and electrohydraulic activation of the brakes can take place by means of the brake cylinder 2.In order to ensure that the brakes are activated even in the event of a fault, such as for example when the high-pressure reservoir 12 fails, the brake system which is illustrated in FIG.", "1 can be changed into a so-called safety mode.", "In this safety mode, the electrovalves 3, 5 and 6 are changed into the respective other state (not illustrated) so that the servovalves 4 and 7 are disconnected from the pedal cylinder 1 and the brake cylinder 2.In the safety mode, a direct connection is brought about between the sides of the pedal cylinder 1 and brake cylinder 2 which face away from the piston rods, via a first electrovalve or solenoid valve 3 and a second electrovalve 6.The connection between the respective working volumes of the cylinders 1 and 2 including these volumes themselves is referred to below as “hydraulic path 13”.", "The side of the pedal cylinder 1 which faces the piston rods is coupled directly to the low-pressure reservoir 13 in the safety mode by the switching over of a third electrovalve 5, the side of the brake cylinder 2 which faces the piston rods also being coupled to said low-pressure reservoir 13.Between the two cylinders 1 and 2 there must be at least one assembly is which throttles the volume flow between the two cylinders.", "A direct hydraulic coupling between the two cylinders is brought about by means of the above-described hydraulic path 13 between the pedal cylinder 1 and brake cylinder 2, so that the brake device 17 can be activated by a pressure on the brake pedal 16.As the pedal cylinder 1 and the brake cylinder 2 operate independently of one another in the regulated mode, their pistons may be in various positions at the changeover into the safety mode (switching over of the valves 3, 5, 6).", "The most important possible cases are illustrated in FIGS.", "2a-2d.", "In FIG.", "2a, the piston of the pedal cylinder 1 is in the respective home position (no activation of the brake), as is the piston of the brake cylinder 2.On the other hand, in FIG.", "2b, the piston of the pedal cylinder 1 is at the respective stop (maximum activation of the brake), as is that of the brake cylinder 2.Both FIGS.", "2a, 2b thus show a hydraulically balanced system for the state of pressure release and pressure loading.", "In FIG.", "2c, the piston of the pedal cylinder 1 is at its stop (brake pedal depressed), and the piston of the brake cylinder 2 is in its home position (brake device loose).", "If the changeover into the safety mode takes place in such a case, there is too little hydraulic fluid in the hydraulic path 13.FIG.", "2d shows the other extreme in which the piston of the pedal cylinder 1 is in its home position (brake pedal released) and the piston of the brake cylinder 2 is in its stop position (brake devices applied), so that at the change-over into the safety mode there is an excess of hydraulic fluid in the hydraulic path.", "The cases illustrated in FIGS.", "2c and 2d delimit the function of the brake system in the safety mode as, when there is a lack of hydraulic fluid, sufficient activation of the brakes is no longer ensured, and when there is an excess of hydraulic fluid the brakes can no longer be released satisfactorily.", "In order to avoid the described problems, according to the invention a balancing device is proposed with which the volume of the hydraulic fluid in the hydraulic path 13 can be set to a desired value at the start of the safety mode.", "In order to achieve this, the system contains a double-acting additional cylinder 8 (cf.", "FIG.", "1) whose piston is prestressed by means of a spring.", "The balancing of that working volume which tends toward expansion (“spring space”) under the effect of the spring is switched by means of an electrovalve 9 in such a way that in the regulated mode (FIG.", "1) it is connected to the low-pressure reservoir 13, and in the safety mode (position of the valve 9 not illustrated in FIG.", "1) to the hydraulic path 13 at a point between the throttle and brake cylinder 2.The connection of the complementary working volume of the additional cylinder 8 is switched via a second electrovalve 10 in such a way that said second electrovalve 10 is connected to the high-pressure reservoir 12 in the regulated mode (FIG.", "1) and to the low-pressure reservoir 13 in the safety mode (position of the valve 10 not illustrated in FIG.", "1).", "Owing to the switching of the additional cylinder 8 described above, a pressure drop is brought about from the working volume facing away from the spring to the spring space in the regulated mode so that the piston of the additional cylinder 8 moves in this direction as far as its stop and remains there during the regulated mode.", "The spring is prestressed in this way.", "If the system changes over from the regulated mode into the safety mode, the pressure difference in the additional cylinder 8 changes the direction as a result of the switching over of the electrovalves 9 and 10.Owing to the effect of the spring in the additional cylinder 8, the spring space expands at the same time.", "If the piston of the brake cylinder 2 is not in its home position at the changeover into the safety mode—similarly to in FIG.", "2d—the volume which is expelled by it will flow off into the spring space of the additional cylinder 8 until the piston of the brake cylinder 2 has assumed its zero position or home position.", "Excess hydraulic volume is carried away in this way.", "The brake system also contains a nonreturn valve 11 which connects the hydraulic path 13 to the low-pressure reservoir 13 at a point between the throttle and brake cylinder 2, the through-flow direction of the nonreturn valve 11 running from the low-pressure reservoir 13 to the hydraulic path 13.The nonreturn valve 11 opens when the piston of the brake cylinder 2 has reached its home position, but the piston of the additional cylinder 8 is not yet at its stop.", "As a result of the opening of the nonreturn valve 11, the spring space of the additional cylinder 8 can then be filled with hydraulic fluid until the piston reaches its stop.", "The volume of the additional cylinder 8 which is expelled on the side facing away from the spring flows off into the low-pressure reservoir 13 here.", "If the piston of the pedal cylinder 1 is not at its home position stop (cf.", "FIG.", "2c) at this time, the nonreturn valve 11 will open at the first pressure release and remain open until the piston of the pedal cylinder 1 has also reached its zero position or home position.", "The brake system is then volumetrically balanced.", "In the case of a fault, the brake system according to the invention is changed over, by switching over the electro valves 3, 5 and 6, into a safety mode in which the pedal cylinder 1 and the brake cylinder 2 are coupled directly hydraulically.", "In order to ensure a defined volume of hydraulic fluid in the hydraulic path here, the additional cylinder 8 is provided, the spring-loaded side of which which expands in the safety mode can carry off excess hydraulic fluid from the brake cylinder 2.Furthermore, hydraulic fluid can be extracted from the low-pressure reservoir 13 via a nonreturn valve 11." ] ]	Patent_10468961
[ [ "Method for producing non-hydrated fexofenadine hydrochloride and a novel crystalline form obtained thereby", "The invention relates to non-hydrated fexofenadine hydrochloride which can be obtained from a fexofenadine base and hydrogen chloride, according to the reaction conditions, either in the form of a novel polymorph (“form A”), in an amorphous form, or in the form of a mixture of different polymorphs.", "Said novel polymorph (“form A”) can be used as a therapeutic active ingredient and can be processed to form a pharmaceutical containing the same and a pharmaceutically acceptable carrier.", "Said pharmaceutical is suitable for use as an antihistaminic agent, an antiallergic agent and/or a bronchodilating agent." ], [ "1-6.", "(canceled) 7.A method of making a non-hydrated fexofenadine hydrochloride in the form of a novel polymorph (“form A”) comprising the steps of: (a) suspending a fexofenadine base in a lower alkyl nitrile to form a suspension, (b) adding a solution of hydrogen chloride in one of a lower alkanol, a di(lower alkyl) ether or a lower alkyl ester of a lower alkanecarboxylic acid to the suspension of step (a) to form a mixture; (c) heating the mixture from step (b) to form a heated mixture; (d) cooling the heated mixture from step (c) to form a cooled mixture; and (e) isolating the non-hydrated fexofenadine hydrochloride in the form of the novel polymorph (“form A”) from the cooled mixture of step (d).", "8.The method of claim 7, wherein the lower alkyl nitrile is acetonitrile.", "9.The method of claim 7, wherein the lower alkanol is methanol.", "10.The method of claim 7, wherein the di(lower alkyl) ether is diethyl ether or diisopropyl ether.", "11.The method of claim 7, wherein the lower alkyl ester of a lower alkanecarboxylic acid is ethyl acetate.", "12.A non-hydrated fexofenadine hydrochloride in the form of a novel polymorph (“form A”), obtainable according to the method of claim 7, wherein the novel polymorph has the following XRD data: rel.", "intensity D/Å (I/Imax)/% 11.8 55 11.2 30 7.5 50 6.6 30 5.9 20 5.6 70 5.4 20 4.9 65 4.7 100 4.6 35 4.4 40 4.3 100 4.1 40 4.0 30 3.4 40 13.A drug composition comprising: a polymorph non-hydrated fexofenadine hydrochloride (“form A”) according to claim 7; and a pharmaceutically acceptable excipient.", "14.A method of using a non-hydrated fexofenadine hydrochloride in the form of a novel polymorph (“form A”), comprising administering the novel polymorph to a patient in need thereof.", "15.A method of making a non-hydrated fexofenadine hydrochloride in an amorphous form comprising the steps of: (a) suspending a fexofenadine base in a lower alkane, a di(lower alkyl) ether or a lower alkyl ester of a lower alkanecarboxylic acid to form a suspension; (b) adding to the suspension from step (a) a solution of hydrogen chloride in one of a lower alkanol, a di(lower alkyl) ether or a lower alkyl ester of a lower alkanecarboxylic acid to form a mixture; (c) heating the mixture from step (b) to form a heated mixture; (d) cooling the heated mixture from step (c) to form a cooled mixture; and (e) isolating the amorphous form of the non-hydrated fexofenadine hydrochloride from the cooled mixture of step (d).", "16.The method of claim 15, wherein the lower alkane is n-hexane or n-heptane.", "17.The method of claim 15, wherein the di(lower alkyl) ether is diethyl ether or diisopropyl ether.", "18.The method of claim 15, wherein the lower alkyl ester of a lower alkanecarboxylic acid is ethyl acetate.", "19.The method of claim 15 wherein the lower alkanol is methanol.", "20.A method of making a non-hydrated fexofenadine hydrochloride in a mixture of different polymorphs comprising the steps of: (a) suspending a fexofenadine base in a lower alkyl nitrile to form a suspension; (b) passing a hydrogen chloride gas into the suspension to form a mixture; (c) heating the mixture from step (b) to form a heated mixture; (d) cooling the heated mixture from step (c) to form a cooled mixture; and (e) isolating the mixture of different polymorphs from the cooled mixture of step (d).", "21.The method of claim 20 wherein the lower alkyl nitrile is acetonitrile." ], [ "The present invention relates to non-hydrated fexofenadine hydrochloride.", "Fexofenadine hydrochloride (4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]butyl]-α,α-dimethylphenylacetic acid hydrochloride) has formula (I) below: and is licensed by the US Food and Drug Administration (FDA) as an antihistamine, antiallergic and bronchodilator under the trade name Allegra®.", "WO-A-95/31437 describes the preparation of hydrated polymorphs, or pseudo-morphs, of fexofenadine hydrochloride (form II and form IV) and their conversion to non-hydrated polymorphous forms (form I and form III) by azeotropic distillation or water-minimizing recrystallization.", "WO-A-00/71124 describes amorphous, presumably non-hydrated fexofenadine hydrochloride and its preparation, a spray drying or freeze drying being carried out as the final stage.", "It has now been found that non-hydrated fexofenadine hydrochloride can be prepared from fexofenadine base and hydrogen chloride in a simple and direct manner, without the need for operations such as azeotropic distillation, water-minimizing recrystallization, spray drying or freeze drying, to give the non-hydrated fexofenadine hydrochloride in the form of a novel polymorph (“form A”) or in amorphous form or in the form of a mixture of different polymorphs, depending on the reaction conditions, wherein (a) fexofenadine base is suspended in a lower alkyl nitrile, a solution of hydrogen chloride in a lower alkanol, in a di(lower alkyl) ether or in a lower alkyl ester of a lower alkanecarboxylic acid is added, and the mixture is heated and then cooled, after which the non-hydrated fexofenadine hydrochloride is isolated in the form of the novel polymorph (“form A”), or (b) fexofenadine base is suspended in a lower alkane, in a di(lower alkyl) ether or in a lower alkyl ester of a lower alkanecarboxylic acid, a solution of hydrogen chloride in a lower alkanol, in a di(lower alkyl) ether or in a lower alkyl ester of a lower alkanecarboxylic acid is added, and the mixture is heated and then cooled, after which the non-hydrated fexofenadine hydrochloride is isolated in amorphous form, or (c) fexofenadine base is suspended in a lower alkyl nitrile, hydrogen chloride gas is passed into the suspension, and the mixture is heated and then cooled, after which the non-hydrated fexofenadine hydrochloride is isolated in the form of a mixture of different polymorphs.", "The compounds and radicals referred to above as “lower” appropriately contain up to eight carbon atoms.", "It is preferable to use acetonitrile as the lower alkyl nitrile, methanol as the lower alkanol, diethyl ether or diisopropyl ether as the di(lower alkyl) ether, ethyl acetate as the lower alkyl ester of a lower alkanecarboxylic acid, and n-hexane or n-heptane as the lower alkane.", "Fexofenadine base (II) is obtainable in known manner from the hydrochloride of the corresponding keto ester, namely ethyl 4-[1-oxo-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]butyl]-α,α-dimethylphenylacetate (III).", "The polymorph of fexofenadine hydrochloride obtainable according to variant (a) of the method according to the invention (“form A”) has a melting range of 153 to 156° C. (DSC) and is characterized by the following XRD data (Table 1): TABLE 1 XRD data of fexofenadine hydrochloride, form A (d = lattice spacing; the relative intensities were taken from the powder diagram obtained using CuK radiation.)", "rel.", "intensity d/Å (I/Imax)/% 11.8 55 11.2 30 7.5 50 6.6 30 5.9 20 5.6 70 5.4 20 4.9 65 4.7 100 4.6 35 4.4 40 4.3 100 4.1 40 4.0 30 3.4 40 This polymorph is novel and also forms a subject of the present invention.", "It can be used as a therapeutic active ingredient and processed to a drug containing the active ingredient and a pharmaceutically acceptable excipient.", "This drug is suitable as an antihistamine, antiallergic and/or bronchodilator.", "Pharmaceutically acceptable excipients which can be used in the preparation of drugs are generally known and familiar to all those skilled in the art.", "By virtue of their different bioavailabilities, rates of release and solubilities, different forms of a pharmaceutical active ingredient, such as novel polymorphs in particular, can be of great benefit to the patients in question since they may allow a lowering of the dosage and/or a lengthening of the dosage intervals, making it possible to reduce the costs of the medication.", "The Examples which follow will illustrate the invention without in any way limiting its scope.", "EXAMPLES The XRD spectra were recorded on a Philips ADP1700 computer-controlled powder diffractometer system with automatic divergence slit and secondary monochromator (graphite).", "The CuKα radiation (λ (CuKα1)=0.15406 nm and λ (CuKα2)=0.15444 nm) from a copper tube (40 kV, 30 mA) was used and the spectra were recorded with Δ(2ΘN)=0.02 for a counting time of 3 s in the range 1.5°≦2Θ≦40°.", "The differential scanning calorimetry (DSC) measurements were made on a METTLER DSC 821e apparatus with a start temperature of 25° C., an end temperature of 250° C. and a heating rate of 10 K min−1.Standard aluminium crucibles with perforated lids were used as the sample vessels.", "The amount of sample was about 5 mg in each case.", "Example 1 Synthesis of Fexofenadine Base 30 g of piperidine derivative III, 1.7 g of sodium borohydride and 7.4 g of sodium hydroxide were suspended in 200 g of ethanol and 44 g of water, refluxed for 3-5 h and then quenched with 10 g of acetone.", "The solvents were stripped off under vacuum and the residue was taken up in 200 g of water/acetone (2:1).", "The pH was adjusted to 5.8 to 6.0 by the addition of acetic acid, causing the fexofenadine base to crystallize out.", "The precipitate was filtered off, washed with water and dried under vacuum at 60° C. to give 22 g (83%) of product.", "Example 2 Synthesis of Fexofenadine Hydrochloride, Form A 86 g of fexofenadine base were suspended in acetonitrile (700 g), and 30 g of a 20.6 percent solution of hydrogen chloride in diisopropyl ether were added at −10 to −12° C. The reaction mixture was heated at the reflux temperature for 1 h and then cooled.", "The hydrochloride was isolated by filtration, washed with acetonitrile and dried under vacuum at 100° C. to give 83 g (90%) of fexofenadine hydrochloride, form A.", "Example 3 Synthesis of Fexofenadine Hydrochloride, Form A 10.0 g of fexofenadine base were suspended in acetonitrile (76 g), and 1.9 g of a 38.6 percent solution of hydrogen chloride in methanol were added at −10 to −12° C. The reaction mixture was heated at the reflux temperature for 1 h and then cooled.", "The hydrochloride was isolated by filtration, washed with acetonitrile and dried under vacuum at 100° C. to give 10.1 g (94%) of fexofenadine hydrochloride, form A.", "Example 4 Synthesis of fexofenadine Hydrochloride, Form A 10.0 g of fexofenadine base were suspended in acetonitrile (76 g), and 3.7 g of a 19.5 percent solution of hydrogen chloride in ethyl acetate were added at −10 to −12° C. The reaction mixture was heated at the reflux temperature for 1 h and then cooled.", "The hydrochloride was isolated by filtration, washed with acetonitrile and dried under vacuum at 100° C. to give 9.8 g (91%) of fexofenadine hydrochloride, form A.", "Example 5 Synthesis of Amorphous Fexofenadine Hydrochloride 10.0 g of fexofenadine base were suspended in n-heptane (90 g), and 4.02 g of a 17.9 percent solution of hydrogen chloride in diisopropyl ether were added at −10 to −12° C. The reaction mixture was heated at the reflux temperature for 1 h and then cooled.", "The hydrochloride was isolated by filtration, washed with n-heptane and dried under vacuum at 100° C. to give 9.7 g (90%) of amorphous fexofenadine hydrochloride.", "Example 6 Synthesis of Amorphous Fexofenadine Hydrochloride 10.0 g of fexofenadine base were suspended in tert-butyl methyl ether (90 g), and 4.0 g of a 17.9 percent solution of hydrogen chloride in diisopropyl ether were added at −10 to −12° C. The reaction mixture was heated at the reflux temperature for 1 h and then cooled.", "The hydrochloride was isolated by filtration, washed with tert-butyl methyl ether and dried under vacuum at 100° C. to give 10.5 g (98%) of amorphous fexofenadine hydrochloride.", "Example 7 Synthesis of Fexofenadine Hydrochloride in the Form of a Mixture of Different Polymorphs 5.1 g of fexofenadine base were suspended in acetonitrile (39 g).", "0.4 g of hydrogen chloride was passed into the suspension at −10 to −12° C., after which the mixture was heated at the reflux temperature for 1.5 h and then cooled.", "The hydrochloride was isolated by filtration, washed with acetonitrile and dried under vacuum at 00° C. to give 5.1 g (92%) of fexofenadine hydrochloride in the form of a mixture of different polymorphs." ] ]	Patent_10468964
[ [ "Resin impregnated filter media", "The present invention discloses a filter medium and a method of manufacturing a filter medium.", "The filter medium is created using a fabric substrate of a woven or non woven material.", "The substrate is impregnated with a thermosetting resin that is subsequently heat cured thereby encapsulating the fibers of the substrate.", "Further a rheology modifier may be mixed with the resin before impregnation to control the flow properties of the resin." ], [ "1.A process of manufacturing a filter medium, comprising: impregnating a substrate with thermosetting resin said substrate being formed from intertwined yarns; and subsequently curing the thermosetting resin by heating the impregnated substrate to the curing temperature of the resin used, to thereby encapsulate the yarns of the substrate with the resin so that the yarns are mutually bonded where they contact each other.", "2.A process according to claim 1 said resin is mixed with a rheology modifier before application to control the viscosity and flow properties of the resin.", "3.A process according to claim 2 wherein the rheology modifier comprises below 5% of the resin composition by weight.", "4.A process according to claim 2 wherein the rheology modifier comprises hydroxyl-ethyl cellulose.", "5.A process according to claim 2 wherein the resin is diluted to below 15% solids content before addition of the rheology modifier.", "6.A process according to claim 2 wherein the thermosetting resin is selected from the group consisting of: phenolic, epoxy, formaldehyde, amino furan, melamine, silicone, unsaturated polyether, polyurethane, polyamide, fluorocarbon based thermosetting resin, cross linked thermoplastic based thermosetting resin and mixtures thereof.", "7.A process according to claim 1 wherein the substrate is selected from the group consisting of: woven fabric, non-woven fabric, spun bonded yarns, thermo-bonded yarns, and melt blown polymeric yarns.", "8.A process according to claim 7 wherein the polymeric yarns are a blend of two or more materials selected from the group consisting of: polyester, polypropylene, polyamide, polyethylene, and polyurethane.", "9.A process according to claim 7 wherein the substrate includes a metal or ceramic mesh screen.", "10.A process according to claim 1 wherein the coated fabric is subjected to an intermediate heating, before curing to a temperature below the curing temperature to remove water or other solvent.", "11.A filter medium comprising a substrate of a fabric impregnated with a thermosetting resin cured by subsequent heating of the impregnated substrate to the curing temperature of the resin.", "12.A filter medium according to claim 11 wherein said resin contains a rheology modifier to control the viscosity and flow properties of the resin.", "13.A filter medium according to claim 12 wherein the rheology modifier comprises below 5% of the resin composition by weight.", "14.A filter medium according to claim 12 wherein the rheology modifier comprises hydroxyl-ethyl cellulose.", "15.A filter medium according to claim 12 wherein the resin is diluted to below 15% solids content before addition of the rheology modifier.", "16.A filter medium according to of claim 11 wherein the thermosetting resin is selected from the group consisting of: phenolic, epoxy, formaldehyde, amino furan, melamine, silicone, unsaturated polyether, polyurethane, polyamide, fluorocarbon based thermosetting resin, cross linked thermoplastic based thermosetting resin and mixtures thereof.", "17.A filter medium according to claim 11 wherein the substrate is selected from the group consisting of: woven fabric, non-woven fabric, spun bonded yarns, thermo-bonded yarns, and melt blown polymeric yarns.", "18.A filter medium according to claim 17 wherein the polymeric yarns are a blend of two or more materials selected from the group consisting of: polyester, polypropylene, polyamide, polyethylene, and polyurethane.", "19.A filter medium according to claim 17 wherein the substrate includes a metal or ceramic mesh screen." ], [ "This invention relates to improved filter media of the kind comprising a woven or non-woven substrate.", "EP-A-0,741,815 discloses a method of making a fabric which is suitable for use as a filter medium by applying a film of a reticular polymer to a suitable substrate from a release sheet.", "The resultant filter medium provides a micro porous filter, which has a good wear resistance so long as the film endures.", "When the film is abraded however, the substrate becomes vulnerable to wear and the filter medium loses its micro-filtration capabilities.", "In order to improve the effective lifetime of filter media, it is desirable to improve the wear resistance of the filter cloths, belts sleeves or the like, and if possible to reduce the rate of deterioration of filtration capacity which tends to occur where a relatively coarse base substrate is surface treated or partially impregnated from one side or the other.", "There is a general tendency for coatings, however applied, to sit on the substrate as a discrete layer, or to penetrate only a comparatively short distance into the substrate, so that when the coated substrate is subjected to abrasion, the filter medium is impaired quickly once the coating has been worn, even if only locally.", "Japanese Patent Application No.", "06301439, published under No.", "08131735, discloses filter medium comprising a blend of two types of fibres, filter fibres and heat fusible fibres.", "The fibres are joined together by partial fusion of the heat fusible fibres as the filter medium is heated and shaped.", "Reinforcing materials are attached to the Intersections of the two types of fibres, and the reinforcement material may be for example a water-soluble phenol, an epoxy resin, unsaturated polyester or a polyamide.", "This type of filter medium is intended for example for cylindrical cartridge filters or the like.", "It is not clear how the reinforcement materials are introduced nor how they are caused to adhere only at the intersections of the two types of fibre used.", "The use of a non-woven substrate comprising a blend of two different fibre tpes raises production costs, as the fibres first have to be blended.", "Also the presence of a fusible component in the fibre blend restricts the utility of the filter medium to lower temperature uses, since high temperatures could cause further fusion of the fusible fibres, leading to blinding of the filter pores by melted fibre material.", "It is an object of the invention to provide a filter medium material and a process for manufacture of a filter medium material with improved wear resistance, and greater dimensional stability and which does not substantially suffer from the disadvantages noted.", "From a first aspect, the invention provides a process for manufacturing a filter medium, comprising impregnating a woven or non-woven subsrate with a thermosetting resin combined with subsequent curing of the resin by heating the impregnated substrate to a curing temperature appropriate for the resin used, whereby the yarns or fibres of the substrate are encapsulated by the resin so that the yarns or fibres are mutually bonded where they cross each other.", "In a preferred process according to the invention, the resin is mixed with a rheology modifier before application to control the viscosity and flow properties of the resin, and may be added to the resin system which is preferably diluted to below 15% solids content, for example 10% solids content, in an amount of below 5% by weight of the rheology modifier for example 3% by weight.", "An example of a rheology modifier, which may be used is hydroxy ethyl cellulose.", "From a second aspect, the invention provides a filter medium, comprising a substrate of woven or non-woven fabric impregnated with a thermosetting resin cured by subsequent heating of the impregnated substrate to a curing temperature appropriate for the resin used, the yarns or fibres of the substrate being encapsulated by the resin so that the yarns or fibres are mutually bonded where they cross each other.", "The thermosetting resin may be any one or a mixture of any two or more selected from:—a phenolic, epoxy, formaldehyde, amino/furan, melamine, silicone, unsaturated polyester, polyurethane, polyamide, fluorocarbon or cross linked thermoplastic based thermosetting resin systems.", "Further, other known additives can be added to the resin to enhance supplementary propeties.", "Examples may include—carbon (to give conductive or anti-static qualities), grafting binders which attach PTFE onto the chain terminator of phenol end groups (to improve cake release), or antibacterial agents such as 3-trimethoxysilyl, poly-dimethyloctadecyl ammonium chloride, and silane quaternary salts (to minimise crystal or fungi build up).", "The thermosetting resin preferably penetrates the substrate, to a depth, which will give the desired balance of dimensional stability and flexibility and effectively encapsulates or coats the fibres or yarns within the impregnated part of the fabric without clogging the void spaces between the woven or felted yarns or fibres.", "The void spaces may then be used if required to hold micro porous materials such as flocculated, coagulated, foamed or other micro porous materials.", "The substrate may be a woven or non woven felt, spun bonded, thermo bonded, or melt blown include polymeric yarns or fibres, and these may be anyone or a blend of two or more of polyester (e.g.", "PET) or polypropylene or other materials, such as polyamide (nylon) or polyethylene or polyurethane.", "The substrate may also include a metal or ceramic mesh or screen.", "The resin may be applied as a liquid, by known means such as knife-coating, spraying or lick coating or by immersion of the base fabric in the coating material.", "The exposure of the fabric to the resin liquid is preferably sufficiently prolonged to ensure the desired depth penetration of the liquid into the base fabric.", "The resin bearing liquid may be an aqueous or non-aqueous dispersion, or emulsion, or the like of the resin in the liquid phase.", "Before curing, the coated fabric is optionally subjected to an intermediate heating, below the curing temperature to drive off moisture or other solvent, and to effect fibre encapsulatlon that is to ensure that the resin adheres to and ‘wets’ the fibres or yarns, without substantially impeding the voids of the fabric structure.", "Curing of the resin is then effected, with typical curing times from 5 minutes to one hour or more, and typical curing temperatures from 100° C. to 200° C. Intermediate heating and curing may be effected during a single pass, or the steps may be carried out separately and not necessarily immediately subsequently.", "Examples of the method according to the invention, and of the filter media produced thereby will now be particularly described by way of example.", "EXAMPLE I A base fabric comprising a needled non-woven felt of PET (polyethylene terephthalate) fibres was lick coated with an aqueous phenolic thermosettable resin.", "The felt was then heated for 10 to 20 minutes to close to 100° C. In order to dry the felt by evaporating the aqueous phase and soften the resin to cause it to wet the fibers effectively, thereby encapsulating the fibres in the resin.", "Following completion of the drying step the coated felt was then heated to 106° C. and maintained at this temperature for 10 minutes, thereby effecting curing of the thermosetting resin.", "Tests showed that the resultant filter medium has only slightly impaired flexibility and air permeability as compared to the untreated felt further, dimensional stability and resistance to abrasion was significantly improved.", "EXAMPLE II A base fabric comprising a woven polypropylene cloth was coated with an aqueous phenolic thermosetting resin.", "The resin was applied using a knife coating mettod.", "The coated cloth was then heated to about 130° C. and maintained at this temperaure for 1 hour, thereby ensuring that the resin wets the fibres effectively, causing encapsulation of the fibres with the resin and also effecting subsequent curing of the resin.", "Again the resulting filter medium had improved dimensional stability and abrasion resistance, and only slightly impaired flexibility and air permeability as compared to the untreated fabric.", "The Method is also applicable to woven fabrics to coat the yarns or fibres comprising the fabric.", "FIG.", "1 illustrates in a considerably magnified view what is believed happens to the structure of the impregnated region of a filter medium produced from a non-woven felt as described by Example I.", "FIG.", "2 is a scanning electron micrograph of a coated polyester needle felt as prepared by the Example I as set out above; FIG.", "3 is a scanning electron micrgraph of uncoated polypropylene cloth before treatment in accordance to the Example II set out above; FIG.", "4 is a scanning electron micrograph of coated woven multi filament polypropylene cloth in accordance with Example II set out above; FIG.", "5 is a magnified view of two adjacent coated fibres within a multi filament yarn and meniscus of the binding resin; FIG.", "6 is micrograph of a cross section through a coated multifilament yarn within a coated woven polypropylene cloth; FIG.", "7 is a graph showing the effect on permeability and filter throughput of a coating according to the invention; FIG.", "8 is a graph showing the improvement in dimensional stability of a polypropylene belt material after treatment by coating according to the invention; and FIG.", "9 is a graph showing the abrasion resistance of a phenolic resin treated woven polypropylene compared to an untreated substrate.", "The impregnated fabric resulting from Example 1 as shown in FIG.", "1 consists of an array of randomly orientated fibres 20, some of which are shown in sectional view.", "The coated fibres each comprise a core 20 consisting of the fibre itself and a coating or sheath 22 of the thermosetting resin produced by wetting of the fibres by the resin during the drying stage of the method described in the examples.", "Where the fibres 20 cross and contact each other, the resin forms bridges or pools 24, which connect the fibres firmly to each other.", "Once the resin has been cured the bonding remains permanent.", "FIG.", "2 shows in a scanning election micrograph, part of a coated polyester needled felt produced by method 1 set out above.", "As shown, the coating 22 tends to collect in pools 24 about the intermeshing zones of the fibres 20, with thickend sheath parts extending along the fibres.", "FIG.", "3 shows uncoated yarns in a polypropylene cloth, which have substantially no medium between yarns to give dimensional stability and to prevent abrasion, which is already evidenced by the presence of fibrils 25 on the yarn surfaces.", "FIG.", "4 shows by contrast shows such yarns after coating, and it will be noted that fibrillation is less marked, and that bridges of resin, e.g.", "26, connect the yarns and inhibit them from moving against each other, increasing stability and hereby reducing abrasion.", "The meniscus of resin between yarns is show enlarged in FIG.", "5.FIG.", "6 shows how resin penetrates between the fibres of a multiflament yarn, reducing internal wear within the yarn.", "FIG.", "7 compares, the filtrate through put (a measure of permeability) of filter cloths which have and which not been impregnated with a phenolic resin by the method of the invention.", "It is noted that the decrease in permeability is marginal.", "FIG.", "8 similarly compares the dimensional stability of treated and untreated polypropylene belt material, when tested by tensioning in the direction of the weft yarns.", "The treated belt (full line) shows greater dimensional stability than the untreated belt, demonstrated in that a greater force is necessary to produce the same “stretch”.", "For example, when the applied stress is 20 kgf/2.5 cm, then the treated belt shows a strain (or elongation) of approximately 7% compared to greater than 12% for the untreated belt.", "Finally FIG.", "9 shows the result of comparative tests of a treated and an untreated woven polypropylene substrate.", "The untreated material showed a marked increase in permeability over 2000 abrasion cycles, signifying a loss of micro filtration, but the treated material exhibited no noticeable deterioration.", "The bonding thus produced increases the dimensional stability hence the propensity of the yarns to be displaced and to rub against each other is reduced lessening internal wear and ultimately yarn breakage within the fabric.", "A similar effect is present in woven base fabrics where the yarns are coated and pools bridges are formed within the weave knuckles or in the case of multi-filament or staple yarns, where parallel fibres contact each other.", "A further benefit is that the resin bridges or pools lock the fibres, and the openings between fibres, relative to one another thus providing more accurate and consistent pore sizes.", "The pore sizes will remain constant throughout the life of the filter media.", "The properties of filter media according to and produced by the method of the invention can be controlled by varying conditions and materials.", "The speed and degree of penetration of the resin into the substrate can be controlled by varying the viscosity of the coating.", "The solids content of the coating may be changed to vary the thickness of the film encapsulating the yarns or fibres.", "The properties of the resin used for coating are preferably controlled depending upon the properties of the substrate by dilution and rheology modifiers (which modify the flow properties of the resin).", "In an example a first sample of a 373 gm-2 nylon 66 monofilament duplex weave cloth was subjected to application of a neat (unmodified) resin comprising 60% solids, 40% solven and having a viscosity of 200 cPs.", "This was found to have an excessive coat weight and low permeability, due to the low surface area monofilament yarns being encapsulated in a thick layer of resin.", "A second sample of the cloth was subjected to application of resin diluted to 10% solids, of lower viscosity (about 20cPs).", "This resin bled through the entire cloth thickness and produced an insufficient coat weight, with the yarns being incompletely encapsulated.", "A third sample of the cloth was subjected to the application of a specially prepared resin mixture.", "This resin sample was prepared by dilution of the resin to 10% solids and then thickening by addition of hydroxy-ethyl cellulose as a rheology modifier (i.e to modify the flow properties of the resin).", "The hydroxyl ethyl cellulose rheology modifier had been prepared previously in accordance to manufactures guidelines and was added slowly to the dilute resin with vigorous stirring to give 3% by weight of rheology modifier in the resin mixture.", "The viscosity of this mixture as applied to the third cloth sample was 4500 cPs.", "The coated fabric was cured in the usual manner in a single pass through an oven at 160° C., with dwell time 10 minutes.", "With use of a rheology modifier and dilution of the resin it was found that the resin system had an optimum solids content for increased abrasion resistance and optimum viscosity for substrate penetration, with minimal loss of substrate flexiblity and permeability.", "Sample 1 Sample 2 Sample 3 substrate 373 gm-2 nylon 66 as sample 1 as sample 1 monofilament duplex weave resin 60% solids 10% solids 10% solids rheology nil Nil hydroxy ethyl modifier cellulose 3% viscosity 200 cPs 20 cPs 4500 cPs permeability low High good encapsulation thick layer coat poor coat bled good weight excessive through fabric encapsulation leaving without exces- insufficient resin sive thickness Drying conditions can determine the way or extent to which the fibres are encapsulated with shorter drying times so that, the coating will migrate less.", "Advantages of the invention for example in relation to EPA 0,741,815 mentioned above include the fact that a single process can provide a coating throughout a variable depth within the substrate, and possibly the whole substrate matrix by if necessary completely soaking the fabric with the liquid phase resin.", "Using the method of the European Patent, if the abrasion resistance of both sides of the fabric are to be improved, the resin has to be applied separately to each side in a repeated process.", "Experimental results show as noted above that the process of the invention does not significantly change the filtrate throughput of the filter medium.", "Air and liquid permeability is only slightly reduced.", "Further the method of the invention can be used with low surface energy materials such as polypropylene due to encapsulation of individual fibres.", "The coating applied by the method known from the said European Patent can be easily peeled off such surface, as it does not adhere to them.", "Because of the depth of penetration, the coating is permanent, with a residue of coating throughout the full depth of the coating and the cloth will continue to resist abrasion during the cloth lifetime.", "The void spaces between the yarns or fibres of the base cloth are not substantially impeded by the coating, as evidenced by the air permeability, and these can be filled to render the filter medium micro porous by coagulated, flocculated, or micro porous foamed materials as known in the art.", "The filter media of the invention may be used In liquid or gas any filtration application as cloths for drum, cartridge, and disc filter sleeves and bags or other filters also conveyor belts, pulp dewatering belts, corrugators belts, tower press belts and filter press cloths." ] ]	Patent_10468969
[ [ "Present invention relates to a vehicle seat support mechanism", "A vehicle seat having a seat cushion and a seat back which are movably connected to one another by a vehicle seat support mechanism and which are also movably mounted to a support floor by said vehicle seat support mechanism, said vehicle seat being movable between a normal in-use position and a stowed position, said slowed position being located rearwards from said normal in-use position and said seat back, when said vehicle seat is in its stowed position, being located at a relatively lower height than when said vehicle seat is in said normal in-use position.", "When the vehicle seat is moved to its stowed position, a seat assembly can be converted from a 3-seater to a 2-seater condition for instance." ], [ "1.A vehicle seat having a seat cushion and a seat back which are movably connected to one another by a vehicle seat support mechanism and which are also movably mounted to a support floor by said vehicle seat support mechanism, said vehicle seat being movable between a normal in-use position and a stowed position, said stowed position being located rearwards from said normal in-use position and said seat back, when said vehicle seat is in its stowed position, being located at a relatively lower height than when said vehicle seat is in said normal in-use position.", "2.A vehicle seat as claimed in claim 1 wherein during movement of said vehicle seat between said normal in-use position and said stowed position, said seat cushion is movable between a normal use position and a stowed position respectively, the seat cushion in said normal use position being located generally horizontal and in said stowed position being located in a generally upright orientation.", "3.A vehicle seat as claimed in claim 2 wherein on movement of said seat cushion between its normal use and stowed positions, said vehicle seat support mechanism is operable to simultaneously move said seat back between an in-use upright position and a stowed upright position.", "4.A vehicle seat as claimed in claim 3 wherein said in-use upright position and said stowed upright position of said seat back have the same general vertical orientation.", "5.A vehicle seat as claimed in claim 1 wherein said vehicle seat support mechanism includes a support mount adapted to be fixedly mounted on said support floor, and further includes first and second links on each said of said seat that pivotally connect said seat back to said mount, said seat back being fixedly connected to one of said links on each side of said seat such that said seat cushion is pivotally connected to said mount by said one link.", "6.A seat assembly for a vehicle comprising at least two side by side seats, a first of said seats being movable to a stowed position and a second of said seats being movable rearwardly, and inwardly, of the vehicle body when said first seat is located in said stowed position.", "7.A seat assembly as claimed in claim 6, wherein said first seat having a seat cushion and a seat back which are movably connected to one another by a vehicle seat support mechanism and which are also movably mounted to a support floor by said vehicle seat support mechanism, said vehicle seat being movable between a normal in-use position and a stowed position, said stowed position being located rearwards from said normal in-use position and said seat back, when said vehicle seat is in its stowed position, being located at a relatively lower height than when said vehicle seat is in said normal in-use position.", "8-9.", "(canceled) 10.A seat assembly as claimed in claim 7, wherein during movement of said vehicle seat between said normal in-use position and said stowed position, said seat cushion is movable between a normal use position and a stowed position respectively, the seat cushion in said normal use position being located generally horizontal and in said stowed position being located in a generally upright orientation.", "11.A vehicle seat as claimed in claim 10, wherein on movement of said seat cushion between its normal use and stowed positions, said vehicle seat support mechanism is operable to simultaneously move said seat back between an in-use upright position and a stowed upright position.", "12.A vehicle seat as claimed in claim 11, wherein said in-use upright position and said stowed upright position of said seat back have the same general vertical orientation.", "13.A vehicle seat comprising: a seat cushion, a seat back, and a means for connecting the seat cushion and the seat back to allow the vehicle seat to be movable between an in-use position and a stowed position.", "14.A method for moving an automobile seat having a seat cushion with an in-use position and a stowed position and a seat back having a stowed position and an in-use position, the method comprising: moving the seat cushion of the automobile seat from its in-use position to its stowed position at a time; and moving the seat back of the automobile seat from its in-use position to its stowed position at the time, wherein the seat back in its stowed position is at a higher relative height than the seat cushion in its stowed position.", "15.The method of claim 14, wherein moving the seat cushion to the stowed position causes the entire seat back to move rearwardly.", "16.The method of claim 14, wherein moving the seat back from its in-use position to its stowed position comprises moving the entire seat back rearward.", "17.The method of claim 14, wherein the stowed position of the seatback is at a lower height than the in-use position of the seat back.", "18.The method of claim 14, further comprising moving a second vehicle seat, that is side-by-side with the automobile seat, rearwardly or inwardly of the vehicle body when the seat cushion is located in the stowed position.", "19.The method of claim 14, wherein moving the seat back from its in-use position to its stowed position comprises pivoting the seat back using first and second links connected to a base; and moving the seat cushion from its in-use position to its stowed position comprises pivoting the seat cushion using the first link.", "20.A method for moving an automobile seat having a seat cushion with an in-use position and a stowed position and a seat back having a stowed position and an in-use position, the method comprising: moving the seat cushion of the automobile seat from its in-use position to its stowed position at a time, moving the seat back from its in-use position to its stowed position comprising pivoting the seat back using first and second links connected to a base; and moving the seat back of the automobile seat from its in-use position to its stowed position at the time, moving the seat cushion from its in-use position to its stowed position comprising pivoting the seat cushion using the first link.", "21.A vehicle seat comprising: a seat back having a first position and a second position; a seat cushion having a first position and a second position; and a mechanism configured to simultaneously move the seat back from its first position to its second position and move the seat cushion from its first position to its second position, the mechanism comprising a first link coupled to the seat cushion, the seat back, and a base, and a second link coupled to the seat back and the base.", "22.The vehicle seat of claim 21, wherein the first position of the seat back is an in-use position and the second position of the seat back is a stowed position; and the first position of the seat cushion is an in-use position and the second position of the seat cushion is a stowed position.", "23.The vehicle seat of claim 21, wherein the second position of the seat back is an in-use position and the first position of the seat back is a stowed position; and the second position of the seat cushion is an in-use position and the first position of the seat cushion is a stowed position.", "24.The vehicle seat of claim 23, wherein the mechanism is configured to simultaneously move the seat back from its second position to its first position and move the seat cushion from its second position to its first position.", "25.An automobile seat comprising: a seat back having a first position and a second position; a seat cushion having a first position and a second position; and a mechanism, joining the seat back and the seat cushion, configured to simultaneously move the seat back from an in-use position to a stowed position and move the seat cushion from an in-use position to a stowed position; wherein the seat back in its stowed position is at a higher relative height than the seat cushion in its stowed position.", "26.The method of claim 25, wherein moving the seat back from its in-use position to its stowed position comprises moving the entire seat back rearward.", "27.An automobile comprising: an automobile seat having; a seat back having a first position and a second position; a seat cushion having a first position and a second position; and a mechanism, joining the seat back and the seat cushion, configured to simultaneously move the seat back from an in-use position to a stowed position and move the seat cushion from an in-use position to a stowed position; and a shelf, the seat back being beneath the shelf when in a stowed position and not beneath the shelf when in an in-use position.", "28.The automobile of claim 27, further comprising a second automobile seat mounted in a side-by-side relationship with the automobile seat.", "29.The automobile of claim 28, wherein the second automobile seat is adapted to move inwardly and rearwardly with respect to the vehicle interior while the automobile seat is in a stowed position." ], [ "The present invention relates to a vehicle seat support mechanism.", "In particular, but not exclusively, one aspect of the present invention relates to a vehicle seat having a seat cushion and a seat back which are movably connected to another by said vehicle seat support mechanism and which are also both movably mounted to a supporting floor by said mechanism.", "Preferably the mechanism is arranged to enable the seat cushion to be moved between a normal use position and a stowed position, the seat cushion in its normal use position being located generally horizontally to enable an occupant of the vehicle to sit thereon and in its stowed position being located in an upright orientation.", "Preferably on movement of the seat cushion between its normal use and stowed positions, the mechanism is operable to simultaneously move the seat back between an in-use upright position to a stowed upright position.", "Preferably the in-use upright position and the stowed upright position of the seat back have the same general vertical orientation.", "Preferably the seat back, when in its stowed position is located at a lower height than when in its in-use upright position.", "Preferably the mechanism includes a support mount adapted to be fixedly mounted on a support floor, such as the vehicle floor, and on each side of the seat, a first link for pivotally connecting the seat back to the mount and a second link for pivotally connecting the seat back to the mount, the seat cushion being fixedly connected to one of said links such that said seat cushion is pivotally connected to said mount by said one link.", "According to another aspect of the invention, there is provided a seat assembly for a vehicle, the seat assembly including at least two side by side seats, a first of said seats being movable to a stowed position and a second of said seats being movable rearwardly and inwardly of the vehicle body when said first seat is located in its stowed position.", "Various aspects of the present invention are hereinafter described with reference to the accompanying drawings, in which: FIG.", "1 is a side view of a vehicle seat frame according to an embodiment of the invention, showing its seat cushion in its normal use position; FIG.", "2 is a perspective view of the seat frame shown in FIG.", "1; FIG.", "3 is a side view of the vehicle seat frame of FIG.", "1 showing the seat cushion in its stowed position; FIG.", "4 is a perspective view of the vehicle seat shown In FIG.", "3; FIG.", "5 is a side view of the vehicle seat of FIG.", "1 showing the seat cushion at a mid-position between the positions shown in FIGS.", "1 and 3; FIG.", "6 is a perspective view of a seat assembly according to an embodiment of the present invention shown in its 3-seater condition; FIG.", "7 is a plan view of the seat assembly as shown in FIG.", "5 FIG.", "8 is a perspective view of the seat assembly of FIG.", "5 shown in its 2-seater condition; and FIG.", "9 is a plan view of the seat assembly as shown in FIG.", "7.Referring initially to FIG.", "1, there is shown a vehicle seat frame 10 having a seat cushion frame 11 and a seat back frame 12.The seat cushion frame 11 and seat back frame 12 are movably connected to one another by a seat support mechanism 16.The mechanism 16 includes a mounting bracket 18 which is normally fixedly attached to the floor (not shown) of the vehicle.", "The bracket 18 may be detachably fixed to the vehicle floor to enable the entire seat 10 to be removed.", "The bracket 18 may be directly fixed to the vehicle floor or may be indirectly fixed to the vehicle floor via a subframe.", "On each side of the seat frame 10 there is provided a linkage assembly 22 which serves to pivotally connect the seat cushion frame 11 to the bracket 18 and movably connect the seat back frame 12 to the bracket 18.As illustrated in FIG.", "1, the linkage assembly 22 includes first and second linkage frames 23, 24 respectively which, on each side of the seat, are connected to the seat back frame 12 and bracket by first, second, third and fourth pivotal connections 26, 27, 28 and 29 respectively.", "Linkage frame 23 includes a pair of link arms 31, 32 which are fixedly connected at one end to a cross-bar 33 and are each pivotally connected to the bracket 18 at their other end by pivotal connection 26.Preferably a shaft 17 is provided which extends between link arms 31, 32 to be rotatably received in bracket 18 and thereby define pivotal connection 26 on each side of the seat 10.The cross-bar 33 is fixedly provided with a pair of link arm extensions 34, 35 which are each pivotally connected to the seat back frame 12 by pivotal connection 27.Each link arm 31, 32 is fixedly secured to the remainder of the seat cushion frame 11 so that, in effect, the seat cushion frame 11 is pivotally connected to bracket 18 via pivotal connections 26.Linkage frame 24 includes a pair of link arms 36, 37 which are one end are fixedly secured to a cross-bar 38 and are each pivotally connected to the bracket 18 at their other end by pivotal connection 29.The cross-bar 38 is rotatably received at each end in the seat back frame 12 to thereby define, on each side of the seat, pivotal connection 28.The linkage frames 23, 24 are arranged so as to abut against one another and thereby define limits pivotal movement of the frames 23, 24 about pivotal connections 26, 29 in both the clockwise and anti-clockwise directions.", "The limit of pivotal movement in the anti-clockwise direction is shown in FIG.", "1 (this defines the normal in-use position of the seat 10) and the limit of pivotal movement in the clockwise direction is shown in FIG.", "3 (this defines the stowed position of the seat 10).", "Preferably the abutment of the frames 23, 24 in the in-use position of the seat is achieved by cross-bar 38 engaging into a recess 40 formed on each link arm extension 34, 35 and the abutment of cross-bar 33 into a recess 42 formed on each link arm 36, 37.Preferably a reinforcement strut 43 extends between arms 36, 37 in the region of recesses 42.Preferably, as shown in FIG.", "1, the geometric shape defined by pivotal connections 26, 27, 28 and 29 is basically rhomboid with the first and second pivotal connections 26, 27 being located higher than respective pivotal connections 28 and 29.This enables the seat back frame 12 to remain in generally the same vertical orientation at its normal in-use and stowed positions.", "In the stowed position, the seat 10 is located at a more rearwards position relative to the front of the vehicle and the seat back frame 12 is also located at a lower height.", "Thus, if the seat 10 is located at the rear of a vehicle having a parcel shelf extending across the rear of the seat when in its normal in-use position, it is possible by pivotally deflecting the seat cushion from 11 (in the clockwise direction) to move the seat 10 rearwardly and downwardly to its stowed position which, conveniently may be located beneath the parcel shelf.", "The seat 10 of the present invention may conveniently be incorporated in a seat assembly 100, as shown in FIGS.", "6 to 9, which enables the seat assembly 100 to be converted between 2-seater and 3-seater conditions.", "As shown in FIGS.", "6 and 7, the seat 10 is located in its normal in-use position.", "The seat 10 has a seat cushion 111 carried by its seat cushion frame 11 and a seat back 112 carried by its seat back frame 12.On either side of the seat 10 there is provided an outboard seat 150.Each seat 150 includes a seat cushion 151 having a pair of holsters 152 located on either side of a control seat zone 153.The seat cushion 111 preferably is provided with no holsters and has a width which is less than the width of its seat back 112.Thus, when the seat 10 is moved to its stowed position, outer marginal regions of the seat back 112 define shoulders 155 (see FIGS.", "6, 7).", "The seats 10, 150 as shown in FIGS.", "6 and 7 are located at a forwardmost position.", "To convert the seat assembly 100 from a 3-seater condition to a 2-seater condition, the seat 10 is first moved to its stowed position.", "This is illustrated in FIGS.", "8 and 9.In reaching this position, seat 10 has moved rearwardly and downwardly.", "Seats 150 are each movably mounted, preferably on rails 160, so as to be movable to a rearmost and innermost position relative to the vehicle body.", "This position is also shown in FIGS.", "6 and 7.Preferably in the rearmost-innermost position of seats 150, the seats 150 are located slightly forward of seat 10 with their seat backs 154 overlapping shoulders 155.This enables seats 150 to positively retain seat 10 in its stowed position.", "When the seats 150 are located in their rearmost-innermost position, they provide the occupants of those seats with more legroom and also more shoulder room." ] ]	Patent_10468972
[ [ "Image pickup apparatus and method", "The present invention relates to an image capturing apparatus and method in which the image recording time is reduced and the memory capacity required for compression is also reduced.", "A number-of-bytes calculation unit 302 determines the number of bytes after compression based on an integrated value of high-frequency integrated data supplied from a high-frequency integration processor.", "Based on the determined number of bytes, a Q-scale calculation unit 303 determines a Q-scale based on which the image data can be compressed one time to a predetermined data size.", "A Q-table generation unit 304 generates a Q-table based on the Q-scale.", "A DCT unit 321 performs a discrete cosine transform on the input image data.", "A quantization processor 322 adjusts the compression ratio of the image data based on the up-to-date Q-table supplied from the Q-table generation unit 304.", "A variable-length coding processor 323 encodes the image data with variable length coding such as Huffman coding, and outputs the resulting compressed image data.", "The present invention is applicable to digital cameras." ], [ "1.An image capturing apparatus having a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data, said image capturing apparatus comprising: high-frequency integration means for integrating a high-frequency component of the obtained image data in the monitoring mode; and compression processing means for compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration of the high-frequency integration means, wherein the high-frequency integration means comprises: extraction means for extracting the high-frequency component of the image data; absolute-value determination means for determining an absolute value of the extracted high-frequency component of the image data; and absolute-value integration means for integrating the absolute value of the high-frequency component of the image data determined by the absolute-value determination means; and the compression processing means comprises: number-of-compressed-bytes calculation means for determining the number of compressed bytes of the photographic image data to be recorded based on the integrated value obtained by integration of the high-frequency integration means; quantization-scale calculation means for determining, based on the number of compressed bytes determined by the number-of-compressed-bytes calculation means, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; quantization-table generation means for generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by the quantization-scale calculation means; and compression means for compressing the photographic image data based on the quantization table generated by the quantization-table generation means.", "2.An image capturing apparatus according to claim 1, wherein the compression processing means further compresses thumbnail image data corresponding to a thumbnail image obtained by reducing the size of a photographic image corresponding to the photographic image data; based on the integrated value obtained by integration of the high-frequency integration means, the number-of-compressed-bytes calculation means further determines the number of compressed bytes of the thumbnail image data to be recorded; based on this number of compressed bytes determined by the number-of-compressed-bytes calculation means, the quantization-scale calculation means further determines a quantization scale based on which the thumbnail image data is compressed one time to a predetermined number of bytes; based on the quantization scale determined by the quantization-scale calculation means, the quantization-table generation means further generates a quantization table for use in compression of the thumbnail image data; and based on the quantization table generated by the quantization-table generation means, the compression means further compresses the thumbnail image data.", "3.An image capturing apparatus according to claim 1, wherein the number-of-compressed-bytes calculation means determines the number of compressed bytes so as to increase as the integrated value of the high-frequency integration means is higher; and the quantization-scale calculation means determines the quantization scale so as to provide a higher compression ratio as the number of bytes when the photographic image data is compressed is greater.", "4.An image capturing apparatus according to claim 1, wherein the high-frequency integration means integrates the high-frequency component of the photographic image data processed by predetermined image signal processing.", "5.An image capturing method for an image capturing apparatus having a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data, said image capturing method comprising: a high-frequency integration step of integrating a high-frequency component of the obtained image data in the monitoring mode; and a compression processing step of compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration performed in the high-frequency integration step, wherein the high-frequency integration step includes: an extraction step of extracting the high-frequency component of the image data; an absolute-value determination step of determining an absolute value of the extracted high-frequency component of the image data; and an absolute-value integration step of integrating the absolute value of the high-frequency component of the image data determined by performing the absolute-value determination step; and the compression processing step includes: a number-of-compressed-bytes calculation step of determining the number of compressed bytes of the photographic image data to be recorded based on the integrated value obtained by integration performed in the high-frequency integration step; a quantization-scale calculation step of determining, based on the number of compressed bytes determined by performing the number-of-compressed-bytes calculation step, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; a quantization-table generation step of generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by performing the quantization-scale calculation step; and a compression step of compressing the photographic image data based on the quantization table generated by performing the quantization-table generation step.", "6.A recording medium having a computer-readable program for an image capturing apparatus recorded therein, said image capturing apparatus having a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data, the program comprising: a high-frequency integration step of integrating a high-frequency component of the obtained image data in the monitoring mode; and a compression processing step of compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration performed in the high-frequency integration step, wherein the high-frequency integration step includes: an extraction step of extracting the high-frequency component of the image data; an absolute-value determination step of determining an absolute value of the extracted high-frequency component of the image data; and an absolute-value integration step of integrating the absolute value of the high-frequency component of the image data determined by performing the absolute-value determination step; and the compression processing step includes: a number-of-compressed-bytes calculation step of determining the number of compressed bytes of the recorded photographic image data based on the integrated value obtained by integration performed in the high-frequency integration step; a quantization-scale calculation step of determining, based on the number of compressed bytes determined by performing the number-of-compressed-bytes calculation step, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; a quantization-table generation step of generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by performing the quantization-scale calculation step; and a compression step of compressing the photographic image data based on the quantization table generated by performing the quantization-table generation step.", "7.A computer-executable program for controlling an image capturing apparatus having a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data, the program comprising: a high-frequency integration step of integrating a high-frequency component of the obtained image data in the monitoring mode; and a compression processing step of compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration performed in the high-frequency integration step, wherein the high-frequency integration step includes: an extraction step of extracting the high-frequency component of the image data; an absolute-value determination step of determining an absolute value of the extracted high-frequency component of the image data; and an absolute-value integration step of integrating the absolute value of the high-frequency component of the image data determined by performing the absolute-value determination step; and the compression processing step includes: a number-of-compressed-bytes calculation step of determining the number of compressed bytes of the photographic image data to be recorded based on the integrated value obtained by integration performed in the high-frequency integration step; a quantization-scale calculation step of determining, based on the number of compressed bytes determined by performing the number-of-compressed-bytes calculation step, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; a quantization-table generation step of generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by performing the quantization-scale calculation step; and a compression step of compressing the photographic image data based on the quantization table generated by performing the quantization-table generation step." ], [ "<SOH> BACKGROUND ART <EOH>In digital still cameras and portable information terminal devices having a digital still camera function, such as video cameras and PDAs, image data corresponding to captured photographic images is recorded as digital data.", "However, since the image data has a large data size and requires a large memory capacity for recording, typically, the image data is compressed according to, for example, a JPEG (Joint Photographic Experts Group) method or the like before it is recorded.", "FIG.", "1 is a block diagram showing an example structure of a JPEG compression unit of the related art for compressing image data according to a JPEG method.", "In FIG.", "1 , image data converted to an image format suitable for compression is input to a JPEG compression unit 1 through an input terminal 11 , and is supplied to a DCT (Discrete Cosine Transform) unit 12 .", "The DCT unit 12 performs a discrete cosine transform on the supplied image data to convert the image data from the time-domain component to the frequency-domain component, and supplies the resulting data to a quantization processor 13 .", "The quantization processor 13 adjusts the compression ratio of the image data based on a Q-table formed of a table of quantized coefficients supplied from a fixed-length Q-(quantized coefficient) table generation unit 17 , and supplies the image data to a variable-length coding processor 14 .", "The variable-length coding processor 14 encodes the image data with variable length coding such as Huffman coding, and the resulting compressed image data is output from an output terminal 19 .", "The variable-length coding processor 14 is also connected with a number-of-bytes calculation unit 15 , and the compressed image data output from the variable-length coding processor 14 is also supplied to the number-of-bytes calculation unit 15 .", "The number-of-bytes calculation unit 15 determines the number of bytes of the compressed image data corresponding to one screen, and supplies the result to a Q-scale calculation unit 16 .", "The Q-scale calculation unit 16 calculates the deviation between the input number of bytes and an expected number of bytes after compressing the photographic image data to determine the amount of adjustment of the compression ratio (Q-scale), and supplies the determined Q-scale to the fixed-length Q-table generation unit 17 .", "The Q-table generation unit 17 generates a new Q-table based on the Q-scale supplied from the Q-scale calculation unit 16 and a predetermined Q-table supplied from a Q-table unit 18 , and supplies the generated Q-table to the quantization processor 13 .", "The quantization processor 13 again adjusts the compression ratio of the image data based on the new Q-table supplied from the Q-table generation unit 17 .", "By repeating the foregoing compression process, the JPEG compression unit 1 can compress the input image data to the predetermined data size.", "However, the above-described method causes a reduction in image quality more than necessary due to block noise or mosquito noise if the compression ratio is set high so that any type of image data can be compressed one time to a predetermined data size.", "Therefore, a problem is that, typically, the compression process must be performed, for example, two or three times in order to compress various types of image data using appropriate compression ratios, thus extending the time required for compression of image data.", "Another problem is that the above-described method requires a memory for storing the original image data to be compressed since the compression process must be repeated, thus increasing the memory capacity required for compression." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 is a block diagram showing an example structure of a JPEG compression unit of the related art for compressing image data according to a JPEG method.", "FIG.", "2 is a block diagram showing an example of the basic structure of an image capturing apparatus according to the present invention.", "FIG.", "3 is a block diagram showing an example structure of internal components of a high-frequency integration processor shown in FIG.", "2 .", "FIG.", "4 is a block diagram showing an example structure of internal components of a JPEG compression processor shown in FIG.", "2 .", "FIG.", "5 is a flowchart illustrating a high-frequency integration process.", "FIG.", "6 is a flowchart illustrating a JPEG compression process.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to an image capturing apparatus and method, and particularly to an image capturing apparatus and method in which image data is compressed one time to a predetermined data size in a still-image recording mode using a compression ratio determined in a monitoring mode in advance based on a high-frequency integrated value of a monitored image signal, thereby reducing the image recording time and also reducing the memory capacity required for compression.", "BACKGROUND ART In digital still cameras and portable information terminal devices having a digital still camera function, such as video cameras and PDAs, image data corresponding to captured photographic images is recorded as digital data.", "However, since the image data has a large data size and requires a large memory capacity for recording, typically, the image data is compressed according to, for example, a JPEG (Joint Photographic Experts Group) method or the like before it is recorded.", "FIG.", "1 is a block diagram showing an example structure of a JPEG compression unit of the related art for compressing image data according to a JPEG method.", "In FIG.", "1, image data converted to an image format suitable for compression is input to a JPEG compression unit 1 through an input terminal 11, and is supplied to a DCT (Discrete Cosine Transform) unit 12.The DCT unit 12 performs a discrete cosine transform on the supplied image data to convert the image data from the time-domain component to the frequency-domain component, and supplies the resulting data to a quantization processor 13.The quantization processor 13 adjusts the compression ratio of the image data based on a Q-table formed of a table of quantized coefficients supplied from a fixed-length Q-(quantized coefficient) table generation unit 17, and supplies the image data to a variable-length coding processor 14.The variable-length coding processor 14 encodes the image data with variable length coding such as Huffman coding, and the resulting compressed image data is output from an output terminal 19.The variable-length coding processor 14 is also connected with a number-of-bytes calculation unit 15, and the compressed image data output from the variable-length coding processor 14 is also supplied to the number-of-bytes calculation unit 15.The number-of-bytes calculation unit 15 determines the number of bytes of the compressed image data corresponding to one screen, and supplies the result to a Q-scale calculation unit 16.The Q-scale calculation unit 16 calculates the deviation between the input number of bytes and an expected number of bytes after compressing the photographic image data to determine the amount of adjustment of the compression ratio (Q-scale), and supplies the determined Q-scale to the fixed-length Q-table generation unit 17.The Q-table generation unit 17 generates a new Q-table based on the Q-scale supplied from the Q-scale calculation unit 16 and a predetermined Q-table supplied from a Q-table unit 18, and supplies the generated Q-table to the quantization processor 13.The quantization processor 13 again adjusts the compression ratio of the image data based on the new Q-table supplied from the Q-table generation unit 17.By repeating the foregoing compression process, the JPEG compression unit 1 can compress the input image data to the predetermined data size.", "However, the above-described method causes a reduction in image quality more than necessary due to block noise or mosquito noise if the compression ratio is set high so that any type of image data can be compressed one time to a predetermined data size.", "Therefore, a problem is that, typically, the compression process must be performed, for example, two or three times in order to compress various types of image data using appropriate compression ratios, thus extending the time required for compression of image data.", "Another problem is that the above-described method requires a memory for storing the original image data to be compressed since the compression process must be repeated, thus increasing the memory capacity required for compression.", "DISCLOSURE OF INVENTION The present invention has been made in view of such situations, and is intended to reduce the image recording time and also reduce the memory capacity required for compression.", "An image capturing apparatus of the present invention has a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data.", "The image capturing apparatus includes high-frequency integration means for integrating a high-frequency component of the obtained image data in the monitoring mode; and compression processing means for compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration of the high-frequency integration means.", "The high-frequency integration means includes extraction means for extracting the high-frequency component of the image data; absolute-value determination means for determining an absolute value of the extracted high-frequency component of the image data; and absolute-value integration means for integrating the absolute value of the high-frequency component of the image data determined by the absolute-value determination means.", "The compression processing means includes number-of-compressed-bytes calculation means for determining the number of compressed bytes of the photographic image data to be recorded based on the integrated value obtained by integration of the high-frequency integration means; quantization-scale calculation means for determining, based on the number of compressed bytes determined by the number-of-compressed-bytes calculation means, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; quantization-table generation means for generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by the quantization-scale calculation means; and compression means for compressing the photographic image data based on the quantization table generated by the quantization-table generation means.", "The compression processing means can further compress thumbnail image data corresponding to a thumbnail image obtained by reducing the size of a photographic image corresponding to the photographic image data.", "Based on the integrated value obtained by integration of the high-frequency integration means, the number-of-compressed-bytes calculation means can further determine the number of compressed bytes of the thumbnail image data to be recorded.", "Based on this number of compressed bytes determined by the number-of-compressed-bytes calculation means, the quantization-scale calculation means can further determine a quantization scale based on which the thumbnail image data is compressed one time to a predetermined number of bytes.", "Based on the quantization scale determined by the quantization-scale calculation means, the quantization-table generation means can further generate a quantization table for use in compression of the thumbnail image data.", "Based on the quantization table generated by the quantization-table generation means, the compression means can further compress the thumbnail image data.", "The number-of-compressed-bytes calculation means can determine the number of compressed bytes so as to increase as the integrated value of the high-frequency integration means is higher, and the quantization-scale calculation means can determine the quantization scale so as to provide a higher compression ratio as the number of bytes when the photographic image data is compressed is greater.", "The high-frequency integration means can integrate the high-frequency component of the photographic image data processed by predetermined image signal processing.", "An image capturing method of the present invention has a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data.", "The image capturing method includes a high-frequency integration step of integrating a high-frequency component of the obtained image data in the monitoring mode; and a compression processing step of compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration performed in the high-frequency integration step.", "The high-frequency integration step includes an extraction step of extracting the high-frequency component of the image data; an absolute-value determination step of determining an absolute value of the extracted high-frequency component of the image data; and an absolute-value integration step of integrating the absolute value of the high-frequency component of the image data determined by performing the absolute-value determination step.", "The compression processing step includes a number-of-compressed-bytes calculation step of determining the number of compressed bytes of the recorded photographic image data based on the integrated value obtained by integration performed in the high-frequency integration step; a quantization-scale calculation step of determining, based on the number of compressed bytes determined by performing the number-of-compressed-bytes calculation step, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; a quantization-table generation step of generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by performing the quantization-scale calculation step; and a compression step of compressing the photographic image data based on the quantization table generated by performing the quantization-table generation step.", "A program of a recording medium of the present invention has a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data.", "The program includes a high-frequency integration step of integrating a high-frequency component of the obtained image data in the monitoring mode; and a compression processing step of compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration performed in the high-frequency integration step.", "The high-frequency integration step includes an extraction step of extracting the high-frequency component of the image data; an absolute-value determination step of determining an absolute value of the extracted high-frequency component of the image data; and an absolute-value integration step of integrating the absolute value of the high-frequency component of the image data determined by performing the absolute-value determination step.", "The compression processing step includes a number-of-compressed-bytes calculation step of determining the number of compressed bytes of the recorded photographic image data based on the integrated value obtained by integration performed in the high-frequency integration step; a quantization-scale calculation step of determining, based on the number of compressed bytes determined by performing the number-of-compressed-bytes calculation step, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; a quantization-table generation step of generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by performing the quantization-scale calculation step; and a compression step of compressing the photographic image data based on the quantization table generated by performing the quantization-table generation step.", "A program of the present invention has a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data.", "The program causes a computer to execute a high-frequency integration step of integrating a high-frequency component of the obtained image data in the monitoring mode, and a compression processing step of compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration performed in the high-frequency integration step, wherein the high-frequency integration step includes an extraction step of extracting the high-frequency component of the image data, an absolute-value determination step of determining an absolute value of the extracted high-frequency component of the image data, and an absolute-value integration step of integrating the absolute value of the high-frequency component of the image data determined by performing the absolute-value determination step; and the compression processing step includes a number-of-compressed-bytes calculation step of determining the number of compressed bytes of the recorded photographic image data based on the integrated value obtained by integration performed in the high-frequency integration step, a quantization-scale calculation step of determining, based on the number of compressed bytes determined by performing the number-of-compressed-bytes calculation step, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes, a quantization-table generation step of generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by the quantization-scale calculation step, and a compression step of compressing the photographic image data based on the quantization table generated by performing the quantization-table generation step.", "In the image capturing apparatus and method, recording medium, and program of the present invention, a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data are provided; the high-frequency component of the image data is extracted; its absolute value is determined and integrated; the number of compressed bytes of the photographic image data to be recorded is determined based on the integrated value; a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes is determined based on the determined number of compressed bytes; a quantization table for use in compression of the photographic image data is generated based on the determined quantization scale; and the photographic image data is compressed based on the generated quantization table.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a block diagram showing an example structure of a JPEG compression unit of the related art for compressing image data according to a JPEG method.", "FIG.", "2 is a block diagram showing an example of the basic structure of an image capturing apparatus according to the present invention.", "FIG.", "3 is a block diagram showing an example structure of internal components of a high-frequency integration processor shown in FIG.", "2.FIG.", "4 is a block diagram showing an example structure of internal components of a JPEG compression processor shown in FIG.", "2.FIG.", "5 is a flowchart illustrating a high-frequency integration process.", "FIG.", "6 is a flowchart illustrating a JPEG compression process.", "BEST MODE FOR CARRYING OUT THE INVENTION FIG.", "2 is a block diagram showing an example of the basic structure of an image capturing apparatus according to the present invention.", "In FIG.", "2, a controller 111 of an image capturing apparatus 100 such as a digital camera includes a microcomputer having a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), and so on, and controls components of the image capturing apparatus 100 to execute a process for photographing an object.", "An operation unit 112 is formed of a shutter button or the like operated by a user of the image capturing apparatus 100 to instruct recording of a still image, and supplies the instruction of the user to the controller 111.A RAM 114 is a storage device using a semiconductor element, and is controlled by a memory controller 115 to temporarily store created photographic image data, etc.", "The controller 111, the RAM 114, and the memory controller 115 are connected with one another via a bus 110 so that control information from the controller 111 and various data can be supplied or acquired.", "In FIG.", "2, light from an object (not shown) is directed through a lens unit 121 into a camera unit 122 formed of a CCD (Charge Coupled Device), a CMOS (Complementary Metal Oxide Semiconductor), or the like on the front surface of which a primary-color filter, (not shown) having a mosaic array of red (R), green (G), and blue (B) filters is placed, so that the light is photoelectrically converted.", "Other than the above filter, the filter placed on the front surface of the CCD or CMOS may also be implemented by, for example, a complementary color filter having a mosaic array of yellow (Ye), cyan (Cy), magenta (Mg), and green (G) filters.", "The camera unit 122 outputs a video signal, which is photoelectrically converted by a light-receiving unit, in a raster scan format, and the output video signal is passed to an analog signal processor 123 including a CDS (Correlated Double Sampling circuit) circuit, an AGC (Automatic Gain Control) circuit, and an A/D (Analog-to-Digital) converter circuit.", "The analog signal processor 123 eliminates noise or adjusts the gain of the video signal, after which the built-in A/D converter circuit converts the input analog signal into a digital signal and outputs the digital signal to a digital signal processor 124.The digital signal processor 124 performs processing on the input digital signal, such as gamma processing, color separation, and color-space conversion for converting from the RGB color space consisting of red, green, and blue signals to the YUV color space consisting of a luminance signal (Y), a color-difference signal (U) from green to red, and a color-difference signal (V) from green to blue.", "The resulting digital signal is supplied to a thumbnail preparation image generation unit 125 as photographic image data containing a luminance signal (hereinafter referred to as a Y signal) and a chroma signal (hereinafter referred to as a C signal).", "In the still-image recording mode, the thumbnail preparation image generation unit 125 creates thumbnail preparation image data, which is image data for creating thumbnail image data, from the photographic image data.", "The thumbnail preparation image data is image data corresponding to an image obtained by reducing the number of horizontal pixels of the photographic image, and is image data prior to the thumbnail image data.", "The thumbnail preparation image generation unit 125 adds the created thumbnail preparation image data to the photographic image data, and supplies the resulting data to a number-of-pixels conversion unit 131 and a JPEG (Joint Photographic Experts Group) compression unit 141 via the bus 110.In the monitoring mode, the thumbnail preparation image generation unit 125 supplies the input image data to an NTSC encoder 151 and a high-frequency integration processor 126.The NTSC encoder 151 converts the supplied image data into NTSC data, and supplies the resulting data to a monitor 152 to display the corresponding image.", "The high-frequency integration processor 126 integrates the high-frequency component of the obtained image data for each screen, and supplies the integrated value to the JPEG compression unit 141.The number-of-pixels conversion unit 131 extracts the thumbnail preparation image data from the supplied photographic image data, and processes the extracted data to create thumbnail image data.", "The number-of-pixels conversion unit 131 supplies the created thumbnail image data to the JPEG compression unit 141 and the NTSC encoder 151 via the bus 110.The JPEG compression unit 141 compresses the photographic image data supplied from the thumbnail preparation image generation unit 125 and the thumbnail image data supplied from the number-of-pixels conversion unit 131 according to the JPEG method, and stores the compressed data into the RAM 114 via the bus 110.The compression ratio is determined based on the integrated value supplied from the high-frequency integration processor 126.The NTSC encoder 151 converts the video signal of the photographic image data supplied from the thumbnail preparation image generation unit 125 into an NTSC signal, and supplies the signal to the monitor 152 to display the corresponding photographic image.", "The JPEG-compressed photographic image data and thumbnail image data stored in the RAM 114 are recorded in an external recording medium 162 such as a memory stick (registered trademark) via the bus 110 and a recording medium interface 161 as, for example, Exif (Exchangeable Image File Format) data.", "A drive 171 is also connected to the bus 110, if necessary, and a magnetic disc 181, an optical disc 182, a magneto-optical disc 183, a semiconductor memory 184, or the like is inserted, as desired, so that a computer program read therefrom is installed to the RAM 114 or the built-in RAM of the controller 111, as required.", "The basic operation of the image capturing apparatus 100 having such a structure is described below.", "When a user of the image capturing apparatus 100 does not operate the shutter button or the like of the operation unit 112 to instruct photography to thereby maintain the image capturing apparatus 100 in a standby state, the controller 111 sets a monitoring mode to control the components.", "In this case, the light incident on the camera unit 122 via the lens unit 121 is photoelectrically converted, and the converted signal is supplied to the analog signal processor 123.In the analog signal processor 123, an undesirable noise signal is eliminated from this analog video signal by the CDS circuit and the gain of the analog video signal is adjusted by the AGC circuit, after which the analog video signal is converted into a digital signal by the A/D converter circuit, and the digital signal is supplied to the digital signal processor 124.The digital video signal is subjected to processing such as gamma processing, color separation, and color-space conversion by the digital signal processor 124, and the resulting signal is supplied to the thumbnail preparation image generation unit 125 as photographic image data.", "The thumbnail preparation image generation unit 125 supplies the supplied photographic image data to the NTSC encoder 151 and the high-frequency integration processor 126.At this time, the thumbnail preparation image generation unit 125 does not generate a thumbnail preparation image from the photographic image data.", "The photographic image data supplied to the NTSC encoder 151 is converted into an NTSC video signal, and the resulting signal is supplied to the monitor 152 to display the corresponding image.", "The high-frequency integration processor 126 to which the photographic image data is supplied extracts the high-frequency component of the acquired photographic image data, and determines its absolute value to integrate for each screen.", "The integrated value for each screen is supplied to the JPEG compression unit 141.In some cases such as a TTL (Through The Lens) system, a high-frequency integrated signal may be used as an AF (Auto Focus) evaluation value.", "The high-frequency integrated signal for AF evaluation employs an image signal which is not subjected to the analog signal processing and the digital signal processing, and an image corresponding to this signal is different in nature from an image corresponding to image data to be recorded actually and is in poor correlation with the JPEG image size.", "Therefore, the image data supplied to the high-frequency integration processor 126 is supplied from the thumbnail preparation image generation unit 125 in which the image data processed by the analog and digital signal processing is acquired.", "The high-frequency integration processor 126 integrates the high-frequency component of the Y signal alone in the acquired image data since a larger proportion of an image signal of the image data is occupied by the Y signal component than the C signal component and a signal containing a fixed Y signal component and a changing C signal component is not conceivable.", "Thus, the high-frequency component of the C signal may, of course, be integrated.", "In the monitoring mode, when a user of the image capturing apparatus 100 operates the shutter button or the like of the operation unit 112, the controller 111 sets a still-image recording mode (capture mode) in which still images are captured to control the components.", "When the operation unit 112 is operated to instruct photography of an object, light from the object is directed through the lens unit 121 into a built-in light-receiving unit such as a CCD of the camera unit 122.The camera unit 122 photoelectrically converts the incident light, and outputs an analog video signal in a raster scan format to the analog signal processor 123.In the analog signal processor 123, this analog video signal from which an undesirable noise signal is eliminated and of which the gain is adjusted is then converted into a digital signal, and the signal is supplied to the digital signal processor 124.The digital signal processor 124 performs processing such as gamma processing, color separation, and color-space conversion on the digital video signal, and supplies the resulting signal to the thumbnail preparation image generation unit 125 as photographic image data.", "The thumbnail preparation image generation unit 125 generates thumbnail preparation image data, which is image data for creating thumbnail image data, from the supplied photographic image data.", "A thumbnail preparation image corresponding to the thumbnail preparation image data is an image obtained by reducing the number of horizontal pixels of a photographic image corresponding to the photographic image data to the number of pixels of the thumbnail image, in which the number of vertical pixels is equal to that of the photographic image.", "The thumbnail preparation image generation unit 125 adds the generated thumbnail preparation image data to the original photographic image data, and supplies the resulting data to the number-of-pixels conversion unit 131 and the JPEG compression unit 141.The number-of-pixels conversion unit 131 extracts the thumbnail preparation image data from the acquired image data, and reduces the number of vertical pixels of a thumbnail preparation image corresponding to the extracted thumbnail preparation image data to generate a thumbnail image.", "The number-of-pixels conversion unit 131 which has generated the thumbnail image supplies thumbnail image data corresponding thereto to the JPEG compression unit 141 and the NTSC encoder 151.The JPEG compression unit 141 compresses the acquired photographic image data and thumbnail image data according to the JPEG method using the compression ratio determined based on the integrated value supplied from the high-frequency integration processor 126, and stores the JPEG-compressed data into the RAM 114.The NTSC encoder 151 to which the thumbnail image data is supplied converts the video signal of the thumbnail image data into an NTSC signal, and supplies the NTSC signal to the monitor 152 to display an image corresponding to the thumbnail image data, which is a still image captured by the image capturing apparatus 100.The RAM 114 is controlled by the memory controller 115 to supply the JPEG-compressed photographic image data and thumbnail image data stored as, for example, Exif format data, and information relating to such image data to the external recording medium 162 such as a semiconductor memory or a magneto-optical disc via the recording medium interface 161 for storage.", "In the foregoing description, the thumbnail preparation image generation unit 125 does not generate a thumbnail preparation image from photographic image data in the monitoring mode; however, the present invention is not limited thereto, and the thumbnail preparation image generation unit 125 may always generate a thumbnail preparation image regardless of modes.", "The high-frequency integration processor 126 is described below.", "FIG.", "3 is a block diagram showing an example structure of internal components of the high-frequency integration processor 126 shown in FIG.", "2.In FIG.", "3, the components of the high-frequency integration processor 126 are controlled by the controller 111 to execute various processes.", "The Y signal of the photographic image data supplied from the thumbnail preparation image generation unit 125 shown in FIG.", "2 is divided into the horizontal component and the vertical component, which are input to the high-frequency integration processor 126 through input terminals 201 and 211, respectively.", "An active pixel area identification signal, an Enable signal, and so on (not shown) supplied from the thumbnail preparation image generation unit 125 shown in FIG.", "2 are also input to the high-frequency integration processor 126.The Y-signal horizontal component input from the input terminal 201 is supplied to a high-pass filter 202.The high-pass filter 202 filters out the low-frequency component of the input Y-signal horizontal component to extract the high-frequency component, and supplies the extracted high-frequency component to an absolute-value determination processor 203.The absolute-value determination processor 203 determines an absolute value of the supplied high-frequency component of the Y-signal horizontal component, and supplies the absolute value to a horizontal high-frequency integration processor 204.The horizontal high-frequency integration processor 204 integrates the obtained absolute value of the high-frequency component of the Y-signal horizontal component to determine the integrated value corresponding to one screen.", "The resulting integrated value is output as horizontal high-frequency integrated data from an output terminal 205.The high-frequency integration processor 126 further includes a high-pass filter 212 through a horizontal high-frequency integration processor 214 corresponding to the high-pass filter 202 through the horizontal high-frequency integration processor 204, respectively, and the high-pass filter 212 through the horizontal high-frequency integration processor 214 perform similar processing to that of the corresponding high-pass filter 202 through horizontal high-frequency integration processor 204 on the Y-signal vertical component input from the input terminal 211.Consequently, the integrated value determined for the Y-signal vertical component is output as vertical high-frequency integrated data from an output terminal 215.In the foregoing description, a photographed nature picture has a very strong correlation between its horizontal high-frequency integrated value and the storage memory size after JPEG compression, and does not have a strong correlation between its vertical high-frequency integrated value and the storage memory size after JPEG compression.", "However, with consideration for photography of a completely horizontal-striped image, the vertical component is also high-frequency integrated.", "Therefore, high-frequency integration on the vertical component is not essential.", "The determined high-frequency integrated value may not be so accurate as long as it allows for prediction of the JPEG compression ratio, and each of the above-noted absolute-value determination processors 203 and 213 may be formed of an absolute-value determination circuit such as an Ex-OR (Exclusive-OR) circuit.", "The JPEG compression unit 141 is described below.", "FIG.", "4 is a block diagram showing an example structure of internal components of the JPEG compression unit 141 shown in FIG.", "2.In FIG.", "4, the components of the JPEG compression unit 141 are controlled by the controller 111 to execute various processes.", "Image data converted to an image format suitable for compression supplied via the bus 110 is input to the JPEG compression unit 141 through an input terminal 311.The horizontal high-frequency integrated data and vertical high-frequency integrated data of the image data supplied from the high-frequency integration processor 126 are further input through an input terminal 301.When acquiring the horizontal and vertical high-frequency integrated data input from the input terminal 301, a number-of-bytes calculation unit 302 determines the number of bytes after compression based on the integrated values, and supplies the determined number of bytes to a Q-scale calculation unit 303.The Q-scale calculation unit 303 calculates the deviation between the number of bytes supplied from the number-of-bytes calculation unit 302 and an expected value to determine a Q-scale based on which the image data can be compressed one time to a predetermined data size, and supplies the determine Q-scale to a Q-table generation unit 304.The Q-table generation unit 304 to which the Q-scale is supplied from the Q-scale calculation unit 303 generates a Q-table for use in quantization based on the Q-scale, and supplies the generated Q-table to a quantization processor 322 of a compression unit 312.The image data input from the input terminal 311 is compressed by the compression unit 312, and is then output from an output terminal 313.The compression unit 312 includes a DCT unit 321 for performing a discrete cosine transform on the input image data, the quantization processor 322 for quantizing a coefficient of the image data converted from the time-domain component to the frequency-domain component, and a variable-length coding processor 323 for encoding the image data with variable length coding.", "When acquiring the image data input from the input terminal 311, the DCT unit 321 performs a discrete cosine transform on the image data, and supplies the resulting data to the quantization processor 322.The quantization processor 322 adjusts the coefficient of the frequency-domain component acquired from the DCT unit based on the up-to-date Q-table supplied from the Q-table generation unit 304 to adjust the compression ratio of the image data, and supplies the result to the variable-length coding processor 323.The variable-length coding processor 323 encodes the acquired image data with variable length coding such as Huffman coding, and outputs the resulting compressed image data from the JPEG compression unit 141 via the output terminal 313.In the foregoing description, the number-of-bytes calculation unit 302 determines the number of bytes after compression based on the high-frequency integrated value determined from the image data processed by signal processing in the monitoring mode.", "This image data has a smaller number of vertical pixels of the corresponding image by a thinning process than the image data to be recorded in the still-image recording mode.", "However, the number-of-bytes calculation unit 302 determines the number of bytes after compression while considering this, and therefore the number of bytes after compression can be determined without error caused by the difference in pixel number.", "A high-frequency integration process executed by the high-frequency integration processor 126 shown in FIG.", "3 is described below with reference to the flowchart of FIG.", "5.When the image capturing apparatus 100 goes to the monitoring mode, the controller 111 executes the high-frequency integration process on the captured image data.", "First, in step S1, the controller 111 controls the high-pass filter 202 of the high-frequency integration processor 126 to cut the low-frequency component of the input Y-signal horizontal component to extract the high-frequency component.", "In step S2, then, the controller 111 controls the absolute-value determination processor 203 to determine an absolute value of the Y-signal horizontal component of which the low-frequency component is cut.", "The absolute-value determination processor 203 is controlled by the controller 111 to determine an absolute value of the high-frequency component of the Y-signal horizontal component.", "In step S3, then, the controller 111 controls the horizontal high-frequency integration processor 204 to integrate the absolute value of the Y-signal horizontal component for one screen.", "Similarly to steps S1 through S3, in step S4, the controller 111 controls the high-pass filter 212 to cut the low-frequency component of the input Y-signal vertical component, and, in step S5, the controller 111 controls the absolute-value determination processor 213 to determine an absolute value of the Y-signal vertical component of which the low-frequency component is cut.", "In step 6, the controller 111 controls the vertical high-frequency integration processor 214 to integrate the absolute value of the Y-signal vertical component for one screen.", "The process proceeds to step S7, in which the controller 111 causes the integrated Y signal for one screen to be output from the output terminals 205 and 215.The process proceeds to step S8, in which the controller 111 determines whether or not the monitoring mode ends.", "If it is determined that the monitoring mode does not end, the process returns to step S1 and the controller 111 repeatedly performs the subsequent steps on newly input image data.", "If it is determined in step S8 that the monitoring mode ends, the controller 111 terminates the high-frequency integration process.", "Accordingly, in the monitoring mode, the high-frequency integrated value of the captured image data is determined.", "A JPEG compression process executed by the JPEG compression unit 141 shown in FIG.", "4 is described below with reference to the flowchart of FIG.", "6.When the image capturing apparatus 100 goes to the still-image recording mode and photographic image data is supplied to the JPEG compression unit 141, the controller 111 starts the JPEG compression process.", "First, in step S21, the controller 111 controls the number-of-bytes calculation unit 302 of the JPEG compression unit 141 to determine the number of compressed bytes of the photographic image data based on the high-frequency integrated data.", "After determining the number of compressed bytes, in step S22, the controller 111 controls the Q-scale calculation unit 303 to determine a Q-scale based on the number of bytes determined by the number-of-bytes calculation unit 302.The Q-scale calculation unit 303 is controlled by the controller 111 to calculate the deviation between the number of bytes supplied from the number-of-bytes calculation unit 302 and an expected value to determine a Q-scale based on which the compression unit 312 can compress the photographic image data one time to a predetermined data size.", "After determining the Q-scale, in step S23, the controller 111 controls the Q-table generation unit 304 to generate a Q-table based on the Q-scale determined by the Q-scale calculation unit 303.In stel S24, the controller 111 controls the DCT unit 321 of the compression unit 312 to perform DCT on the photographic image data input from the input terminal 311.In step S25, the controller 111 controls the quantization process 322 to quantize a DCT coefficient of the DCT image data based on the Q-table generated in step S23 to adjust the compression ratio.", "In step S26, the controller 111 controls the variable-length coding processor 323 to encode the quantized DCT coefficient with variable length coding to compress the photographic image data.", "In step S27, the controller 111 causes the compressed photographic image data to be output from the output terminal 313.With the JPEG compression process in this way, one-time compression is only required for compressing the photographic image data to a predetermined data size, thus reducing the time required for compression and further reducing the memory capacity required for compression.", "While a compression process performed on photographic image data has been described, this is not a limited form, and a compression process performed on thumbnail image data corresponding to the photographic image data can be performed in a similar way.", "In the foregoing description, Exif format data is created; however, the present invention is not limited thereto, and any format data may be created.", "Although the compression format of photographic image data has been discussed in the context of JPEG format, the present invention is not limited thereto, and any format may be used.", "The foregoing processes may be executed by either hardware or software.", "If the series of processes is executed by software, a program constituting the software is installed from a network or a recording medium to a computer incorporated in dedicated hardware or a general-purpose personal computer capable of executing various functions by installing various programs.", "This recording medium is formed of not only a packaged media such as, as shown in FIG.", "2, the magnetic disc 181 (including a floppy disc), optical disc 182 (including a CD-ROM (Compact Disk-Read Only Memory) and a DVD (Digital Versatile Disk)), magneto-optical disc 183 (including an MD (Mini-Disk)) or semiconductor memory 184 having a program recorded therein, which is distributed separately from the apparatus to offer the program to users, but is also formed of a built-in ROM of the controller 111 having a program recorded therein which is offered to users as is incorporated in the apparatus in advance.", "In this document, the steps describing a program recorded in a recording medium include not only operations performed in a time-series manner according to the order described but also operations performed in a parallel or discrete manner although they are not necessarily performed in a time-series manner.", "INDUSTRIAL APPLICABILITY According to the image capturing apparatus and method of the present invention, therefore, the image recording time can be reduced and the memory capacity required for compression can also be reduced." ] ]	Patent_10468981
[ [ "Managing coherence via put/get windows", "A method and apparatus for managing coherence between two processors of a two processor node of a multi-processor computer system.", "Generally the present invention relates to a software algorithm that simplifies and significantly speeds the management of cache coherence in a message passing parallel computer, and to hardware apparatus that assists this cache coherence algorithm.", "The software algorithm uses the opening and closing of put/get windows to coordinate the activated required to achieve cache coherence.", "The hardware apparatus may be an extension to the hardware address decode, that creates, in the physical memory address space of the node, an area of virtual memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements." ], [ "1.A method of simplifying and speeding the management of cache coherence in a message passing parallel supercomputer including two or more non-coherent processor elements, where one processor element is primarily performing calculations, while the other processor element is performing message passing activities, the method comprising the steps: opening and closing a put/get window; performing activities to achieve cache coherence; and using said opening and closing of the put/get window to coordinate the activities to achieve cache coherence.", "2.A method according to claim 1, wherein the method is implemented by a software algorithm.", "3.A method according to claim 1, wherein said using step includes the step of ensuring that data being sent is not in the cache of either processor, and that the data being received is also not in the cache of either processor.", "4.A method according to claim 3, wherein the ensuring step includes the step of loading data into cache by issuing a software command.", "5.A program storage device readable by machine, tangibly embodying a program of instructions executable by the machine to perform method steps for simplifying and speeding the management of cache coherence in a message passing parallel supercomputer including two or more non-coherent processor elements, where one processor element is primarily performing calculations, while the other processor element is performing message passing activities, the method steps comprising: opening and closing a put/get window; performing activities to achieve cache coherence; and using said opening and closing of the put/get window to coordinate the activities to achieve cache coherence.", "6.A program storage device according to claim 5, wherein said using step includes the step of ensuring that data being sent is not in the cache of either processor, and that the data being received is also not in the cache of either processor.", "7.A program storage device according to claim 6, wherein the ensuring step includes the step of loading data into cache by issuing a software command.", "8.A system to simplify and speed the management of cache coherence in a message passing parallel supercomputer including two or more non-coherent processor elements, where one processor element is primarily performing calculations, while the other processor element is performing message passing activities, the system comprising: means for opening and closing a put/get window; means for performing activities to achieve cache coherence; and means for using said opening and closing of the put/get window to coordinate the activities to achieve cache coherence.", "9.A system according to claim 8, wherein said using means includes means for ensuring that data being sent is not in the cache of either processor, and that the data being received is also not in the cache of either processor.", "10.A system according to claim 9, wherein the ensuring means includes means for loading data into cache by issuing a software command.", "11.Hardware apparatus to assist achieving cache coherence in a message passing parallel computer including two or more non-coherent processing elements, where one processing element is principally performing calculations, while the second processing element is performing message passing activities, the hardware apparatus comprising: a memory controller to create, in the physical memory address space of the node, an area of virtual memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements.", "12.Hardware apparatus according to claim 11, wherein the memory controller allows garbage data, which the processor will never use, to be pulled into the processor's cache, thereby evicting just the modified data and displacing unmodified data with optimal performance.", "13.Hardware apparatus according to claim 12, wherein the garbage data does not actually need to be fetched from memory, rather, the memory controller need only instantly reply.", "14.Hardware apparatus according to claim 13, wherein only actually modified data is written to memory from cache, while clean data is simply instantly discarded.", "15.Hardware apparatus according to claim 14, wherein, when the total size of the put/get window exceeds the size of the processor's cache, cleaning the cache in this manner provides an upper bound on the total amount of work that is required to ensure that no data from the communication area remains in the cache.", "16.A method of operating computer hardware apparatus to assist achieving cache coherence in a message passing parallel computer including two or more non-coherent processing elements, where one processing element is principally performing calculations, while the second processing element is performing message passing activities, the method comprising the steps: using a memory controller to create, in the physical memory address space of the node, an area of virtual memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements.", "17.A method according to claim 16, wherein the memory controller allows garbage data, which the processor will never use, to be pulled into the processor's cache, thereby evicting just the modified data and displacing unmodified data with optimal performance.", "18.A method according to claim 17, wherein the garbage data does not actually need to be fetched from memory, rather, the memory controller need only instantly reply.", "19.A method according to claim 18, wherein only actually modified data is written to memory from cache, while clean data is simply instantly discarded.", "20.A method according to claim 19, wherein, when the total size of the put/get window exceeds the size of the processor's cache, cleaning the cache in this manner provides an upper bound on the total amount of work that is required to ensure that no data from the communication area remains in the cache." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention This invention relates to the field of distributed-memory message-passing parallel computer design and system software, as applied for example to computation in the field of life sciences.", "2.Background Art In provisional patent application No.", "60/271,124 titled “A Novel Massively Parallel Supercomputer,” therein is described a massively parallel supercomputer architecture in the form of a three-dimensional torus designed to deliver processing power on the order of teraOPS (trillion operations per second) for a wide range of applications.", "The architecture comprises 65,536 processing nodes organized as a 64×32×32 three-dimensional torus, with each processing node connected to six (6) neighboring nodes.", "Each processing node of the supercomputer architecture is a semiconductor device that includes two electronic processors (among other components).", "One of these processors is designated the “Compute Processor” and, in the common made operation, is dedicated to application computation.", "The other processor is the “I/O Processor,” which, in the common mode of operation, is a service processor dedicated to performing activities in support of message-passing communication.", "Each of these processors contains a separate first-level cache (L1) which may contain a copy of data stored in a common memory accessed by both processors.", "If one processor changes its L1 copy of a memory location, and the other processor has a copy of the same location, the two copies become “coherent” if they are made to be the same.", "Message passing is a commonly-known form of computer communication wherein processors explicitly copy data from their own memory to that of another node.", "In the dual-processor node disclosed in the above-identified provisional patent application No.", "60/271,124, the I/O Processor is principally used to facilitate message passing between the common memory of a node and the common memory of other nodes.", "Therefore, it both produces data (when a message is received) that is consumed by the Compute Processor, and consumes data (in order to send a message) that is produced by the Compute Processor.", "As a result, it is very common for both processors to have a copy of the same memory location in their L1s.", "If the messages passed are small and many, then the problem is exacerbated.", "Thus, there is a clear need to find a way to make the L1s of each processor coherent, without extensive circuitry, and with minimal impact on performance.", "As massively parallel computers are scaled to thousands of processing nodes, typical application messaging traffic involves an increasing number of messages, where each such message contains information communicated by other nodes in the computer.", "Generally, one node scatters locally-produced messages to some number of other nodes, while receiving some number of remotely produced messages into its local memory.", "Overall performance for these large-scale computers is often limited by the message-passing performance of the system.", "For such data transfers, a common message-passing interface, described in the literature (see for example http://www.mpi-forum.org/docs/docs.html, under MPI-2), is known as “one-sided communication.” One-sided communication uses a “put/get” message-passing paradigm, where messages carry the source (for get) or the destination (for put) memory address.", "In parallel supercomputers operating on a common problem, puts and gets are typically assembled in batches and issued together.", "This keeps the independently operating processors in rough synchronization, maximizing performance.", "The time during which puts and gets occur is termed the put/get window.", "This window extends both in time (when it occurs) and in memory (over the range of memory addresses carried by the put or get messages).", "FIG.", "2 shows a put/get window 30 having a number of distinct messages.", "Put/get windows extend the concept of coherence to processors on different processing nodes of the massively parallel supercomputer.", "Implementations of put/get windows must insure that all messages put to a window during the time it is open are received into the memory of the window before the window is closed.", "Similarly, a get on the memory of the window is only allowed during the time the window is open.", "Therefore, put/get windows are simply a mechanism for a node to synchronize with remote processors operating on its memory.", "The management of a put/get window is currently accomplished by either buffering the put/get messages or by using explicit synchronization messages.", "Buffering the messages consumes memory, which is always in limited supply.", "Explicit synchronization for each window suffers from the long latency of round-trip messages between all the nodes accessing the window.", "Therefore, on large-scale machines such as the one described in copending patent application No.", "______ (attorney Docket 15275), these approaches do not scale well because of limited memory for buffering, and because the number of nodes accessing any particular window often scales along with the number of processing nodes in the computer.", "A long-standing problem in the field of computer design, is how to keep these L1 caches coherent.", "Typical solutions employ techniques known as “snooping” the memory bus of the other processor, which can be slow and reduce the performance of each processor.", "Alternatively, the processor that contains an old copy in L1 of the data in the common memory, can request a new copy, or mark the old copy obsolete, but this requires knowledge of when the copy became invalid.", "Sometime this knowledge is incomplete, forcing unnecessary memory operations, further reducing performance.", "Other computers make use of “interlocks,” whereby one processor is granted permission to use certain data while the other processor cannot, but this permission involves interactions between the two processors, which usually requires additional complex circuitry in the semiconductor device, reducing the performance of the two processors.", "Still other solutions in common practice disable all caching for areas of memory intended to be shared.", "This practice penalizes all memory accesses to these areas, not just those to the shared data." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>An object of this invention is to provide an improved procedure for managing coherence in a parallel processing computer system.", "Another object of the present invention is to achieve coherency between the first-level caches of the processors of a multi-processor node without extensive circuitry and with minimal impact on the performance of each processor.", "A further object of the invention is to provide a method and apparatus, working in conjunction with software algorithms, to accomplish efficient high speed message-passing communications between processors or a direct memory access (DMA) device, which maintains coherence without significantly reducing performance.", "These and other objectives are attained with the method and apparatus of the present invention.", "In accordance with a first aspect, the invention provides a software algorithm that simplifies and significantly speeds the management of cache coherence in a message passing massively parallel supercomputer (such as the one described in copending patent application No.", "______ (attorney Docket 15275)) containing two or more non-coherent processing elements (or even a DMA controller) where one processing element is primarily performing calculations, while the other element is performing message passing activities.", "In such a massively parallel supercomputer, algorithms often proceed as a series of steps, where each step consists of a computation phase followed by a communication phase.", "In the communication phase, the nodes exchange data produced by the computation phase and required for the next step of the algorithm.", "Because of the nature of the algorithms, the phases are usually tightly synchronized, so that the communication happens all at once over the entire machine.", "Therefore, the cost of managing the synchronization of put/get windows can be amortized over a large number of nodes at the start and end of each communication phase.", "Briefly, a global operation can be used to open many put/get windows at the start of a communication phase, and a second global operation can be used to close the windows at the end of the communication phase.", "Because the I/O Processor cannot actually send or receive the messages until after cache coherence has been guaranteed, the invention provides a mechanism to ensure that the data being “put” (sent) is not in the cache of either processor, and that the data being “gotten” (received) is also not in the cache of either processor.", "By coordinating these activities upon opening and closing the “Put/Get Window”, the invention reduces the total amount of work required to achieve coherence and allow that work to be amortized over a large number of individual messages.", "Also, since both processing elements within a node must perform this work, the invention enables this to happen concurrently.", "Further, when required, these activities can be coordinated over a large number of independent nodes in the massively parallel machine by employing the Global Barrier Network described in copending patent application No.", "______ (attorney Docket 15275).", "In accordance with a second aspect, the invention provides a hardware apparatus that assists the above-described cache coherence software algorithm, and limits the total time (or latency) required to achieve cache coherence over the Put/Get Window.", "This apparatus is a simple extension to the hardware address decoder that creates, in the physical memory address space of the node, an area of memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements.", "This further speeds the coherence activities because it allows garbage data (which the processor will never use) to be pulled into the processor's cache, thereby evicting just the modified data and displacing unmodified data with optimal performance.", "The performance is faster because this garbage data does not actually need to be fetched from memory, rather, the memory controller need only instantly reply.", "The performance is also faster because only modified data is written to memory from cache, while clean data is simply instantly discarded.", "Further, for the case where the total size of the “Put/Get Window” exceeds, perhaps greatly, the size of the processor's cache, cleaning the cache in this manner provides an upper bound on the total amount of work that is required to ensure that no data from the communication area remains in the cache.", "It may be noted that, independent of the above-described software algorithms, this hardware device is useful for computer systems in general which employ a Least Recently Used cache replacement policy.", "Also, two specific software instructions may be used in the preferred implementation of the invention.", "One instruction, termed “data cache block flush and invalidate”, may be used to write data from the memory area of the first processor into the shared memory area, while at the same time, preventing the first processor from using data the data written in its memory area.", "A second software instruction, termed “data cache block zero”, may be used to write data from the memory area of the first processor into the shared memory.", "By using these, or similar software instructions, the method and apparatus of the invention, working in conjunction with software algorithms, achieve high speed message passing communications between nodes, while maintaining coherence without significantly reducing performance.", "Further benefits and advantages of the invention will become apparent from a consideration of the following detailed description, given with reference to the accompanying drawings, which specify and show preferred embodiments of the invention." ], [ "CROSS-REFERENCE TO RELATED APPLICATIONS The present invention claims the benefit of commonly-owned, co-pending U.S.", "Provisional Patent Application Ser.", "No.", "60/271,124 filed Feb. 24, 2001 entitled MASSIVELY PARALLEL SUPERCOMPUTER, the whole contents and disclosure of which is expressly incorporated by reference herein as if fully set forth herein.", "This patent application is additionally related to the following commonly-owned, co-pending United States Patent Applications filed on even date herewith, the entire contents and disclosure of each of which is expressly incorporated by reference herein as if fully set forth herein.", "U.S. patent application Serial No.", "(YOR920020027US1, YOR920020044US1 (15270)), for “Class Networking Routing”; U.S. patent application Serial No.", "(YOR920020028US1 (15271)), for “A Global Tree Network for Computing Structures”; U.S. patent application Serial No.", "(YOR920020029US1 (15272)), for ‘Global Interrupt and Barrier Networks”; U.S. patent application Serial No.", "(YOR920020030US1 (15273)), for ‘Optimized Scalable Network Switch”; U.S. patent application Serial No.", "(YOR920020031US1, YOR920020032US1 (15258)), for “Arithmetic Functions in Torus and Tree Networks’; U.S. patent application Serial No.", "(YOR920020033US1, YOR920020034US1 (15259)), for ‘Data Capture Technique for High Speed Signaling”; U.S. patent application Serial No.", "(YOR920020035US1 (15260)), for ‘Managing Coherence Via Put/Get Windows’; U.S. patent application Serial No.", "(YOR920020036US1, YOR920020037US1 (15261)), for “Low Latency Memory Access And Synchronization”; U.S. patent application Serial No.", "(YOR920020038US1 (15276), for ‘Twin-Tailed Fail-Over for Fileservers Maintaining Full Performance in the Presence of Failure”; U.S. patent application Serial No.", "(YOR920020039US1 (15277)), for “Fault Isolation Through No-Overhead Link Level Checksums’; U.S. patent application Serial No.", "(YOR920020040US1 (15278)), for “Ethernet Addressing Via Physical Location for Massively Parallel Systems”; U.S. patent application Serial No.", "(YOR920020041US1 (15274)), for “Fault Tolerance in a Supercomputer Through Dynamic Repartitioning”; U.S. patent application Serial No.", "(YOR920020042US1 (15279)), for “Checkpointing Filesystem”; U.S. patent application Serial No.", "(YOR920020043US1 (15262)), for “Efficient Implementation of Multidimensional Fast Fourier Transform on a Distributed-Memory Parallel Multi-Node Computer”; U.S. patent application Serial No.", "(YOR9-20010211US2 (15275)), for “A Novel Massively Parallel Supercomputer”; and U.S. patent application Serial No.", "(YOR920020045US1 (15263)), for “Smart Fan Modules and System”.", "BACKGROUND OF THE INVENTION 1.Field of the Invention This invention relates to the field of distributed-memory message-passing parallel computer design and system software, as applied for example to computation in the field of life sciences.", "2.Background Art In provisional patent application No.", "60/271,124 titled “A Novel Massively Parallel Supercomputer,” therein is described a massively parallel supercomputer architecture in the form of a three-dimensional torus designed to deliver processing power on the order of teraOPS (trillion operations per second) for a wide range of applications.", "The architecture comprises 65,536 processing nodes organized as a 64×32×32 three-dimensional torus, with each processing node connected to six (6) neighboring nodes.", "Each processing node of the supercomputer architecture is a semiconductor device that includes two electronic processors (among other components).", "One of these processors is designated the “Compute Processor” and, in the common made operation, is dedicated to application computation.", "The other processor is the “I/O Processor,” which, in the common mode of operation, is a service processor dedicated to performing activities in support of message-passing communication.", "Each of these processors contains a separate first-level cache (L1) which may contain a copy of data stored in a common memory accessed by both processors.", "If one processor changes its L1 copy of a memory location, and the other processor has a copy of the same location, the two copies become “coherent” if they are made to be the same.", "Message passing is a commonly-known form of computer communication wherein processors explicitly copy data from their own memory to that of another node.", "In the dual-processor node disclosed in the above-identified provisional patent application No.", "60/271,124, the I/O Processor is principally used to facilitate message passing between the common memory of a node and the common memory of other nodes.", "Therefore, it both produces data (when a message is received) that is consumed by the Compute Processor, and consumes data (in order to send a message) that is produced by the Compute Processor.", "As a result, it is very common for both processors to have a copy of the same memory location in their L1s.", "If the messages passed are small and many, then the problem is exacerbated.", "Thus, there is a clear need to find a way to make the L1s of each processor coherent, without extensive circuitry, and with minimal impact on performance.", "As massively parallel computers are scaled to thousands of processing nodes, typical application messaging traffic involves an increasing number of messages, where each such message contains information communicated by other nodes in the computer.", "Generally, one node scatters locally-produced messages to some number of other nodes, while receiving some number of remotely produced messages into its local memory.", "Overall performance for these large-scale computers is often limited by the message-passing performance of the system.", "For such data transfers, a common message-passing interface, described in the literature (see for example http://www.mpi-forum.org/docs/docs.html, under MPI-2), is known as “one-sided communication.” One-sided communication uses a “put/get” message-passing paradigm, where messages carry the source (for get) or the destination (for put) memory address.", "In parallel supercomputers operating on a common problem, puts and gets are typically assembled in batches and issued together.", "This keeps the independently operating processors in rough synchronization, maximizing performance.", "The time during which puts and gets occur is termed the put/get window.", "This window extends both in time (when it occurs) and in memory (over the range of memory addresses carried by the put or get messages).", "FIG.", "2 shows a put/get window 30 having a number of distinct messages.", "Put/get windows extend the concept of coherence to processors on different processing nodes of the massively parallel supercomputer.", "Implementations of put/get windows must insure that all messages put to a window during the time it is open are received into the memory of the window before the window is closed.", "Similarly, a get on the memory of the window is only allowed during the time the window is open.", "Therefore, put/get windows are simply a mechanism for a node to synchronize with remote processors operating on its memory.", "The management of a put/get window is currently accomplished by either buffering the put/get messages or by using explicit synchronization messages.", "Buffering the messages consumes memory, which is always in limited supply.", "Explicit synchronization for each window suffers from the long latency of round-trip messages between all the nodes accessing the window.", "Therefore, on large-scale machines such as the one described in copending patent application No.", "______ (attorney Docket 15275), these approaches do not scale well because of limited memory for buffering, and because the number of nodes accessing any particular window often scales along with the number of processing nodes in the computer.", "A long-standing problem in the field of computer design, is how to keep these L1 caches coherent.", "Typical solutions employ techniques known as “snooping” the memory bus of the other processor, which can be slow and reduce the performance of each processor.", "Alternatively, the processor that contains an old copy in L1 of the data in the common memory, can request a new copy, or mark the old copy obsolete, but this requires knowledge of when the copy became invalid.", "Sometime this knowledge is incomplete, forcing unnecessary memory operations, further reducing performance.", "Other computers make use of “interlocks,” whereby one processor is granted permission to use certain data while the other processor cannot, but this permission involves interactions between the two processors, which usually requires additional complex circuitry in the semiconductor device, reducing the performance of the two processors.", "Still other solutions in common practice disable all caching for areas of memory intended to be shared.", "This practice penalizes all memory accesses to these areas, not just those to the shared data.", "SUMMARY OF THE INVENTION An object of this invention is to provide an improved procedure for managing coherence in a parallel processing computer system.", "Another object of the present invention is to achieve coherency between the first-level caches of the processors of a multi-processor node without extensive circuitry and with minimal impact on the performance of each processor.", "A further object of the invention is to provide a method and apparatus, working in conjunction with software algorithms, to accomplish efficient high speed message-passing communications between processors or a direct memory access (DMA) device, which maintains coherence without significantly reducing performance.", "These and other objectives are attained with the method and apparatus of the present invention.", "In accordance with a first aspect, the invention provides a software algorithm that simplifies and significantly speeds the management of cache coherence in a message passing massively parallel supercomputer (such as the one described in copending patent application No.", "______ (attorney Docket 15275)) containing two or more non-coherent processing elements (or even a DMA controller) where one processing element is primarily performing calculations, while the other element is performing message passing activities.", "In such a massively parallel supercomputer, algorithms often proceed as a series of steps, where each step consists of a computation phase followed by a communication phase.", "In the communication phase, the nodes exchange data produced by the computation phase and required for the next step of the algorithm.", "Because of the nature of the algorithms, the phases are usually tightly synchronized, so that the communication happens all at once over the entire machine.", "Therefore, the cost of managing the synchronization of put/get windows can be amortized over a large number of nodes at the start and end of each communication phase.", "Briefly, a global operation can be used to open many put/get windows at the start of a communication phase, and a second global operation can be used to close the windows at the end of the communication phase.", "Because the I/O Processor cannot actually send or receive the messages until after cache coherence has been guaranteed, the invention provides a mechanism to ensure that the data being “put” (sent) is not in the cache of either processor, and that the data being “gotten” (received) is also not in the cache of either processor.", "By coordinating these activities upon opening and closing the “Put/Get Window”, the invention reduces the total amount of work required to achieve coherence and allow that work to be amortized over a large number of individual messages.", "Also, since both processing elements within a node must perform this work, the invention enables this to happen concurrently.", "Further, when required, these activities can be coordinated over a large number of independent nodes in the massively parallel machine by employing the Global Barrier Network described in copending patent application No.", "______ (attorney Docket 15275).", "In accordance with a second aspect, the invention provides a hardware apparatus that assists the above-described cache coherence software algorithm, and limits the total time (or latency) required to achieve cache coherence over the Put/Get Window.", "This apparatus is a simple extension to the hardware address decoder that creates, in the physical memory address space of the node, an area of memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements.", "This further speeds the coherence activities because it allows garbage data (which the processor will never use) to be pulled into the processor's cache, thereby evicting just the modified data and displacing unmodified data with optimal performance.", "The performance is faster because this garbage data does not actually need to be fetched from memory, rather, the memory controller need only instantly reply.", "The performance is also faster because only modified data is written to memory from cache, while clean data is simply instantly discarded.", "Further, for the case where the total size of the “Put/Get Window” exceeds, perhaps greatly, the size of the processor's cache, cleaning the cache in this manner provides an upper bound on the total amount of work that is required to ensure that no data from the communication area remains in the cache.", "It may be noted that, independent of the above-described software algorithms, this hardware device is useful for computer systems in general which employ a Least Recently Used cache replacement policy.", "Also, two specific software instructions may be used in the preferred implementation of the invention.", "One instruction, termed “data cache block flush and invalidate”, may be used to write data from the memory area of the first processor into the shared memory area, while at the same time, preventing the first processor from using data the data written in its memory area.", "A second software instruction, termed “data cache block zero”, may be used to write data from the memory area of the first processor into the shared memory.", "By using these, or similar software instructions, the method and apparatus of the invention, working in conjunction with software algorithms, achieve high speed message passing communications between nodes, while maintaining coherence without significantly reducing performance.", "Further benefits and advantages of the invention will become apparent from a consideration of the following detailed description, given with reference to the accompanying drawings, which specify and show preferred embodiments of the invention.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 shows a two processor node embodying this invention.", "FIG.", "2 illustrates a put/get window that may be used in the practice of this invention.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to a method and apparatus for managing coherence of a multi-processor computer system.", "FIG.", "1 illustrates a node 10 that may embody this invention.", "Each of the processors 12, 14 of node 10 has a respective cache memory area 16, 20, and the two processors share a third memory area 22.Generally the present invention relates to a software algorithm that simplifies and significantly speeds the management of cache memory coherence in a message passing parallel computer, and to hardware apparatus that assists this cache coherence algorithm.", "The software algorithm uses the opening and closing of put/get windows to coordinate the activities required to achieve cache coherence.", "The hardware apparatus may be an extension to the hardware address decode, that creates, in the physical memory address space of the node, an area of physical memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements.", "As indicated above, this invention utilizes a principal referred to as “put/get” data transfer.", "As parallel multi-computers are scaled to increasing numbers of nodes, typical application messaging traffic involves an increasing number of messages, where each such message contains a piece of work performed by other nodes in the multi-computer.", "Generally, one node scatters locally produced work items to numerous other nodes (a “put”), while assembling numerous remotely produced work items into its local memory (a “get”).", "Overall performance for these multi-computers is often gated by the message passing performance of the system.", "For such data transfers, a particularly efficient message-passing interface, described in the literature (see for example http://www.mpi-forum.org/docs/docs.html, under MPI-2), is known as One-Sided Communication.", "One-Sided Communication uses a “put/get” message-passing paradigm, where messages carry the source (for “get”) or destination (for “put”) memory address.", "In parallel supercomputers operating on a common problem, typically puts and gets are assembled in batches and issued simultaneously.", "This keeps independently operating processors in rough synchronization, allowing good performance on a common problem.", "This time during which puts and gets occur is termed the put/get window.", "This window extends both in time (when it occurs) and in memory (over which range of memory addresses does the data in the put or get reside).", "FIG.", "2 shows a put/get window 30 having a number of distinct messages.", "In such a massively parallel supercomputer, algorithms often proceed as a series of steps, where each step consists of a computation phase followed by a communication phase.", "In the communication phase, the nodes exchange data produced by the computation phase and required for the next step of the algorithm.", "Because of the nature of the algorithms, the phases are usually tightly synchronized, sot that the communication happens all at once over the entire machine.", "Therefore, the cost of managing the synchronization of put/get windows can be amortized over a large number of nodes at the start and end of each communication phase.", "Briefly, a global operation can be used to open many put/get windows at the start of a communication.", "The present invention utilizes this put/get window to provide a simple means to manage memory coherence.", "In accordance with a first aspect, a software algorithm is provided that simplifies and significantly speeds the management of cache coherence in a message passing massively parallel supercomputer (such as the one described in copending patent application No.", "______ (attorney Docket 15275)) containing two or more non-coherent processing elements (or even a DMA controller) where one processing element is primarily performing calculations, while the other element is performing message passing activities.", "Briefly, this algorithm uses the opening and closing of “Put/Get Windows” to coordinate the activities required to achieve memory coherence.", "Because the messages cannot actually be sent or received until after cache coherence has been guaranteed, this invention provides a mechanism to ensure that the data being “put” (sent) is not in the cache of either processor, and that the data being “gotten” (received) is also not in the cache of either processor.", "By coordinating these activities upon opening and closing the “Put/Get Window”, this invention reduces the total amount of work required to achieve coherence and allow that work to be amortized over a large number of individual messages.", "Also, since both processing elements within a node must perform this work, this invention enables this to happen concurrently.", "Further, when required, these activities can be coordinated over a large number of independent nodes in the massively parallel machine by employing the Global Barrier Network described in copending patent application No; ______ (attorney Docket 1527).", "This algorithm is assisted by the hardware, described below, but even in the absence of the apparatus benefits message-passing computers in general.", "Without the apparatus, a special reserved area of physical memory, equal in size to the processor's cache may be utilized, albeit at reduced performance by loading from this physical area into cache by issuing a DCBT (Data Cache Block Touch) instruction for each cache line of the reserved physical area.", "In accordance with a second aspect of the invention, a novel hardware apparatus is provided that assists the above-described cache coherence algorithm, and limits the total time (or latency) required to achieve cache coherence over the Put/Get Window.", "This apparatus is a simple extension to the hardware address decoder that creates, in the physical memory address space of the node, an area of virtual memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements.", "This further speeds the coherence activities because it allows garbage data (which the processor will never use) to be pulled into the processor's cache, thereby evicting just the modified data and displacing unmodified data with optimal performance.", "The performance is faster because this garbage data does not actually need to be fetched from memory, rather, the memory controller need only instantly reply.", "The performance is also faster because only actually modified data is written to memory from cache, while clean data is simply instantly discarded.", "Further, for the case where the total size of the “Put/Get Window” exceeds, perhaps greatly, the size of the processor's cache, cleaning the cache in this manner provides an upper bound on the total amount of work that is required to ensure that no data from the communication area remains in the cache.", "For example, assuming a fully associative cache, if the communication area is 16 Megabytes (common occurrence), traditional cache flush techniques would require (16 MB/32 B per cache line equals) 524,288 DCBF instructions, while the algorithm described here would require at most 1,024 DCBT instructions if the processor's cache was 32 Kilobytes in size with 32 byte cache lines.", "It may be noted that, independent of the above-described software algorithm, this hardware device is useful for computer systems in general which employ a Least Recently Used cache replacement policy.", "Two specific software embodiments are described below.", "The first embodiment may be preferred if the size of the message being received is smaller than the size of L1, while the second embodiment may be preferred if the size of the message received is larger than L1.First Embodiment If the size of the message being received is smaller than the size of L1.In this case, the invention makes use of a software instruction termed “data cache block flush and invalidate” (DCBF), whereby a contiguous range of memory is written from L1 back to the common memory if it has been modified in L1.DCBF is a PowerPC BookE instruction; similar instructions exist for other processors.", "At the same time, the data in the cache is marked as invalid, and cannot be used without reloading contents of the common memory.", "A DCBF is issued for every line in the address window.", "More specifically, when the window is opened for puts or gets, software, (in the communication library) instructs the receiving processor (the Compute Processor in our dual processor node) to flush the contents of L1 in the address window, as described above.", "This simple operation insures that the data in common memory are the same as the data in the compute processor's cache, and further, because of the invalidate, allows an opportunity for the I/O processor to change the contents of the common memory, because the entire contents of L1 is replaced quickly from the reserved area.", "The software then instructs the I/O processor to proceed until all expected messages arrive.", "The software then allows the computer processor to continue to process instructions, and closes the put/get window using a global synchronization operation such as the global barrier described in copending application copending application D#15272 Global Interrupt and Barrier Networks.", "Second Embodiment If the size of the message received is larger than the size of L1.In this case, the invention makes use of an instruction termed “data cache block zero” (DCBZ), on a reserved contiguous physical address range equal in size to L1.DCBZ creates a new cache line with contents of zero.", "If a new cache line is not available, then another cache line in L1 (for example, the least recently used line), has its data written back to the common memory, and is then zero'ed with the address given by the DCBZ instruction.", "DCBZ is a PowerPC BookE instruction; similar instructions exist for other processors.", "The software executes DCBZ to each line of the reserved area consecutively, where a line of the reserved area is equal in size to a cache line and like-aligned.", "This causes all lines in the L1 to be flushed, i.e., all modified lines are written back to common memory, because the entire contents of L1 is replaced quickly from the reserved area.", "The software then allows the compute processor to continue to process instructions, and closes the put/get window using a global synchronization operation such as the global barrier described in copending application copending application D#15272 Global Interrupt and Barrier Networks.", "It may be notes that the reserved physical space need not exist in physical memory, only that accesses to the space must not cause access violations.", "All writes to this reserved memory space must be acknowledged by the memory controller.", "All reads to this reserved space must immediately return an arbitrary (i.e.", "“garbage”) value to the requesting processor's L1.Note further that such an apparatus also provides the most efficient means for an un-privileged (a.k.a.", "user-space) program to flush and invalidate the entire contents of the L1 cache.", "It may also be noted that if DCBF instructions are slower than DCBZ, then the operating system may use the DCBZ instruction for messages smaller then L1 and vice-versa.", "Using this invention, the I/O Processor need not flush its cache at all if the communication memory space is marked write-through to its L1 cache.", "The making of the above-mentioned global “and” in a short interval of time, which allows the put/get window to be made temporarily narrow, is discussed in detail in related patent application no.", "(Attorney Docket: 15258).", "While it is apparent that the invention herein disclosed is well calculated to fulfill the objects previously stated, it will be appreciated that numerous modifications and embodiments may be devised by those skilled in the art, and it is intended that the appended claims cover all such modifications and embodiments as fall within the true spirit and scope of the present invention." ] ]	Patent_10468995
[ [ "Omega aminoalkylamides of R-2 aryl propionic acids as inhibitors of the chemotaxis of polymorphonucleate and mononucleate cells", "(R)-2-Arylpropionamide compounds of formula (I) are described.", "The process for their preparation and pharmaceutical preparations thereof are also described.", "The 2-Arylpropionamides of the invention are useful in the prevention and treatment of tissue damage due to the exacerbate recruitment of polymorphonuclear leukocytes (leukocytes PMN) and of monocytes at the inflammatory sites.", "In particular, the invention relates to the R enantiomers of omega-aminoalkylamides of 2-aryl propionic acids, of formula (I), for use in the inhibition of the chemotaxis of neutrophils and monocytes induced by the C5a fraction of the complement and by other chemotactic proteins whose biological activity is associated with activation of a 7-TD receptor.", "Selected compounds of formula (I) are dual inhibitors of both the C5a-induced chemotaxis of neutrophils and monocytes and the IL-8-induced chemotaxis of PMN leukocytes.", "The compounds of the invention are used in the treatment of psoriasis, ulcerative cholitis, glomerular nephritis, acute respiratory insufficiency, idiopathic fibrosis, rheumatoid arthritis and in the prevention and the treatment of injury caused by ischemia and reperfusion." ], [ "1.", "(R)-2-Aryl-propionamide compounds of formula (I).", "and pharmaceutically acceptable salts thereof, wherein Ar represents a substituted or non-substituted aryl group; R represents hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, optionally substituted by a CO2R3 group, wherein R3 represents hydrogen or a linear or branched C1-C6 alkyl group or a linear or branched C2-C6 alkenyl group; X represents: linear or branched C1-C6 alkylene, C4-C6 alkenylene, C4-C6 alkynylene, optionally substituted by a CO2R3 group or by a CONHR4 group wherein R4 represents hydrogen, linear or branched C2-C6 alkyl or an OR3 group, R3 being defined as above; a (CH2)m—B—(CH2)n, group, optionally substituted by a CO2R3 or CONHR4 group, as defined above, wherein B is an oxygen or sulfur atom, m is zero or an integer from 2 to 3 and n is an integer from 2 to 3; or B is a CO, SO or CONH group, m is an integer from 1 to 3 and n is an integer from 2 to 3; or X together with the nitrogen atom of the omega-amino group to which it is bound and with the R1 group forms a non-aromatic nitrogen containing 3-7 membered heterocyclic, monocyclic or polycyclic ring wherein the nitrogen atom has a substituent Rc, where Rc represents hydrogen, C1-C4 allyl, C1-C4 hydroxyalkyl, C1-C4 acyl, substituted or non-substituted phenyl, diphenylmethyl; R1 and R2 are independently hydrogen, linear or branched C1-C6 alkyl, optionally interrupted by an O or S atom, a C3-C7 cycloalkyl C3-C6 alkenyl, C3-C6-alkynyl, aryl-C1-C3-alkyl, hydroxy-C2-C3-alkyl group; or R1 and R2 together with the N atom to which they are bound, form a nitrogen containing 3-7 membered heterocyclic ring of formula (II) wherein Y represents a single bond, CH2, O, S, or a N-Rc group as defined above and p represents an integer from 0 to 3; or, R1 being as defined above, R2 represents a group of formula (III): wherein Ra is hydrogen and Rb is hydrogen, hydroxy, C1-C4-alkyl or an NRdRe group wherein Rd and Re are independently hydrogen, C1-C4-alkyl or phenyl; or Ra and Rb, together with the nitrogen atoms to which they are bound, form a 5-7 membered heterocyclic ring, monocyclic or fused with a benzene, pyridine or pyrmidine ring; with the proviso that when Ar is a 4-diphenyl residue and X is an ethylene or propylene residue, R1 and R2 are not ethyl; with the further proviso that, when Ar is a 4-(2-fluoro)diphenyl residue, and X is butylene substituted by a CO2H group, Ra and Rb are not hydrogen, or R1 and R2 are not hydrogen; and with the further proviso that, when Ar is phenyl and X is butylene, R1 and R2 together are not a N-(2-methoxy phenyl) piperazine.", "2.", "(R)-2-Aryl-propionamide compounds of formula (I).", "and pharmaceutically acceptable salts thereof, wherein Ar represents a substituted or non-substituted aryl group; R represents hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, optionally substituted by a CO2R3 group, wherein R3 represents hydrogen or a linear or branched C1-C6 alkyl group or a linear or branched C2-C6 alkenyl group; X represents: linear or branched C1-C6 alkylene, C4-C6 alkenylene, C4-C6 alkynylene, optionally substituted by a CO2R3 group Or by a CONHR4 group wherein R4 represents hydrogen, linear or branched C2-C6 alkyl or an OR3 group, R3 group, R3 being defined as above; a (CH2)m—B—(CH2)n, group, optionally substituted by a CO2R3 or CONHR4 group, as defined above, wherein B is an oxygen or sulfur atom, m is zero or an integer from 2 to 3 and n is an integer from 2 to 3; or B is a CO, SO or CONH group, m is an integer from 1 to 3 and n is an integer from 2 to 3; or X together with the nitrogen atom of the omega-amino group to which it is bound and with the R1 group forms a non-aromatic nitrogen containing 3-7 membered heterocyclic, monocyclic or polycyclic ring wherein the nitrogen atom has a substituent Rc, where Rc represents hydrogen, C1-C4 alkyl, C1-C4 hydroxylalyl, C1-C4 acyl, substituted or non-substituted phenyl, diphenylmethyl; R1 and R2 are independently hydrogen, linear or branched C1-C6 alkyl optionally interrupted by an O or S atom, a C3-C7 cycloalkyl, C3-C6 alkenyl, C3-C6-alkyl, aryl-C1-C3-alkyl, hydroxy-C2-C3-alkyl group; or R1 and R2 together with the N atom to which they are bound, form a 3-7 membered nitrogen heterocyclic ring of formula (II) wherein Y represents a single bond, CH2, O, S, or a N-Rc group as defined above and p represents an integer from 0 to 3; or, R1 being as defined above, R2 represents a group of formula (III): wherein Ra is hydrogen and Rb is hydrogen, hydroxy, C1-C4-alkyl or an NRdRe group wherein Rd and Re, are each independently, hydrogen, C1-C4-alkyl or phenyl; or Ra and Rb, together with the nitrogen atoms to which they are bound, form a 5-7 membered heterocyclic ring, monocyclic or fused with a benzene, pyridine or pyrmidine ring, with the proviso that Ar is not a dihydropyrrole residue; leukocytes and monocytes.", "3.Compounds according to claim 1, wherein Ar is chosen from: a) an Ara, mono- or poly-substituted aryl group of (±)2-aryl-propionic acids selected from alminoprofen, benoxaprofen, carprofen, fenbufen, fenoprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, loxoprofen, R-naproxen, pirprofen and its dehydro and dihydro derivatives, pranoprofen, surprofen, tiaprofenic acid, zaltoprofen; b) an aryl-hydroxymethyl-aryl group of formula (IVa), both as diastereoisomeric mixture and as single S′ and/or R′ 0diastereoisomers wherein, when Ar2 is phenyl Ar1 is selected from the group consisting of phenyl and thien-2-yl while when Ar1 is phenyl, Ar2 is selected from the group consisting of phenyl, 4-thienyl pyridyl; c) an aryl of formula (IVb): Φ-Arb (IVb) wherein Arb is a phenyl mono- and poly-substituted by hydroxy, mercapto, C1-C3-alcoxy, C1-C3-alkylthio, chlorine, fluorine, trifluoromethyl, nitro, amino, C1-C7-acylamino optionally substituted; and Φ is hydrogen; a linear or branched C1-C5 alkyl, C2-C5-alkenyl or C2-C5-alkynyl residue optionally substituted by C1-C3-alkoxycarbonyl, substituted or non-substituted phenyl, 2-, 3- or 4-pyridyl, quinolin-2-yl; a C3-C6-cycloalkyl; 2-furyl; 3-tetrahydrofuryl; 2-thiophenyl; 2-tetrahydrothiophenyl or a residue of formula (IVc) A-(CH2)q- (IVc) wherein A is a C1-C5-dialkylamino group, a C1-C8-(alcanoyl, cycloalcanoyl, arylalcanoyl)-C1-C5-alkylamino group, for example dimethylamino, diethylamino, methyl-N-ethyl-amino, acetyl-N-methyl-amino, pivaloyl-N-ethyl-amino; a nitrogen containing 5-7 membered monocyclic ring optionally containing one or two double bonds and optionally another heteroatom separated by at least 2 carbon atoms from the N atom, so as to form, for example, a 1-pyrrolidino, 2,5-dihydro-pyrrol-1-yl, 1-pyrrol, 1-piperidino, 1-piperazino-4-non-substituted or 4-substituted (methyl, ethyl, 2-hydroxyethyl, benzyl, benzyhydril or phenyl), 4-morpholino, 4-3,5-dimethyl-morpholino, 4-thiomorpholino group; or alternatively, a residue of formula (IVd) wherein Rg is hydrogen, C1-C3-alkyl or the residue of a C1-C3-alcanoic acid; q is zero or the integer 1, d) a 2-(phenylamino)-phenyl of formula (IVe): wherein the substituents P1 and P2 indicate that the two phenyl groups bear, each independently, mono- or poly-substitutions with C1-C4-alkyl, C1-C3-alcoxy groups, chlorine, fluorine and/or trifluoromethyl.", "4.Compounds according to any of claims 1 to 3, wherein Ar is chosen from: 4-isobutylphenyl, 4-(2-methyl)allyl-phenyl, 3-phenoxyphenyl, 3-benzoyl-phenyl, 3-acetyl-phenyl, the single diastereoisomers (R), (S) and the diastereoisomeric mixture (R,S) of 3-C6H5—CH(OH)-phenyl, 3-CH3—CH(OH)-phenyl, 5-C6H5-CH(OH)-thienyl, 4-thienyl-CH(OH)-phenyl, 3-(pyrid-3-yl)-CH(OM-phenyl, 5-benzoyl-thien-2-yl, 4 thienoyl-phenyl, 3-nicotinoyl-phenyl, 2-fluoro-4-phenyl, 6-metoxy-2-naphthyl, 5-benzoyl-2-acetoxy-phenyl and 5-benzoyl-2-hydroxy-phenyl.", "5.Compounds according to any of claims 1 to 3, wherein Ar is phenyl 3-substituted by a group selected form: isoprop-1-en-1-yl, isopropyl, pent-2-en-3-yl, pent-3-yl, 1-phenylethylen-1-yl, α-methylbenzyl.", "6.Compounds according to claim 3, wherein the Ar group of formula IVc is selected from: 4-(pyrrolidin-1-yl)-methyl-phenyl, 3-chloro-4(pyrrolidin-1-yl)-methyl-phenyl, 3-chloro-4-(2,5-dihydro-1-H-pyrrol-1-yl)-methyl-phenyl, 3 chloro-4-(thiomorpholin-4-yl)phenyl, 3-chloro-piperidin-1-yl)-phenyl, 4-((N-ethyl-N-quinolin-2-yl-methylamino)-methyl)phenyl, 3-chloro-4-(morpholin-4-yl)-phenyl.", "7.Compounds according to claim 3, wherein the Ar group of formula IVe is selected from: 2-(2,6-dichloro-phenyl-amino)-phenyl, 2-(2,6-dichloro-phenyl-amino)-5-chloro-phenyl, 2-(2,6-dichloro-3-methyl-phenyl-amino)-phenyl, 2-(3-trifluoromethyl-phenyl-amino)-phenyl.", "8.Compounds according to any of claims 1 to 7, wherein: R is hydrogen, X is: a linear alkylene optionally substituted at C1 by a —CO2R3 group as defined above; a linear alkylene optionally substituted at C1 by a —CONHR4 group wherein R4 is OH; 2-butynylene, cis-2-butenylene, trans-2-butenylene; 3-oxa-pentylene, 3-thio-pentylene, 3-oxa-hexylene, 3-thio-hexylene; (CH2)m—CO—NH—(CH2)n-wherein m and n are each independently an integer from 2 to 3; (CHR′)—CONH—(CH2)n wherein n is an integer from 2 to 3 and R″ is a methyl, in absolute configuration R or S; or X, together with the N atom of the omega-amino group, forms a nitrogen containing cycloaliphatic ring selected from 1-methyl-piperidin-4-yl and 1,5-tropan-3-yl.", "9.Compounds according to any of claims 1 to 8, wherein NR1R2 represents an NH2 group, dimethylamino, diethylamino, diisopropylamino, 1-piperidinyl, 4-morpholyl, 4-thiomorpholyl or, R1 and R2 together form a residue of guanidine, aminoguanidine, hydroxyguanidine, 2-amino-3,4,5,6-tetrahydropyrimidyl, 2-amino-3,5-dihydro-imidazolyl.", "10.Compounds according to any of claims 1 to 9, selected from: (R)-2-[(4-isobutyl)phenyl]-N-(3-dimethylaminopropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(4-dimethylaminobutyl)-propionamide hydrochloride; (R)-2-[(4-isobutyl)phenyl]-N-(3-N-morpholinylpropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(2-dimethylaminoethyl)propionamide; (R)-2-[(4-isobutyl)phenyl)-propionyl]-N-[2-(4methyl-piperazin-1-yl)ethyl]propionamide; (R)-N-(exo-8-methyl-8-aza-bicyclo[3,2,1]oct-3-yl)-2-[(4-isobutylphenyl)-propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(3-N-thiomorpholinylpropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[4-(N′-methyl)piperidinyl]propionamide hydrochloride; (R),(S′)-2-[(4-isobutyl)phenyl]-N-(1-carboxy-2-dimethylaminoethyl)-propionamide; (R),(S′)-2-[(4-isobutyl)phenyl]-N-[(1-carboxy-4-piperidin-1-yl)butyl]propionamide; (R),(S′)-2-[(4-isobutyl)phenyl]-N-(1-carboxy-4-aminobutyl)propionamide; (R)-2-(4-isobutyl)phenyl-N-[2-dimethylaminoethyl)aminocarbonylmethyl)]propionamide hydrochloride; 2-(2,6-dichlorophenylamino)-phenyl-N-(3-dimethylaminopropyl)propionamide; (R),(R′,S′)-3-[3-(α-methyl)benzyl]phenyl-N-(3-dimethylaminopropyl)-propionamide; (R-2-[(3-isopropyl)phenyl]-N-(3-dimethylaminopropyl)propionamide; (R)2-[3-pent-3-yl)phenyl]-N-(3-dimethylaminopropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(3-guanidylpropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[(3-hydroxy-guanidyl)propyl]propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[(3-amino-guanidyl)propyl]propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[3-(2-amino-2-imidazoline) propyl]propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[N-methyl-N-(2-hydroxyethyl)aminoethoxy]propionamide; (R),(S′)-2-[(4-isobutyl)phenyl]-N-[1-carboxy-5-aminopentyl]propionamide.", "11.", "(R)2-(4-isobutylphenyl)-N-(3-dimethylaminopropyl)propionamide hydrochloride 12.", "(R)2-(4-isobutylphenyl)-N-3-(1-piperidinylpropyl)propionamide hydrochloride 13.Compounds according to any of claims 1 to 9 wherein R1 and R2 are groups different from hydrogen.", "14.Compounds according to claim 13, wherein X is a linear C2-C4 alkylene.", "15.Process for the preparation of (R)-2-aryl propionamide compounds of formula (I) according to claim 1 wherein Ar, X, R, R1 and R2 have the meanings as defined in claim 1, comprising reaction of an activated form of an R-2-arylpropionic acid of formula (V) with an amine of formula (VI) wherein AT is a residue activating the carboxy group of the R-2-arylpropionic acid; with the proviso that Ar is not substituted by a dihydropyrrole residue.", "16.Compounds according to claim 1, for use as medicaments.", "17.Compounds according to claim 2, for use in the treatment of psoriasis, pemphigus and pemphigoid, rheumatoid arthritis, intestinal chronic inflammatory patologies including ulcerative colitis, acute respiratory distress syndrome, idiopathic fibrosis, cystic fibrosis, chronic obstructive pulmonary disease and glomerulonephritis.", "18.Compounds according to claim 2 for use in the prevention and the treatment of injury caused by ischemia and reperfusion.", "19.Compounds according to claims 13 and 14, for use as inhibitors of both the C5a-induced chemotaxis of polymorphonucleate leukocytes and monocytes, and the interleukin 8-induced chemotaxis of polymorphonucleate leukocytes.", "20.Pharmaceutical compositions containing a compound according to any of claims 1 to 14, in admixture with a suitable carrier thereof." ], [ "<SOH> INTRODUCTION AND BACKGROUND OF THE INVENTION <EOH>Animal studies show that some aminoalkylester and amide prodrugs of racemic ibuprofen and naproxen, in particular some N-(3-diethylaminopropyl)amides, exhibit analgesic and antiinflammatory activity significantly better than the parent compounds, even though “in vitro” they have been found to be poor inhibitors of the synthesis of prostaglandins.", "All these prodrugs, except a glycine amide, have also been found to be significantly less irritating to the gastric mucosa than their precursor free acids.", "(Shanbhag V R et al., J. Pharm.", "Sci., 81, 149, 1992 and references 8-19) therein cited.", "Piketoprofen [(±)2-(3-benzoylphenyl)-N-(4-methyl-2-pyridinyl)propionamide] and Amtolmetin Guacil (also named guaiacol ester of tolmetinglycinamide, Eufans) are further examples of non steroidal antiinflammatory (NSAI) prodrugs in current therapeutic use.", "Moderate antiinflammatory activity, minor side effects and good gastro-intestinal tolerance are reported for a series or N-[2-(1-piperidinyl)propyl]amides of some NSAI drugs such as racemic ibuprofen, indomethacin, p-chlorobenzoic acid, acetylsalicyclic acid, diacetylgentisic acid and adamantane-1-carboxylic acid (Nawladonski F. and Reewuski, Pol.", "J.", "Chem., 52, 1805, 1978).", "Other amides of racemic 2-arylpropionic acids have been disclosed by S. Biniecki et al., [PL 114050 (31.01.1981)], H. Akguen et al., [Arzneim-Forsch., 46, 891, 1986] and by G. L. Levitt et al., [Russ.", "J. Org.", "Chem., 34, 346, 1998].", "Anti-inflammatory and analgesic potencies “in vivo”, comparable and sometimes greater than those of the precursor free acids, along with decreased number of gastric lesions, have been reported for some N-3-[(1-piperidinyl)propyl]amides of racemic ketoprofen and flurbiprofen and for certain Mannich bases obtained reacting their amides with formaldehyde and secondary amines such as morpholine, piperidine, dicyclohexylamine, dimethylamine, diethylamine, dibenzylamine and dibutylamine (N. Kawathekar et al., Indian J. Pharm.", "Sci., 60, 346, 1998).", "International patent application, WO 00/40088, has recently reported that the mere conversion to an amide derivative of a 2-arylacetic and/or 2-arylpropionic acid is enough to change a selective COX-1 inhibitor into a COX-2 selective inhibitor which explains the decreased gastrolesivity of said amides, for a long time believed to be only NSAI prodrugs.", "In the past, inhibition of the cyclooxygenase enzymes was known to be proper of the S enantiomer of 2-arylpropionic acids alone, joined together with the portion of R CoA-thioester suffering bioconversion “in vivo”.", "Therefore, the poor correlation between enzymatic inhibition “in vitro” and analgesic effects “in vivo” found for certain R,S 2-arylpropionic acids (Brune K. et al., Experientia, 47, 257, 1991) has induced to presume that alternative mechanisms, such as inhibition of transcription of the kB-nuclear transcription factor (NF-kB) and/or inhibition of neutrophil chemotaxis induced by interleukin 8 (IL-8), can be operating.", "R enantiomers of flurbiprofen, ketoprofen, naproxen, thiaprofen and phenoprofen are, in fact, disclosed in WO 00/40088 as inhibitors of the NF-kB transcription factor activation and claimed to be useful in the treatment of NF-kB dependent diseases (asthma, tumor, shock, Crohn'disease and ulcerative colitis, arteriosclerosis, etc).", "IL-8 is an important mediator of inflammation and has been shown to be a potent chemotactic/cell activator for polymorphonucleate neutrophils and basophils (PMNs), and T lymphocytes.", "Cellular sources of IL-8 include monocytes, PMNs, endotelial cells, epithelial cells, and keratinocytes when stimulated by factors such as lipopolysaccaride, IL-1 and TNF-α.", "On the other hand, the complement fragment C5a, in addition to being a direct mediator of inflammation, has been found to induce both IL-8 synthesis and high level of IL-8 release from monocytes.", "The quantity of IL-8 recovered from C5a activated monocytes in peripheral blood mononuclear cells is up to 1,000 fold greater than that released from comparable numbers of PMNs under similar conditions.", "Therefore IL-8 released from C5a-activated monocytes may play a significant role in expanding and prolonging cellular infiltration and activation at the sites of infection, inflammation, or tissue injury (Ember J.", "A. et al., Am.", "J.", "Pathol., 144, 393, 1994).", "In response to immunologic and infective events, activation of the complement system mediates amplification of inflammatory response both via direct membrane action and via release of a series of peptide fragments, generally known as anaphylatoxins, generated by enzymatic cleavage of the C3, C4 and C5 complement fractions.", "These peptides include C3a, C4a, both made of 77 aminoacids; in turn, C5 convertase cleaves the C5 complement fraction to give the glycoprotein C5a of 74 aminoacids.", "Anaphilatoxins contribute to the spreading of the inflammatory process by interaction with individual cell components; their common properties are cellular release of vasoactive amines and lysosomal enzymes, contraction of smooth muscle and increased vascular permeability.", "Moreover, C5a causes chemotaxis and aggregation of neutrophils, stimulates the release of leukotrienes and of oxidized oxygen species, induces the transcription of IL-1 in macrophages and the production of antibodies.", "The C5a peptide fragment of the complement has been defined as the “complete” pro-inflammatory mediator.", "On the contrary, other inflammatory mediators such as selected cytokines (IL-8, MCP-1 and RANTES, for example) are highly selective towards self-attracted cells, while histamine and bradykinin are only weak chemotactic agents.", "Convincing evidences support the involvement of C5a, “in vivo”, in several pathological conditions including ischemia/reperfusion, autoimmune dermatitis, membrane-proliferative idiopathic glomerulonephritis, airway iperresponsiveness and chronic inflammatory diseases, ARDS and COPD, Alzheimer'disease, juvenile rheumatoid arthritis (N. P. Gerard, Ann.", "Rev.", "Immunol., 12, 755, 1994).", "In view of the neuro-inflammatory potential of C5a/C5a-desArg generated by both local complement production and amyloid activation joined with astrocyte and microglia chemotaxis and activation directly induced by C5a, complement inhibitors have been proposed for the treatment of neurological diseases such as Alzheimer'disease (McGeer & McGeer P. L., Drugs, 55, 738, 1998).", "Therefore, the control of the local synthesis of complement fractions is considered of high therapeutic potential in the treatment of shock and in the prevention of rejection (multiple organ failure and hyperacute graft rejection) (Issekutz A. C. et al., Int.", "J. Immunopharmacol, 12, 1, 1990; Inagi R. et at., Immunol.", "Lett., 27, 49, 1991).", "More recently, inhibition of complement fractions has been reported to be involved in the prevention of native and transplanted kidney injuries taking account of complement involvement in the pathogenesis of both chronic interstitial and acute glomerular renal injuries.", "(Sheerin N. S. & Sacks S. H., Curr.", "Opinion Nephrol.", "Hypert., 7, 395, 1998).", "Genetic engineering and molecular biology studies led to the cloning of complement receptors (CRs) and to the production of CRs agonists and antagonists.", "The recombinant soluble receptor CR1 (sCR1), that blocks enzymes activating C3 and C5, has been identified as a potential agent for the suppression of C activation on ischemia/reperfusion injury (Weisman H. F. et al., Science, 239, 146, 1990; Pemberton M. et al., J.", "Immunol., 150, 5104, 1993).", "The cyclic peptide F-[OPdChWR], is reported to antagonize the C5a binding to its CD38 receptor on PMNs and to inhibit C5a-dependent chemotaxis and cytokine production by macrophages and rat neutropenia induced by C5a and LPS stimulation (Short A. et al., Br.", "J.", "Pharmacol., 126, 551, 1999; Haynes D. R. et al., Biochem.", "Pharmacol., 60, 729, 2000).", "Both C5aR antagonist CGS 27913 and its dimer CGS 32359 are reported to inhibit, “in vitro”, C5a binding to neutrophil membranes, intracellular Ca 2+ mobilization, lysozyme release, neutrophil chemotaxis and dermal edema in rabbits (Pellas T. C. et al., J.", "Immunol., 160, 5616, 1998).", "Finally, selection from phage libraries with the “phage display” technique has led to the isolation of a specific C5aR antagonist able to decrease inflammatory responses in diseases mediated by immuno-complexes and in ischemia and reperfusion injuries (Heller T. et al., J.", "Immunol., 163, 985, 1999).", "Despite their therapeutic potential, only two of the above discussed C5a antagonists have demonstrated activity “in vivo”; furthermore, their use is therapeutically limited by their peptidic nature.", "(Pellas T. C., Wennogle P., Curr.", "Pharm.", "Des., 10, 737, 1999).", "Characteristic neutrophil accumulation can be observed in some pathologic conditions, for example in the highly inflamed and therapeutically recalcitrant areas of psoriatic lesions.", "Neutrophils are chemotactically attracted and activated by the sinergistic action of chemokines, IL-8 and Gro-a released by the stimulated keratinocytes, and of the C5a/C5a-desArg fraction produced via the alternative complement pathway activation (T. Terui et al., Exp.", "Dermatol., 9, 1, 2000).", "In many circustances it is, therefore, highly desirable to combine inhibition of the chemotaxis induced by C5a and inhibition of the chemotaxis induced by IL-8 in one single agent.", "Non-peptidic antagonists of complement fractions have also been prepared, for example substituted-4,6-diamino-quinolines.", "In particular, [N,N″-bis-(4-amino-2-methyl-6-quinolyl)]urea and [6-N-2-chlorocynnamoyl)-4,6-diamino-2-methylquinoline] have been found selective C5R antagonists, their IC 50 ranging between 3.3 and 12 μg/mL (Lanza T. J. et al., J. Med.", "Chem., 35, 252, 1992).", "Some serine-protease inhibitors [nafamostat mesilate (FUT 175) and certain analogs] have been recently reported to be inhibitors of both complement activation and C3a/C5a production (Ueda N. et al., Inflammation Res.", "49, 42, 2000).", "U.S. Pat.", "No.", "6,069,172 reports the use of pharmaceutical formulations of R(−)ketoprofen ammonium salts for the inhibition of neutrophil chemotaxis induced by IL-8.WO 00/24710 discloses N-acylsulfonamides of R(−)2-aryl-propionic acids as inhibitors of IL-8 dependent polymorphonucleate leukocytes chemotaxis.", "Two recent patent applications [WO 01/58852 and WO 01/79189] disclose certain R-2-aryl-propionamides and R-2-(aminophenyl)propionamides useful for preventing leukocyte activation induced by IL-8.We have recently observed that the mere formal reduction of the hetero-aromatic ring of certain R 2-aryl-N-(pyridinyl)propionamides causes marked loss of potency (1 or 2 logarithmic order) in the capacity to inhibit PMN neutrophil chemotaxis induced by IL-8.Unexpectedly, the related R 2-aryl-N-(piperidinyl)propionamides have been found to be potent inhibitors of chemotaxis of human PMN leukocytes and monocytes induced by the C5a fraction of the complement.", "These unexpected findings have originated a novel family of omega-aminoalkylamides of R-2-aryl-propionic acids which are able to inhibit the chemotactic activity induced by C5a and other chemotactic proteins whose biological activity is associated with activation of a 7-membered-domain receptor (7-TD) homologous to the receptor of C5a (for example, the C3a receptor and the CXCR2 receptor; Neote K. et al., Cell, 72, 415, 1993; Tometta M. A., J.", "Immunol., 158, 5277, 1997).", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "<SOH> BRIEF DESCRIPTION OF THE INVENTION <EOH>It is the object of the present invention a novel class of omega-aminoalkylamides of R-2-aryl-propionic acids and pharmaceutical compositions containing them.", "The position “omega” in the alkyl chain refers to the furthest carbon atom starting from the N atom of the amide group to which said alkyl is linked.", "Such amides are useful in the inhibition of the chemotactic activation induced by C5a and by other chemotactic proteins whose biological activity is associated with the activation of 7-transmembrane domains (7-TD) receptors homologous to the C5a receptor.", "In particular such amides are useful in the inhibition of the chemotactic activation of polymorphonucleate leukocytes, monocytes and lymphocytes T induced by the fraction C5a of the complement and in the treatment of pathologies related to said activation." ], [ "The present invention relates to omega-aminoalkylamides of (R)2-aryl-propionic acids as inhibitors of the chemotaxis of polymorphonucleate and mononucleate cells.", "In particular, the invention relates to inhibitors of the C5a—induced chemotaxis of polymorphonucleate leukocytes and monocytes, which are used in the treatment of pathologies including psoriasis, rheumatoid arthritis and injury caused by ischemia and reperfusion.", "INTRODUCTION AND BACKGROUND OF THE INVENTION Animal studies show that some aminoalkylester and amide prodrugs of racemic ibuprofen and naproxen, in particular some N-(3-diethylaminopropyl)amides, exhibit analgesic and antiinflammatory activity significantly better than the parent compounds, even though “in vitro” they have been found to be poor inhibitors of the synthesis of prostaglandins.", "All these prodrugs, except a glycine amide, have also been found to be significantly less irritating to the gastric mucosa than their precursor free acids.", "(Shanbhag V R et al., J. Pharm.", "Sci., 81, 149, 1992 and references 8-19) therein cited.", "Piketoprofen [(±)2-(3-benzoylphenyl)-N-(4-methyl-2-pyridinyl)propionamide] and Amtolmetin Guacil (also named guaiacol ester of tolmetinglycinamide, Eufans) are further examples of non steroidal antiinflammatory (NSAI) prodrugs in current therapeutic use.", "Moderate antiinflammatory activity, minor side effects and good gastro-intestinal tolerance are reported for a series or N-[2-(1-piperidinyl)propyl]amides of some NSAI drugs such as racemic ibuprofen, indomethacin, p-chlorobenzoic acid, acetylsalicyclic acid, diacetylgentisic acid and adamantane-1-carboxylic acid (Nawladonski F. and Reewuski, Pol.", "J.", "Chem., 52, 1805, 1978).", "Other amides of racemic 2-arylpropionic acids have been disclosed by S. Biniecki et al., [PL 114050 (31.01.1981)], H. Akguen et al., [Arzneim-Forsch., 46, 891, 1986] and by G. L. Levitt et al., [Russ.", "J. Org.", "Chem., 34, 346, 1998].", "Anti-inflammatory and analgesic potencies “in vivo”, comparable and sometimes greater than those of the precursor free acids, along with decreased number of gastric lesions, have been reported for some N-3-[(1-piperidinyl)propyl]amides of racemic ketoprofen and flurbiprofen and for certain Mannich bases obtained reacting their amides with formaldehyde and secondary amines such as morpholine, piperidine, dicyclohexylamine, dimethylamine, diethylamine, dibenzylamine and dibutylamine (N. Kawathekar et al., Indian J. Pharm.", "Sci., 60, 346, 1998).", "International patent application, WO 00/40088, has recently reported that the mere conversion to an amide derivative of a 2-arylacetic and/or 2-arylpropionic acid is enough to change a selective COX-1 inhibitor into a COX-2 selective inhibitor which explains the decreased gastrolesivity of said amides, for a long time believed to be only NSAI prodrugs.", "In the past, inhibition of the cyclooxygenase enzymes was known to be proper of the S enantiomer of 2-arylpropionic acids alone, joined together with the portion of R CoA-thioester suffering bioconversion “in vivo”.", "Therefore, the poor correlation between enzymatic inhibition “in vitro” and analgesic effects “in vivo” found for certain R,S 2-arylpropionic acids (Brune K. et al., Experientia, 47, 257, 1991) has induced to presume that alternative mechanisms, such as inhibition of transcription of the kB-nuclear transcription factor (NF-kB) and/or inhibition of neutrophil chemotaxis induced by interleukin 8 (IL-8), can be operating.", "R enantiomers of flurbiprofen, ketoprofen, naproxen, thiaprofen and phenoprofen are, in fact, disclosed in WO 00/40088 as inhibitors of the NF-kB transcription factor activation and claimed to be useful in the treatment of NF-kB dependent diseases (asthma, tumor, shock, Crohn'disease and ulcerative colitis, arteriosclerosis, etc).", "IL-8 is an important mediator of inflammation and has been shown to be a potent chemotactic/cell activator for polymorphonucleate neutrophils and basophils (PMNs), and T lymphocytes.", "Cellular sources of IL-8 include monocytes, PMNs, endotelial cells, epithelial cells, and keratinocytes when stimulated by factors such as lipopolysaccaride, IL-1 and TNF-α.", "On the other hand, the complement fragment C5a, in addition to being a direct mediator of inflammation, has been found to induce both IL-8 synthesis and high level of IL-8 release from monocytes.", "The quantity of IL-8 recovered from C5a activated monocytes in peripheral blood mononuclear cells is up to 1,000 fold greater than that released from comparable numbers of PMNs under similar conditions.", "Therefore IL-8 released from C5a-activated monocytes may play a significant role in expanding and prolonging cellular infiltration and activation at the sites of infection, inflammation, or tissue injury (Ember J.", "A. et al., Am.", "J.", "Pathol., 144, 393, 1994).", "In response to immunologic and infective events, activation of the complement system mediates amplification of inflammatory response both via direct membrane action and via release of a series of peptide fragments, generally known as anaphylatoxins, generated by enzymatic cleavage of the C3, C4 and C5 complement fractions.", "These peptides include C3a, C4a, both made of 77 aminoacids; in turn, C5 convertase cleaves the C5 complement fraction to give the glycoprotein C5a of 74 aminoacids.", "Anaphilatoxins contribute to the spreading of the inflammatory process by interaction with individual cell components; their common properties are cellular release of vasoactive amines and lysosomal enzymes, contraction of smooth muscle and increased vascular permeability.", "Moreover, C5a causes chemotaxis and aggregation of neutrophils, stimulates the release of leukotrienes and of oxidized oxygen species, induces the transcription of IL-1 in macrophages and the production of antibodies.", "The C5a peptide fragment of the complement has been defined as the “complete” pro-inflammatory mediator.", "On the contrary, other inflammatory mediators such as selected cytokines (IL-8, MCP-1 and RANTES, for example) are highly selective towards self-attracted cells, while histamine and bradykinin are only weak chemotactic agents.", "Convincing evidences support the involvement of C5a, “in vivo”, in several pathological conditions including ischemia/reperfusion, autoimmune dermatitis, membrane-proliferative idiopathic glomerulonephritis, airway iperresponsiveness and chronic inflammatory diseases, ARDS and COPD, Alzheimer'disease, juvenile rheumatoid arthritis (N. P. Gerard, Ann.", "Rev.", "Immunol., 12, 755, 1994).", "In view of the neuro-inflammatory potential of C5a/C5a-desArg generated by both local complement production and amyloid activation joined with astrocyte and microglia chemotaxis and activation directly induced by C5a, complement inhibitors have been proposed for the treatment of neurological diseases such as Alzheimer'disease (McGeer & McGeer P. L., Drugs, 55, 738, 1998).", "Therefore, the control of the local synthesis of complement fractions is considered of high therapeutic potential in the treatment of shock and in the prevention of rejection (multiple organ failure and hyperacute graft rejection) (Issekutz A. C. et al., Int.", "J. Immunopharmacol, 12, 1, 1990; Inagi R. et at., Immunol.", "Lett., 27, 49, 1991).", "More recently, inhibition of complement fractions has been reported to be involved in the prevention of native and transplanted kidney injuries taking account of complement involvement in the pathogenesis of both chronic interstitial and acute glomerular renal injuries.", "(Sheerin N. S. & Sacks S. H., Curr.", "Opinion Nephrol.", "Hypert., 7, 395, 1998).", "Genetic engineering and molecular biology studies led to the cloning of complement receptors (CRs) and to the production of CRs agonists and antagonists.", "The recombinant soluble receptor CR1 (sCR1), that blocks enzymes activating C3 and C5, has been identified as a potential agent for the suppression of C activation on ischemia/reperfusion injury (Weisman H. F. et al., Science, 239, 146, 1990; Pemberton M. et al., J.", "Immunol., 150, 5104, 1993).", "The cyclic peptide F-[OPdChWR], is reported to antagonize the C5a binding to its CD38 receptor on PMNs and to inhibit C5a-dependent chemotaxis and cytokine production by macrophages and rat neutropenia induced by C5a and LPS stimulation (Short A. et al., Br.", "J.", "Pharmacol., 126, 551, 1999; Haynes D. R. et al., Biochem.", "Pharmacol., 60, 729, 2000).", "Both C5aR antagonist CGS 27913 and its dimer CGS 32359 are reported to inhibit, “in vitro”, C5a binding to neutrophil membranes, intracellular Ca2+ mobilization, lysozyme release, neutrophil chemotaxis and dermal edema in rabbits (Pellas T. C. et al., J.", "Immunol., 160, 5616, 1998).", "Finally, selection from phage libraries with the “phage display” technique has led to the isolation of a specific C5aR antagonist able to decrease inflammatory responses in diseases mediated by immuno-complexes and in ischemia and reperfusion injuries (Heller T. et al., J.", "Immunol., 163, 985, 1999).", "Despite their therapeutic potential, only two of the above discussed C5a antagonists have demonstrated activity “in vivo”; furthermore, their use is therapeutically limited by their peptidic nature.", "(Pellas T. C., Wennogle P., Curr.", "Pharm.", "Des., 10, 737, 1999).", "Characteristic neutrophil accumulation can be observed in some pathologic conditions, for example in the highly inflamed and therapeutically recalcitrant areas of psoriatic lesions.", "Neutrophils are chemotactically attracted and activated by the sinergistic action of chemokines, IL-8 and Gro-a released by the stimulated keratinocytes, and of the C5a/C5a-desArg fraction produced via the alternative complement pathway activation (T. Terui et al., Exp.", "Dermatol., 9, 1, 2000).", "In many circustances it is, therefore, highly desirable to combine inhibition of the chemotaxis induced by C5a and inhibition of the chemotaxis induced by IL-8 in one single agent.", "Non-peptidic antagonists of complement fractions have also been prepared, for example substituted-4,6-diamino-quinolines.", "In particular, [N,N″-bis-(4-amino-2-methyl-6-quinolyl)]urea and [6-N-2-chlorocynnamoyl)-4,6-diamino-2-methylquinoline] have been found selective C5R antagonists, their IC50 ranging between 3.3 and 12 μg/mL (Lanza T. J. et al., J. Med.", "Chem., 35, 252, 1992).", "Some serine-protease inhibitors [nafamostat mesilate (FUT 175) and certain analogs] have been recently reported to be inhibitors of both complement activation and C3a/C5a production (Ueda N. et al., Inflammation Res.", "49, 42, 2000).", "U.S. Pat.", "No.", "6,069,172 reports the use of pharmaceutical formulations of R(−)ketoprofen ammonium salts for the inhibition of neutrophil chemotaxis induced by IL-8.WO 00/24710 discloses N-acylsulfonamides of R(−)2-aryl-propionic acids as inhibitors of IL-8 dependent polymorphonucleate leukocytes chemotaxis.", "Two recent patent applications [WO 01/58852 and WO 01/79189] disclose certain R-2-aryl-propionamides and R-2-(aminophenyl)propionamides useful for preventing leukocyte activation induced by IL-8.We have recently observed that the mere formal reduction of the hetero-aromatic ring of certain R 2-aryl-N-(pyridinyl)propionamides causes marked loss of potency (1 or 2 logarithmic order) in the capacity to inhibit PMN neutrophil chemotaxis induced by IL-8.Unexpectedly, the related R 2-aryl-N-(piperidinyl)propionamides have been found to be potent inhibitors of chemotaxis of human PMN leukocytes and monocytes induced by the C5a fraction of the complement.", "These unexpected findings have originated a novel family of omega-aminoalkylamides of R-2-aryl-propionic acids which are able to inhibit the chemotactic activity induced by C5a and other chemotactic proteins whose biological activity is associated with activation of a 7-membered-domain receptor (7-TD) homologous to the receptor of C5a (for example, the C3a receptor and the CXCR2 receptor; Neote K. et al., Cell, 72, 415, 1993; Tometta M. A., J.", "Immunol., 158, 5277, 1997).", "BRIEF DESCRIPTION OF THE INVENTION It is the object of the present invention a novel class of omega-aminoalkylamides of R-2-aryl-propionic acids and pharmaceutical compositions containing them.", "The position “omega” in the alkyl chain refers to the furthest carbon atom starting from the N atom of the amide group to which said alkyl is linked.", "Such amides are useful in the inhibition of the chemotactic activation induced by C5a and by other chemotactic proteins whose biological activity is associated with the activation of 7-transmembrane domains (7-TD) receptors homologous to the C5a receptor.", "In particular such amides are useful in the inhibition of the chemotactic activation of polymorphonucleate leukocytes, monocytes and lymphocytes T induced by the fraction C5a of the complement and in the treatment of pathologies related to said activation.", "DETAILED DESCRIPTION OF THE INVENTION The following paragraphs provide definitions of outstanding chemical moieties that make up the compounds according to the invention and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.", "The term “alkyl” refers to monovalent alkyl groups having preferably 1 to 6 carbon atoms.", "These terms are exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and the like.", "“Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g.", "phenyl) or multiple condensed rings (e.g.", "naphthyl).", "Preferred aryl include phenyl, biphenyl, naphthyl, phenantrenyl and the like.", "“Alkenyl” refers to alkenyl groups preferably having from 2 to 5 carbon atoms and having one or more sites of alkenyl unsaturation.", "Preferred alkenyl groups include ethenyl.", "(—CH═CH2), n-2-propenyl (allyl, —CH2CH═CH2) and the like.", "“Alkylene”, “Alkenylene”, Alkynylene” refer to groups disubstituted at both ends.", "Preferred groups include methylene, ethylene, propylene, and like.", "“Substituted or non-substituted”: unless otherwise constrained by the definition of the individual substituent, the above set out groups, like “alkyl”, “alkenyl”, “aryl” groups etc.", "can optionally be substituted with from 1 to 5 substituents selected from the group consisting of “C1-C6-alkyl”, “C1-C6-alkyl aryl”, “C1-C6-alkyl heteroaryl”, “C2-C6-alkenyl”, primary, secondary or tertiary amino groups or quarternary ammonium moieties, “acyl”, “acyloxy”, “acylamino”, “aminocarbonyl”, “alkoxycarbonyl”, “aryl”, “heteroaryl”, carboxyl, cyano, halogen, hydroxy, mercapto, nitro, sulfoxy, sulfonyl, alkoxy, thioalkoxy, trihalomethyl and the like.", "Within the framework of this invention, said “substitution” is meant to also comprise situations where neighbouring substituents undergo ring closure, in particular when vicinal functional substituents are involved, thus forming e.g.", "lactams, lactons, cyclic anhydrides or cycloalkanes, but also acetals, thioacetals, aminals formed by ring closure for instance in an effort to obtain a protective group.", "“Pharmaceutically acceptable salts” refers to salts or complexes of the below-identified compounds of formula I that retain the desired biological activity.", "Examples of such salts include, but are not restricted to, acid addition salts formed with inorganic acids (e.g.", "hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene sulfonic acid, naphthalene disulfonic acid, and poly-galacturonic acid.", "Examples of salts also include acid addition salts formed with inorganic bases such as sodium hydroxyde and with organic bases such as tromethamine, L-lysine, L-arginine and the like.", "The present invention provides (R)-2-aryl-propionamide compounds of formula (I), wherein Ar represents a substituted or non-substituted aryl group; R represents hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, optionally substituted by a CO2R3 group, wherein R3 represents hydrogen or a linear or branched C1-C6 alkyl group or a linear or branched C2-C6 alkenyl group; X represents: linear or branched C1-C6 alkylene, C4-C6 alkenylene, C4-C6 alkynylene, optionally substituted by a CO2R3 group or by a CONHR4 group wherein R4 represents hydrogen, linear or branched C2-C6 alkyl or an OR3 group, R3 being defined as above; a (CH2)m—B—(CH2)n, group, optionally substituted by a CO2R3 or CONHR4 group, as defined above, wherein B is an oxygen or sulfur atom, m is zero or an integer from 2 to 3 and n is an integer from 2 to 3; or B is a CO, SO or CONH group, m is an integer from 1 to 3 and n is an integer from 2 to 3; or X together with the nitrogen atom of the omega-amino Croup to which it is bound and with the R1 group forms a non-aromatic nitrogen containing 3-7 membered heterocyclic, monocyclic or polycyclic ring wherein the nitrogen atom has a substituent Rc, where Rc represents hydrogen, C1-C4 alkyl, C1-C4 hydroxylalkyl, C1-C4 acyl, substituted or non-substituted phenyl, diphenylmethyl; R1 and R2 are independently hydrogen, linear or branched C1-C6 alkyl, optionally interrupted by an O or S atom, a C3-C7 cycloalkyl, C3-C6 alkenyl, C3-C6-alkynyl, aryl-C1-C3-alkyl, hydroxy-C2-C3-alkyl group; or R1 and R2 together with the N atom to which they are bound, form a nitrogen containing 3-7 membered heterocyclic ring of formula (II) wherein Y represents a single bond, CH2, O, S, or a N-Rc group as defined above and p represents an integer from 0 to 3; or, R1 being as defined above, R2 represents a group of formula (III): wherein Ra is hydrogen and Rb is hydrogen, hydroxy, C1-C4-alkyl or an NRdRe group wherein Rd and Re, are each independently, hydrogen, C1-C4-alkyl or phenyl; or Ra and Rb, together with the nitrogen atoms to which they are bound, form a 5-7 membered heterocyclic ring, monocyclic or fused with a benzene, pyridine or pyrimidine ring; with the proviso that when Ar is a 4-diphenyl residue and X is an ethylene or propylene residue, R1 and R2 are not ethyl; with the further proviso that, when Ar is a 4-(2-fluoro)diphenyl residue, and X is butylene substituted by a CO2H group, Ra and Rb are not hydrogen; and with the further proviso that, when Ar is phenyl and X is butylene, R1 and R2 together are not a N-(2-methoxy phenyl)piperazine.", "In addition, the present invention further provides (R)-2-aryl-propionamide compounds of formula (I) wherein Ar represents a substituted or non-substituted aryl group; R represents hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, optionally substituted by a CO2R3 group, wherein R3 represents hydrogen or a linear or branched C1-C6 alkyl group or a linear or branched C2-C6 alkenyl group; X represents: linear or branched C1-C6 alkylene, C4-C6 alkenylene, C4-C6 alkynylene, optionally substituted by a CO2R3 group or by a CONHR4 group wherein R4 represents hydrogen, linear or branched C2-C6 alkyl or an OR3 group, R3 being defined as above; a (CH2)m—B—(CH2)n, group, optionally substituted by a CO2R3 or CONHR4 group, as defined above, wherein B is an oxygen or sulfur atom, m is zero or an integer from 2 to 3 and n is an integer from 2 to 3; or B is a CO, SO or CONH group, m is an integer from 1 to 3 and n is an integer from 2 to 3; or X together with the nitrogen atom of the omega-amino group to which it is bound and with the R1 group forms a non-aromatic nitrogen containing 3-7 membered heterocyclic, monocyclic or polycyclic ring wherein the nitrogen atom has a substituent Rc, where Rc represents hydrogen, C1-C4 alkyl, C1-C4 hydroxylalkyl, C1-C4 acyl, substituted or non-substituted phenyl, diphenylmethyl; R1 and R2 are independently hydrogen, linear or branched C1-C6 alkyl, optionally interrupted by an O or S atom, a C3-C7 cycloalkyl, C3-C6 alkenyl, C3-C6-alkynyl, aryl-C1-C3-alkyl, hydroxy-C2-C3-alkyl group; or R1 and R2 together with the N atom to which they are bound, form a 3-7 membered nitrogen heterocyclic ring of formula (II) wherein Y represents a single bond, CH2, O, S, or a N-Rc group as defined above and p represents an integer from 0 to 3; or, R1 being as defined above, R2 represents a group of formula (III): wherein Ra is hydrogen and Rb is hydrogen, hydroxy, C1-C4-alkyl or an NRdRe group wherein Rd and Re, are each independently, hydrogen, C1-C4-alkyl or phenyl; or Ra and Rb, together with the nitrogen atoms to which they are bound, form a 5-7 membered heterocyclic ring, monocyclic or fused with a benzene, pyridine or pyrimidine ring; for use as inhibitors of the C5a-induced chemotaxis of polymorphonucleate leukocytes and monocytes.", "Pharmaceutically acceptable salts of the compounds of formula (I) are also within the scope of the present invention.", "Examples of aryl groups preferably comprise: a) an Ara mono- or poly-substituted aryl group, or the most common heterocyclic rings found 2-aryl-propionic acids in current therapeutic use: alminoprofen, benoxaprofen, carprofen, fenbufen, fenoprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, loxoprofen, naproxen, pirprofen and its dehydro and dihydro derivatives, pranoprofen, surprofen, tiaprofenic acid, zaltoprofen; b) an aryl-hydroxymethyl-aryl group of formula (IVa) deriving from the reduction of the phenone carbonyl of 2-aryl-propionic acids: ketoprofen, surprofen, thiaprofenic acid, both as single (S′,R) and/or (R′,R) diastereoisomer and as diastereoisomeric mixture, wherein, when Ar2 is phenyl, Ar1 is selected from the group consisting of phenyl and thien-2-yl and, when Ar1 is phenyl, Ar2 is selected from the group consisting of phenyl, 4-thienyl, pyridyl, c) an aryl of formula (IVb): φ-Arb (IVb) wherein Arb is a phenyl mono- and poly-substituted by optionally substituted hydroxy, mercapto, C1-C3-alcoxy, C1-C3-alkylthio, chlorine, fluorine, trifluoromethyl, nitro, amino, C1-C7-acylamino optionally substituted; and φ is hydrogen; a linear or branched C1-C5 alkyl, C2-C5-alkenyl or C2-C5-alkynyl residue by C1-C3-alkoxycarbonyl, substituted or non-substituted phenyl, 2-, 3- or 4-pyridyl, quinolin-2-yl; a C3-C6-cycloalkyl group; 2-furyl; 3-tetrahydrofuryl; 2-thiophenyl; 2-tetrahydrothiophenyl or a residue of formula (IVc) A-(CH2)q- (IVc) wherein A is a C1-C5-dialkylamino group, a C1-C8-(alcanoyl, cycloalcanoyl, arylalcanoyl)-C1-C5-alkylamino group, for example dimethyamino, diethylamino, methyl-N-ethyl-amino, acetyl-N-methyl-amino, pivaloyl-N-ethyl-amino; a nitrogen containing 5-7 membered monocyclic ring optionally containing one or two double bonds and optionally an additional heteroatom separated by at least 2 carbon atoms from the atom of N, so as to form, for example, a 1-pyrrolidino, 2,5-dihydro-pyrrol-1-yl, 1-pyrrol, 1-piperidino, 1-piperazino-4-non-substituted or 4-substituted (methyl, ethyl, 2-hydroxyethyl, benzyl, benzhydril or phenyl), 4-morpholino, 4-3,5-dimethyl-morpholino, 4-thiomorpholino group; or alternatively, a residue of formula (IVd) wherein Rg is hydrogen, C1-C3-alkyl or the residue of a C1-C3-alcanoic acid; q is zero or the integer 1, d) a 2-(phenylamino)-phenyl of formula (IV e): wherein P1 and P2 indicate that the two phenyl groups may be substituted independently, with one or more C1-C4-alkyl groups, C1-C3-alkoxy groups, chlorine, fluorine and/or trifluoromethyl.", "Preferred compounds of the invention are compounds wherein: R is hydrogen, X is: a linear alkylene optionally substituted at C1 by a —CO2R3 group as defined above; a linear alkylene optionally substituted at C1 by a —CONHR4 group wherein R4 is OH; 2-butynylene, cis-2-butenylene, trans-2-butenylene; 3-oxa-pentylene, 3-thio-pentylene, 3-oxa-hexylene, 3-thio-hexylene; (CH2)m—CO—NH—(CH2)n-wherein m and n are each independently an integer from 2 to 3; (CHR′)—CONH—(CH2)n wherein n is an integer from 2 to 3 and R′ is a methyl, in absolute configuration R or S; or X, together with the N atom of the omega-amino group, forms a nitrogen containing cycloaliphatic ring, preferably 1-methyl-piperidin-4-yl or 1,5-tropan-3-yl.", "Preferred compounds are also those wherein NR1R2 represents an NH2 group, dimethylamino, diethylamino, diisopropylamino, 1-piperidinyl, 4-morpholyl, 4-thiomorpholyl or R1 and R2 together form a residue of guanidine, aminoguanidine, hydroxyguanidine, 2-amino-3,4,5,6-tetrahydropyrimidyl, 2-amino-3,5-dihydro-imidazolyl.", "Examples of particularly preferred aryl groups comprise: 4-isobutylphenyl, 4-cyclohexylmethylphenyl, 4-(2-methyl)allyl-phenyl, 3-phenoxyphenyl, 3-benzoyl-phenyl, 3-acetyl-phenyl, the single diastereoisomers (R) (S) and the diastereoisomeric mixture (R,S) of 3-C6H5—CH(OH)-phenyl, 3-CH3—CH(OH)-phenyl, 5-C6H5—CH(OH)-thienyl, 4-thienyl-CH(OH)-phenyl, 3-(pyrid-3-yl)-CH(OH)-phenyl, 5-benzoyl-thien-2-yl, 4 thienoyl-phenyl, 3-nicotinoyl-phenyl, 2-fluoro-4-phenyl, 6-metoxy-2-naphthyl, 5-benzoyl-2-acetoxy-phenyl and 5-benzoyl-2-hydroxy-phenyl.", "Particularly preferred aryl groups of formula (IVb) are phenyl groups 3-substituted by: isoprop-1-en-1-yl, isopropyl, pent-2-en-3-yl; pent-3-yl; 1-phenylethylen-1-yl; α-methylbenzyl.", "Particularly preferred aryls of formula (IVc) are 4-(pyrrolidin-1-yl)-methyl-phenyl, 3-chloro-4-(pyrrolidin-1-yl)-methyl-phenyl, 3-chloro-4-(2,5-dihydro-1-H-pyrrol-1-yl)-methyl-phenyl, 3 chloro-4-(thiomorpholin-4-yl)phenyl; 3-chloro-4-(piperidin-1-yl)-phenyl, 4-((N-ethyl-N-quinolin-2-yl-methylamino)-methyl)phenyl, 3-chloro-4-(morpholin-4-yl)-phenyl.", "Particularly preferred aryls of formula (IVe) are 2-(2,6-dichloro-phenyl-amino)-phenyl; 2-(2,6-dichloro-phenyl-amino)-5-chloro-phenyl; 2-(2,6-dichloro-3-methyl-phenyl-amino)-phenyl; 2-(3-trifluoromethyl-phenyl-amino)-phenyl.", "Particularly preferred compounds of the invention are: (R)-2-[(4-isobutyl)phenyl]-N-(3-dimethylaminopropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(4-dimethylaminobutyl)-propionamide hydrochloride; (R)-2-[(4-isobutyl)phenyl]-N-(3-N-morpholinylpropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(2-dimethylaminoethyl)propionamide; (R)-2-[(4-isobutyl)phenyl)-propionyl]-N-[2-(4-methyl-piperazin-1-yl)ethyl]propionamide; (R)-N-(exo-8-methyl-8-aza-bicyclo[3,2,1]oct-3-yl)-2-[(4-isobutylphenyl)-propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(3-N-thiomorpholinylpropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[4-(N′-methyl)piperidinyl]propionamide hydrochloride; (R),(S′)-2-[(4-isobutyl)phenyl]-N-(1-carboxy-2-dimethylaminoethyl)-propionamide; (R),(S′)-2-[(4-isobutyl)phenyl]-N-[(1-carboxy-4-piperidin-1-yl)butyl]propionamide; (R),(S′)-2-[(4-isobutyl)phenyl]-N-(1-carboxy-4-aminobutyl)propionamide; (R)-2-(4-isobutyl)phenyl-N-[2-(dimethylaminoethyl)aminocarbonylmethyl]propionamide hydrochloride; 2-(2,6-dichlorophenylamino)-phenyl-N-(3-dimethylaminopropyl)propionamide; (R),(R′,S′)-3-[3-(α-methyl)benzyl]phenyl-N-(3-dimethylaminopropyl)-propionamide; (R)-2-[(3-isopropyl)phenyl]-N-(3-dimethylaminopropyl)propionamide; (R)-2-[3-(pent-3-yl)phenyl]-N-(3-dimethylaminopropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(3-guanidylpropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[(3-hydroxy-guanidyl)propyl]propionamide; (R)-2-[(4-isobutyl)plenyl]-N-[(3-amino-guanidyl)propyl]propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[3-(2-amino-2-imidazoline)propyl]propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[N-methyl-N-(2-hydroxyethyl)aminoethoxy]propionamide; (R),(S′)-2-[(4-isobutyl)phenyl]-N-[1-carboxy-5-aminopentyl]propionamide.", "The preparation of the compounds of formula (I) has been carried out using known methods such as the reaction of an activated form of an R-2-arylpropionic acid of formula (V) with an amine of formula (VI) in non-racemizing conditions, preferably in the presence of a molar excess of a base: wherein: AT is the residue activating the carboxy group.", "Examples of activated forms of 2-arylpropionic acids of formula (V, AT=OH) are chlorides (AT=Cl), imidazolides (AT=1-imidazole), phenol esters such as p-nitrophenol (AT=p-NO2-C6H4O—) or activated forms obtained by reaction in the presence of 1-hydroxybenzotriazole (HOBZ) or of a carbodiimide, for example dicyclohexylcarbodiimide.", "Ar, R, X, R1 and R2 are as defined above, optionally protected, where necessary.", "The reaction of the activated form of a 2-aryl-propionic acid of formula (V) with a protected amine of formula (VI), is usually carried out at room temperature, using conventional protic or aprotic solvents and/or their mixtures, preferably anhydrous solvents, for example esters such as methyl acetate, ethyl acetate, ethyl formate, nitrites such as acetonitrile, linear or cyclic ethers such as ethyl ether, sulfolane, dioxane, tetrahydrofuran, amides such as dimethylformamide, formamide, halogenated solvents such as dichloromethane, aromatic hydrocarbons such as toluene, chlorobenzene or hetero-aromatic hydrocarbons such as pyridine and picoline.", "The reactions may be carried out in the presence of a base; preferred inorganic bases are alkaline and alkaline-earth carbonates and bicarbonates, such as for instance finely ground potassium carbonate, potassium bicarbonate, and magnesium and/or calcium carbonate.", "The obtained protected amides may be converted into amides of formula (I) by cleaving the protective groups and any ester groups that might be present.", "A particularly preferred ester of this kind is the allyl ester, which is removable in highly selective conditions, for example through the transfer of the allyl group to a morpholine molecule, which, in the presence of Pd(0) as catalyst, acts as transferor of H and as nucleophile acceptor according to the procedure disclosed in J. Org.", "Chem., 54, 751 1989.Amides of formula (I) wherein R2 is a group of formula (III) can be prepared by reaction of primary and secondary amines of formula (I) with an isothioureide or the corresponding isothio-uronium salts of formula (III) wherein Alk is a C1-C3-alkyl and Ra and Rb are as defined above.", "The prepararation of hydroxy-isothioureas of formula (IIIa), wherein Ra is OH and Rb is H, is described in Bernd Clement, Arch.", "Pharm.", "(Wheineim) 319, 968 (1986); other compounds of formula IIIa are known compounds or can be prepared by the conventional methods for alkylation in basic medium of the corresponding linear and/or cyclic thioureas and of thiosemicarbazides.", "The compounds of formula IIIa are isolated as isothio-uronium salts and may be reacted with the amines of formula Ie according to the method disclosed by Bodansky M. et al., J.", "Am.", "Chem.", "Soc., 86, 4452, 1964.Alternatively, an excess of a solvent such as ethyl acetate (AcOEt) is added to an aqueous solution or suspension of the isothio-uronium salt of formula IIIa and under vigorous stirring the salt is neutralized by adding the equivalent base solution (NaOH N, potassium carbonate N), to yield the corresponding isothioureide.", "Amides of formula (Ia) wherein Ar1, Ar2, X, R, R1 and R2 have the meanings disclosed above, can undergo reduction of the phenone carbonyl group to give a diastereoisomeric pair of R′, S′ alcohols optionally separated by fractioned crystallization and/or preparative chromatography to provide the individual diastereoisomers of formula (Ib): The convention has been adopted of indicating the absolute configuration S′ to the most polar diastereoisomer.", "Compounds of formula (I) may be converted into pharmaceutically acceptable salts through salification of the basic or acid groups which are present in their stricture, using respectively pharmaceutically acceptable acids or bases.", "Examples of salts with pharmaceutically acceptable bases are those with alkaline or alkaline-earth metals, preferably lithium, sodium and magnesium, or with organic bases, such as tromethamine, D-glucosamine, lysine, arginine.", "The compounds of formula (I) are generally isolated in the form of their addition salts with both organic and inorganic pharmaceutically acceptable acids.", "Examples of these acids are: hydrochloric, nitric, sulfuric, phosphoric, formic, acetic, trifluoroacetic, propionic, maleic and succinic, malonic and methansulfonic, D and L-tartaric acids.", "The R enantiomers of the 2-arylpropionic acids of formula (Va): wherein Ar is as defined above, are weak inhibitors of cycloxygenases and are usually known compounds.", "The acids of formula (Vb): wherein φ and Arb are as defined above, are obtained by alkylation with stannanes of a polysubstitute 2-phenyl-propionic acid bearing, in ortho or meta or para, a perfluorobutanesulfonate group, as described herein below.", "The compounds of formula (Vb) are disclosed in International patent application WO 01/58852.In particular, 2-[3′-isopropyl)phenyl]-propionic, 2-[3′-(α-methyl)benzyl)phenyl]-propionic and 2-[3′-(3-isopentyl)phenyl]-propionic acids, are among the preferred precursors of the amides of formula (I).", "Each 2-arylpropionic acid can be prepared by total and stereospecific synthesis or by conversion of the racemate into one of the individual enantiomers after conversion into 2-aryl-2-propyl-ketenes, as disclosed by Larse R. D. et al., J.", "Am.", "Chem.", "Soc., 111, 7650, 1989, and by Myers A. G., ibidem, 119, 6496, 1997.Stereoselective syntheses of 2-arylpropionic acids are usually directed to the S enantiomers, but may be easily modified in order to obtain R enantiomers via a convenient choice of the chiral auxiliary agent.", "The use of arylalkylketones as reactants in the synthesis of α-arylalcanoic acids, is described for examplein B. M. Trost and J. H. Rigby, J. Org.", "Chem., 14, 2926, 1978; the arylation of Meldrum acids, is described in J. T. Piney and R. A. Rowe, Tetrah.", "Lett., 21, 965, 1980; the use of tartaric acid as chiral auxiliary agent, in G. Castaldi et al., J. Org.", "Chem., 52, 3019, 1987; the use of α-hydroxyesters as chiral reactants is reported in R. D. Larsen et al., J.", "Am.", "Chem.", "Soc., 111, 7650, 1989 and U.S. Pat.", "No.", "4,940,813 and the references cited therein.", "A process for the preparation of 2-(2-OH-phenyl)-propionic acids and their esters is disclosed in Italian patent No.", "1,283,649.A tested and efficient method for the preparation of the R enantiomer of the (R,S)-2-(5-benzoyl-2-acetoxy)-propionic acid and of the acids of formula (Vb) disclosed above consists in the conversion of the chlorides of said prop-1-ketene acids by reaction with a tertiary amine, such as dimethyl-ethyl-amine, followed by the reaction of the ketene with R(−)-pantolactone, which yields the esters of R-enantiomers of said acids with R-dihydro-3-hydroxy-4,4-dimethyl-2(3H)-furan-2-one.", "The subsequent saponification of the ester with LiOH yields the corresponding free acid.", "A general procedure for the preparation of R(−)-2-arylpropionic acids of formula (Vb) includes the reaction of hydroxyarylketones of formula (Vc) mono or polysubstituted with a perfluorobutanesulfonylfluoride to yield perfluorobutanesufonic esters of formula (Vd) where n is an integer from 1 to 9.The compounds of formula (Vd) are subjected to Willoerodt re-arrangement to obtain, after esterification and methylation on the alpha carbon, arylpropionic derivates of formula (Ve) where n is an integer from 1 to 9 and R3 represents a C1-C4 alkyl or a C2-C4 alkenyl.", "The compounds of formula (Ve) are reacted with the appropriate tributylstannane of formula Bu3SnR5 where R5 is a linear or branched C1-C6 alkyl, a linear or branched C2-C6 alkenyl or a linear or branched C2-C6 alkynyl, non-substituted or substituted with an aryl group, to obtain the corresponding (R,S)-2-arylpropionates of formula (Vf).", "The alkenyl or alkynyl groups can be hydrogenated in catalytic hydrogenation conditions to obtain the corresponding saturated alkyl groups.", "The compounds of formula (Vf) are submitted to the de-racemization process as disclosed above of conversion of the corresponding acid chlorides into ketenes which, by reaction with R(−)-pantonolactone and subsequent hydrolysis, are converted into pure R enantiomers.", "The amines of formula (VI) are known products, mostly commercially available or can be prepared by known methods.", "The synthesis of 4-dialkylamino-2-butynyl-amine and, from this, of cis- and trans-4-dialkylamino-2-butenylamine is reported in R. Dalhome et al., J. Med.", "Chem., 9, 843, 1966 and T. Singh et al.", "ibidem, 12, 368, 1969, respectively.", "α-Amino acids with an amino group of formula —NR1′R2′ bound to the terminal carbon atom are prepared by known methods starting from {overscore (ω)}-hydroxy-α-amino acids, the carboxy and amino groups of which have been conveniently protected.", "The alcoholic group is transformed into a bromide through reaction with triphenylphosphine and CBr4 (R G Weiss et al., J. Org.", "Chem.", "36, 403, 1971 and M.", "Kang., ibidem, 64, 5528, 1966) followed by reaction of the halide thus obtained with at least 2M excess of the desired amine (i.e.", "dimethylamine, piperidine).", "Commercially available substrates for this purpose are serine and homoserine: superior homologs are obtained starting from commercially available dicarboxylic α-amino-acids protected at C1 and at the amino group, the free carboxy group of which is selectively reduced to alcohol by reduction in THF at room temperature with an excess of diborane.", "The present invention provides compounds of formula (I), which are R enantiomers of 2-arylpropionamides, for use as medicaments.", "The compounds of the invention of formula (I) were evaluated “in vitro” for their ability to inhibit chemotaxis of polymorphonucleate leukocytes (hereinafter referred to as PMNs) and monocytes, induced by the fractions of the complement C5a and C5a-desArg.", "For this purpose, to isolate the PMNs from heparinized human blood, taken from healthy adult volunteers, mononucleates were removed by means of sedimentation on dextrane (according to the procedure disclosed by W. J. Ming et al., J.", "Immunol., 138, 1469, 1987) and red blood cells by a hypotonic solution.", "The cell vitality was calculated by exclusion with Trypan blue, whilst the ratio of PMNs was estimated on the cytocentrifugate after staining with Diff Quick.", "The fractions hr-C5a and hrC5a-desArg (Sigma) were used as stimulating agents in chemotaxis experiments, obtaining practically identical results.", "Lyophilized C5a was dissolved in a volume of HBSS containing 0.2% BSA so as to obtain a stock solution having a concentration of 10−5 M, to be diluted in HBSS to a concentration of 10−9 M, for the chemotaxis assays.", "In the chemotaxis experiments, the PMNs were incubated with the compounds of the invention of formula (I) for 15′ at 37° C. in an atmosphere containing 5% CO2.The chemotactic activity of the C5a was evaluated on human circulating polymorphonucleates (PMNs) resuspended in HBSS at a concentration of 1.5×106 PMNs per ml.", "During the chemotaxis assay (according to W. Falket et al.", "J. Immunol.", "Methods, 33, 239, 1980) PVP-free filters with a porosity of 5 mcm and microchambers suitable for carrying out the test were used.", "The compounds of the invention in formula (I) were evaluated at a concentration ranging between 10−6 and 10−10 M; for this purpose they were added, at the same concentration, both to the lower pores and the upper pores of the microchamber.", "The wells in the lower part contain the solution of C5a or the simple carrier, those in the upper part contain the suspension in PMNs.", "Inhibition of C5a-induced chemotactic activity by the individual compounds of the invention of formula (I) was evaluated by incubating the microchamber for the chemotaxis for 60 min at 37° C. in an atmosphere containing 5% CO2.Evaluation of the ability of the compounds of the invention of formula (I) to inhibit C5a-induced chemotaxis of human monocytes was carried out according to the method reported above (Van Damme J. et al., Eur.", "J.", "Immunol., 19, 2367, 1989).", "Inhibition of C5a-induced chemotactic activity by the individual compounds of the invention of formula (I) towards human monocytes was evaluated at a concentration ranging between 10−6 and 10−10 M by incubating the microchamber for the chemotaxis for 120 min.", "at 37° C. in an atmosphere containing 5% CO2.The compounds of the invention were also evaluated in their ability to inhibit IL-8-induced chemotaxis of human PMNs.", "For this purpose, recombinant human interleukin-8 (rhIL-8, Pepro Tech) was used: the lyophilized protein was dissolved in HBSS (Hank's balanced salts solution) at the concentration of 100 mcg/mL and then diluted down to a concentration of 10 ng/mL in the chemotaxis experiments.", "R(−)-2-[(4′-isobutyl)phenyl]-propionyl methansulfonamide (ED50=10−9 M) described in WO 00/24710, was used as reference standard.", "Results on inhibition of the chemotaxis induced by C5a and by IL-8 are listed in Table I.", "Results show that different structures of the amide group can lead to different selectivity in the compounds of the present invention.", "A selected number of compounds are dual inhibitors, inhibiting chemotaxis induced both by C5a and by IL-8, others are selective inhibitors of the chemotaxis induced by C5a.", "For example, N-(1-methyl-pyrid-4-yl)amides, β-tropylamides, N-(H2N-alkyl)-amides of formula (I) are all selective inhibitors of C5a-induced chemotaxis of PMN and of monocytes in the concentration range between 10−6 and 10−8 M. All these compounds have shown poor activity as inhibitors of interleukin-8-induced chemotaxis in the same concentration range.", "A selected number of compounds of the invention are able of inhibiting also interleukin 8-induced chemotaxis of PMN leukocytes and lymphocytes T, in addition to the C5a-induced chemotaxis of PMN leukocytes and monocytes in the concentration range between 10−6 and 10−8 M. More particularly, the compounds of formula (I) wherein R1 and R2 are different from hydrogen, exert both activities of inhibition of C5a-induced chemotaxis and IL-8-induced chemotaxis.", "Both activities are present in compounds wherein the distance between the terminal basic N and the amide N is between 2 and 4 C atoms, with an optimum for n=3.In this structural framework, it can be stated that the compounds of the invention exert the dual role of inhibitors of C5a-induced chemotaxis and IL-8-induced chemotaxis.", "The compounds of formula (I), evaluated ex vivo in blood in toto according to the procedure disclosed by Patrignani et al., in J. Pharmacol.", "Exper.", "Ther., 271, 1705, 1994, were found to be totally ineffective as inhibitors of COX enzymes.", "In almost all cases, the compounds of formula (I) do not interfere with the production of PGE2 induced in murine macrophages by lipopolysaccharides stimulation (LPS, 1 μg/mL) at a concentration ranging between 10−5 and 10−7 M. Inhibition of the production of PGE2 which may be recorded, is mostly at the limit of statistical significance, and more often is below 15-20% of the basal value.", "In consideration of the experimental evidence discussed above and of the role of complement activation, through its fraction C5a, in pathologies such as psoriasis (R. J. Nicholoff et al., Am.", "J.", "Pathol., 138, 129, 1991), pemphigus and pemphigoid, rheumatoid arthritis (M. Selz et al., J. Clin.", "Invest., 87, 463, 1981), intestinal chronic inflammatory pathologies such as ulcerative colitis (Y. R. Mahida et al., Clin.", "Sci., 82, 273, 1992), acute respiratory distress syndrome, cystic fibrosis and idiopathic fibrosis (E. J. Miller, previously cited, and P. C. Carre et al., J. Clin.", "Invest., 88, 1882, 1991), Chronic Obstructive Pulmonary Disease (COPD), glomerilonephritis (T. Wada et al., J. Exp.", "Med., 180, 1135, 1994) as well as in the prevention and treatment of injury caused by ischemia and reperfusion, the compounds of the present invention are particularly useful to attain these therapeutic purposes.", "The present invention thus provides the compounds of formula (I) for use in the treatment of psoriasis, pemphigus and pemphigoid, rheumatoid arthritis, intestinal chronic inflammatory patologies including ulcerative colitis, acute respiratory distress syndrome, systemic and pulmonary idiopathic fibrosis, cystic fibrosis, chronic obstructive pulmonary disease, glomerulonephritis and in the prevention and in the treatment of injury caused by ischemia and reperfusion.", "The invention further provides the use of the compounds of formula (I) in the manufacture of medicaments for the treatment and prevention of said pathologies.", "The compounds of the invention, together with a conventionally employed adjuvant, carrier, diluent or excipient may be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.", "Such pharmaceutical compositions and unit dosage forms thereof may comprise ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.", "When employed as pharmaceuticals, the amides of this invention are typically administered in the form of a pharmaceutical composition.", "Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.", "Generally, the compounds of this invention are administered in a pharmaceutically effective amount.", "The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.", "The pharmaceutical compositions of the invention can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal.", "Depending on the intended route of delivery, the compounds are preferably formulated as either injectable or oral compositions.", "The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders.", "More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing.", "The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.", "Typical unit dosage forms include prefilled, premeasured ampoules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.", "In such compositions, the amide compound is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.", "Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like.", "Liquid forms, including the injectable compositions described herebelow, are always stored in the absence of light, so as to avoid any catalytic effect of light, such as hydroperoxide or peroxide formation.", "Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatine; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.", "Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art.", "As above mentioned, the amide derivative of formula I in such compositions is typically a minor component, frequently ranging between 0.05 to 10% by weight with the remainder being the injectable carrier and the like.", "The mean daily dosage will depend upon various factors, such as the seriousness of the disease and the conditions of the patient (age, sex and weight).", "The dose will generally vary from 1 mg or a few mg up to 1500 mg of the compounds of formula (I) per day, optionally divided into multiple administrations.", "Higher dosages may be administered also thanks to the low toxicity of the compounds of the invention over long periods of time.", "The above described components for orally administered or injectable compositions are merely representative.", "Further materials as well as processing techniques and the like are set out in Part 8 of “Remington's Pharmaceutical Sciences Handbook”, 18th Edition, 1990, Mack Publishing Company, Easton, Pa., which is incorporated herein by reference.", "The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems.", "A description of representative sustained release materials can also be found in the incorporated materials in the Remington's Handbook as above.", "The present invention shall be illustrated by means of the following examples which are not construed to be viewed as limiting the scope of the invention.", "In the description of the compounds of the invention of formula (I), the convention has been adopted of indicating the absolute configurations of any additional chiral substituents, optionally present in the structure of said compounds, with prime signs (e.g., R′, S′, S″ etc.).", "Examples of abbreviations are: AcOH for acetic acid, AcOEt for ethyl acetate, BOC for N-tert-butoxycarbonyl-, DCC for dicyclohexylcarbodiimide, DCU for dicylohexylurea, DMF for dimethylformamide, EtOH for ethanol, Et2O for diethyl ether, HOBZ for 1-hydroxybenzothiazole, hr for hour, hrs for hours, MeOH for methanol, r.t. for room temperature, THF for tetrahydrofuran, Z for N-benzyloxycarbonyl.", "Preparations: Intermediate compounds, which are used in the Examples herebelow, have been prepared according to the following procedures.", "1-amino,4-dimethylamino-butane Dimethylamine hydrochloride (1.2 g; 12.5 mmol) and, 1 hr later, 4-bromobutylphtalimide (3.5 g; 12.4 mmol) are added to a suspension of K2CO3 (4.3 g; 31 mmol), in acetone (5 mL) at 25° C.; the suspension is then refluxed overnight.", "After cooling at r.t., the mixture is filtered and evaporated to dryness; silica gel flash chromatography of the residue oil (eluent CHCl3/CH3OH 8:2) yields N-(4-dimethylamino-butyl)-phtalimide as a white solid (2.2 g; 8.94 mmol).", "A solution of said compound in EtOH, treated with a 35% aqueous hydrazine (0.45 mL), is heated at reflux temperature until all the reagents are disappeared (˜2 hrs), filtered and evaporated to dryness.", "Final crystallization from CH2Cl2/CH3OH (98:2) yields 0.85 g (7.32 mmol; 82% yield) of 1-amino, 4-dimethylamino-butane as a white solid.", "1H-NMR (CDCl3): δ 7.75 (m, 2H); 7.65 (m, 2H); 2.72 (m, 2H); 2.35 (t, 2H, J=7 Hz); 2.23 (s, 6H); 1.75 (m, 2H); 1.56 (bs, 2H, NH2); 1.48 (m, 2H).", "1-amino,4-methylamino-butane A lot of 1-amino, 4-methylamino-butane is obtained using methylamine instead of dimethylamine in the previous procedure.", "1-(3-aminopropyl)-thiamorpholine A solution of 3-BOC-aminopropyl bromide (3.07 g; 12.9 mmol) and thiamorpholine (2.6 mL; 25.8 mmol) in CH2Cl2 (25 mL) is heated at the reflux temperature for 24 h. The mixture is cooled at r.t., filtered, washed with water (2×50 mL), dried over Na2SO4 and evaporated to dryness in vacuum.", "Purification by flash chromatography on silica gel (eluent CHCl3/CH3OH 9:1) yields 1-(3-BOC-aminopropyl)-thiamorpholine (3.1 g; 11.96 mmol), as a transparent oil.", "Cleavage of the protective group is performed dissolving 1.4 g (5.4 mmol) of said compound in 3N aqueous HCl (6 mL) at rt.", "; 18 hrs later, the solution, made alkaline by addition of aqueous 2N NaOH until to reach pH=8, is extracted with CH2Cl2 (2×10 mL).", "The combined extracts, dried over Na2SO4, are evaporated to dryness to give 1-(3-aminopropyl)-thiamorpholine as a transparent oil (0.63 g; 3.96 mmol).", "1H-NMR (CDCl3): δ 7.75 (m, 2H); 7.65 (m, 2H); 2.72 (m, 2H); 2.35 (t, 2H, J=7 Hz); 2.23 (s, 6H); 1.75 (m, 2H); 1.56 (bs, 2H, NH2); 1.48 (m, 2H).", "1-(3-aminopropyl),4-methyl-piperazine(isolated as the hydrochloride salt) 1H-NMR (D2O): δ 3.75 (m, 7H); 3.45 (m, 3H); 3.15 (m, 2H); 3.05 (m, 4H); 2.20 (m, 2H) is obtained using 4-methyl-piperazine instead of thiamorpholine in the same procedure.", "1-(3-aminopropyl)-piperidine 1H-NMR (CDCl3): δ 2.85 (t, 2H, J=8 Hz); 2.45 (m, 6H); 1.90 (bs, 2H, NH2); 1.8-1.62 (m, 6H); 1.55 (m, 2H) is obtained using piperidine instead of thiamorpholine in the same procedure.", "1-BOC-propane-1,3-diamine An aqueous solution (5 mL) of NaN3 (1.4 g; 21.5 mmol) and 2-3 drops of Aliquat 336 are added to a stirred solution of 3-BOC-amino-propyl bromide (5 g; 21.5 mmol) in toluene (10 mL); the mixture is heated at the reflux temperature for 4 hrs.", "After cooling at r.t., the organic phase is separated, dried over Na2SO4, and evaporated to dryness in vacuum to give 3-BOC-amino-propyl azide (3.75 g; 18.3 mmol) as a transparent oil (yield 85%).", "A triphenylphosphine (4.8 g; 18.3 mmol) solution in THF (15 mL) is added dropwise to a stirred solution of the above azide in THF (30 mL)/H2O (0.3 mL; 18.3 mmol); the stirring is continued for 24 hrs at r.t. After removal of the solvents to dryness in vacuum, the residue is taken up with a few of EtOH to separate a white precipitate of triphenylphosphine oxide by stirring for 6 hrs at r.t.", "The final EtOH removal to dryness, at low pressure, gives 3.22 g (18 mmol) of 1-BOC-propane-1,3-diamine as a pale yellow oil.", "1H-NMR (CDCl3): δ 4.90 (bs, 1H, CONH); 3.25 (m, 2H), 2.85 (t, 2H, J=7 Hz); 1.75 (t, 2H, J=7 Hz); 1.60 (bs, 2H, NH2); 1.55 (s, 9H).", "3-(BOC-methylamino)-propylamine It is obtained by use of 3-(BOC-methylamino)-propyl bromide in the previous procedure.", "Methyl (S)-2-amino-3-dimethylamino-propionate A 2M solution of dimethylamine in THF (2.5 mL) is added dropwise to a stirred solution of methyl (S) 2-BOC-amino-3-bromo-propionate (0.45 g; 1.42 mmol) (Weiss R. G. et al., J. Org.", "Chem, 36, 403, 1971; Kang M. et al., ibidem, 61, 5528, 1996) in anhydrous THF (10 mL) at 25° C. The mixture is stirred overnight at r.t. and evaporated to dryness in vacuum.", "The residue is partitioned between Et2O (30 mL) and aqueous 0.5 N NaOH (2×5 mL); the ethereal extracts are combined, washed with brine, dried over Na2SO4 and evaporated to dryness to obtain 0.34 g (1.22 mmol) of methyl (S)-2-amino-3-dimethylamino-propionate as a pale yellow oil.", "1H-NMR (CDCl3): δ 7.45 (m, 5H); 5.73 (bs, 1H, CONH); 5.15 (s, 2H), 4.32 (m, 1H); 3.82 (s, 3H); 2.75 (m, 2H); 2.22 (s, 6H).", "A stirred solution of said methyl ester (0.34 g; 1.22 mmol) in acetonitrile (12 mL) is treated with trimethylsilyl iodide (0.21 mL; 1.46 mmol) at r.t.; 3 hrs later, the mixture is quenched with MeOH (0.24 mL; 5.9 mmol) and evaporated in vacuum to dryness.", "The residue is taken up with Et2O (2×10 mL); the ethereal extracts are re-extracted with a 30% aqueous AcOH (2×5 mL), collected, made basic up to pH=8 and extracted with CH2Cl2 (2×10 mL).", "The dichloromethane extracts are combined, dried over Na2SO4, evaporated to dryness to yield 0.16 g (1.1 mmol) of methyl (S)2-amino-3-dimethylamino-propionate.", "1H-NMR (CDCl3): δ 4.32 (m, 1H); 3.82 (s, 3H); 3.24 (bs, 2H, NH2); 2.75 (m, 2H), 2.22 (s, 6H).", "Methyl(S)-2-amino-5-(piperidin-1-yl)-pentanoate Under stirring and with external cooling to maintain the reaction temperature between 20-25° C., 0.03 molar equivalents of 1 N B2H6 (diborane) solution in THF are added to a 0.01 M solution of (S)2-BOC-amino-1,5-pentadioic acid 1-hemi-methyl ester in THF (15 mL); 2 hrs later, the diborane excess is destroyed by cautious addition of water.", "After concentration to a small volume under vacuum, the solution is diluted with AcOEt (25 mL).", "The organic phase is washed with 5% aqueous NaHCO3, brine and water to neutrality, dried over Na2SO4 and evaporated to dryness.", "The crude residue of methyl (S)2-BOC-amino-5-hydroxy-pentanoate is treated with triphenylphosphine and CBr4 to obtain a crude sample of methyl (S)2-BOC-amino-5-bromo-pentanoate.", "Reaction of the latter compound with piperidine in THF provides methyl (S)2-BOC-amino-5-(piperidin-1-yl)-pentanoate that by treatment with a trifluoroacetic acid in dichloromethane, affords methyl (S)-2-amino-5-(piperidin-1-yl)-pentanoate bis-trifluoroacetate salt.", "1H-NMR (CDCl3): δ 4.32 (m, 1H); 3.82 (s, 3H); 3.54 (m, 1H); 2.85 (t, 2H, J=7 Hz); 2.45 (m, 6H), δ 1.85 (bs, 2H, NH2); δ 1.75-1.6 (m, 6H), δ 1.5 (m, 2H).", "5-BOC-ornithine-methyl ester hydrochloride Maintaining the reaction temperature around 0-5° C. by external cooling, solid 2-Z,5-BOC-ornithine (1 g 2.7 mmol; commercial reagent) and, 15 min.", "later, methyl iodide (0.34 mL, 5.4 mmol) are added to a stirred suspension of finely powdered K2CO3 (0.38 g; 2.7 mmol) in dry DMF (20 mL).", "The mixture is stirred for an additional hr at 0-5° C. and at r.t. for 1 hr, then diluted with EtOAc (40 ml) and filtered.", "The clear solution is washed with water (40 ml) and brine (3×30 ml); dried over Na2SO4 and evaporated to dryness.", "Following purification by silica gel flash chromatography (eluent CHCl3/CH3OH 8:2) yields 2-Z,5-BOC-ornithine methyl ester (0.8 g; 2.1 mmol).", "Hydrolytic cleavage of the Z protecting group (carried out according to the procedure of Meienhofer J. et. al, Tetrahedron.", "Lett., 3259, 1974) yields 5-BOC-ornithine methyl ester hydrochloride (0.73 g; 2.0 mmol) as a white solid.", "1H-NMR (CDCl3): δ 9.25 (bs, 3H, NH3+); 5.40 (bs, 1H CONH+EE); 4.40 (m, 1H); 3.8 (s, 3H); 3.0 (m, 2H); 1.8 (m, 4H); 1.4 (s, 9H).", "Exo-8-methyl-8-aza-bicyclo[3,2,1]octan -3-amine(β-1H,5H-tropanamine) A sample is prepared starting from tropinone according to the procedure of Burks J. E. et al., Org.", "Proc.", "Res.", "Dev., 1, 198, 1997.4-(N,N-dimethylamino)aniline 4-nitroaniline (1.83 g; 13.24 mmol) is added portionwise to cooled (T=+4° C.) formic acid (3 mL; 66.2 mmol).", "Formaldehyde (37 wt.", "% solution in water; 2.72 mL; 29.13 mmol) is added and the resulting mixture refluxed for 24 h. After cooling at room temperature 6N HCl is added (2.2 mL) and the formed precipitate is filtered off.", "The filtrate is diluted with 1N NaOH (5 mL) and extracted with CH2Cl2 (3×20 mL); the organic collected extracts are dried over Na2SO4 and evaporated under vacuum to give a solid residue which, after treatment with a mixture of diisopropyl ether/acetone 1:1 and filtration, gives 4-nitro-N,N-dimethylaniline as a yellow powder (1.65 g; 9.93 mmol).", "Iron powder (2.145 g; 38.3 mmol) and 37% HCl (28 μl) are suspended in 96% ethyl alcohol (35 mL) and the mixture refluxed for 30′; at the end 4-nitro-N,N-dimethylaniline (0.64 g; 3.84 mmol) is added and the mixture left under reflux and stirring for 2 h. The hot mixture is filter over a Celite pad and, after cooling at room temperature, the filtrate is evaporated under vacuum.", "The oily residue is diluted with CH2Cl2 (25 mL) and washed with 1N NaOH (3×25 mL), dried over Na2SO4 and evaporated under vacuum to give 4-(N,N-dimethylamino)aniline as pale yellow oil (0.44 g; 3.26 mmol).", "1H-NMR (CDCl3): δ 7.10 (d, 2H, J=8 Hz); 6.60 (d, 2H, J=8 Hz); 3.55 (bs, 2H, NH2); 2.25 (s, 6H).", "According the same procedure 4-(N,N-dimethylaminomethyl)aniline is prepared as pale yellow oil.", "1H-NMR (CDCl3): δ 7.12 (d, 2H, J=8 Hz); 6.64 (d, 2H, J=8 Hz); 3.50 (bs, 2H, NH2); 3.28 (s, 2H); 2.25 (s, 6H).", "N,N-dimethylbutin-2-yl diamine Propargyl bromide (1.3 mL, 17.4 mmol) is dissolved in DMF (30 mL) and potassium phtalimide (3.4 g; 18.4 mmol) is added.", "The mixture is refluxed for 5 h. After cooling at room temperature the mixture is diluted with diethyl ether, washed with water (3×50 mL), dried over Na2SO4 and evaporated under vacuum to give N-propargyl phtalimide as white solid (3.15 g; 17 mmol).", "N-propargyl phtalimide (0.64 g; 3.4 mmol) is dissolved in 1,4-dioxane (20 mL), then dimethylamine (8.5 mL; 17 mmol), copper (I) chloride (0.35 g) and paraformaldehyde (1 g) are added.", "The solution is refluxed for 3 h. After cooling at room temperature the formed precipitate is filtered off and the filtrate is evaporated under vacuum to give a green oily residue that, after dissolution in CH2Cl2, is washed with sat.", "sol.", "NaHCO3 (2×30 mL) and water (2×30 mL).", "The organic phase is dried over Na2SO4 and evaporated under vacuum.", "The crude product is purified by treatment with diethyl ether to give N-phtalimido-N′,N′-dimethylbutin-2-yl-1,4-diamine as pale yellow solid (0.5 g; 2.05 mmol).", "A suspension of N-phtalimido-N′,N′-dimethylbutin-2-yl-1,4-diamine (0.5 g; 2.05 mmol) in ethyl alcohol (10 mL) is treated with hydrazine hydrate (98 μL; 2 mmol)) and the mixture is refluxed overnight.", "After cooling at room temperature the precipitate is filtered off and the filtrate is evaporated under vacuum; the crude residue is treated with acetone at room temperature to give, after removal of the formed precipitate, the pure product N,N-dimethylbutin-2-yl-1,4-diamine as red oil (0.2 g; 1.78 mmol).", "1H-NMR (CDCl3): δ 3.52 (m, 2H); 3.27 (m, 2H); 2.35 (s, 6H); 1.90-1.65 (bs, 2H, NH2).", "2-(amineoxy)-N-methyl-N-(2-hydroxyethyl)]ethylamine a) (Z-amineoxy)-acetic acid Maintaining the reaction temperature around 0-5° C. by external cooling, benzylchloroformate (1.41 mL, 10 mmol) and aqueous 4N NaOH (2.23 mL) are, dropwise and alternately, added to a solution in aqueous 2N NaOH:(5 mL) of 2.18 g (10 mmol) of carboxymethoxylamine hemihydrochloride [(commercial reagent) also named (amineoxy)acetic acidhydrochloride].", "Stirring is continued for 15 min before removal of any organic impurities with Et2O (2×15 mL); then addition of crushed ice and acidification until pH=2 with 37% HCl yields a solid that is filtered, washed with cold water and dried under vacuum at T=40° C. to give 2.62 g (8.2 mmol) of (Z-amineoxy)-acetic acid.", "b) 2-(Z-amineoxy)-N-methyl-N-(2-hydroxyethyl)acetamide Thionyl chloride (0.78 mL, 9 mmol) is added to a stirred solution of (Z-amineoxy)-acetic acid (2.62 g, 8.2 mmol) in MeOH (10 mL).", "The mixture is maintained overnight at room temperature to give a crude sample of (Z-amineoxy)-acetyl chloride after the usual solvent evaporation under high-vacuum conditions.", "Without any further purification, a solution of said compound in CH2Cl2 (10 mL) is dropwise added at r.t. into a stirred solution of 2-methylaminoethanol (1.44 mL, 18 mmol) in CH2Cl2 (5 mL); 18 hrs later, the reaction mixture is diluted with aqueous 1N HCl (15 mL).", "The organic phase is separated; washed with water (2×15 mL), dried over Na2SO4 and evaporated to yield 2-(Z-amineoxy)-N-methyl-N-(2-hydroxyethyl)acetamide (2.64 g, 7 mmol) as a transparent oil.", "c) 2-(Z-amineoxy)-N-methyl-N-(2-hydroxyethyl)ethylamine The selective reduction with diborane of the 2-(Z-amineoxy)-N-methyl-N-(2-hydroxyethyl)acetamide, carried out according to the Brown procedure (J.", "Am.", "Chem.", "Soc.", "86, 3566, 1964 and J. Org.", "Chem., 38, 912, 1973) yields 2.1 g (5.8 mmol) of 2-(Z-amineoxy)-N-methyl-N-(2-hydroxyethyl)ethylamine, as an oil.", "d) 2-(amineoxy)-N-methyl-N-(2-hydroxyethyl)ethylamine Benzyloxycarbonyl hydrogenolytic cleavage, carried out in the presence of ammonium formate according to Makowski procedure (Liebigs Ann.", "Chem., 1457, 1985) gives 2-(amineoxy)-N-methyl-N-(2-hydroxyethyl)ethylamine (1.06 g, 4.64 mmol) as a transparent oil.", "1H-NMR (CDCl3): δ 5.28 (bs, 2H, ONH2); 4.67 (t, 2H, J=7 Hz); 3.40 (m, 2H); 2.75 (t, 2H, J=7 Hz); 2.42 (t, 2H, J=7 Hz); 2.21 (s, 3H); 1.8 (bs, 1H, OH).", "2-aryl-propionyl chlorides of formula V (general procedure) A solution of 72.8 mmol of a 2-arylpropionic acid of formula V [for example, (R)-2-(4-isobutylphenyl)propionic acid, (R) (−).ibuprofen, 72.8 mmol] in thionyl chloride (37.5 mL) is refluxed for 3 hrs.", "The mixture is cooled at r.t.; the excess reagent is evaporated to dryness in vacuum; then, twice in succession, small amounts of anhydrous dioxane are added and evaporated to dryness under high vacuum conditions to fully eliminate any residual thionyl chloride.", "The final oily residue is used in the following reactions.", "IR (film) cm−1: 1800 (ClC═O) (S)2-(4-isobutylphenyl)]-N-(3-dimethylaminopropyl)-propionamide hydrochloride Using the previous procedure, (S)(+) ibuprofen (Fluka reagent) is converted into its propionyl chloride, whose treatment with 3-dimethylaminopropylamine, in the procedure of the example 1, allows to obtain a sample of (S)2-(4-isobutylphenyl)]-N-(3-dimethylaminopropyl)-propionamide hydrochloride m.p.", "97-98° C., [α]D=+27(c=1; CH3OH).", "1H-NMR (D2O): δ 7.45-7.21 (m, 4H); 3.75 (q, 1H, J1=7 Hz, J2=7 Hz); 3.45-3.15 (m, 2H); 2.95 (t, 2H, J=8 Hz); 2.85 (s, 6H); 2.52 (d, 2H, J=7 Hz); 1.98 (m, 1H); 1.47 (d, 3H, J=7 Hz); 0.90 (d, 6H, J=7 Hz).", "EXAMPLE 1 (R)2-(4-isobutylphenyl)-N-(3-dimethylaminopropyl)propionamide hydrochloride With external cooling, keeping the reaction temperature below 40° C., a solution of (R)2-(4-isobutylphenyl)-propionyl chloride (16.35 g; 72.8 mmol) in CH2Cl2 (10 mL) is slowly added to a stirred solution of 3-dimethylaminopropylamine (19 mL; 152 mmol).", "After a night at r.t., the reaction mixture is diluted with water (100 mL), the organic phase is separated, washed with water (50 mL) and dried over Na2SO4.After solvent removal at low pressure, 20 g (68.8 mmol) of crude (R)2-(4-isobutylphenyl)-N-(3-dimethylaminopropyl)propionamide are obtained as a pale yellow oil.", "A stirred solution of a portion of said amide (58 mmol) in isopropyl alcohol (200 mL) is treated with aqueous 37% HCl (6 mL), slowly added at r.t.; after 2 hrs, the reaction mixture is evaporated to dryness, at low pressure.", "The residual water is eliminated by azeotropic removal through the addition of small amounts of anhydrous isopropyl alcohol, in vacuum.", "Final crystallization from AcOEt (300 mL) separates a white powder that is filtered, washed with dry AcOEt and dried for 24 h under vacuum conditions at T=40° C. to obtain 18 g (55 mmol) of (R)2-(4-isobutylphenyl)-N-(3-dimethylaminopropyl) propionamide hydrochloride.", "m.p.", "95-98° C., [α]D=−26 (c=1.6; CH3OH).", "1H-NMR (D2O): δ 7.5-7.2 (m, 4H); 3.75 (q, 1H, J1=7 Hz, J2=7 Hz); 3.45-3.15 (m, 2H); 3.05 (t, 2H, J=8 Hz); 2.80 (d, 6H, J=4.5 Hz); 2.55 (d, 2H, J=7 Hz); 1.95 (m, 1H); 1.45 (d, 3H, J=7 Hz); 0.93 (d, 6H, J=7 Hz).", "EXAMPLE 2 Using 2-dimethylaminoethylamine and 4-dimethylaminobutylamine instead of 3-dimethylpropylamine in the procedure of the example 1, the following compounds are obtained: (R)-2-(4-isobutylphenyl)-N-(2-dimethylaminoethyl)propionamide.HCl m.p.", "90-93° C.; [α]D=−16 (c=1; CH3OH).", "1H-NMR (CDCl3): δ 12.25 (bs, 1H, NH+); 7.82 (bs, 1H, CONH); 7.45 (d, 2H, J=8 Hz); 7.05 (d, 2H, J=8 Hz); 3.85 (m, 2H); 3.70 (m, 1H); 3.10 (m, 2H); 2,80 (s, 3H); 2.75 (s, 3H); 2.55 (d, 2H, J=7 Hz); 1.97 (m, 1H); 1.65 (d, 3H, J=7 Hz); 0.98 (d, 6H, J=7 Hz).", "(R)2-(4-isobutylphenyl)-N-(4-dimethylaminobutyl)propionamide.HCl m.p.", "95-97° C.; [α]D=−16 (c=0.52; CH3OH).", "1H-NMR (CDCl3): δ 7.25 (d, 2H, J=8 Hz); 7.10 (d, 2H, J=8 Hz); 6.18 (bs, 1H, CONH); 3.60 (q, 1H, J1=7 Hz, J2=7 Hz); 3.25-3.15 (m, 2H); 2.95 (m, 2H); 2.75 (s, 6H); 2.45 (d, 2H, J=7 Hz); 1.85 (m, 1H); 1.65 (m, 4H); 1.48 (d, 3H, J=7 Hz); 0.93 (d, 6H, J=7 Hz).", "EXAMPLE 3 (R)2-(4-isobutylphenyl)-N-2-(N-morpholinyl ethyl)propionamide.HCl Using 1-aminoethyl-morpholine in the procedure of the example 1, crude (R)2-(4-isobutylphenyl)-N-[2-(1-morpholinyl)ethyl]propionamide is obtained.", "A solution of 4.2N acetyl chloride in absolute EtOH (3 mL) is added dropwise to a stirred solution of said amide (0.416 g, 1.3 mmol) in absolute EtOH (5 mL).", "The mixture is stirred for additional 2 hrs at r.t. before removal of solvents at low pressure.", "The residue is taken up with ethyl ether to separate 0.39 g (1.1 mmol) of (R)2-(4-isobutylphenyl)-N-[2-(1-morpholinyl)ethyl]propionamide hydrochloride as a white solid, that is filtered and washed with the same solvent.", "m.p.", "123-125° C.; [α]D=−36.3 (c=0.5; CH3OH).", "1H-NMR (CDCl3): δ 12.55 (bs, 1H, NH+); 7.80 (bs, 1H, CONH); 7.45 (d, 2H, J=8 Hz); 7.05 (d, 2H, J=8 Hz); 4.25 (m, 2H); 3.95 (m, 1H); 3.70 (m, 4H); 3.41 (m, 1H); 3.05 (m, 3H); 2.75 (m, 2H); 2.45 (d, 2H, J=7 Hz); 1.97 (m, 1H); 1.65 (d, 3H, J=7 Hz)l 0.95 (d, 6H, J=7 Hz EXAMPLE 4 The use in the procedure of the Example 3 of the following amines: 1-(3-aminopropyl)morpholine, 1-(3-aminopropyl)-4-thiomorpholine, 1-(2-aminoethyl)-piperazine-4-methyl, 1-(3-aminopropyl)-piperazine-4-methyl, 1-(3-aminopropyl)piperidine, and exo-8-methyl-8-aza-bicyclo[3,2,1]octan-3-amine instead of 1-(3-aminopropyl)morpholine gives: (R)2-(4-isobutylphenyl)-N-3-(N-morpholinyl propyl)propionamide.HCl m.p.", "90-93° C. [α]D=−22.6 (c=0.5; CH3OH).", "1H-NMR (CDCl3): δ 12.55 (bs, 1H, NH+); 7.80 (bs, 1H, CONH); 7.45 (d, 2H, J=8 Hz); 7.05 (d, 2H, J=8 Hz); 4.25 (m, 2H); 3.95 (m, 1H); 3.70 (m, 4H); 3.41 (m, 1H); 3.05 (m, 3H); 2.75 (m, 2H); 2.45 (d, 2H, J=7 Hz); 2.15 (m, 2H); 1.97 (m, 1H); 1.65 (d, 3H, J=7 Hz); 0.95 (d, 6H, J=7 Hz).", "(R)2(4-isobutylphenyl)-N-3-(N-thiomorpholinyl propyl)propionamide HCl m.p.", "70-73° C.; [α]D=−23 (c=0.5; CH3OH).", "1H-NMR (D2O): δ 8.15 (bs, 1H, CONH); 7.40 (m, 4H); 3.82 (q, 1H, J=7 Hz); 3.65 (m, 2H); 3.41 (m, 1H); 3.25 (m, 1H); 3.15-2.80 (m, 8H); 2.45 (d, 2H, J=7 Hz); 1.95 (m, 3H); 1.55 (d, 3H, J=7 Hz); 0.95 (d, 6H, J=7 Hz).", "(R)2-(4-isobutylphenyl)-N-[2-(4-methyl-piperazin-1-yl)ethyl]propionamide hydrochloride m.p.", "above 240° C.; [α]D=−33.7 (c=0.5; CH3OH).", "1H-NMR (DMSO-d6): δ 7.15 (m, 4H); 4.45 (M, 1H); 4.13 (m, 2H); 3.02 (m, 3H); 2.75 (m, 4H); 2.38 (d, 2H, J=7 Hz); 1.85 (m, 1H); 1.30 (d, 3H, J=7 Hz); 0.81 (d, 6H, J=7 Hz).", "(R)2-(4-isobutylphenyl)-N-[3-(4-methyl-piperazin-1-yl)propyl]propionamide bis-hydrochloride m.p.", "216-220° C.; [α]D=−20.5 (c=0.5; CH3OH).", "1H-NMR (D2O): δ 7.25 (m, 4H); 3.75 (m, 1H); 3.55 (m, 8H); 3.25 (m, 2H); 3.15 (m, 1H); 3.00 (s, 3H); 2.48 (d, 2H, J=7 Hz); 1.95 (m, 3H); 1.45 (d, 3H, J=7 Hz); 0.90 (d, 6H, J=7 Hz).", "(R)2-(4-isobutylphenyl)-N-[3-(1-piperidinyl)propyl]propionamide hydrochloride m.p.", "76-80° C.; [α]D=−29 (c=0.5; CH3OH).", "1H-NMR (CDCl3): δ 11.4 (bs, 1H, NH+); 7.45 (d, 2H, J=8 Hz); 7.35 (bs, 1H, CONH); 7.05 (d, 2H, J=8 Hz); 3.85 (q, 1H, J=7 Hz); 3.45 (m, 4H); 2.75 (m, 2H); 2.52 (m, 4H); 2.25 (m, 2H); 2.05 (m, 2H); 1.97 (m, 3H); 1.60 (d, 3H, J=7 Hz); 0.97 (d, 6H, J=7 Hz).", "(R)2-(4-isobutylphenyl)-N-(exo-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)propionamide hydrochloride m.p.", "72-75° C.; [α]D=−3.3 (c=0.5; CH3OH).", "1H-NMR (CDCl3): δ 7.15 (d, 2H, J=8 Hz); 7.05 (d, 2H, J=8 Hz); 6.15 (bs, 1H, CONH); 4.34 (m, 1H); 3.75 (m, 2H); 3.47 (q, 1H, J=7 Hz); 2.72 (s, 3H); 2.60-2.38 (m, 4H); 2.30-1.98 (m, 6H); 1.92 (m, 2H); 1.45 (d, 3H, J=7 Hz); 0.9 (d, 6H, J=7 Hz).", "EXAMPLE 5 (R)2-(4-isobutylphenyl)-N-(3-aminopropyl)propionamide hydrochloride A solution of 3-BOC-aminopropylamine (3.22 g; 18 mmol) in CH2Cl2 (10 mL) is added dropwise to a stirred suspension of (R)(−) ibuprofen (3 g; 17.5 mmol), DCC (3.8 g; 18 mmol) and HOBZ (2.8 g; 18 mmol) in CH2Cl2 (50 mL) at 25° C. The stirring is continued for 18 hrs at r.t.; after DCU removal by filtration, the reaction mixture is evaporated to dryness in vacuum.", "The residue oil is more times taken up with acetonitrile; finally the collected extracts are filtered, evaporated to dryness to give a crude sample of (R)2-(4-isobutylphenyl)-N-3-(BOC-aminopropyl)propionamide that is crystallized from hot MeOH (50 mL) to obtain 3.4 g (9.25 mmol,.", "53% yield) of pure (R)2-(4-isobutylphenyl)-N-3-(BOC-aminopropyl)propionamide by cooling at T=+4° C. for 18 hrs A suspension of said compound in 10 mL of aqueous 3N HCl is stirred at r.t. for 48 hrs to give (R)2-(4-isobutylphenyl)-N-3-(aminopropyl)propionamide hydrochloride (1.9 g; 6.3 mmol); m.p.", "160-163° C.; [α]D=−31 (c=0.5; CH3OH).", "1H-NMR (CDCl3): δ 8.2 (bs, 1H, NH3+); 7.18 (d, 2H, J=8 Hz); 7.05 (d, 2H, J=8 Hz); 6.83 (bs, 1H, CONH); 3.65 (q, 1H, J=7 Hz); 3.30 (m, 2H); 3.00 (m, 2H); 2.40 (d, 2H, J=7 Hz); 1.95-1.74 (m, 3H); 1.45 (d, 3H, J=7 Hz); 0.92 (d, 6H, J=7 Hz).", "EXAMPLE 6 (R)2-(4-isobutylphenyl)-N-(1-methyl-piperidin-4-yl)propionamide hydrochloride Ammonium formate (15.4 g; 240 mmol) and 10% Pd/C (3.14 g; 29 mmol) are added to a solution of 1-methyl-4-piperidone (3.26 mL; 26.5 mmol) in aqueous methanol (80 mL, CH3OH/H2O 9:1); the mixture is stirred for 24 h. at r.t.;.", "catalyst removal by filtration over Celite and solvent evaporation to dryness at low pressure give a pale yellow residue of 1-methyl-4-aminopiperidine.", "Dropwise addition of 37% HCl (4.6 mL) to a stirred solution of said amine in EtOH (50 mL) separates a white precipitate of 1-methyl-4-aminopiperidine hydrochloride that is filteted 18 hrs later, after cooling for 18 hrs at T=+4° C. Finally, an aqueous solution of the hydrochloride treated with an excess of 0.1 N NaOH (≈10 mL) is extracted with CH2Cl2 (3×10 mL).", "After the usual work-up, solvent evaporation to dryness yields pure 1-methyl-4-aminopiperidine (1.4 g; 12.4 mmol).", "1H-NMR (CDCl3): δ 2.85 (m, 2H); 2.58 (m, 1H); 2.25 (s, 3H); 2.01 (m, 2H); 1.85 (m, 2H); 1.63 (bs, 2H, NH2); 1.47 (m, 2H).", "At room temperature, a solution of (R)2-(4-isobutylphenyl)-propionyl chloride (1.12 g; 5 mmol) in CH2Cl2 (20 mL) is slowly added dropwise to a solution of 1-methyl-4-aminopiperidine (1.1 g; 10 mmol) in CH2Cl2 (10 mL).", "After 3 hrs., the reaction mixture is diluted again with CH2Cl2 (10 mL), washed with 1 N HCl (25 mL) and with brine, dried over Na2SO4 to give after solvent removal to dryness (R)2-(4-isobutylphenyl)-N-(1-methyl-piperidin-4-yl)propionamide hydrochloride as a glass solid (1.2 g; 3.5 mmol).", "[α]D=−11 (c=0.5; CH3OH).", "1H-NMR (D2O): δ 7.28 (m, 5H); 3.95 (m, 1H); 3,75 (q, 1H, J=7 Hz); 3.54 (m, 2H); 3.15 (m, 2H); 2.90 (s, 3H); 2.53 (d, 2H, J=7 Hz); 2.28-2.05 (m, 2H); 1.95-1.65 (m, 4H); 1.45 (d, 3H, J=7 Hz); 0.95 (d, 6H, J=7 Hz).", "EXAMPLE 7 (R),(S)2-(4-isobutylphenyl)-N-(1-carboxy-2-dimethylamino-ethyl)propionamide sodium salt A solution of (S)methyl 3-dimethylamino-2-amino-propanoate (0.16 g; 1.1 mmol) in CH2Cl2 (2 mL) is added dropwise to a stirred suspension of (R)(−)ibuprofen (0.23 g; 1.1 mmol), DCC (0.23 g; 1.1 mmol) and HOBZ (0.17 g; 1.1 mmol) in CH2Cl2 (5 mL) at room temperature.", "The stirring is continued for 18 hrs at r.t.; after DCU removal by filtration, the reaction mixture is evaporated to dryness in vacuum.", "The residue is more times taken up with acetonitrile; then, the collected extracts are filtered and evaporated to dryness in vacuum.", "Following purification by flash chromatography on silica gel (eluent CH2Cl2/CH3OH 95:5) yields 0.3 g (0.88 mmol) of methyl (S),(R)3-dimetylamino-2-[2-(4-isobutylphenyl)propionyl]amino-propanoate (80% yield) as a transparent oil.", "A stirred solution of said ester (0.3 g; 0.88 mmol) in dioxane (2 mL) is treated with a stechiometrical amount of aqueous N NaOH (0.88 mL) and maintained for 18 hrs.", "at r.t., before dilution with cooled water (20 mL).", "The frozen solution is lyophilized to yield 0.307 g (0.88 mmol) of (R),(S)2-(4-isobutylphenyl)-N-(1-carboxy-2-dimethylamino-ethyl)propionamide sodium salt, as a white solid m.p.", "above 240° C.; [α]D=−25 (c=0.5; CH3OH) 1H-NMR (CDCl3): δ 7.35 (m, 4H); 6.25 (bs, 1H, CONH); 4.72 (m, 1H); 3.60 (m, 1H); 2.51 (d, 2H, J=7 Hz); 2.30 (d, 2H, J=7 Hz); 2.22 (m, 6H); 1.55 (d, 3H, J=7 Hz); 0.95 (d, 6H, J=7 Hz).", "EXAMPLE 8 (R),(S)2-(4-isobutylphenyl)-N-(1-carboxy-2-piperidin-1-yl-butyl)propionamide sodium salt; and (R),(S)2-(4-isobutylphenyl)-N-(1-ethoxycarbonyl-2-piperidin-1-yl-butyl)propionamide are obtained using (S)methyl-5-(piperidin-1-yl)-2-amino-pentanoate in the procedure of the example 7 instead of (S)methyl3-dimethylamino-2-amino-propanoate.", "EXAMPLE 9 R-2-[(4′-isobultylphenyl]-N-[2-(dimethylaminoethyl)aminocarbonylmethyl]-propionamide hydrochloride HOBZ (0.607 g; 4.49 mmol) is added to a stirred solution of (R)(−)ibuprofen (1.01 g; 4.9 mmol) in DMF (4 mL) at T=0° C. and left under stirring for 30 min.", "Then a mixture of N-(3-dimethylaminopropyl)glycinamide hydrochloride (0.64 g; 4.47 mmol) in DMF (8 mL) and triethylamine (0.6 mL; 4.45 mmol) is added and N,N-dicyclohexylcarbodiimide (1 g; 4.85 mmol), in small portions, is also added.", "The mixture is stirred for 2 hrs at T=0° C. and then for 18 hrs at r.t. After DCU filtration most of DMF is then removed by distillation at low pressure.", "The residue is taken up with water and extracted with Et2O (3×25 mL); the organic extracts are combined, dried over Na2SO4, and evaporated a low pressure to yield a transparent oil (1 g; 3.43 mmol).", "Then a solution of this compound in dioxane (3.5 mL) is treated with 1N NaOH (3.5 mL), stirred for 24 hrs at r t., diluted with water (10 mL) and then acidified with 2N HCl, and extracted with CH2Cl2 (3×10 mL).", "Then, the organic extracts are combined, dried over Na2SO4, evaporated at low pressure to yield R-2-[(4′-isobutyl)phenyl]-N-[2-(dimethylaminoethyl)aminocarbonylmethyl]-propionamide hydrochloride (0.68 g; 2.04 mmol), as a pale yellow oil.", "[α]D=−25 (c=0.5; CH3OH).", "1H-NMR (CDCl3): δ 7.24 (m, 2H); 7.10 (m, 2H); 6.10 (bs, 1H, CONH); 3.55 (m, 1H); 3.30 (m, 2H); 2.45 (d, 2H, J=7 Hz); 2.35 (m, 2H); 2.18 (s, 6H); 1.85 (m, 1H); 1.52 (d, 3H, J=7 Hz); 0.90 (d, 6H, J=7 Hz).", "EXAMPLE 10 (R)-2-[2-(2,6-dichlorophenylamino)-phenyl]-N-3-(dimethylaminopropyl)propionamide A suspension of (R)2-[2-(2,6-dichlorophenylamino)]phenyl]propionic acid (0.15 g; 0.48 mmol), DCC (0.173 g; 0.84 mmol) and HOBZ (0.075 g; 0.56 mmol) in CH2Cl2 (6 m L) is stirred for 4 hrs at r.t.; then, a solution of 3-(dimethylamino) propylamine (0.06 ml; 0.48 mmol) in CH2Cl2 (5 mL) is added dropwise.", "The stirring is continued for 18 hrs at r.t., then the separated DCU is filtered and the solvent removed at low pressure.", "The residue is taken up with acetonitrile twice, the extracts are combined, filtered to totally eliminate DCU, and evaporated at low pressure.", "Purification by flash chromatography (eluent CH2Cl2/CH3OH 95:5) yields (R)2-[2-(2,6-dichlorophenylamino)-phenyl]-N-3-(dimethylaminopropyl)propionamide (0.141 g; 0.36 mmol; 75% yield), as a transparent oil.", "[α]D=−30 (c=1; CH3OH).", "1H-NMR (D2O): δ 7.38 (m, 4H); 7.15 (m, 1H); 7.05 (m, 1H); 6.60 (m, 1H+CONH); 4.25 (dd, 2H, J1=7 Hz, J2=3 Hz); 3.30 (m, 2H); 2.35 (m, 2H); 2.10 (s, 6H); 1.65 (m, 2H); 1.65 (d, 3H, J=7 Hz).", "EXAMPLE 11 The following amides are obtained using (R),(R′,S′)-2-[3-(α-hydroxybenzyl)phenyl]propionic acid, 2-[3′-(α-hydroxyethyl)phenyl]propionic acid and (R),(R′,S′)2-[3′-(α-hydroxy, α-methylbenzyl)phenyl]propionic acid as starting material instead of (R)2-[2-(2,6-dichlorophenylamino)]phenyl]propionic acid in the procedure of example 10.", "(R),(R′,S′)2-[3-(α-hydroxybenzyl)phenyl]-N-3-(dimethylaminopropyl)propionamide as a colourless oil [α]D=−24 (c=1; CH3OH).", "1H-NMR (CDCl3): δ 7.41-7.3 (m, 3H); 7.31-7.14 (m, 6H); 5.75 (s, 1H); 4.02 (bs, 1H, OH) 3.31 (m, 2H); 2.38 (t, 2H, J=8 Hz); 2.15 (s, 6H); 1.75 (m, 2H); 3.68 (q, 1H, J=7 Hz); 1.4 (d, 3H, J=7 Hz).", "(R),(R′,S′)2-[3′-(α-hydroxy, α-methylbenzyl)phenyl]-N-3-(dimethylaminopropyl)propionamide as a colourless oil [α]D=−28 (c=1; CH3OH).", "1H-NMR (CDCl3): δ 7.41-7.3 (m, 3H); 7.31-7.14 (m, 6H); 4.02 (bs, 1H, OH) 3.31 (m, 2H); 2.38 (t, 2H, J=8 Hz); 2.15 (s, 6H); 1.75 (m, 2H); 3.68 (q, 1H, J=7 Hz); 1.4 (d, 3H, J=7 Hz).", "(R),(R′,S′)2-3-(α-hydroxyethyl)phenyl]-(3-dimethylaminopropyl)propionamide 1H-NMR (DMSO-d6): δ 8.12 (bs, 1H, CONH); 7.31 (s, 1H); 7.25-7.10 (m, 3H); 5.1 (bs, 1H, OH); 4.7 (m, 1H); 3.62 (m, 1H); 3.10 (m, 2H); 2.91 (m, 2H); 3.65 (s, 6H); 1.73 (m, 2H); 1.30 (m, 6H) EXAMPLE 12 (R),(R′,S′)2-[3′-(α-methylbenzyl)phenyl]-N-3-(dimethylaminopropyl)propionamide as a pale yellow oil (1.2 g, 3.52 mmol) [α]D=−30 (c=1; CH3OH).", "1H-NMR (CDCl3): δ 7.38-7.13 (m, 9H); 6.60 (bs, 1H, CONH) 4.20 (m, 1H); 3.78 (m, 1H); 3.27 (m, 2H); 2.30 (m, 2H); 2.12 (s, 6H); 1.72 (d, 3H, J=7 Hz); 1.65 (m, 2H); 1.55 (d, 3H, J=7 Hz) is prepared using the (R),(R′,S′)2-[3-(α-methylbenzyl)phenyl]propionyl chloride in the procedure of the example 1 instead of the (R)2-(4-isobutylphenyl)-propionyl chloride.", "The alternative use of (R)2-(3-isopropylphenyl)propionyl chloride, (R)2-(3-isobutylphenyl), (R)2-[3-(styren-1-yl)phenyl]propionyl chloride, (R)2-[3′-(pent-3-yl)phenyl]propionyl chloride in the procedure of the example 1 gives: (R)2-(3-isopropylphenyl)-N-3-(dimethylaminopropyl)propionamide 1H-NMR (CDCl3): δ 7.21-7.13 (m, 4H); 6.95 (bs, 1H, CONH) 3.53 (m, 1H); 3.30 (m, 2H); 2.90 (m, 1H); 2.37 (m, 2H); 2.15 (s, 6H); 1.65 (d, 3H, J=7 Hz); 1.23 (d, 3H, J=7 Hz).", "(R)2-(3-isobutylphenyl)-N-3-(dimethylaminopropyl)propionamide [α]D=−30 (c=1; CH3OH).", "1H-NMR (CDCl3): δ 7.21-7.13 (m, 4H); 6.85 (bs, 1H, CONH) 3.53 (m, 1H); 3.25 (m, 2H); 2.48 (d, 2H, J=7 Hz); 2.30 (t, 2H, J=7 Hz); 209 (s, 6H); 1.9 (m, 1H); 1.55 (m, 2H); 1.45 (d, 3H, J=7 Hz); 0.95 (d, 3H, J=7 Hz).", "(R)2-[3-(styren-1-yl)-phenyl]-N-3-(dimethylaminopropyl)propionamide [α]D=−31 (c=1; CH3OH).", "hu 1H-NMR (CDCl3): δ 7.8-7.13 (m, 9H); 6.95 (bs, 1H, CONH) 5.0 (s, 2H); 3.53 (m, 1H); 3.30 (m, 2H); 2.37 (m, 2H); 2.15 (s, 6H).", "(R)2-[3′-(pent-3-yl)phenyl]-N-3-(dimethylaminopropyl)propionamide [α]D=−28 (c=1; CH3OH).", "1H-NMR (CDCl3): δ 7.25 (m, 3H); 7.12 (m, 1H); 7.08 (bs, 1H, CONH) 3.65 (m, 1H); 3.5-3.13 (m, 2H); 2.75 (m, 2H); 2.55 (s, 6H); 2.35 (m, 1H); 1.95 (m, 2H); 1.70 (m, 2H); 1.58 (m, 2H); 1.50 (d, 3H, J=7 Hz); 0.76 (t, 6H, J=7 Hz).", "(R)-2-[(3-benzoyl)phenyl]-N-(3-diethylaminopropyl)propionamide [α]D=−11.5 (c=3;CH3OH) 1H-NMR (CDCl3): δ 7.8 (m, 3H); 7.70-7.55 (m, 3H); 7.50-7.28 (m, 3H); 7.25 (bs, 1H, CONH); 3.75 (m, 1H); 3.50-3.20 (m, 2H); 3.3.15-2.80 (m, 6H); 2.05 (m, 2H); 1.65 (d, 3H, J=7 Hz); 1.70-1.53 (m, 3H); 1.50-1.45 (m, 3H).", "(R)-2-[(3-benzoyl)phenyl]-N-(3-dimethylaminopropyl)propionamide [α]D=−20 (c=1; CH3OH) 1H-NMR (CDCl3): δ 7.88-7.78 (m, 3H); 7.75-7.58 (m, 3H); 7.55-7.46 (m, 3H); 7.25 (bs, 1H, CONH); 3.62 (m, 1H); 3.28 (m, 2H); 2.35 (m, 2H); 2.12 (s, 6H); 1.68-1.53 (m, 5H).", "EXAMPLE 13 (R)2-(4-isobutylphenyl)-N-3(guanidinylpropyl)propionamide hydrochloride (R)2-[(4-isobutylphenyl)-N-3-(aminopropyl)propionamide hydrochloride of example 5 is converted into the free amine and treated with isothiouronium chloride according to the procedure of Bodanszky M. et al., (J.", "Am.", "Chem.", "Soc., 86, 4452, 1964) to obtain (R)2-(4-isobutylphenyl)-N-3(guanidinylpropyl)propionamide hydrochloride m.p.", "142-146° C.; [α]D=−24 (c=1; CH3OH).", "1H-NMR (D2O): δ 7.2 (d, 2H, J=8 Hz); 7.1 (d, 2H, J=8 Hz); 6.8 (bs, 1H, CONH); 3.6 (q, 1H, J=7 Hz); 3.55 (m, 2H); 2.95 (m, 2H); 2.4 (d, 2H, J=7 Hz); 2.0-1.8 (m, 3H); 1.5 (d, 3H, J=7 Hz); 0.9 (d, 6H, J=7 Hz).", "Alternative use in the same procedure of the N-hydroxy-carbamidothioic acid methylester hydrochloride salt and of the N-amino-carbamidothioic acid methylester gives: (R)2-(4-isobutylphenyl)-N-[3-(hydroxyguanidinyl)propyl]propionamide.HCl (R)2-(4-isobutylphenyl)-N-[3-(aminoguanidinyl)propyl]propionamide.HCl EXAMPLE 14 (R)2-(4-isobutylphenyl)-N-[3-(imidazolin-2-yl)aminopropyl]propionamide The (R)2-[(4-isobutylphenyl)-N-3-(aminopropyl)propionamide hydrochloride (see example 5) is converted in the free amine and treated with 2-methylthio-2-imidazoline iodohydrate (commercial reactant) according to the above cited Bodanszky procedure (J.", "Am.", "Chem.", "Soc., 86, 4452, 1964) to give (R)2-(4′-isobutylphenyl)-N-[3-(imidazolin-2-yl)aminopropyl]propionamide m.p.", "155-168° C.; [α]D=−15 (c=1; CH3OH).", "1H-NMR (D2O): δ 7.2 (d, 2H, J=8 Hz); 7.1 (d, 2H, J=8 Hz); 6.8 (bs, 1H, CONH); 3.6 (q, 1H, J=7 Hz); 3.55 (m, 2H); 3.40 (s, 4H); 2.90 (m, 2H); 2.35 (d, 2H, J=7 Hz); 2.0-1.8 (m, 3H); 1.55 (d, 3H, J=7 Hz); 1.0 (d, 6H, J=7 Hz).", "The use of 2-methylthio-tetrahydropyrimidine in the above procedure yields: (R)2-(4-isobutylphenyl)-N-[3-(tetrahydropyrimidine-2-yl)aminopropyl]propionamide.", "1H-NMR (D2O): δ 7.2 (d, 2H, J=8 Hz); 7.1 (d, 2H, J=8 Hz); 6.8 (bs, 1H, CONH); 3.6 (q, 1H, J=7 Hz); 3.55 (m, 2H); 3.40 (s, 4H); 2.90 (m, 2H); 2.35 (d, 2H, J=7 Hz); 2.0-1.8 (m, 5H); 1.55 (d, 3H, J=7 Hz); 1.0 (d, 6H, J=7 Hz).", "EXAMPLE 15 (R),(S′)2-(4-isobutylphenyl)-N-[(1-carboxy-4-amino)butyl]propionamide A solution of (R)2-(4-isobutylphenyl)propionyl chloride (0.54 g; 2.42 mmol) in CH2Cl2 (10 mL) is slowly added dropwise to a suspension of 5-BOC-ornithine methyl ester hydrochloride (0.69 g; 2.42 mmol) and triethylamine (0.68 mL; 4.84 mmol) in CH2Cl2 at 25° C. The mixture is kept under stirring overnight at r.t., then diluted with water (10 mL).", "The organic phase is separated and washed with a saturated solution of NaHCO3 (10 mL), dried over Na2SO4, and evaporated to obtain a crude product, which is purified by flash chromatography (eluent CHCl3/CH3OH 9:1) to yield (R),(S)2-(4-isobutylphenyl)propionyl-(5-BOC)ornithine methyl ester as a transparent oil (0.6 g; 1.4 mmol).", "Treatment of said compound with HCl 3N (8 mL) for 18 h at r.t. followed by solvent evaporation yields (R),(S′)2-(4-isobutylphenyl)-N-[(1-methoxycarbonyl-4-amino)butyl]propionamide hydrochloride (0.41 g, 1.25 mmol).", "To a solution of said hydrochloride in dioxane 4N NaOH (0.625 mL; 2.5 mmol) is added at r.t. the mixture is stirred overnight and evaporated to dryness at low pressure.", "The residue is taken up with EtOAc (15 mL); the organic phase is washed with a saturated NaCl solution (2×15 mL) and dried over Na2SO4.AcOEt evaporation yields (R),(S′)2-(4-isobutylphenyl)-N-[(1-carboxy-4-amino)butyl]propionamide as a white solid, m.p.", "above 240° C.; [α]D=−29 (c=0.5; CH3OH).", "1H-NMR (DMSO-d6): δ 7.3 (d, 2H); δ 7.1 (d, 2H); 6.25 (bs, 1H, CONH); 4.20 (m, 1H); 3.70 (m, 1H); 3.50 (m, 2H); 2.5 (d, 2H); 1.9 (m, 1H); 1.8 (m, 4H); 1.6 (d, 3H); 0.95 (d, 6H, J=7 Hz).", "(R),(S′)2-(4′-isobutylphenyl)-N-(1-carboxy-5-aminopentyl)propionamide hydrochloride Prepared using the corresponding (L)-lysine derivative instead of the ornithine derivative.", "[α]D=−28.3 (c=1; CH3OH) 1H-NMR (DMSO-d6): δ 12.62 (bs, 1H, COOH); 8.25 (d, 1H, CONH, J=8 Hz); 7.75 (bs, 3H, NH3+); 7.25 (d, 2H, J=8 Hz); 7.06 (d, 2H, J=8 Hz); 4.15 (m, 1H); 3.70 (m, 1H); 2.63 (m, 2H); 2.38 (d, 2H, J=7 Hz); 1.92-2.78 (m, 1H); 1.70-1.38 (m, 4H); 1.35 (d, 3H, J=7 Hz); 1.20 (m, 2H); 0.92 (d, 6H, J=7 Hz).", "EXAMPLE 16 (R)2-(4-isobutylphenyl)-N-[(N′-methyl,N′2-hydroxyethyl)-aminoethoxy]propionamide A solution of (R)2-(4-isobutylphenyl)propionyl chloride (0.42 g; 1.875 mmol) in CH2Cl2 (10 mL) is slowly added dropwise to a solution of 0.85 g (3.75 mmol) of 2-(amineoxy)-N-methyl-N-(2-hydroxyethyl)ethylamine in CH2Cl2 (10 mL) at 25° C. The mixture is kept under stirring at room temperature for 3 h, and then diluted with H2O (10 mL).", "The two phases are then shaken and the organic phase is separated, washed with water (5 mL), dried over Na2SO4 and evaporated to yield 0.59 g (1.43 mmol).", "of (R)2-(4-isobutylphenyl)-N-2-[(N′-methyl,N′2-hydroxyethyl)-aminoethoxy]propionamide as an oil.", "[α]D=−35 (c=1; CH3OH).", "1H-NMR (CDCl3): δ 7.25 (m, 4H); 6.15 (bs, 1H, CONH); 4.67 (t, 2H, J=7 Hz; 3.40 (m, 2H); 2.75 (t, 2H, J=7 Hz); 2.55 (d, 2H, J=7 Hz); 2.35 (bs, 1H, OH); 2.42 (t, 2H, J=7 Hz); 2.21 (s, 3H); 1.95 (m, 1H); 1.53 (d, 3H, J=7 Hz); 1.00 (d, 6H, J=7 Hz).", "EXAMPLE 17 R-2-[(4-isobutyl)phenyl]-N-[4-(dimethylamino)-2-butinyl]propionamide R(−)-ibuprofen (0.34 g; 1.65 mmol) is dissolved in dry CH2Cl2; DCC (0.37 g; 1.8 mmol) and HOBZ (0.24 g; 1.78 mmol) are added and the solution is left at r.t under stirring.", "for 3 hrs.", "N,N-dimethylbutin-2-yl-1,4-diamine (0.2 g; 1.78 mmol) dissolved in dry CH2Cl2 (2 mL) is added to the solution and the resulting mixture is stirred overnight.", "After 18 hrs, DCU is filtered off and the filtrate is diluted with CH2Cl2, washed with sat.", "sol.", "NaHCO3 (2×10 mL), water (2×10 mL) and brine, dried over Na2SO4 and evaporated under vacuum to give a red oily crude residue.", "The following purification by flash chromatography gives R(−)-2-[(4′-isobutyl)phenyl]-N-[4-(dimethylamino)-2-butinyl]propionamide as a yellow oil (0.347; 1.155 mmol).", "[α]D=+4.4 (c=0.5;CH3OH) 1H-NMR (CDCl3): δ 7.15-7.10 (m, 2H); 7.09-7.05 (m, 2H); 5.45 (bs, 1H, CONH); 4.05 (m, 2H); 3.55 (m, 1H); 3.15 (s, 2H); 2.47 (d, 2H, J=7 Hz); 2.22 (s, 6H); 1.85 (m, 1H); 1.48 (d, 3H, J=7 Hz); 0.91 (d, 6H, J=7 Hz).", "EXAMPLE 18 R-Z-2-[(4-isobutyl)phenyl]-N-[4-(dimethylamino)-2-butenyl]propionamide R-2-[(4′-isobutyl)phenyl]-N-[4-dimethylamino-2-butinyl]propionamide of example 17 (0.08 g; 0.27 mmol) is dissolved in abs.", "EtOH (5 mL) and 5% Palladium on calcium carbonate (Lindlar catalyst; 0.08 g) is added.", "The mixture is hydrogenated under atmospheric pressure at r.t. for 2 hrs, then is filtered over a Celite pad.", "The filter cake is deeply washed with EtOH, the filtrate is evaporated under vacuum to give pure R-Z-2-[(4-isobutyl)phenyl]-N-[4-(dimethylamino)-2-butenyl]propionamide as pale yellow oil (0.07 g; 0.23 mmol) [α]D=−26.5 (c=1.1;CH3OH) 1H-NMR (CDCl3): δ 7.20-7.12 (d, 2H, J=8 Hz); 7.10-7.05 (d, 2H, J=8 Hz); 5.95 (bs, 1H, CONH); 5.67-5.55 (m, 2H); 3.93-3.85 (m, 2H); 5.02 (m, 1H); 3.05 (d, 2H J=8 Hz); 2.47 (d, 2H, J=7 Hz); 2.25 (s, 6H); 1.93 (m, 1H); 1.55 (d, 3H, J=7 Hz); 0.95 (d,6H, J=7 Hz).", "EXAMPLE 19 R-2-[(4-isobutyl)phenyl]-N-[4-(dimethylaminomethyl)phenyl]propionamide R(−)Ibuprofen (0.31 g; 1.5 mmol) is dissolved in thionyl chloride (5 mL) and the solution is refluxed for 90′.", "The complete disappearance of starting carboxylic acid is monitored by IR; after cooling at room temperature and solvent stripping by 1.4-dioxane additions, the oily residue is diluted with dry DMF (5 mL) and added dropwise to a stirred solution of 4-(N,N-dimethylaminomethyl)aniline (0.27 g; 1.8 mmol) in dry DMF (3 mL) at room temperature.", "The solution is left under stirring overnight; the solvent evaporated under vacuum and the residue purified by flash chromatography to give R2-[(4-isobutyl)phenyl]-N-[4-(dimethylaminomethyl)phenyl]propionamide as a pale yellow oil (0.406 g; 1.2 mmol).", "[α]D=−98 (c=1;CH3OH) 1H-NMR (CDCl3): δ 7.40-7.18 (m, 9H); 3.75 (m, 1H); 3.47 (s, 2H); 2.50 (d, 2H, J=7 Hz); 2.17 (s, 6H); 1.95 (m, 1H); 1.56 (d, 3H, J=7 Hz); 0.94 (d, 6H, J=7 Hz).", "Following the same procedure R-2-[(4-isobutyl)phenyl]-N-[4-(dimethylamino)phenyl]propionamide has been prepared.", "[α]D=−131 (c=0.25;CH3OH) 1H-NMR (CDCl3): δ 7.28-7.25 (m, 4H); 7.22-7.15 (m, 2H); 6.83-6.79 (bs, 1H, CONH); 6.73-6.65 (m, 2H); 3.72 (m, 1H); 2.80 (s, 6H); 2.48 (d, 2H, J=7 Hz); 1.85 (m, 1H); 1.52 (d, 3H, J=7 Hz); 0.97 (d, 6H, J=7 Hz).", "TABLE I % Inhibition % Inhibition of IL-8 of C5a induced induced (10 ng/ (1 ng/ mL) PMNs mL) PMNs Example Structure Chemotaxis Chemotaxis (R),(S′)-2-(4′-isobutylphenyl)-N-(1-carboxy-5- aminopentyl)propionamide hydrochloride 10−8 M 5 ± 8 10−5 M 49 ± 3 (S′),(R)-2-(4-isobutylphenyl)-N-[1-carboxy-4-(1- piperidinyl)butyl]propionamide sodium salt 56 ± 9 33 ± 15 (R)-2-(4-isobutylphenyl)-N-(2-dimethylamino- ethyl) propionamide hydrochloride 56 ± 13 62 ± 12 (R)-2-(4-isobutylphenyl)-N-(3-dimethylamino- propyl) propionamide hydrochloride 51 ± 15 65 ± 14 (R)-2-(4-isobutylphenyl)-N-(3-aminopropyl) propionamide hydrochloride 2 ± 7 84 ± 8 (R)-2-(4-isobutylphenyl)-N-(4-dimethylamino- butyl) propionamide hydrochloride 34 ± 6 55 ± 8 (R)-2-(4-isobutylphenyl)-N-(1-methyl-piperidin- 4-yl)propionamide hydrochloride 4 ± 9 48 ± 8 (R)-2-(4-isobutylphenyl)-N-(exo-8-methyl-8-aza- bicyclo[3.2.1]oct-3-yl)propionamide hydrochloride 3 ± 8 57 ± 6 (R)-2-(4-isobutylphenyl)-N-3-(N-morpholinyl propyl)propionamide hydrochloride 55 ± 12 24 ± 11 (R)-2-(4-isobutylphenyl)-N-3-(1-piperidinylpropyl) propionamide hydrochloride 46 ± 8 76 ± 6 (R)-2-(4-isobutyl)phenyl-N-[2-(dimethylaminoethyl) aminocarbonylmethyl]propionamide hydrochloride 31 ± 6 68 ± 4 (R)-2-(3-isopropylphenyl)-N-3- (dimethylaminopropyl) propionamide 48 ± 2 (c = 10−6 M) 42 ± 18 (R)-2-(3-isopropylphenyl)-N-3- (dimethylaminopropyl) propionamide 5 ± 6 42 ± 18 (R)-2-(3-benzoylphenyl)-N-3-(dimethylamino propyl) propionamide 53 ± 8 56 ± 2 (R)-2-[2-(2,6-dichlorophenylamino)phenyl]-N-3- (dimethylaminopropyl)propionamide 58 ± 5 (c = 10−6 M) 41 ± 2 (R)-2-[2-(2,6-dichlorophenylamino)-phenyl]-N-3- (dimethylaminopropyl)propionamide 1 ± 13 41 ± 2" ] ]	Patent_10469094
[ [ "Nozzle for injecting sublimable solid particles entrained in gas for cleaning a surface", "Disclosed is a nozzle for injecting sublimable solid particles, which is capable of minimizing consumption of the carrier gas and also maximizing cleaning efficiency.", "The nozzle comprises a base block having a space in which carrier gas is supplied through a gas supplying pipe; a sub-block having a space in which cleaning medium decompressed by a regulator is supplied through a cleaning medium supplying pipe; a first venturi block having a venturi path for adiabatically expanding the carrier gas supplied from the space of the base block, and a cleaning medium injection path communicating the venturi path and the space of the sub-block and the carrier gas passed through the venturi path; and a second venturi block having a venturi path for adiabatically expanding the mixed gas of the carrier gas and the cleaning medium." ], [ "1.A nozzle for injecting sublimable solid particles entrained in gas for cleaning a surface, comprising: a base block having a space in which carrier gas is supplied through a gas supplying pipe; a sub-block having a space in which cleaning medium decompressed by a regulator is supplied through a cleaning medium supplying pipe; a first venturi block having a venturi path for adiabatically expanding the carrier gas supplied from the space of the base block, and a cleaning medium injection path is adjacent to a throttle portion so as to connect the throttle portion of the venturi path with the space of the sub-block to mix the cleaning medium of the sub-block and the carrier gas passed through the venturi path; and a second venturi block having a venturi path for adiabatically expanding the mixed gas of the carrier gas and the cleaning medium, wherein the venturi path of the first venturi block has an acute angle with respect to the cleaning medium injection path.", "2.The nozzle of claim 1, further comprising an intermediate block having a path, which is disposed between the first and second venturi blocks, for promoting the mixture of the carrier gas and the cleaning medium in the mixed gas moving from the path of the first venturi block to the path of the second venturi block and thus inducing growth of snow particles.", "3.The nozzle of claim 1, wherein the first and second venturi blocks respectively have a plurality of venturi paths disposed in parallel, and the sub-block has the same number of cleaning injection paths as the number of venturi paths.", "4.The nozzle of claim 1, wherein the venturi path of the first venturi block has an angle of 15 to 60° with respect to the cleaning medium injection path.", "5.The nozzle of claim 1, wherein the carrier gas supplying pipe has a slit at an end thereof so that the carrier gas is injected at a desired angle range when supplied to the space of the base block.", "6.The nozzle of claim 5, wherein the carrier gas injected through the slit has an angle of 116°.", "7.The nozzle of claim 1, wherein the cleaning medium supplied to the first venturi path has a pressure of 10 to 50 psi, and the carrier supplied to the space of the base block has a pressure of 60 to 100 psi.", "8.The nozzle of claim 1, wherein the cleaning medium is CO2+ or Ar+.", "9.The nozzle of claim 1, wherein the carrier gas is N2 gas or clean dry air." ], [ "<SOH> BACKGROUND ART <EOH>In order to clean pollutants such as fine particles on a surface of a wafer, an LCD, a color filter or various glass substrates, there has been proposed various techniques.", "Particularly, in the semiconductor industry, high-pressure liquid is used independently or used in a state of being combined with brushes to remove the polluted fine particles from a surface of a semiconductor wafer.", "These processes achieved partial success in removing the pollutant.", "However, the brushes scratch the surface of the substrate, and also it may generate undesirable static electricity.", "And, the high-pressure liquid is apt to cut the soft surface of the substrate.", "Further, the high-pressure liquid has a drawback that it is not easy to withdraw the liquid from the brushes and high-pressure liquid cleaning system.", "Meanwhile, it is well known that solid and gas phase carbon dioxide (CO 2 snow) can remove the polluted fine particles from the surface of the substrate without the above-mentioned drawback.", "One of the techniques is disclosed in U.S. Pat.", "No.", "5,125,979.In the above mentioned technique, there are provided a small expansion chamber and a large expansion chamber which are communicated with each other through a venturi interposed therebetween.", "At an outlet of the large expansion chamber is provided an accelerating chamber for accelerating an injecting speed of a cleaning medium.", "The cleaning medium is supplied from a cleaning medium supplying source to the small expansion chamber, and then adiabatically expanded while being supplied through the venturi to the large expansion chamber, thereby forming Co 2 snow having snow particles of about 46%.", "The Co 2 snow is accelerated by inert gas introduced to the accelerating chamber, and then injected through a nozzle to a desired position in which the cleaning process is performed.", "That is, in the technique, the cleaning medium is transformed into the Co 2 snow, while passing through the venturi.", "Then, the particles of the Co 2 snow grow, while the Co 2 snow passes through the large expansion chamber.", "The cleaning medium injected through the nozzle cleans the pollutant on the surface of the substrate and then sublimed.", "However, in the technique, since the cleaning medium of Co 2 is transformed into the Co 2 snow, while passing through one venturi, a solidification rate of the cleaning medium is low.", "Furthermore, since the cleaning process is typically performed at a high presser, there is a problem that a large quantity of cleaning medium is needed to remove the polluted fine particles under the same conditions.", "To solve the problem, the applicant had proposed Korean Patent application No.", "2000-8560 filed on Feb. 22, 2000, entitled “Nozzle for cleaning components of semiconductor fabricating equipment”.", "As shown in FIGS.", "1 to 3 , the nozzle for cleaning components of a semiconductor fabricating equipment has first and second venturi blocks 51 and 53 , which are disposed in series, to provide a wider cleaning surface than a single nozzle, thereby maximizing the cleaning efficiency.", "A carrier gas supplying pipe 61 is connected to a base block 55 in which the first venturi block 51 is disposed.", "A cleaning medium supplying pipe 59 is connected to a sub-block 57 disposed at an upper side of the base block 55 .", "The cleaning medium supplying pipe 59 is connected to a cleaning medium chamber 13 b in which high-pressure Co 2 is stored in liquid phase.", "The cleaning medium supplying pipe 59 is controlled by a regulator 11 to have a lower pressure of 100˜120 psi.", "Since the cleaning medium of Co 2 is reduced from the high pressure to the low pressure, the particles of snow state are formed in the cleaning medium.", "The cleaning medium controlled to have the above-mentioned pressure is supplied through the cleaning medium supplying pipe 59 and the sub-block 57 to a fan-shaped space 51 a formed in the base block 55 .", "The carrier gas supplying pipe 61 is connected to a carrier gas chamber 13 a to supply carrier gas such as N 2 to the base block 55 .", "The carrier gas is stored in the carrier gas chamber 13 a at a high pressure.", "As shown in FIG.", "2 , at an distal end of the carrier gas supplying pipe 61 , there is formed a slot 61 a for uniformly injecting the carrier gas into a plurality of venturi paths formed at the first venturi block 51 .", "At the sub-block 57 , there is formed a path 57 a perpendicular to the fan-shaped space 51 a to be communicated with the space 51 a .", "The cleaning medium supplying pipe 59 has a circular portion on which the cleaning medium is dashed, so that the cleaning medium is uniformly injected into the fan-shaped space 51 a.", "Accordingly, the cleaning medium supplied through the cleaning medium supplying pipe 59 to the space 51 a of the base block 55 is mixed with the carrier gas injected from the carrier gas supplying pipe 61 in the space 51 a so as to firstly induce the solidification of the cleaning medium.", "The mixed gas is adiabatically expanded, while passing through the venturi paths formed in the first venturi block 51 , whereby a temperature and a pressure of the mixed gas are sharply reduced.", "Since the cleaning medium is adiabatically expanded in the first venturi block 51 , the solidification of the cleaning medium is further promoted.", "Further, the cleaning medium is adiabatically expanded again, while passing through the second venturi block 53 , and thus, the solidification of the cleaning medium is promoted once more.", "However, the conventional technique as described above has a structure that the cleaning medium supplying path is perpendicular to the carrier gas supplying path at a place where the cleaning medium and the carrier gas is mixed.", "Therefore, since the carrier gas having the higher pressure than the cleaning medium is flowed back to the cleaning medium supplying path, there is a problem that the cleaning medium supplying path is clogged.", "Furthermore, since the carrier gas is supplied at the high pressure, there is another problem that the consumption of the carrier gas is increased." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.", "1 is a perspective view a conventional nozzle for cleaning a component of a semiconductor; FIG.", "2 is an exploded perspective view of the nozzle of FIG.", "1 ; FIG.", "3 is a cross-sectional view taken along the line III-III of FIG.", "1 ; FIG.", "4 is a perspective view of a nozzle for injecting sublimable solid particles entrained in gas for cleaning a surface according to the present invention; FIG.", "5 is an exploded perspective view of the nozzle of FIG.", "4 ; FIG.", "6 is a plan view of the nozzle in which a sub-block is removed according to the present invention; and FIG.", "7 is a cross-sectional view taken along the line VII-VII of FIG.", "4 .", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to a nozzle for injecting sublimable solid particles such as Co2 snow, Ar snow, etc., to clean a surface of a wafer or FPD (Flat Panel Display) and the like.", "BACKGROUND ART In order to clean pollutants such as fine particles on a surface of a wafer, an LCD, a color filter or various glass substrates, there has been proposed various techniques.", "Particularly, in the semiconductor industry, high-pressure liquid is used independently or used in a state of being combined with brushes to remove the polluted fine particles from a surface of a semiconductor wafer.", "These processes achieved partial success in removing the pollutant.", "However, the brushes scratch the surface of the substrate, and also it may generate undesirable static electricity.", "And, the high-pressure liquid is apt to cut the soft surface of the substrate.", "Further, the high-pressure liquid has a drawback that it is not easy to withdraw the liquid from the brushes and high-pressure liquid cleaning system.", "Meanwhile, it is well known that solid and gas phase carbon dioxide (CO2 snow) can remove the polluted fine particles from the surface of the substrate without the above-mentioned drawback.", "One of the techniques is disclosed in U.S. Pat.", "No.", "5,125,979.In the above mentioned technique, there are provided a small expansion chamber and a large expansion chamber which are communicated with each other through a venturi interposed therebetween.", "At an outlet of the large expansion chamber is provided an accelerating chamber for accelerating an injecting speed of a cleaning medium.", "The cleaning medium is supplied from a cleaning medium supplying source to the small expansion chamber, and then adiabatically expanded while being supplied through the venturi to the large expansion chamber, thereby forming Co2 snow having snow particles of about 46%.", "The Co2 snow is accelerated by inert gas introduced to the accelerating chamber, and then injected through a nozzle to a desired position in which the cleaning process is performed.", "That is, in the technique, the cleaning medium is transformed into the Co2 snow, while passing through the venturi.", "Then, the particles of the Co2 snow grow, while the Co2 snow passes through the large expansion chamber.", "The cleaning medium injected through the nozzle cleans the pollutant on the surface of the substrate and then sublimed.", "However, in the technique, since the cleaning medium of Co2 is transformed into the Co2 snow, while passing through one venturi, a solidification rate of the cleaning medium is low.", "Furthermore, since the cleaning process is typically performed at a high presser, there is a problem that a large quantity of cleaning medium is needed to remove the polluted fine particles under the same conditions.", "To solve the problem, the applicant had proposed Korean Patent application No.", "2000-8560 filed on Feb. 22, 2000, entitled “Nozzle for cleaning components of semiconductor fabricating equipment”.", "As shown in FIGS.", "1 to 3, the nozzle for cleaning components of a semiconductor fabricating equipment has first and second venturi blocks 51 and 53, which are disposed in series, to provide a wider cleaning surface than a single nozzle, thereby maximizing the cleaning efficiency.", "A carrier gas supplying pipe 61 is connected to a base block 55 in which the first venturi block 51 is disposed.", "A cleaning medium supplying pipe 59 is connected to a sub-block 57 disposed at an upper side of the base block 55.The cleaning medium supplying pipe 59 is connected to a cleaning medium chamber 13b in which high-pressure Co2 is stored in liquid phase.", "The cleaning medium supplying pipe 59 is controlled by a regulator 11 to have a lower pressure of 100˜120 psi.", "Since the cleaning medium of Co2 is reduced from the high pressure to the low pressure, the particles of snow state are formed in the cleaning medium.", "The cleaning medium controlled to have the above-mentioned pressure is supplied through the cleaning medium supplying pipe 59 and the sub-block 57 to a fan-shaped space 51a formed in the base block 55.The carrier gas supplying pipe 61 is connected to a carrier gas chamber 13a to supply carrier gas such as N2 to the base block 55.The carrier gas is stored in the carrier gas chamber 13a at a high pressure.", "As shown in FIG.", "2, at an distal end of the carrier gas supplying pipe 61, there is formed a slot 61a for uniformly injecting the carrier gas into a plurality of venturi paths formed at the first venturi block 51.At the sub-block 57, there is formed a path 57a perpendicular to the fan-shaped space 51a to be communicated with the space 51a.", "The cleaning medium supplying pipe 59 has a circular portion on which the cleaning medium is dashed, so that the cleaning medium is uniformly injected into the fan-shaped space 51a.", "Accordingly, the cleaning medium supplied through the cleaning medium supplying pipe 59 to the space 51a of the base block 55 is mixed with the carrier gas injected from the carrier gas supplying pipe 61 in the space 51a so as to firstly induce the solidification of the cleaning medium.", "The mixed gas is adiabatically expanded, while passing through the venturi paths formed in the first venturi block 51, whereby a temperature and a pressure of the mixed gas are sharply reduced.", "Since the cleaning medium is adiabatically expanded in the first venturi block 51, the solidification of the cleaning medium is further promoted.", "Further, the cleaning medium is adiabatically expanded again, while passing through the second venturi block 53, and thus, the solidification of the cleaning medium is promoted once more.", "However, the conventional technique as described above has a structure that the cleaning medium supplying path is perpendicular to the carrier gas supplying path at a place where the cleaning medium and the carrier gas is mixed.", "Therefore, since the carrier gas having the higher pressure than the cleaning medium is flowed back to the cleaning medium supplying path, there is a problem that the cleaning medium supplying path is clogged.", "Furthermore, since the carrier gas is supplied at the high pressure, there is another problem that the consumption of the carrier gas is increased.", "DISCLOSURE OF THE INVENTION Therefore, an object of the present invention is to provide a nozzle for injecting sublimable solid particles, which is capable of preventing the clogging of the cleaning medium supplying path due to backflow of carrier gas, and also minimizing consumption of the carrier gas.", "Another object of the present invention is to provide a nozzle for injecting sublimable solid particles, in which a desired staying space for carrier gas and cleaning medium is secured at a place that the carrier gas and the cleaning medium are mixed, thereby uniformly mixing the carrier gas and the cleaning medium, and which stabilizes growth of particles and flow of the carrier gas and the cleaning medium to maximize solidification of the cleaning medium, thereby improving cleaning efficiency.", "The present invention provides nozzle for injecting sublimable solid particles entrained in gas for cleaning a surface, comprising a base block having a space in which carrier gas is supplied through a gas supplying pipe; a sub-block having a space in which cleaning medium decompressed by a regulator is supplied through a cleaning medium supplying pipe; a first venturi block having a venturi path for adiabatically expanding the carrier gas supplied from the space of the base block, and a cleaning medium injection path communicating the venturi path and the space of the sub-block to mix the cleaning medium of the sub-block and the carrier gas passed through the venturi path; and a second venturi block having a venturi path for adiabatically expanding the mixed gas of the carrier gas and the cleaning medium, wherein the venturi path of the first venturi block has an acute angle with respect to the cleaning medium injection path.", "The nozzle of the present invention comprises an intermediate block having a path, which is disposed between the first and second venturi blocks, for promoting the mixture of the carrier gas and the cleaning medium in the mixed gas moving from the path of the first venturi block to the path of the second venturi block and thus inducing growth of snow particles.", "Herein, the first and second venturi blocks respectively have a plurality of venturi paths disposed in parallel, and the sub-block has the same number of cleaning medium injection paths as the number of venturi paths.", "Further, the venturi path of the first venturi block has an angle of 15 to 60° with respect to the cleaning medium injection path.", "Therefore, it is prevented that the carrier gas is flowed back to the cleaning medium injection path.", "More preferably, the injection hole in which the cleaning medium decompressed by the regulator is mixed with the carrier gas has a diameter of 0.1 to 0.3 mm.", "Therefore, since the pressure of the cleaning medium injected through the injection path is higher than that of the carrier gas, it is efficiently prevented that the carrier gas is flowed back.", "the carrier gas supplying pipe has a slit at an end thereof so that the carrier gas is injected at a desired angle range when supplied to the space of the base block.", "The carrier gas injected through the slit has an angle of 116°.", "Further, the cleaning medium supplied to the first venturi path has a pressure of 110 to 170 psi, and the carrier gas supplied to the space of the base block has a pressure of 60 to 100 psi.", "The cleaning medium is Co2+ or Ar+, and the carrier gas is N2 gas or clean dry air.", "BRIEF DESCRIPTION OF DRAWINGS FIG.", "1 is a perspective view a conventional nozzle for cleaning a component of a semiconductor; FIG.", "2 is an exploded perspective view of the nozzle of FIG.", "1; FIG.", "3 is a cross-sectional view taken along the line III-III of FIG.", "1; FIG.", "4 is a perspective view of a nozzle for injecting sublimable solid particles entrained in gas for cleaning a surface according to the present invention; FIG.", "5 is an exploded perspective view of the nozzle of FIG.", "4; FIG.", "6 is a plan view of the nozzle in which a sub-block is removed according to the present invention; and FIG.", "7 is a cross-sectional view taken along the line VII-VII of FIG.", "4.BEST MODE FOR CARRYING OUT THE INVENTION The present invention will now be described in further detail with reference to the drawings.", "As shown in FIGS.", "4 and 5, a nozzle according to the present invention includes a base block 1, a sub-block 2, a first venturi block 3 and a second venturi block 4.A intermediate block 5 may be disposed between the first and second venturi blocks 3 and 4.The intermediate block 5 has a mixing space for assuring mixture of cleaning medium and carrier gas and stabilizing flow of the cleaning medium and carrier gas.", "The base block 1 is connected through a carrier gas supplying pipe 12 to a carrier gas chamber 13a in which carrier gas such as N2 gas or clean dryd air.", "The carrier gas supplied to the base block 1 typically has a pressure of 60 to 100 psi.", "The carrier gas supplying pipe 12 has a nozzle body 7 at an end thereof.", "As shown in FIG.", "5, the nozzle body 7 is disposed at the base block 1 so as to inject the carrier gas into a space 6 formed in the base block 1.The nozzle body-7 is formed with a slit 7a for injecting the carrier gas at an injecting angle a of about 116° into the space of the base block 1.As shown in FIG.", "6, the space 6 formed in the base block 1 is formed into a sector having an angle of 116°.", "The sub-block 2 is positioned at an upper side of the base block 1 to be connected to the first venturi block 3.The sub-block 1 is connected through a cleaning medium supplying pipe 14 to a Co2 cleaning medium chamber 13b in which cleaning medium for generating solidifying particles like Co2 particles.", "When the cleaning medium is injected through a nozzle body 9, which is disposed at an end of the cleaning medium supplying pipe 14, to an injection space 10, the cleaning medium is firstly decompressed to a pressure of about 110 to 170 psi by a regulator 11 or other valve means disposed at the cleaning medium supplying pipe 14 and then injected into the injection space 10 of the sub-block 1 in a liquid Co2 state.", "Meanwhile, a cryogenic heat exchanger (not shown) is disposed between the cleaning medium chamber 13b and the regulator 11.Therefore, the cleaning medium of Co2 gas is transformed into liquid Co2 due to temperature drop while passing through the cryogenic heat exchanger, and transformed again into liquid Co2 having a lower pressure while passing through the regulator 11, and then introduced into the sub-block 2.Solidified particles like Co2 particles is generated at a place where the cleaning medium is mixed with the carrier gas.", "As shown in FIGS.", "5 and 7, the injection space 10 of the sub-block 2 is formed into the sector, and disposed to be perpendicular to the nozzle body 9.The nozzle body 9 is positioned at an upper side of the space 10.The nozzle body 9 has an arc-shaped distribution surface 9a at an end thereof so that the cleaning medium injected through a nozzle hole 9b of the nozzle body 9 is injected into a lower portion of the injection space 10.Therefore, the cleaning medium injected from the nozzle body 9 is dashed on the distribution surface 9a, and thus injected at an angle of about 146° into the lower portion of the injection space 10.In order to obtain such injection angle of the cleaning medium, the distribution surface 9a has an angle of about 75° with respect to the distribution surface 9a.", "As shown in drawing, the first venturi block 3 is connected to the base block 1 and the sub-block 2.The first venturi block 3 has multiple, preferably, 10 or more venturi paths 15 disposed in parallel and multiple cleaning medium injection paths 16 for connecting each venturi path 15 to the injection space 10 of the sub-block 2.The venturi paths 15 are connected with the space 6 formed in the base block 1.The carrier gas supplied from the space 6 formed in the base block 1 is adiabatically expanded and a flow rate of the carrier gas is increased, while the carrier gas passes through a throttle portion of the venturi path 15.Therefore, in the venturi path 15, the cleaning medium supplied from the sub-block 2 through the cleaning medium injection path 16 to the venturi path 15 can be rapidly mixed with the carrier gas supplied from the base block 1 to the venturi path 15.Each of the cleaning medium injection paths 16 has an angle of 15° to 60°, preferably 450 with respect to each of the venturi paths 15.The venturi paths 15 respectively have a diameter of about 2 mm at a place where the venturi paths 15 are contacted with the cleaning medium injection paths 16.The throttle portion formed in the venturi path 15 has a diameter of about 0.3 to 1 mm.", "An inlet and an outlet of the venturi path 15 respectively have a diameter of 1 to 3 mm.", "However, it is preferable that the diameter of the outlet is smaller than that of the inlet.", "In addition, since the cleaning medium injection paths 16 respectively have the above-mentioned angle with respect to the venturi paths 15 and the pressure of the carrier gas is about 10 to 50 psi lower than that of the cleaning medium of liquid Co2 state, a clogging phenomenon of the pipe due to back pressure or solidified particles is prevented.", "Meanwhile, the carrier gas can be uniformly supplied to the venturi paths 15 formed in the first venturi block 3 according to a structure of the slit 7a of the nozzle body 7 disposed in base block ion solution, and the cleaning medium can be uniformly supplied to the cleaning medium supplying path 16 according to a structure of the distribution surface 9a and the space 10 of the nozzle body 9.The cleaning medium is adiabatically expanded while passing through the venturi paths 15 of the first venturi block 3, and the carrier gas has the increased flow rate after passing through the throttle portion, whereby a mixing rate of the carrier gas and the cleaning medium is increased.", "Preferably, the cleaning medium path 16 has a larger diameter of an upper portion thereof than that of a lower portion thereof, and the diameter of the lower portion is 0.1 to 0.3 mm.", "Therefore, the snow solidification of the cleaning medium is improved.", "The intermediate block 5 is connected to the first venturi block 3.The intermediate block 5 has a mixing space 18 and a path 19 communicated with a venturi path 17 of the second venturi block 4.The mixing space 18 secures the sure mixing of the cleaning medium and the carrier gas and also stabilizes the flow of the cleaning medium and the carrier gas so as to induce the growth of the snow particles.", "In the mixing space 18, a turbulent flow is stably transformed into a laminar flow, while the carrier gas and the cleaning medium are completely mixed and, at the same time, the snow particles are grown, thereby maintaining the uniform injection conditions.", "Preferably, each of the paths 19 has the same diameter as an inlet of the venturi path 15.The second venturi block 4 has the same number of venturi paths 17 as the number of venturi paths 15 of the first venturi block 3 and the number of the paths of the intermediate block 5.The second venturi block 4 is connected to the intermediate block 5 so that the venturi paths 17 are exactly met with each other.", "As described above, since there are provided 10 or more venturi paths 17 of the second venturi block 4 through which the mixture of the cleaning medium and the carrier gas is injected into the outside, the nozzle of the present invention has an increased injection surface area.", "The mixed gas of the cleaning medium and the carrier gas, of which the snow particles are grown, is adiabatically expanded again, while passing through the venturi paths 17 of the second venturi block 4.Thus, a size of the sublimable solid particle can be maximized.", "Meanwhile, in the embodiment of the present invention, as described above, the intermediate block 5 is disposed between the first venturi block 3 and the second venturi block 4.Further, since the diameter of the cleaning medium path 16 through which the cleaning medium of the liquid Co2 state is passed is reduced, the pressure of the cleaning medium, which is higher than the supplying pressure of the carrier gas, is applied to the cleaning medium path, thereby solving the problem of back pressure.", "Furthermore, since a proper amount of snow particles are formed, it is possible to inject the cleaning medium with the low pressure carrier gas.", "As described above, the mixing places of the paths 15, 16, 17 and 19 formed in the first and second venturi block 3 and 4 and the intermediate block 5 respectively have a diameter of 2 mm.", "The throttle portion of each venturi path 15 and 17 has a diameter of 0.3 to 1 mm.", "The cleaning medium path 16 has a diameter of 0.1 to 0.3 mm.", "The inlet and outlet have a diameter of 1 to 3 mm.", "According to the nozzle of the present invention, at the place in which the cleaning medium and the carrier gas are mixed, the cleaning medium supplying paths have an acute angle with respect to the venturi paths.", "The clogging of the cleaning medium supplying path due to the backflow of the carrier gas is prevented by pressure gradient according to a size of the cleaning medium path.", "In addition, due to the first and second venturi blocks and the intermediate block, the flow and the solidified particles of the cleaning medium are stabilized and the solidification rate is maximized.", "Therefore, the consumption of the cleaning medium can be reduced, while a time of cleaning process can be remarkably reduced by the increasing in the injection surface area of the nozzle.", "INDUSTRIAL APPLICABILITY As described above, the cleaning medium is transformed into the liquid Co2 by the first decompression using the regulator and other valve and the temperature drop using the cryogenic heat exchanger.", "The liquid Co2 is phase-changed to form the sublimable solid particles like the Co2 particles, while passing through the cleaning medium path 16.Further, while the cleaning medium is passed through the nozzle of the present invention, an opportunity of the adiabatic expansion of the cleaning medium is maximized, and the flow rate of the cleaning medium is increased by the carrier gas.", "Meanwhile, in the embodiment, the Co2 is used as the cleaning medium.", "However, it is obvious to those skilled in the art that other medium like Ar+ can be used as the cleaning medium." ] ]	Patent_10469109
[ [ "Pumping unit driven by a linear electric motor", "The present invention disclose a pumping unit driven by a linear motor, comprising a guide rail support in the form of a frame structure, a mover of the linear motor provided with slide blocks, said slide blocks engages guide slide ways used for said mover and mounted on inner sides of said guide rail support, six stators of the linear motor, said stators are mounted on inner sides of said guide rail support, a chain wheel disposed at the top of said guide rail support, a chain, said chain is connected to an upper end of said mover of the linear motor at one end, passes said chain wheel and is then connected to a pumping slick rod of a wellhead device at the other end thereof, a counterweight, and a counterweight chain, said counterweight chain is connected to a lower end of said mover at one end, passes a bottom chain wheel disposed at the bottom of the guide rail support and a top chain wheel disposed at the top of the guide rail support sequentially and is then connected to said counterweight at the other end thereof.", "Since the pumping unit is disposed at a side of the wellhead, it is convenient for operations and eliminates possibility of occurring of accident without changing the original wellhead." ], [ "1.A pumping unit driven by a linear motor, comprising: a guide rail support in the form of a frame structure; a mover of the linear motor provided with slide blocks, said slide blocks engages guide slide ways used for said mover and mounted on inner sides of said guide rail support; stators of the linear motor, said stators are mounted on inner sides of said guide rail support; a chain wheel disposed at the top of said guide rail support; a chain, said chain is connected to an upper end of said mover of the linear motor at one end, passes said chain wheel and is then connected to a pumping slick rod of a wellhead device at the other end thereof; a counterweight; and a counterweight chain, said counterweight chain is connected to a lower end of said mover at one end, passes a bottom chain wheel disposed at the bottom of the guide rail support and a top chain wheel disposed at the top of the guide rail support sequentially and is then connected to said counterweight at the other end thereof.", "2.The pumping unit driven by a linear motor according to the claim 1, wherein said chain wheel, said bottom chain wheel and said top chain wheel are replaced by pulleys, and said chain and counterweight chain are replaced by wire ropes accordingly.", "3.The pumping unit driven by a linear motor according to the claim 1 or 2, wherein said slide blocks of said mover of the linear motor used for engaging said slide ways are rollers." ], [ "<SOH> BACKGROUND ART <EOH>Presently, most of oil well pumping units are driven by a rotating motor vie a speed reducer and a conversion device for converting rotary movement into linear movement, thereby driving the pumping slick rod and the pumping rod to reciprocate up and down and driving the downhole oil extraction pump to extract oil.", "The pumping unit driven by a rotating motor is large in volume, complex in structure and high in electrical energy consumption.", "The Chinese Utility Model No.", "96217 638.9 disclosed a pumping unit driven by a linear motor.", "The technical scheme of the Utility Model is that the linear motor is connected to and drives the pumping rod and the oil extraction pump, and the speed reducer and the conversion device for converting rotary movement into linear movement are omitted, thus eliminating the disadvantages of the conventional pump unit such as large volume, complex structure and high energy consumption.", "This pumping unit is advantageous because it is driven by a linear motor.", "The pumping unit comprises a downhole oil extraction pump, a pumping rod, a driving device, a headframe and a counterweight.", "A linear motor is used as the driving device, stators of the linear motor together with longitudinal limit posts are fixed on the wellhead device, positioning blocks are fixed to an upper end and a lower end of the mover of the linear motor, the mover is slidably against to the side longitudinal limit posts, buffer springs are fixed to the wellhead device and the limit posts respectively.", "The mover of the motor is hollow inside, and the pumping rod passes through the center of the mover.", "The pumping rod together with a clipping seat is fixed to the mover of the linear motor.", "The headframe is provided with pulleys of a weight-balancing device.", "A wire rope is connected to the clipping seat and the counterweight at both ends thereof via the pulleys.", "Since stators of the linear motor together with the limit posts are fixed above the wellhead device, many inevitable disadvantages in the pump unit are as follows: 1.Vibration generates while the mover of the linear motor reciprocates up and down, thus causing the pumping unit to vibrate with the linear motor.", "The stators of the linear motor and the limit posts are fixed to the wellhead device, the mover of the linear motor causes the wellhead device to vibrate while it moves, thus causing leakage of crude oil and natural gas from the wellhead device.", "Even if the leakage of the crude oil and the natural gas is not caused by the vibration, it generally occurs, during the mechanical pumping of the oil.", "If leakage of the natural gas occurs, since the linear motor is fixed above the wellhead device, the leaked natural gas will enter the linear motor directly, thus causing potential safety hazard.", "2.The linear motor is fixed above the wellhead device so that it occupies a space around the wellhead, operations such as replacement of the packing set box and the packing set will become difficult.", "3.The linear motor is fixed above the wellhead device, the vibration of the motor causes the downhole casing connected to wellhead device to vibrate and deform.", "4.The linear motor is fixed above the wellhead device which can not endure the higher outside pressure.", "If the linear motor is fixed above the wellhead, a wellhead device which can endure higher outside pressure should be designed renewedly to match the linear motor, this will increase the cost of the pumping unit." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Accordingly, an object of the present invention is to move the linear motor away from the wellhead device so as to resolve the above problems, i.e.", "the potential safety hazard occurs when the natural gas leaks; the linear motor occupies much space around wellhead because it is fixed above the wellhead device, thus causing many operations difficult; the linear motor causes the down hole casing to vibrate.", "In addition, the wellhead device can endure higher outside pressure without changing the original wellhead device in site.", "The headframe is replaced by a guide rail support of the linear motor.", "The object is accomplished by providing a pumping unit driven by a linear motor, comprising a guide rail support, a mover of the linear motor, stators of the linear motor, a chain, chain wheel and a counterweight, the guide rail support is in the form of a frame structure, stators of the linear motor are mounted on inner sides of the guide rail support, and guide slide-ways used for the mover of the linear motor are fixed on inner sides of the guide rail support.", "The mover of the linear motor is provided with slide blocks engaging with the guide slide ways.", "The chain wheel is disposed on the top of the guide rail support.", "The chain passes the chain wheel and is connected to the mover of the linear motor at one end and connected to a pumping slick rod of the wellhead device at the other end thereof.", "A counterweight chain is connected to a lower end of the mover of the linear motor at one end, passes a bottom chain wheel disposed at the bottom of the guide rail support and a top chain wheel disposed at the top of the guide rail support sequentially, and is then connected to a counterweight at the other end thereof.", "In another embodiment of the present invention, pulleys can replace the chain wheels respectively and the chains can be replaced by wire ropes accordingly.", "The advantages of the present invention are as follows: an upper end of the mover of the linear motor is connected to one end of the chain, the chain passes the chain wheel and is connected to the pumping slick rod at the other end thereof.", "The wellhead device and the pumping slick rod are away from the linear motor, and the linear motor is moved away from the above of the wellhead, when leakage of the natural gas occurs, the leaked natural gas can not enter the linear motor directly, thus eliminating the potential safety hazard.", "The linear motor is not disposed above the wellhead device, and the original wellhead device can be used, so that it is still convenient for a worker to operate around the wellhead by comparison with the pumping unit driven by a rotating motor in the prior art.", "In addition, even if the linear motor generates vibration during operating, the downhole casing will not be affected and the wellhead device can endure high pressure.", "The structure of the pumping unit driven by a linear motor is also simplified by replacing the headframe by the guide rail support of the linear motor." ], [ "CROSS-REFERENCE TO RELATED APPLICATIONS This application relates to and claims priority from PCT/CN02/00111 filed Feb. 25, 2002 and parent Application Serial No.", "01204055.X filed Feb. 26, 2001.FIELD OF THE INVENTION The present invention generally relates to a pumping unit, more particularly to an oil well pumping unit disposed on the ground and driven by a linear motor.", "BACKGROUND ART Presently, most of oil well pumping units are driven by a rotating motor vie a speed reducer and a conversion device for converting rotary movement into linear movement, thereby driving the pumping slick rod and the pumping rod to reciprocate up and down and driving the downhole oil extraction pump to extract oil.", "The pumping unit driven by a rotating motor is large in volume, complex in structure and high in electrical energy consumption.", "The Chinese Utility Model No.", "96217 638.9 disclosed a pumping unit driven by a linear motor.", "The technical scheme of the Utility Model is that the linear motor is connected to and drives the pumping rod and the oil extraction pump, and the speed reducer and the conversion device for converting rotary movement into linear movement are omitted, thus eliminating the disadvantages of the conventional pump unit such as large volume, complex structure and high energy consumption.", "This pumping unit is advantageous because it is driven by a linear motor.", "The pumping unit comprises a downhole oil extraction pump, a pumping rod, a driving device, a headframe and a counterweight.", "A linear motor is used as the driving device, stators of the linear motor together with longitudinal limit posts are fixed on the wellhead device, positioning blocks are fixed to an upper end and a lower end of the mover of the linear motor, the mover is slidably against to the side longitudinal limit posts, buffer springs are fixed to the wellhead device and the limit posts respectively.", "The mover of the motor is hollow inside, and the pumping rod passes through the center of the mover.", "The pumping rod together with a clipping seat is fixed to the mover of the linear motor.", "The headframe is provided with pulleys of a weight-balancing device.", "A wire rope is connected to the clipping seat and the counterweight at both ends thereof via the pulleys.", "Since stators of the linear motor together with the limit posts are fixed above the wellhead device, many inevitable disadvantages in the pump unit are as follows: 1.Vibration generates while the mover of the linear motor reciprocates up and down, thus causing the pumping unit to vibrate with the linear motor.", "The stators of the linear motor and the limit posts are fixed to the wellhead device, the mover of the linear motor causes the wellhead device to vibrate while it moves, thus causing leakage of crude oil and natural gas from the wellhead device.", "Even if the leakage of the crude oil and the natural gas is not caused by the vibration, it generally occurs, during the mechanical pumping of the oil.", "If leakage of the natural gas occurs, since the linear motor is fixed above the wellhead device, the leaked natural gas will enter the linear motor directly, thus causing potential safety hazard.", "2.The linear motor is fixed above the wellhead device so that it occupies a space around the wellhead, operations such as replacement of the packing set box and the packing set will become difficult.", "3.The linear motor is fixed above the wellhead device, the vibration of the motor causes the downhole casing connected to wellhead device to vibrate and deform.", "4.The linear motor is fixed above the wellhead device which can not endure the higher outside pressure.", "If the linear motor is fixed above the wellhead, a wellhead device which can endure higher outside pressure should be designed renewedly to match the linear motor, this will increase the cost of the pumping unit.", "SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to move the linear motor away from the wellhead device so as to resolve the above problems, i.e.", "the potential safety hazard occurs when the natural gas leaks; the linear motor occupies much space around wellhead because it is fixed above the wellhead device, thus causing many operations difficult; the linear motor causes the down hole casing to vibrate.", "In addition, the wellhead device can endure higher outside pressure without changing the original wellhead device in site.", "The headframe is replaced by a guide rail support of the linear motor.", "The object is accomplished by providing a pumping unit driven by a linear motor, comprising a guide rail support, a mover of the linear motor, stators of the linear motor, a chain, chain wheel and a counterweight, the guide rail support is in the form of a frame structure, stators of the linear motor are mounted on inner sides of the guide rail support, and guide slide-ways used for the mover of the linear motor are fixed on inner sides of the guide rail support.", "The mover of the linear motor is provided with slide blocks engaging with the guide slide ways.", "The chain wheel is disposed on the top of the guide rail support.", "The chain passes the chain wheel and is connected to the mover of the linear motor at one end and connected to a pumping slick rod of the wellhead device at the other end thereof.", "A counterweight chain is connected to a lower end of the mover of the linear motor at one end, passes a bottom chain wheel disposed at the bottom of the guide rail support and a top chain wheel disposed at the top of the guide rail support sequentially, and is then connected to a counterweight at the other end thereof.", "In another embodiment of the present invention, pulleys can replace the chain wheels respectively and the chains can be replaced by wire ropes accordingly.", "The advantages of the present invention are as follows: an upper end of the mover of the linear motor is connected to one end of the chain, the chain passes the chain wheel and is connected to the pumping slick rod at the other end thereof.", "The wellhead device and the pumping slick rod are away from the linear motor, and the linear motor is moved away from the above of the wellhead, when leakage of the natural gas occurs, the leaked natural gas can not enter the linear motor directly, thus eliminating the potential safety hazard.", "The linear motor is not disposed above the wellhead device, and the original wellhead device can be used, so that it is still convenient for a worker to operate around the wellhead by comparison with the pumping unit driven by a rotating motor in the prior art.", "In addition, even if the linear motor generates vibration during operating, the downhole casing will not be affected and the wellhead device can endure high pressure.", "The structure of the pumping unit driven by a linear motor is also simplified by replacing the headframe by the guide rail support of the linear motor.", "BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION These and/or other advantages of the present invention will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompany drawings, in which: FIG.", "1 is a schematic view of the structure of a pumping unit driven by a linear motor according to an embodiment of the present invention.", "DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS FIG.", "1 shows a structure of the pumping unit driven by a linear motor according to an embodiment of the present invention.", "A wellhead device 1 and a pumping slick rod 2 are necessary for an oil well, their detailed descriptions are omitted because they are substantially identical with that in prior art.", "In FIG.", "1, reference numerical 3 denotes a chain, 4 denotes a chain wheel, 5 denotes a top chain wheel, 6 denotes a counterweight chain, 7 denotes a counterweight, 8 denotes a mover of the linear motor, 9 denote stators of the linear motor, 10 denotes a bottom chain wheel disposed at bottom of the guide rail support, and 11 denotes a guide rail support.", "The structure of the present invention is explained in detail with reference to embodiments taken in conjunction with the drawing as follows.", "Embodiment 1 The guide rail support 11 of the pumping unit driven by a linear motor is formed by two channel beams, between the channel beams are provided with crossbeams, thus forming a frame structure.", "The frame structure can be formed by the channel beams and the crossbeams through welding or screwed connection.", "In order to improve stability of the frame structure, the guide rail support can be formed by flanged beams and steel plates instead of the channel beams and the crossbeams through welding.", "Four guide slide ways formed by angle bars through welding are provided on inner sides of the guide rail support.", "The guide slide ways are used for controlling sliding movement of the mover 8 of the linear motor, the mover 8 is connected to one end of the counterweight chain 6 at a lower end thereof, and the counterweight chain 6 passes a bottom chain wheel 10 disposed at the bottom of the guide rail support and a top chain wheel 5 disposed at the top of the support sequentially and is then connected to the counterweight 7 at the other end thereof.", "The counterweight 7 should be selected to meet the requirements of the oil well.", "Embodiment 2 The guide rail support 11 of the pumping unit driven by a linear motor in the embodiment 2 is substantially identical with that in the embodiment 1, except for the following aspects: there are two pulleys 4 at the top of the guide rail support 11, the diameter of the pulleys is 500 mm, the chain 3 is replaced by a wire rope 3 whose diameter is 25 mm, and the counterweight chain 6 is also replaced by a counterweight wire rope 6.One end of the counter weight wire rope 6 is connected to a lower end of the mover 8 of the linear motor, and the other end thereof passes the bottom chain wheel 10 disposed at bottom of the guide rail support and the top chain wheel 5 disposed at top of the support sequentially and is then connected to the counterweight 7.In other words, in the embodiment 2, the chain and the counterweight chain in the embodiment 1 are replaced by wire ropes, and the chain wheel, the bottom chain wheel and the top chain wheel in the embodiment 1 are replaced by pulleys accordingly.", "Of course, only the chain and chain wheel can be replaced by wire rope and pulley respectively, and the counterweight chain and the bottom and top chain wheels are not changed, vice versa.", "In any case, the effect of the present invention will not be affected." ] ]	Patent_10469112
[ [ "Creep resistant magnesium alloy", "A magnesium based alloy consists of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, and 0-0.1% oxidation inhibiting element(s) the remainder being magnesium except for incidental impurities." ], [ "1.A magnesium based alloy consisting of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, and 0-0.1% oxidation inhibiting element(s) the remainder being magnesium except for incidental impurities.", "2.A magnesium alloy consisting of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, 0-0.1% oxidation inhibiting element(s), no more than 0.15% titanium, no more than 0.15% hafnium, no more than 0.1% aluminium, no more than 0.1% copper, no more than 0.1% nickel, no more than 0.1% silicon, no more than 0.1% silver, no more than 0.1% yttrium, no more than 0.1% thorium, no more than 0.01% iron, no more than 0.005% strontium, the balance being magnesium except for incidental impurities.", "3.An alloy as claimed in claim 1 wherein the magnesium content is 95.5-97% by weight.", "4.An alloy as claimed in claim 1 wherein the neodymium content is 1.6-1.8% by weight.", "5.An alloy as claimed in claim 1 wherein the content of rare earth(s) other than neodymium is 0.9-1.1% by weight.", "6.An alloy as claimed in claim 1 which contains a plurality of rare earth elements other than neodymium and in which cerium comprises over half the weight of the rare earth elements other than neodymium.", "7.An alloy as claimed in claim 1 wherein the zirconium content is greater than 0.4% by weight.", "8.An alloy as claimed in claim 1 wherein the zinc content is 0.4-0.6% by weight.", "9.A magnesium based alloy having a microstructure comprising equiaxed grains of magnesium based solid solution separated at the grain boundaries by a generally contiguous intergranular phase, the grains containing a uniform distribution of nano-scale precipitate platelets on more than one habit plane containing magnesium and neodymium, the intergranular phase consisting almost completely of rare earth elements, magnesium and a small amount of zinc, and the rare earth elements being substantially cerium and/or lanthanum.", "10.A method of producing a magnesium alloy article, the method comprising subjecting to a T6 heat treatment an article cast from an alloy as claimed in claim 1.11.A method of manufacturing a magnesium alloy article, the method comprising the steps of: (a) solidifying in a mould a casting of an alloy as claimed in claim 1, (b) heating the solidified casting at a temperature of 500-550° C. for a first period of time, (c) quenching the casting, and (d) ageing the casting at a temperature of 200-230° C. for a second period of time.", "12.A method of manufacturing a casting made from magnesium alloy comprising the steps of: (i) melting an alloy as claimed in claim 1 to form a molten alloy, (ii) introducing the molten alloy into a sand mould or permanent mould and allowing the molten alloy to solidify, (iii) removing the resultant solidified casting from the mould, and (iv) maintaining the casting within a first temperature range for a first period of time during which a portion of an intergranular phase of the casting is dissolved, and subsequently maintaining the casting within a second temperature range lower than the first temperature range for a second period of time during which nano-scale precipitate platelets are caused to precipitate within grains of the casting and at grain boundaries.", "13.A method as claimed in claim 12 wherein the first temperature range is 500-550° C., the second temperature range is 200-230° C., the first period of time is 6-24 hours, and the second period of time is 3-24 hours.", "14.An engine block for an internal combustion engine produced by a method as claimed in claim 10.15.An engine block for an internal combustion engine formed from a magnesium alloy as claimed in claim 1." ], [ "<SOH> BACKGROUND TO THE INVENTION <EOH>Magnesium alloys have been used for many years in applications where the material of construction is required to exhibit a high strength to weight ratio.", "Typically a component made from a magnesium alloy could be expected to have a weight about 70% of an aluminium (Al) alloy component of similar volume.", "The aerospace industry has accordingly been a significant user of magnesium alloys and magnesium alloys are used for many components in modern defence aircraft and spacecraft.", "However, one limitation preventing wider use of magnesium alloys is that, when compared to aluminium alloys, they typically have poorer resistance to creep at elevated temperatures.", "With the increasing needs to control international fuel consumption and reduce harmful emissions into the atmosphere, automobile manufacturers are being pressured into developing more fuel efficient vehicles.", "Reducing the overall weight of the vehicles is a key to achieving this goal.", "A major contributor to the weight of any vehicle is the engine itself, and the most significant component of the engine is the block, which makes up 20-25% of the total engine weight.", "In the past significant weight savings were made by introducing an aluminium alloy block to replace the traditional grey iron block, and further reductions of the order of 40% could be achieved if a magnesium alloy that could withstand the temperatures and stresses generated during engine operation was used.", "However, the development of such an alloy, which combines the desired elevated temperature mechanical properties with a cost effective production process, is necessary before a viable magnesium engine block manufacturing line could be considered.", "In recent years, the search for an elevated temperature magnesium alloy has focused primarily on the high pressure die casting (HPDC) processing route and several alloys have been developed.", "HPDC was considered to be the best option for achieving the high productivity rates required to counteract the probable high cost of the base magnesium alloy.", "However, HPDC is not necessarily the best process for the manufacture of an engine block and, in reality, the majority of blocks are still precision cast by gravity or low pressure sand casting.", "There are two major classes of magnesium sand casting alloys.", "(A) Alloys based on the magnesium-aluminium binary system, often with small additions of zinc (Zn) for improved strength and castability.", "These alloys have adequate room temperature mechanical properties, but do not perform well at elevated temperatures and are inappropriate at temperatures in excess of 150° C. These alloys do not contain expensive alloying elements and are widely used in areas where high temperature strength is not a requirement.", "(B) Alloys able to be grain refined by the addition of zirconium (Zr).", "The major alloying elements in this group are zinc, yttrium (Y), silver (Ag), thorium (Th), and the rare earth (RE) elements such as neodymium (Nd).", "Throughout this specification the expression “rare earth” is to be understood to mean any element or combination of elements with atomic numbers 57 to 71, ie.", "lanthanum (La) to lutetium (Lu).", "With the right choice of alloying additions, alloys in this group can have excellent room and elevated temperature mechanical properties.", "However, with the exception of zinc, the alloying additions within this group, including the grain refiner, are expensive with the result that the alloys are generally restricted to aeronautical applications.", "The magnesium alloy ML10, developed in the USSR, has been used for many years for cast parts intended for use in aircraft at temperatures up to 250° C. ML10 is a high strength magnesium alloy developed on the basis of the Mg—Nd—Zn—Zr system.", "ML19 alloy additionally contains yttrium.", "A paper by Mukhina et al entitled “Investigation of the Microstructure and Properties of Castable Neodymium and Yttrium-Bearing Magnesium Alloys at Elevated Temperatures” published in “Science and Heat Treatment” Vol 39, 1997, indicated typical compositions (% by weight) of ML10 and ML19 alloys are: ML10 ML19 Nd 2.2-2.8 1.6-2.3 Y Nil 1.4-2.2 Zr 0.4-1.0 0.4-1.0 Zn 0.1-0.7 0.1-0.6 Mg Balance Balance with impurity levels of: Fe <0.01 Si <0.03 Cu <0.03 Ni <0.005 Al <0.02 Be <0.01 Alternatives which have been developed are alloys known to those in the art as QE22 (an Mg—Ag—Nd—Zr system alloy) and EH21 (an Mg—Nd—Zr—Th system alloy).", "However, these alternatives are expensive to manufacture as they contain significant quantities of silver and thorium respectively.", "Heat resistant grain refined magnesium alloys can be strengthened by a T6 heat treatment which comprises an elevated temperature solution treatment, followed by quenching, followed by an artificial aging at an elevated temperature.", "In heating before quenching the excess phases pass into solid solution.", "In the aging process refractory phases, in the form of finely dispersed submicroscopic particles, are segregated and these create microheterogeneities inside the grains of the solid solution, blocking diffusion and shear processes at elevated temperatures.", "This improves the mechanical properties, namely the ultimate long term strength and the creep resistance of the alloys at high temperature.", "To date, a sand casting magnesium alloy having desired elevated temperature (eg 150-200° C.) properties at a reasonable cost has been unavailable.", "At least preferred embodiments of the present invention relate to such an alloy and the present invention is particularly, but not exclusively, directed to application with precision casting operations." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In a first aspect the invention provides a magnesium based alloy consisting of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, and 0-0.1% oxidation inhibiting element(s), the remainder being magnesium except for incidental impurities.", "In a second aspect, the present invention provides a magnesium alloy consisting of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, 0-0.1% oxidation inhibiting element, no more than 0.15% titanium, no more than 0.15% hafnium, no more than 0.1% aluminium, no more than 0.1% copper, no more than 0.1% nickel, no more than 0.1% silicon, no more than 0.1% silver, no more than 0.1% yttrium, no more than 0.1% thorium, no more than 0.01% iron, no more than 0.005% strontium, the balance being magnesium except for incidental impurities.", "Preferably, alloys according to the second aspect of the present invention: (a) contain less than 0.1% titanium, more preferably less than 0.05% titanium, more preferably less than 0.01% titanium, and most preferably substantially no titanium; (b) contain less than 0.1% hafnium, more preferably less than 0.05% hafnium, more preferably less than 0.01% hafnium, and most preferably substantially no hafnium; (c) contain less than 0.05% aluminium, more preferably less than 0.02% aluminium, more preferably less than 0.01% aluminium, and most preferably substantially no aluminium; (d) contain less than 0.05% copper, more preferably less than 0.02% copper, more preferably less than 0.01% copper, and most preferably substantially no copper; (e) contain less than 0.05% nickel, more preferably less than 0.02% nickel, more preferably less than 0.01% nickel, and most preferably substantially no nickel; (f) contain less than 0.05% silicon, more preferably less than 0.02% silicon, more preferably less than 0.01% silicon, and most preferably substantially no silicon; (g) contain less than 0.05% silver, more preferably less than 0.02% silver, more preferably less than 0.01% silver, and most preferably substantially no silver; (h) contain less than 0.05% yttrium, more preferably less than 0.02% yttrium, more preferably less than 0.01% yttrium, and most preferably substantially no yttrium; (i) contain less than 0.05% thorium, more preferably less than 0.02% thorium, more preferably less than 0.01% thorium, and most preferably substantially no thorium; (j) contain less than 0.005% iron, most preferably substantially no iron; and (k) contain less than 0.001% strontium, most preferably substantially no strontium.", "Preferably, alloys according to the present invention contain at least 95% magnesium, more preferably 95.5-97% magnesium, and most preferably about 96.3% magnesium.", "Preferably, the neodymium content is greater than 1.5%, more preferably greater than 1.6%, more preferably 1.6-1.8% and most preferably about 1.7%.", "The neodymium content may be derived from pure neodymium, neodymium contained within a mixture of rare earths such as a misch metal, or a combination thereof.", "Preferably, the content of rare earth(s) other than neodymium is 0.9-1.1%, more preferably about 1%.", "Preferably, the rare earth(s) other than neodymium are cerium (Ce), lanthanum (La), or a mixture thereof.", "Preferably, cerium comprises over half the weight of the rare earth elements other than neodymium, more preferably 60-80%, especially about 70% with lanthanum comprising substantially the balance.", "The rare earth(s) other than neodymium may be derived from pure rare earths, a mixture of rare earths such as a misch metal or a combination thereof.", "Preferably, the rare earths other than neodymium are derived from a cerium misch metal containing cerium, lanthanum, optionally neodymium, a modest amount of praseodymium (Pr) and trace amounts of other rare earths.", "The habit plane of the precipitating phase in Mg—Nd—Zn alloys is related to the zinc content, being prismatic at very low levels of Zn and basal at levels in excess of about 1 wt %.", "The best strength results are obtained at zinc levels which promote a combination of the two habit planes.", "Preferably, the zinc content is less than 0.65%, more preferably 0.4-0.6%, more preferably 0.45-0.55%, most preferably about 0.5%.", "Reduction in iron content can be achieved by addition of zirconium which precipitates iron from molten alloy.", "Accordingly, the zirconium contents specified herein are residual zirconium contents.", "However, it is to be noted that zirconium may be incorporated at two different stages.", "Firstly, on manufacture of the alloy and secondly, following melting of the alloy just prior to casting.", "The elevated temperature properties of alloys of the present invention are reliant on adequate grain refinement and it is therefore necessary to maintain a level of zirconium in the melt beyond that required for iron removal.", "For desired tensile and compressive strength properties the grain size is preferably less than 200 μm and more preferably less than 150 μm.", "The relationship between creep resistance and grain size in alloys of the present invention is counter-intuitive.", "Conventional creep theory will predict that the creep resistance will decrease as the grain size decreases.", "However, alloys of the present invention have shown a minimum in creep resistance at a grain size of 200 μm and improvements in creep resistance at smaller grain sizes.", "For optimum creep resistance the grain size is preferably less than 100 μm and more preferably about 50 μm.", "Preferably, the zirconium content will be the minimum amount required to achieve satisfactory iron removal and adequate grain refinement for the intended purpose.", "Typically, the zirconium content will be greater than 0.4%, preferably 0.4-0.6%, more preferably about 0.5%.", "Manganese is an optional component of the alloy which may be included if there is a need for additional iron removal over and above that achieved by zirconium, especially if the zirconium levels are relatively low, for example below 0.5 wt %.", "Elements which prevent or at least inhibit oxidation of molten alloy, such as beryllium (Be) and calcium (Ca), are optional components which may be included especially in circumstances where adequate melt protection through cover gas atmosphere control is not possible.", "This is particularly the case when the casting process does not involve a closed system.", "Ideally, the incidental impurity content is zero but it is to be appreciated that this is essentially impossible.", "Accordingly, it is preferred that the incidental impurity content is less than 0.15%, more preferably less than 0.1%, more preferably less than 0.01%, and still more preferably less than 0.001%.", "In a third aspect, the present invention provides a magnesium based alloy having a microstructure comprising equiaxed grains of magnesium based solid solution separated at the grain boundaries by a generally contiguous intergranular phase, the grains containing a uniform distribution of nano-scale precipitate platelets on more than one habit plane containing magnesium and neodymium, the intergranular phase consisting almost completely of rare earth elements, magnesium and a small amount of zinc, and the rare earth elements being substantially cerium and/or lanthanum.", "The grains may contain clusters of small spherical and globular precipitates.", "The spherical clusters may comprise fine rod-like precipitates.", "The globular precipitates may be predominantly zirconium plus zinc with a Zr:Zn atomic ratio of approximately 2:1.The rod-like precipitates may be predominantly zirconium plus zinc with a Zr:Zn atomic ratio of approximately 2:1.The expression “generally contiguous” as used in this specification is intended to mean that at least most of the intergranular phase is contiguous but that some gaps may exist between otherwise contiguous portions.", "In a fourth aspect, the present invention provides a method of producing a magnesium alloy article, the method comprising subjecting to a T6 heat treatment an article cast from an alloy according to the first, second or third aspect of the present invention.", "In a fifth aspect, the present invention provides a method of manufacturing a magnesium alloy article, the method comprising the steps of: (a) solidifying in a mould a casting of an alloy according to the first, second or third aspects of the present invention, (b) heating the solidified casting at a temperature of 500-550° C. for a first period of time, (c) quenching the casting, and (d) ageing the casting at a temperature of 200-230° C. for a second period of time.", "Preferably, the first period of time is 6-24 hours and the second period of time is 3-24 hours.", "In a sixth aspect, the present invention provides a method of manufacturing a casting made from magnesium alloy comprising the steps of: (i) melting an alloy according to the first, second or third aspects of the present invention to form a molten alloy, (ii) introducing the molten alloy into a sand mould or permanent mould and allowing the molten alloy to solidify, (iii) removing the resultant solidified casting from the mould, and (iv) maintaining the casting within a first temperature range for a first period of time during which a portion of an intergranular phase of the casting is dissolved, and subsequently maintaining the casting within a second temperature range lower than the first temperature range for a second period of time during which nano-scale precipitate platelets are caused to precipitate within grains of the casting and at grain boundaries.", "The first temperature range is preferably 500-550° C., the second temperature range is preferably 200-230° C., the first period of time is preferably 6-24 hours, and the second period of time is preferably 3-24 hours.", "In a seventh aspect, the present invention provides an engine block for an internal combustion engine produced by a method according to the fourth, fifth or sixth aspect of the present invention.", "In an eighth aspect, the present invention provides an engine block for an internal combustion engine formed from a magnesium alloy according to the first, second or third aspects of the present invention.", "Specific reference is made above to engine blocks but it is to be noted that alloys of the present invention may find use in other elevated temperature applications as well as low temperature applications.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates to magnesium (Mg) alloys and, more particularly, to magnesium alloys which are resistant to creep at high temperatures.", "BACKGROUND TO THE INVENTION Magnesium alloys have been used for many years in applications where the material of construction is required to exhibit a high strength to weight ratio.", "Typically a component made from a magnesium alloy could be expected to have a weight about 70% of an aluminium (Al) alloy component of similar volume.", "The aerospace industry has accordingly been a significant user of magnesium alloys and magnesium alloys are used for many components in modern defence aircraft and spacecraft.", "However, one limitation preventing wider use of magnesium alloys is that, when compared to aluminium alloys, they typically have poorer resistance to creep at elevated temperatures.", "With the increasing needs to control international fuel consumption and reduce harmful emissions into the atmosphere, automobile manufacturers are being pressured into developing more fuel efficient vehicles.", "Reducing the overall weight of the vehicles is a key to achieving this goal.", "A major contributor to the weight of any vehicle is the engine itself, and the most significant component of the engine is the block, which makes up 20-25% of the total engine weight.", "In the past significant weight savings were made by introducing an aluminium alloy block to replace the traditional grey iron block, and further reductions of the order of 40% could be achieved if a magnesium alloy that could withstand the temperatures and stresses generated during engine operation was used.", "However, the development of such an alloy, which combines the desired elevated temperature mechanical properties with a cost effective production process, is necessary before a viable magnesium engine block manufacturing line could be considered.", "In recent years, the search for an elevated temperature magnesium alloy has focused primarily on the high pressure die casting (HPDC) processing route and several alloys have been developed.", "HPDC was considered to be the best option for achieving the high productivity rates required to counteract the probable high cost of the base magnesium alloy.", "However, HPDC is not necessarily the best process for the manufacture of an engine block and, in reality, the majority of blocks are still precision cast by gravity or low pressure sand casting.", "There are two major classes of magnesium sand casting alloys.", "(A) Alloys based on the magnesium-aluminium binary system, often with small additions of zinc (Zn) for improved strength and castability.", "These alloys have adequate room temperature mechanical properties, but do not perform well at elevated temperatures and are inappropriate at temperatures in excess of 150° C. These alloys do not contain expensive alloying elements and are widely used in areas where high temperature strength is not a requirement.", "(B) Alloys able to be grain refined by the addition of zirconium (Zr).", "The major alloying elements in this group are zinc, yttrium (Y), silver (Ag), thorium (Th), and the rare earth (RE) elements such as neodymium (Nd).", "Throughout this specification the expression “rare earth” is to be understood to mean any element or combination of elements with atomic numbers 57 to 71, ie.", "lanthanum (La) to lutetium (Lu).", "With the right choice of alloying additions, alloys in this group can have excellent room and elevated temperature mechanical properties.", "However, with the exception of zinc, the alloying additions within this group, including the grain refiner, are expensive with the result that the alloys are generally restricted to aeronautical applications.", "The magnesium alloy ML10, developed in the USSR, has been used for many years for cast parts intended for use in aircraft at temperatures up to 250° C. ML10 is a high strength magnesium alloy developed on the basis of the Mg—Nd—Zn—Zr system.", "ML19 alloy additionally contains yttrium.", "A paper by Mukhina et al entitled “Investigation of the Microstructure and Properties of Castable Neodymium and Yttrium-Bearing Magnesium Alloys at Elevated Temperatures” published in “Science and Heat Treatment” Vol 39, 1997, indicated typical compositions (% by weight) of ML10 and ML19 alloys are: ML10 ML19 Nd 2.2-2.8 1.6-2.3 Y Nil 1.4-2.2 Zr 0.4-1.0 0.4-1.0 Zn 0.1-0.7 0.1-0.6 Mg Balance Balance with impurity levels of: Fe <0.01 Si <0.03 Cu <0.03 Ni <0.005 Al <0.02 Be <0.01 Alternatives which have been developed are alloys known to those in the art as QE22 (an Mg—Ag—Nd—Zr system alloy) and EH21 (an Mg—Nd—Zr—Th system alloy).", "However, these alternatives are expensive to manufacture as they contain significant quantities of silver and thorium respectively.", "Heat resistant grain refined magnesium alloys can be strengthened by a T6 heat treatment which comprises an elevated temperature solution treatment, followed by quenching, followed by an artificial aging at an elevated temperature.", "In heating before quenching the excess phases pass into solid solution.", "In the aging process refractory phases, in the form of finely dispersed submicroscopic particles, are segregated and these create microheterogeneities inside the grains of the solid solution, blocking diffusion and shear processes at elevated temperatures.", "This improves the mechanical properties, namely the ultimate long term strength and the creep resistance of the alloys at high temperature.", "To date, a sand casting magnesium alloy having desired elevated temperature (eg 150-200° C.) properties at a reasonable cost has been unavailable.", "At least preferred embodiments of the present invention relate to such an alloy and the present invention is particularly, but not exclusively, directed to application with precision casting operations.", "SUMMARY OF THE INVENTION In a first aspect the invention provides a magnesium based alloy consisting of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, and 0-0.1% oxidation inhibiting element(s), the remainder being magnesium except for incidental impurities.", "In a second aspect, the present invention provides a magnesium alloy consisting of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, 0-0.1% oxidation inhibiting element, no more than 0.15% titanium, no more than 0.15% hafnium, no more than 0.1% aluminium, no more than 0.1% copper, no more than 0.1% nickel, no more than 0.1% silicon, no more than 0.1% silver, no more than 0.1% yttrium, no more than 0.1% thorium, no more than 0.01% iron, no more than 0.005% strontium, the balance being magnesium except for incidental impurities.", "Preferably, alloys according to the second aspect of the present invention: (a) contain less than 0.1% titanium, more preferably less than 0.05% titanium, more preferably less than 0.01% titanium, and most preferably substantially no titanium; (b) contain less than 0.1% hafnium, more preferably less than 0.05% hafnium, more preferably less than 0.01% hafnium, and most preferably substantially no hafnium; (c) contain less than 0.05% aluminium, more preferably less than 0.02% aluminium, more preferably less than 0.01% aluminium, and most preferably substantially no aluminium; (d) contain less than 0.05% copper, more preferably less than 0.02% copper, more preferably less than 0.01% copper, and most preferably substantially no copper; (e) contain less than 0.05% nickel, more preferably less than 0.02% nickel, more preferably less than 0.01% nickel, and most preferably substantially no nickel; (f) contain less than 0.05% silicon, more preferably less than 0.02% silicon, more preferably less than 0.01% silicon, and most preferably substantially no silicon; (g) contain less than 0.05% silver, more preferably less than 0.02% silver, more preferably less than 0.01% silver, and most preferably substantially no silver; (h) contain less than 0.05% yttrium, more preferably less than 0.02% yttrium, more preferably less than 0.01% yttrium, and most preferably substantially no yttrium; (i) contain less than 0.05% thorium, more preferably less than 0.02% thorium, more preferably less than 0.01% thorium, and most preferably substantially no thorium; (j) contain less than 0.005% iron, most preferably substantially no iron; and (k) contain less than 0.001% strontium, most preferably substantially no strontium.", "Preferably, alloys according to the present invention contain at least 95% magnesium, more preferably 95.5-97% magnesium, and most preferably about 96.3% magnesium.", "Preferably, the neodymium content is greater than 1.5%, more preferably greater than 1.6%, more preferably 1.6-1.8% and most preferably about 1.7%.", "The neodymium content may be derived from pure neodymium, neodymium contained within a mixture of rare earths such as a misch metal, or a combination thereof.", "Preferably, the content of rare earth(s) other than neodymium is 0.9-1.1%, more preferably about 1%.", "Preferably, the rare earth(s) other than neodymium are cerium (Ce), lanthanum (La), or a mixture thereof.", "Preferably, cerium comprises over half the weight of the rare earth elements other than neodymium, more preferably 60-80%, especially about 70% with lanthanum comprising substantially the balance.", "The rare earth(s) other than neodymium may be derived from pure rare earths, a mixture of rare earths such as a misch metal or a combination thereof.", "Preferably, the rare earths other than neodymium are derived from a cerium misch metal containing cerium, lanthanum, optionally neodymium, a modest amount of praseodymium (Pr) and trace amounts of other rare earths.", "The habit plane of the precipitating phase in Mg—Nd—Zn alloys is related to the zinc content, being prismatic at very low levels of Zn and basal at levels in excess of about 1 wt %.", "The best strength results are obtained at zinc levels which promote a combination of the two habit planes.", "Preferably, the zinc content is less than 0.65%, more preferably 0.4-0.6%, more preferably 0.45-0.55%, most preferably about 0.5%.", "Reduction in iron content can be achieved by addition of zirconium which precipitates iron from molten alloy.", "Accordingly, the zirconium contents specified herein are residual zirconium contents.", "However, it is to be noted that zirconium may be incorporated at two different stages.", "Firstly, on manufacture of the alloy and secondly, following melting of the alloy just prior to casting.", "The elevated temperature properties of alloys of the present invention are reliant on adequate grain refinement and it is therefore necessary to maintain a level of zirconium in the melt beyond that required for iron removal.", "For desired tensile and compressive strength properties the grain size is preferably less than 200 μm and more preferably less than 150 μm.", "The relationship between creep resistance and grain size in alloys of the present invention is counter-intuitive.", "Conventional creep theory will predict that the creep resistance will decrease as the grain size decreases.", "However, alloys of the present invention have shown a minimum in creep resistance at a grain size of 200 μm and improvements in creep resistance at smaller grain sizes.", "For optimum creep resistance the grain size is preferably less than 100 μm and more preferably about 50 μm.", "Preferably, the zirconium content will be the minimum amount required to achieve satisfactory iron removal and adequate grain refinement for the intended purpose.", "Typically, the zirconium content will be greater than 0.4%, preferably 0.4-0.6%, more preferably about 0.5%.", "Manganese is an optional component of the alloy which may be included if there is a need for additional iron removal over and above that achieved by zirconium, especially if the zirconium levels are relatively low, for example below 0.5 wt %.", "Elements which prevent or at least inhibit oxidation of molten alloy, such as beryllium (Be) and calcium (Ca), are optional components which may be included especially in circumstances where adequate melt protection through cover gas atmosphere control is not possible.", "This is particularly the case when the casting process does not involve a closed system.", "Ideally, the incidental impurity content is zero but it is to be appreciated that this is essentially impossible.", "Accordingly, it is preferred that the incidental impurity content is less than 0.15%, more preferably less than 0.1%, more preferably less than 0.01%, and still more preferably less than 0.001%.", "In a third aspect, the present invention provides a magnesium based alloy having a microstructure comprising equiaxed grains of magnesium based solid solution separated at the grain boundaries by a generally contiguous intergranular phase, the grains containing a uniform distribution of nano-scale precipitate platelets on more than one habit plane containing magnesium and neodymium, the intergranular phase consisting almost completely of rare earth elements, magnesium and a small amount of zinc, and the rare earth elements being substantially cerium and/or lanthanum.", "The grains may contain clusters of small spherical and globular precipitates.", "The spherical clusters may comprise fine rod-like precipitates.", "The globular precipitates may be predominantly zirconium plus zinc with a Zr:Zn atomic ratio of approximately 2:1.The rod-like precipitates may be predominantly zirconium plus zinc with a Zr:Zn atomic ratio of approximately 2:1.The expression “generally contiguous” as used in this specification is intended to mean that at least most of the intergranular phase is contiguous but that some gaps may exist between otherwise contiguous portions.", "In a fourth aspect, the present invention provides a method of producing a magnesium alloy article, the method comprising subjecting to a T6 heat treatment an article cast from an alloy according to the first, second or third aspect of the present invention.", "In a fifth aspect, the present invention provides a method of manufacturing a magnesium alloy article, the method comprising the steps of: (a) solidifying in a mould a casting of an alloy according to the first, second or third aspects of the present invention, (b) heating the solidified casting at a temperature of 500-550° C. for a first period of time, (c) quenching the casting, and (d) ageing the casting at a temperature of 200-230° C. for a second period of time.", "Preferably, the first period of time is 6-24 hours and the second period of time is 3-24 hours.", "In a sixth aspect, the present invention provides a method of manufacturing a casting made from magnesium alloy comprising the steps of: (i) melting an alloy according to the first, second or third aspects of the present invention to form a molten alloy, (ii) introducing the molten alloy into a sand mould or permanent mould and allowing the molten alloy to solidify, (iii) removing the resultant solidified casting from the mould, and (iv) maintaining the casting within a first temperature range for a first period of time during which a portion of an intergranular phase of the casting is dissolved, and subsequently maintaining the casting within a second temperature range lower than the first temperature range for a second period of time during which nano-scale precipitate platelets are caused to precipitate within grains of the casting and at grain boundaries.", "The first temperature range is preferably 500-550° C., the second temperature range is preferably 200-230° C., the first period of time is preferably 6-24 hours, and the second period of time is preferably 3-24 hours.", "In a seventh aspect, the present invention provides an engine block for an internal combustion engine produced by a method according to the fourth, fifth or sixth aspect of the present invention.", "In an eighth aspect, the present invention provides an engine block for an internal combustion engine formed from a magnesium alloy according to the first, second or third aspects of the present invention.", "Specific reference is made above to engine blocks but it is to be noted that alloys of the present invention may find use in other elevated temperature applications as well as low temperature applications.", "DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION EXAMPLE 1 Samples were gravity cast from six alloy compositions (see Table 1) into a stepped plate mould having step thicknesses from 5 mm to 25 mm to form castings as illustrated in FIG.", "1.The rare earths other than neodymium were added as a Ce-based misch metal which contained cerium, lanthanum and some neodymium.", "The extra neodymium and the zinc were added in their elemental forms.", "The zirconium was added through a proprietary Mg—Zr master alloy.", "Standard melt handling procedures were used throughout preparation of the cast plates.", "Individual samples were then subjected to T6 heat treatment no.", "3 of Table 2 which was determined to provide the best results.", "The solution heat treatment was carried out in a controlled atmosphere environment to prevent oxidation of the surface layers during the heat treatment.", "The resulting heat treated samples were then examined and tested to determine hardness, tensile strength, creep properties, corrosion resistance, fatigue performance and bolt load retention behaviour.", "Details are as shown in Tables 1 and 2 below.", "TABLE 1 Compositions Evaluated Wt % RE Composition other Wt % Wt % No.", "Wt % Zn Wt % Nd than Nd Zr Total RE Comparative - A 0.42 1.40 1.33 0.47 2.73 Comparative - B 0.85 2.04 1.13 0.503 3.17 Comparative - C 0.88 1.68 0.82 0.519 2.50 Inventive - 1 0.41 1.63 0.8 0.495 2.43 Inventive - 2 0.67 1.64 0.81 0.459 2.45 Inventive - 3 0.55 1.70 0.94 0.55 2.64 TABLE 2 T6 Heat Treatments Evaluated Heat Solution Treatment No.", "Treatment Quench Type Ageing 0 525° C. 80° C. Water 215° C. 8 hrs 16 hours 1 525° C. 80° C. Water 215° C. 8 hrs 4 hours 2 525° C. 80° C. Water 215° C. 4 hrs 150 mins 3 525° C. 80° C. Water + Aquaquench 215° C. 8 hrs 4 hours 4 525° C. Air 215° C. 8 hrs 4 hours 5 525° C. 80° C. Water + Aquaquench 215° C. 8 hrs 8 hours 6 525° C. 80° C. Water + Aquaquench 215° C. 8 hrs 150 mins 7 525° C. 80° C. Water + Aquaquench 215° 4 hours 4 hrs The following conclusions were drawn from analysis of the results.", "Micrographs showed that Comparative Composition B had the greatest amount of intermetallic phase at the grain boundaries and triple points, which is consistent with it having the highest total rare earth content.", "Comparative Composition C and Inventive Composition 1 had the least amounts of intermetallic phase, which is also consistent with them having a low total rare earth content.", "Micrographs of Inventive Composition 2 clearly showed a much larger and more variable grain size than any of the other compositions.", "This may be due to the slightly lower Zr content of this composition.", "All six compositions had the clouds of precipitates located approximately at the centre of the grains which are described elsewhere in this specification as being a Zr—Zn compound.", "Hardness measurements were carried out and Inventive Compositions 1 and 2 were consistently as good as or better than Inventive Composition 3, indicating that Zn levels of 0.4-0.6 wt % are acceptable.", "Comparative Composition C gave consistently low hardness values, indicating that the combination of high Zn and low rare earth is less suitable.", "Comparative Compositions A and B were very similar to the Inventive Compositions, which could indicate that the deleterious effect of a high Zn content can be compensated for by very high rare earth contents.", "However, this is commercially unattractive because of the high cost of rare earth metals.", "The tensile properties were determined at room temperature, 100° C., 150° C. and 177° C. The composition variants were chosen so that the effects of several interactions could be investigated, and the following observations have been made.", "Inventive Composition 1, which is similar to Inventive Composition 3 in Nd content but lower in Zn and other rare earth elements, has mechanical properties as good as or better than Inventive Composition 3, indicating that a low Zn and/or rare earth content is not necessarily detrimental to mechanical properties.", "Comparative Composition A and Inventive Composition 1 have very similar low Zn contents, whilst Comparative Composition A has a lower Nd content, a higher other rare earth content and a higher total rare earth content.", "At room temperature Inventive Composition 1 had the better proof stress and slightly higher elongation, which is consistent with there being extra Nd to provide strengthening and less Ce/La grain boundary intermetallic phase.", "At elevated temperature the room temperature trend was maintained.", "Inventive Compositions 1 and 2 and Comparative Composition C were compositionally very similar except for Zn content which was higher in Comparative Composition C. Comparative Composition C had slightly higher Nd and other rare earth contents than Inventive Compositions 1 or 2.At both room and elevated temperatures it was found that as the Zn content was increased the proof stress decreased and the elongation increased.", "The most significant drop in proof stress occurred between 0.4 and 0.67% Zn.", "Comparative Compositions B and C both had very similar (high) Zn contents with Comparative Composition B having a higher total rare earth content (from higher Nd and higher Ce/La) than Comparative Composition C. Comparative Composition B was consistently better than Comparative Composition C in terms of both proof stress and elongation at all temperatures; two properties which have a significant effect on creep behaviour.", "Creep tests were carried out on all compositions at a constant load of 90 MPa and at temperatures of 150° C. and 177° C. The steady state creep rates are listed in Table 3.TABLE 3 Steady State Creep Rates (s−1) 90 MPa 150° C. 90 MPa 177° C. Comparative Composition A 7.05 × 10−11 3.6 × 10−10 Comparative Composition B 2.66 × 10−11 1.67 × 10−10 Comparative Composition C 4.07 × 10−11 2.5 × 10−10 Inventive Composition 1 5.56 × 10−11 5.31 × 10−10 Inventive Composition 2 2.59 × 10−11 3.6 × 10−10 Inventive Composition 3 2.80 × 10−11 1.40 × 10−10 The stress to give a value of 0.1% creep strain after 100 hours is often quoted when comparing various creep resistant magnesium alloys.", "None of the six compositions had creep strains of this order after 100 hours at 150° C. and 90 MPa.", "Similarly, at 177° C., no composition exceeded this value after 100 hours, although creep strains in excess of that were reached at much longer test times.", "At 150° C. all six compositions would be acceptable in terms of their creep behaviour.", "The zinc effect noticed in the tensile results was also evident in the creep results at 150° C., particularly with respect to the primary creep extension where Inventive Composition 1 was better than Inventive Composition 2, which was in turn better than Comparative Composition C. The secondary creep rates were similar in these three compositions.", "Comparative Composition B, which had the highest Zn content but also a high rare earth content was also acceptable, indicating again that the deleterious effects of the high Zn content can be counteracted by high rare earth contents.", "Comparative Composition A had a higher primary response than Inventive Composition 1 and a slightly higher steady state creep rate, which indicates that although a Nd level of 1.4% is acceptable, 1.5% would be a preferable minimum and 1.6% even more preferable.", "EXAMPLE 2 Experimental Procedure Samples of an alloy designated SC1 (96.3% Mg, 1.7% Nd, 1.0% RE (Ce:La of ˜70:30), 0.5% Zn and 0.5% Zr) were prepared from gravity cast stepped plates, as shown in FIG.", "1.The Ce and La were added as a Ce-based misch metal which also contained some Nd.", "The extra Nd and the Zn were added in their elemental forms.", "The zirconium was added through a proprietary Mg—Zr master alloy.", "The mechanical properties presented here were determined from samples cut from the 15 mm step, where the grain size achieved was approximately 40 μm.", "Standard melt handling procedures and controlled environment heat treatment conditions were used throughout the preparation of the cast plates.", "MICROSTRUCTURE—Samples for metallographic examination were polished with diamond pastes to 1 μm followed by 0.05 μm colloidal silica.", "Etching was carried out in a solution of nitric acid in ethylene glycol and water for approximately 12 seconds.", "TENSION AND COMPRESSION TESTS—The tensile properties were measured in accordance with ASTM E8 at 20, 100, 150 and 177° C. in air using an Instron Testing Machine.", "Samples were held at temperature for 10 minutes prior to testing.", "The test specimens had a rectangular cross section (6 mm×3 mm), with a gauge length of 25 mm (FIG.", "2(a)).", "The compressive yield strength was determined in accordance with ASTM E9 at the same temperatures using cylindrical samples 15 mm in diameter and 30 mm long.", "The elastic modulus of the alloy was determined at room and elevated temperatures using a Piezoelectric Ultrasonic Composite Oscillator Technique (PUCOT) [Robinson, W H and Edgar A IEEE Transactions on Sonics and Ultrasonics, SU-21(2) 1974 98-105].", "CREEP TESTS—The creep behaviour was determined on constant load machines at temperatures of 150 and 177° C. and stresses of 46, 60, 75 and 90 MPa, in temperature controlled silicone oil baths.", "The test samples were the same geometry as those used in the tensile testing, and the extension during creep was measured directly from the gauge lengths of the samples.", "FATIGUE TESTS—The fatigue strengths at 106 and 107 cycles were determined at 25 and 120° C. in air.", "The specimens had a circular cross-section, 5 mm in diameter and a 10 mm gauge length (FIG.", "2(b)), polished to 1 μm finish which corresponds approximately to the surface finish at the main bearing—the most highly stressed part of an engine block.", "Specimens were loaded axially in fully reversed tension-compression (ie.", "at zero mean stress) and the test frequency was 60 Hz, corresponding to nominal service conditions.", "There are several procedures for assessing the fatigue strength at a given life and here the staircase method was used (BS 3518 Part 5).", "BOLT LOAD RETENTION (BLR) TESTS—Bolt load retention testing can be used to simulate the relaxation that may occur in service under a compressive loading.", "The test method [Pettersen K and Fairchild S SAE Technical Paper 970326] involves applying an initial load (in this case 8 kN) through an assembly consisting of two identical bosses, 15 mm thick and 16 mm outside diameter, made of the test material and a high strength M8 bolt instrumented with strain gauges (FIG.", "3).", "The change in load over 100 h at an elevated temperature (150° C. and 177° C.) is measured continuously.", "The two significant loads, in terms of defining the BLR behaviour, are the initial load at ambient temperature, PI, and the load at the completion of the test after returning to ambient conditions, PF.", "The ratio of these two values (PF/PI) is a measure of the bolt load retention behaviour of an alloy.", "There is often an initial increase in load as the bolted assembly is heated to the test temperature.", "This is the result of the combined thermal expansion of the bolted assembly and the yield deformation in the alloy bosses.", "THERMAL CONDUCTIVITY—The thermal conductivity was measured on samples 30 mm in diameter and 30 mm long.", "CORROSION RESISTANCE—The corrosion resistance of SC1 was compared to that of AZ91, using standard saline immersion tests at room temperature.", "The tests were carried out over a period of seven days in a saline environment (3.5% NaCl solution) with the pH stabilised to 11.0 using 1M NaOH solution.", "The corrosion products were removed from the test coupons using a chromic acid wash followed by an ethanol rinse.", "Results and Discussion MICROSTRUCTURE—Being a sand casting alloy, SC1 requires a T6 treatment (solution heat treatment in a controlled atmosphere, cold or warm water quench, and elevated temperature anneal) to fully develop its mechanical properties.", "The recommended heat treatment regime is a balance between mechanical property requirements and commercially acceptable holding times after casting.", "The T6 microstructure of SC1, which is shown in FIG.", "4, consists of grains of an α-Mg phase (A) locked by a magnesium-rare earth intermetallic phase (B) at grain boundaries and triple points.", "Clusters of rod-like precipitates (C) are present within the central regions of most grains.", "The intermetallic phase, B, has a stoichiometry close to Mg12(La0.43Ce0.57) TENSILE AND COMPRESSIVE STRENGTHS—FIG.", "5(a) shows both the tensile properties (the 0.2% proof strength and the ultimate tensile strength) and the compressive yield strength as a function of temperature.", "FIG.", "5(b) shows the tensile elongation, also as a function of temperature.", "It is significant to note that the mechanical properties of SC1 are extremely stable at elevated temperatures, with the proof strengths in both tension and compression being relatively unchanged between room temperature and 177° C. The room temperature properties of SC1 are nowhere near as high as most other magnesium sand casting alloys but it is the stability of these properties up to 177° C. which makes this alloy particularly attractive for engine block applications.", "The results of the elastic modulus determination are shown in Table 4, and it is of note that the elastic modulus shows a drop of less than 10% at 177° C. over the room temperature value.", "TABLE 4 Elastic Modulus of SC1 as determined using a PUCO technique.", "Young's Modulus (GPa) 25° C. 100° C. 177° C. 45.8 ± 0.3 43.9 ± 0.3 41.9 ± 0.3 CREEP AND BOLT LOAD RETENTION BEHAVIOUR—The microstructure of SC1 is extremely stable at temperatures up to 177° C., and this is an important factor, together with the form and distribution of the grain boundary intermetallic phase, in achieving the requisite creep resistance.", "The use of a creep stress, being the stress to produce a creep strain of 0.1% after 100 hours at temperature, as a measure of creep resistance is an arbitrary one, but it is nonetheless a useful method for comparing alloy behaviour.", "Using this concept, the behaviour of SC1 may be compared to that of A319 (FIG.", "6) and it is clear that the two alloys are very similar in their creep responses in the temperature range 150 to 177° C. More importantly, however, it should be noted that the stresses required to produce a creep strain of 0.1% in SC1 after 100 hours at both 150 and 177° C. are approaching the tensile yield strengths (0.2% offset) of the material.", "Typical bolt load retention curves for SC1, A319 and AE42 at 150° C. and 8 kN load are shown in FIG.", "7(a).", "SC1 is in the T6 condition, A319 is as sand cast and AE42 is high pressure die cast (ie.", "all three alloys are in their normal operating condition).", "The increase in load occurring at the commencement of the test is the net result of the thermal expansion of the bolted assembly less the yield deformation in the alloy bosses.", "Two significant loads are the initial load at ambient temperature, PI (8 kN in this case), and the load at the completion of the test after returning to ambient conditions, PF.", "The ratio of these two values is taken as a measure of the bolt load retention behaviour of an alloy, and has been used in this case to compare SC1 with die cast AE42 at 150 and 177° C. (FIG.", "7(b)).", "The bolt load retention behaviour at elevated temperatures again reflects the high temperature stability of this alloy and it is clear that SC1 is as good as the aluminium alloy A319 and superior to AE42 in this respect.", "FATIGUE PROPERTIES—An engine block is continually subjected to cyclic stresses during service and it is necessary, therefore, to ensure that the material chosen for the block can withstand this fatigue loading.", "The fatigue strengths of SC1 at 106 and 107 cycles were determined at both 24 and 120° C., and the figures quoted in Table 5 are the stresses giving a 50% probability of fracture.", "The limits represent the stresses for the 10% and 90% probabilities of fracture.", "It should be noted that these results are for a maximum of 107 cycles, rather than the 5×107 specified in the design criteria.", "Nonetheless, the strengths are sufficiently high for the alloy to be considered to have met the target.", "TABLE 5 Fatigue Strengths of SC1 at two temperatures (R = −1).", "Fatigue Strength (MPa) Temperature 106 cycles 107 cycles 24° C. ˜80 75 ± 18 120° C. 74 ± 9 71 ± 7 ˜denotes 12 samples only tested, rather than the 15 required by the standard CORROSION—The corrosion behaviour of the alloy, both internally and externally, is of paramount importance.", "Corrosion on the internal surfaces may be controlled by the use of an appropriate engine coolant combined with careful design to ensure compatibility of all the metal components in contact with the coolant liquid.", "The corrosion resistance of the external surfaces will depend to a large extent on the composition of the alloy itself.", "There is no one test which can determine the corrosion resistance of an alloy in all environments and therefore SC1 has been compared to AZ91 using a standard saline immersion test.", "Both the alloys were in the T6 heat treated condition, and the mean weight loss rates over this time were found to be 0.864 mg/cm2/day for SC1 and 0.443 mg/cm2/day for AZ91E.", "THERMAL CONDUCTIVITY—The thermal conductivity of SC1 was found to be 102 W/mK, which is slightly less than that originally specified in the design criteria.", "However, with this information available, it is not difficult to modify the design of an engine block to accommodate this thermal conductivity value.", "CONCLUSION SC1 is able to meet the following specifications: 0.2% proof strength of 120 MPa at room temperature and 110 MPa at 177° C. Creep resistance comparable to that of A319 at temperatures of 150° C. and 177° C. Fatigue limit in excess of 50 MPa at room temperature.", "This combination of superior elevated temperature mechanical properties and calculated cost effectiveness suggests SC1 would make a commercially viable option as an engine block material.", "In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, ie.", "to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.", "It is to be clearly understood that although prior art publication(s) are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art in Australia or in any other country." ] ]	Patent_10469113
[ [ "Alzheimer's disease diagnosis based on mitogen-activated protein kinase phosphorylation", "A method of diagnosing Alzheimer's disease in a patient comprises determining whether the phosphorylation level of an indicator protein in cells of the patient after stimulus with an activator compound is abnormally elevated as compared to a basal phosphorylation level, the indicator protein being e.g.", "Erk1/2 and the activator compound being e.g.", "bradykinin." ], [ "1.A method of diagnosing Alzheimer's disease in a subject, said method comprising: (a) measuring a basal level of phosphorylation of an indicator protein in cells from the subject; (b) contacting cells from the subject with an activator compound, the activator compound and indicator protein being selected such that the activator elicits a differential response of activated phosphorylation of the indicator protein in cells of the subject as compared to an activated phosphorylation response in cells from a non-Alzheimer's control subject at a predetermined time after the contacting is initiated; (c) measuring an activated phosphorylation level of the indicator protein in said subject cells at the predetermined time after contacting is initiated; and (d) calculating a ratio of the activated phosphorylation level determined in step (c) to the basal phosphorylation level of step (a); and (e) comparing the calculated ratio of step (d) to previously determined activated phosphorylation ratios measured from known Alzheimer's disease cells and from known non-Alzheimer's disease cells at said predetermined time; wherein if the calculated ratio is not statistically different from the previously determined ratios for said known Alzheimer's disease cells, the diagnosis is positive, and/or if the calculated ratio is not statistically different from the previously determined ratios for said known non-Alzheimer's disease cells, the diagnosis is negative.", "2.A method of diagnosing Alzheimer's disease in a subject comprising: (a) in cells of the subject, measuring a background phosphorylation level of an indicator calcium signaling pathway protein whose phosphorylation is associated with IP-3R-sensitive Ca2+ elevation in the cells; (b) stimulating cells of the subject by contact with and IP3R agonist that elicits a differential response in the phosphorylation level of the indicator protein in cells of Alzheimer's subjects as compared to the level in a non-Alzheimer's control cell; (c) thereafter, measuring a response phosphorylation level of the indicator protein in the contacted cells, and (d) determining whether the response phosphorylation level of the indicator protein as compared to the background level matches the response level known for a cell from an Alzheimer's subjects or from a healthy control cell.", "3.The method of claim 2, comprising first measuring the background phosphorylation level of the indicator protein in a culture of cells, then adding the IP3R agonist to the culture, and measuring the response phosphorylation level.", "4.The method of claim 2, comprising measuring the background level in a first aliquot of cells, stimulating a similar aliquot of the cells, and measuring the response level in the aliquot.", "5.The method of claim 2, wherein the measuring comprises an immunoassay 6.The method of claim 2, wherein the measuring comprises an immunoassay of disrupted cells.", "7.The method of claim 2, wherein the IP3-R agonist is selected from the group consisting of bradykinin, bombesin, choleystokinin, thrombin, prostaglandin F2α and vasopressin.", "8.The method of claim 2, wherein said cells are selected from the group consisting of fibroblasts, buccal mucosal cells, neurons, and blood cells.", "9.A method of diagnosing Alzheimer's disease in a subject, comprising: (a) obtaining cells from said subject; (b) measuring the basal level of phosphorylation of an indicator protein in said cells; (c) contacting said cells with an activator of phosphorylation of the indicator protein; (d) measuring the phosphorylation level of the indicator protein in said cells at a predetermined time after initiation of the contacting; and (e) calculating a first ratio of the level measured in step (d) to the level measured in step (b) and comparing said first ratio to a previously determined second ratio of said levels obtained at said predetermined time from known Alzheimer's disease cells and a third ratio of said levels obtained at said predetermined time from known non-Alzheimer's disease cells; wherein (i) if the first ratio of step (e) is statistically not different from the previously determined second ratio, the diagnosis is positive, and (ii) if the first ratio of step (e) is not statistically different from the previously determined third ratio, the diagnosis is negative.", "10.The method of claim 9, wherein the predetermined time of step (d) is a time when the difference between the second ratio and the third ratio of step (e) is greatest.", "11.The method of claim 1, wherein the activator is selected from the group consisting of bradykinin, bombesin, cholecystokinin, thrombin, prostaglandin F2α and vasopressin.", "12.The method of claim 1 wherein the measuring is by immunoassay, and wherein said subject's cells are contacted with an antibody specific for the phosphorylated indicator protein, permitting the antibody to bind to the indicator protein, and detecting the antibody bound to the indicator protein.", "13.The method of claim 12 wherein said immunoassay is a radioimmunoassay a Western blot assay, an immunofluorescence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay.", "14-17 (canceled).", "18.A method of diagnosing Alzheimer's disease in a subject, comprising: contacting cells from the subject with an agent that triggers intracellular calcium release via the inositol 1,4,5-trisphosphate (IP3) receptor, measuring the amount of phosphorylation of a MAPK protein in the subject's cells at one or more time points after the contacting step, and comparing the amount of phospliorylation of the MAPK protein in the subject's cells at the one or more time points with the amount of phosphorylation in cells from a non-Alzheimer's control subject at the same time points after contacting the control cells with the agent, wherein increased phosphorylation of the MAPK protein in the subject's cells compared to the control cells is diagnostic of Alzheimer's disease.", "19.The method of claim 18, wherein the agent is bradykinin or a bradykinin receptor agonist.", "20.The method of claim 18, wherein the agent is bombesin.", "21.The method of claim 18, wherein the agonist is one which induces IP3-mediated Ca2+ release.", "22.The method of claim 18, wherein the MAPK protein is Erk1/2.23.The method of claim 18, wherein the amount of phosphorylation is measured at a single time point after the contacting step.", "24.The method of claim 18, wherein the measuring comprises measuring the amount of phosphorylation in a first aliquot of the subject's cells at a first time point after the contacting step, and measuring the amount of phosphorylation in a second aliquot of the subject's cells at a second time point after the contacting step.", "25.The method of claim 18, wherein the time points are selected from one or more of about 0.5 minutes, 1 minute, 2 minutes, 2.5 minutes, 5 minutes, 10 minutes, 20, and 30 minutes.", "26.he method of claim 18, wherein the cells are from peripheral tissue.", "27.The method of claim 18, wherein the cells are skin fibroblasts.", "28.The method of claim 18, wherein the measuring step comprises detecting phosphorylation in a lysate of the subject's cells.", "29.The method of claim 18, wherein the measuring step is carried out in vitro.", "30.The method of claim 18, wherein the measuring step comprises gel electrophoresis.", "31.The method of claim 18, wherein the measuring step comprises Western blotting.", "32.The method of claim 31, wherein the Western blotting comprises using an anti-phospho-MAP kinase antibody.", "33.The method of claim 18, wherein the increased phosphorylation is an elevation in the amount of phosphorylated protein at a single time point.", "34.The method of claim 18, wherein the increased phosphorylation is an increase in duration of the phosphorylated protein.", "35.The method of claim 18, wherein the subject lacks clinical manifestations of Alzheimer's disease.", "36.The method of claim 18, further comprising contacting the subject's cells with one or more inhibitors selected from the group consisting of an inhibitor of protein kinase C activity, an inhibitor of PI-3 kinase activity, an inhibitor of C-SRC protein tyrosine kinase activity, an inhibitor of the IP-3 receptor and an inhibitor of a protein phosphatase.", "37.The method of claim 18, wherein the increased phosphorylation is inhibited by contacting the subject's cells with an inhibitor selected from the group consisting of an inhibitor of protein kinase C activity, C-src protein tyrosine kinase activity, PI-3 kinase activity, and the IP-3 receptor.", "38.The method of claim 37, wherein said inhibitor is selected from the group consisting of BiSM-1, PP1, and 2-aminoethoxydiphenyl borate.", "39-57.", "(Canceled)." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The invention provides a diagnostic and screening test for Alzheimer's disease (“AD”).", "An example of the test involves detecting abnormally enhanced phosphorylation of extracellular signal-regulated kinase type 1 or 2 (“Erk1/2”) in skin fibroblasts from AD patients after stimulating the cells with agonist such as bradykinin or other agents that stimulate the inositol 1,4,5-trisphosphate (IP3) receptor, in comparison to cells from age-matched controls.", "Enhanced phosphorylation may be measured by Western blot using antibodies specific for the phosphorylated protein or other similar approaches.", "Accumulating evidence indicates that the early pathogenesis of Alzheimer's disease (AD) involves perturbation of intracellular calcium homeostasis and increased levels of oxidative stress that contribute to excitatory toxicity and neuronal death in the AD brain (Putney, 2000; Yoo et al., 2000; Sheehan et al., 1997).", "Studies have reported enhanced elevation of intracellular Ca 2+ levels in AD brains as well as in peripheral cells in response to activation of bradykinin receptors and inactivation of a K + channel (Ito et al., 1994; Etcheberrigaray et al., 1994; Hirashima, et al., 1996; Gibson et al., 1996; Etcheberrigaray et al., 1998).", "Critical proteins such as amyloid precursor protein (APP), presenilin 1 and presenilin 2, mutations of which are associated with the pathogenesis of AD, have been reported to induce dysregulation of both the IP3 receptor (IP3R) and the ryanodine receptor-(RYR-) mediated intracellular Ca 2+ homeostasis (Yoo et al., 2000; Leissring et al., 1999; 2000; Mattson et al., 2000; Barrow et al., 2000).", "The alteration in cytosolic Ca 2+ concentration is thought to contribute to the pathophysioloy of AD, including increased production of the neurotoxic 42 amino acid β-amyloid peptide (APβ) involved in plaque formation, hyperphosphorylation of tau protein involved in formation of neurfibrillay tangles, and enhanced general vulnerability of neurons to cell death.", "Bradykinin (BK) is a potent vasoactive nonapeptide that is generated in the course of various inflammatory conditions.", "BK binds to and activates specific cell membrane BK receptor(s), thereby triggering a cascade of intracellular events leading to the phosphorylation of proteins known as “mitogen activated protein kinase” (MAPK; see below).", "Phosphorylation of proteins, the addition of a phosphate group to a Ser, Thr or Tyr residue, is mediated by a large number of enzymes known collectively as protein kinases.", "Phosphorylation normally modifies the function of, and usually activates, a protein.", "Homeostasis requires that phosphorylation be a transient process, which is reversed by phosphatase enzymes that dephosphorylate the substrate.", "Any aberration in phosphorylation or dephosphorylation disrupts biochemical pathways and multiple cellular functions.", "Such disruptions may be the basis for certain brain diseases.", "Increased intracellular Ca 2+ levels in response to BK is mediated at least by the “type 2” BK receptor (BKb2R), a G-protein-coupled receptor.", "Stimulation of BKb2R by BK activates phospholipase C (PLC) resulting in production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), second messengers involved in regulation of intracellular Ca 2+ levels and activation of protein kinase C (PKC).", "The PLC/phospholipid/PKC pathway also interact with the Ras signaling pathway that activates the MAPK pathway.", "MAPK (or MAP kinase) refers to an enzyme family termed “mitogen activated protein kinase,” an important member of which is the “extracellular signal-regulated kinase” type 1 or 2 (“Erk1/2”) (Berridge, 1984; Bassa et al., 1999).", "Erk1/2 receive signals from multiple signal transductional pathways and is part of a pathway that leads to cell proliferation and differentiation by regulation of gene expression through a number of transcriptional factors including cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB).", "Erk1/2 phosphorylates tau protein at multiple Ser/Thr sites including Ser262 and Ser356 (Reynolds et al., 2000), which are in microtubule-binding regions of tau.", "Phosphorylation of Ser262 markedly compromises the ability of tau to assemble and stabilize microtubules (Biemat et al., 1993; Lu et al., 1993).", "Increased oxidative stress, aberrant expression of amyloid precursor protein (APP), and exposure to APβ cause activation of MAPK (McDonald et al., 1998; Ekinci and Shea, 1999; Grant et al., 1999) and enhanced tau phosphorylation (Greenberg et al., 1994).", "Young L T et al., Neurosci Lett, 1988, 94:198-202 studied IP3 receptor binding sites in autopsied brains from 10 subjects with AD and 10 age-matched controls.", "In the parietal cortex and hippocampus, there was a 50-70% loss of [ 3 H]-IP3 binding whereas no significant changes were observed in frontal, occipital and temporal cortices, caudate or amygdala.", "Scatchard analysis confirmed a reduction in receptor density rather than a change in affinity.", "Also, many neurotransmitters, hormones and growth factors act at membrane receptors to stimulate the phosphodiesterase hydrolysis of phosphatidyl-inositol 4,5-bisphosphate (PIP2) generating the comessengers IP3 and diacylglycerol (DAG).", "DAG stimulates PKC while IP3 was initially postulated to activate specific receptors leading to release of intracellular calcium, probably from the endoplasmic reticulum.", "Though earlier reports had detected 32 P-IP3 binding to liver and adrenal microsomes and to permeabilized neutrophils and liver cells, Solomon Snyder's group was the first to localize, isolate, analyze and later clone, IP3 receptors.", "Worley P F et al., Nature 1987;325:159-161, demonstrated high affinity, selective binding sites for 3 H- and 32 P-labelled IP3 in the brain at levels 100-300 times higher than those observed in peripheral tissues.", "These receptors were considered physiologically relevant because the potencies of various myoinositol analogues at the IP3 binding site corresponded to their potencies in releasing calcium from microsomes.", "Brain autoradiograms demonstrated discrete, heterogeneous localization of IP3 receptors.", "In 1988, this group (Supattapone S et al., J Biol Chem, 1988, 263:1530-1534, reported the solubilization, purification to homogeneity, and characterization of an IP3 receptor from rat cerebellum.", "The purified receptor was a globular protein that migrated in electrophoresis as one protein band with an Mr of 260 kDa.", "In a review, Snyder et al.", "( Cell Calcium, 1989, 10:337-342) noted that immunohistochemical studies with antisera to the purified receptor protein localized the receptor to a subdivision of the rough endoplasmic reticulum occurring in synaptic areas and in close association with the nuclear membrane.", "The IP3 receptor protein was selectively phosphorylated by cAMP-dependent protein kinase.", "This phosphorylation decreased 10-fold the potency of IP3 in releasing calcium from brain membranes.", "Ferris C D et al., Proc Natl Acad Sci USA, 1991 88:2232-2235 later studied phosphorylation of IP3 receptors with purified receptor protein reconstituted in liposomes (to remove detergent that can inhibit protein kinases).", "The IP3 receptor was stoichiometrically phosphorylated by protein kinase C (PKC) and CaM kinase II as well as by protein kinase A (PKA).", "IP3 receptors are regulated by phosphorylation catalyzed by the three enzymes which was additive and involved different peptide sequences.", "Phosphorylation by (1) PKC which was stimulated by Ca 2+ and DAG, and (2) by CaM kinase II which required Ca 2+ , provided a means whereby Ca 2+ and DAG, formed during inositol phospholipid turnover, regulate IP3 receptors.", "Chadwick C C et al., Proc Natl Acad Sci USA, 1990 87:2132-2136, described the isolation from smooth muscle of an IP3 receptor that was an oligomer of a single polypeptide with a Mr of 224 kDa.", "Furuichi T, et al., FEBS Lett, 1990 267:85-88 examined distribution of IP3 receptor mRNA in mouse tissues.", "The concentration of was greatest in cerebellar tissue.", "Moderate amounts of IP3 receptor mRNA were present in other brain tissue: thymus, heart, lung, liver, spleen, kidney, and uterus.", "Small amounts of IP3 receptor mRNA were observed in skeletal muscle and testicular tissue.", "Based on in situ hybridization, a considerable amount of IP3 receptor mRNA was located in smooth muscle cells, such as those of the arteries, bronchioles, oviduct and uterus.", "Ferris C D et al., J Biol Chem, 1992, 267:7036-7041, demonstrated serine autophosphorylation of the purified and reconstituted IP3 receptor and found serine protein kinase activity of the IP3 receptor toward a specific peptide substrate.", "The investigators concluded that the IP3 receptor protein and the phosphorylating activity reside in the same molecule.", "Ross C A et al.", "( Proc Natl Acad Sci USA, 1992, 89:4265-4269), cloned three IP3R cDNAs, designated IP3R-II, -III, and -IV, from a mouse placenta cDNA library.", "All three displayed strong homology in membrane-spanning domains M7 and M8 to the originally cloned cerebellar IP3R-I, with divergences predominantly in cytoplasmic domains.", "Levels of mRNA for the three additional IP3Rs in general were substantially lower than for IP3R-I, except for the gastrointestinal tract where levels were comparable.", "Cerebellar Purkinje cells expressed at least two and possibly three distinct IP3Rs, suggesting heterogeneity of IP3 action within a single cell.", "Sharp A H, Neuroscience, 1993, 53:927-42, examined in detail the distribution of IP3 receptors in the rat brain and spinal cord using immunohistochemical methods.", "IP3 receptors are present in neuronal cells, fibers and terminals in a wide distribution of areas throughout the CNS, including the olfactory bulb, thalamic nuclei and dorsal hom of the spinal cord, in circumventricular organs and neuroendocrine structures such as the area postrema, choroid plexus, subcommisural organ, pineal gland and pituitary.", "Ca2+ release mediated by the phosphoinositide second messenger system is important in control of diverse physiological processes.", "Studies of IP3 receptors in lymphocytes (T cells) by Snyder's group localized these receptors to the plasma membrane.", "Capping of the T cell receptor-CD3 complex, which is associated with signal transduction, was accompanied by capping of IP3 receptors.", "The IP3 receptor on T cells appears to be responsible for the entry of Ca 2+ that initiates proliferative responses (Khan, A A et al., Science, 1992, 257:815-818) Further with regard to IP3, Wilcox R A et al., Trends Pharmacol Sci, 1998, 19:467-475, noted that receptor-mediated activation of PLC to generate IP3 is a ubiquitous signalling pathway in mammalian systems.", "A family of three IP3 receptor subtype monomers form functional tetramers, which act as IP3 effectors, providing a ligand-gated channel that allows Ca 2+ ions to move between cellular compartments.", "As IP3 receptors are located principally, although not exclusively, in the endoplasmic reticular membrane, IP3 is considered to be a second messenger that mobilizes Ca 2+ from intracellular stores contributing to a variety of physiological and pathophysiological phenomena.", "Patel S et al., Cell Calcium, 1999, 25:247-264, reviewed the molecular properties of IP3 receptors.", "Several Ca 2+ -binding sites and a Ca 2+ -calmodulin-binding domain were mapped within the type I IP3 receptor, and studies on purified cerebellar IP3 receptors suggested a second Ca 2+ -independent calmodulin-binding domain.", "Overexpression of IP3 receptors provided further clues to the regulation of individual IP3 receptor isoforms present within cells, and the role that they play in the generation of IP3-dependent Ca 2+ signals.", "IP3 receptors may be involved in cellular processes such as proliferation and apoptosis.", "Abdel-Latif AA.", "Exp Biol Med (Maywood) 2001 March;226(3):153-63 reviewed evidence, both from nonvascular and vascular smooth muscle, for cross talk between the cyclic nucleotides, cAMP and cGMP via their respective protein kinases, and the Ca 2+ -dependent- and Ca 2+ -independent-signaling pathways involved in agonist-induced contraction.", "These included the IP3-Ca 2+ -CaM-myosin light chain kinase (MLCK) pathway and the Ca 2+ -independent pathways, including PKC, MAP kinase, and Rho-kinase.", "Mikoshiba K et al., Sci STKE 2000 Sep. 26;2000(51):P, described the regulated release of calcium from intracellular stores by the IP3 receptor and the relationship of this release mechanism to calcium influx from the extracellular milieu through store-operated calcium channels.", "They disclosed a model of functional and physical coupling of intracellular and plasma membrane calcium channels.", "Although AD is well known for its severe brain damage and memory loss, pathological changes are manifest elsewhere in the body and can be detected at the cellular level.", "Skin fibroblasts lying in the deep layer of skin reveal characteristic cellular and molecular abnormalities of AD damage.", "Skin fibroblasts are readily obtained and cultured for diagnostic purposes (U.S. Pat.", "No.", "6,107,050, “Diagnostic Test for Alzheimer's Disease,” issued Aug. 22, 2000, which is incorporated herein by reference).", "However, there is a need for simpler, more economical, accurate and reliable methods for diagnosis of Alzheimer's disease.", "It is known e.g.", "from U.S. Pat.", "No.", "6,107,050, Alkon et al., that differential effects of an activator of intracellular Ca2+ release can be measured.", "Both healthy and Alzheimer's cell types exhibit a release of calcium from storage, but Alzheimer's cells exhibit a much greater release.", "Known methods for measuring the release of Ca2+(i) include fluorescent indicators, absorbance indicators or a Ca2+“patch clamp” electrode, and others, and such methods may be used for diagnostic purposes.", "However, there is a tremendous need for more effective techniques for measuring the differential effects of IP3R activators, for diagnostic, research, and clinical purposes." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a method of diagnosing Alzheimer's disease in a patient comprising detecting the presence or absence of an abnormally elevated level of a phosphorylated indicator protein in cells of the patient after activating the cells with a compound that stimulates phosphorylation of the indicator protein, the presence of such an elevated level indicating a positive diagnosis for Alzheimer's disease.", "The diagnostic method comprises measuring a phosphorylation level of an indicator protein in cells of the patient at a predetermined time after stimulating the cells with an activator compound, and determining comparing the stimulis abnormally elevated as compared to a basal phosphorylation level without stimulus.", "The invention provides a method of diagnosing Alzheimer's disease in a subject, said method comprising: (a) measuring a basal level of phosphorylation of an indicator protein in cells from the subject; (b) contacting cells from the subject with an activator compound, the activator compound and indicator protein being selected such that the activator elicits a differential response of activated phosphorylation of the indicator protein in cells of the subject as compared to an activated phosphorylation response in cells from a non-Alzheimer's control subject at a predetermined time after the contacting is initiated; (c) measuring an activated phosphorylation level of the indicator protein in said subject cells at the predetermined time after contacting is initiated; and (d) calculating a ratio of the activated phosphorylation level determined in step (c) to the basal phosphorylation level of step (a); and (e) comparing the calculated ratio of step (d) to previously determined activated phosphorylation ratios measured from known Alzheimer's disease cells and from known non-Alzheimer's disease cells at said predetermined time; wherein if the calculated ratio is not statistically different from the previously determined ratios for said known Alzheimer's disease cells, the diagnosis is positive, and/or if the calculated ratio is not statistically different from the previously determined ratios for said known non-Alzheimer's disease cells, the diagnosis is negative.", "The invention further provides a method of diagnosing Alzheimer's disease in a subject comprising: in cells of the subject, measuring a background phosphorylation level of an indicator calcium signalling pathway protein whose phosphorylation is associated with IP-3R-sensitive Ca 2+ elevation in the cells; stimulating cells of the subject by contact with an IP3R agonist that elicits a differential response in the phosphorylation level of the indicator protein in cells of Alzheimer's subjects as compared to the level in a non-Alzheimer's control cell; thereafter, measuring a response phosphorylation level of the indicator protein in the contacted cells, and determining whether the response phosphorylation level of the indicator protein as compared to the background level matches the response level known for a cell from an Alzheimer's subjects or from a healthy control cell.", "The methods may comprise first measuring the background phosphorylation level of the indicator protein in a culture of cells, then adding the IP3R agonist to the culture, and measuring the response phosphorylation level.", "Or the method may comprise measuring the background level in a first aliquot of cells, stimulating a similar aliquot of the cells, and measuring the response level in the aliquot.", "The activator compound may be an IP3-R agonist selected from the group consisting of bradykinin, bombesin, cholecystokinin, thrombin, prostaglandin F 2α and vasopressin.", "The cells may be selected from the group consisting of fibroblasts, buccal mucosal cells, neurons, and blood cells.", "According to the invention, a method of diagnosing Alzheimer's disease in a subject comprises (a) obtaining cells from said subject; (b) measuring the basal level of phosphorylation of an indicator protein in said cells; (c) contacting said cells with an activator of phosphorylation of the indicator protein; (d) measuring the phosphorylation level of the indicator protein in said cells at a predetermined time after initiation of the contacting; and (e) calculating a first ratio of the level measured in step (d) to the level measured in step (b) and comparing said first ratio to a previously determined second ratio of said levels obtained at said predetermined time from known Alzheimer's disease cells and a third ratio of said levels obtained at said predetermined time from known non-Alzheimer's disease cells; wherein (i) if the first ratio of step (e) is statistically not different from the previously determined second ratio, the diagnosis is positive, and (ii) if the first ratio of step (e) is not statistically different from the previously determined third ratio, the diagnosis is negative.", "The predetermined time of step (d) may be a time when the difference between the second ratio and the third ratio of step (e) is greatest.", "In the inventive methods, the measuring may comprise an immunoassay of disrupted cells, and the subject's cells may be contacted with an antibody specific for the phosphorylated indicator protein, permitting the antibody to bind to the indicator protein, and detecting the antibody bound to the indicator protein.", "The immunoassay may be a radioimmunoassay, a Western blot assay, an immunofluorescence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay.", "A further inventive method of diagnosing Alzheimer's disease in a subject comprises: (a) incubating cells from said subject with a compound in a diluent, wherein the compound stimulates calcium signaling pathway-mediated phosphorylation of an indicator protein, thereby producing stimulated cells; (b) before, at the same time or after step (a) incubating cells of the same type from the subject with a control compound or with said diluent, thereby producing unstimulated control cells; (c) comparing a level of the phosphorylated indicator protein in the stimulated cells to a level of phosphorylated indicator protein in the unstimulated control cells, wherein an increase in the level of the phosphorylated indicator protein in stimulated cells as compared to the unstimulated cells indicates the presence of Alzheimer's disease.", "The comparing step (c) may include the following steps: (i) contacting a protein sample from said stimulated and/or said unstimulated cells with an antibody which recognizes the phosphorylated indicator protein; and (ii) detecting the binding of said antibody to said indicator protein.", "The method may further comprise contacting a protein sample from said stimulated and/or said unstimulated cells with an antibody which recognizes an unphosphorylated form of said indicator protein, and detecting the binding of said antibody and unphosphorylated indicator protein, and normalizing the level of protein.", "The comparing step may further include the step of obtaining a protein sample from said stimulated and said unstimulated cells.", "A method of diagnosing the presence of Alzheimer's disease in a subject may comprise the steps of: a) stimulating cells from said subject with an activator compound that increases phosphorylation of an indicator protein, and b) comparing the level of unphosphorylated indicator protein and phosphorylated indicator protein in stimulated cells to the level of unphosphorylated indicator protein and phosphorylated indicator protein in unstimulated cells of the same type from said subject, wherein an increase in the relative level of phosphorylated indicator protein in stimulated cells as compared to unstimulated cells indicates the presence of Alzheimer's disease.", "The invention provides a method of diagnosing Alzheimer's disease in a subject, comprising: contacting cells from the subject with an agent that triggers intracellular calcium release via the inositol 1,4,5-trisphosphate (IP3) receptor, measuring the amount of phosphorylation of a MAPK protein in the subject's cells at one or more time points after the contacting step, and comparing the amount of phosphorylation of the MAPK protein in the subject's cells at the one or more time points with the amount of phosphorylation in cells from a non-Alzheimer's control subject at the same time points after contacting the control cells with the agent, wherein increased phosphorylation of the MAPK protein in the subject's cells compared to the control cells is diagnostic of Alzheimer's disease.", "In the methods of the invention, the agent may be bradykinin or a bradykinin receptor agonist, or bombesin, and may be an agonist which induces IP3-mediated Ca 2+ release.", "The amount of phosphorylation may be measured at a single time point after the contacting step.", "According to the invention, the measuring may comprise measuring the amount of phosphorylation in a first aliquot of the subject's cells at a first time point after the contacting step, and measuring the amount of phosphorylation in a second aliquot of the subject's cells at a second time point after the contacting step.", "The time points may be about 0.5 minutes or shorter, 1 minute, 2 minutes, 2.5 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, or 1 hour, or longer for some combinations of cell types, activators, and indicator proteins.", "The cells are typically from peripheral tissue, such as skin fibroblasts.", "The measuring step in the inventive methods optionally comprises detecting phosphorylation in a lysate of the subject's cells, in vitro, and may comprise gel electrophoresis, Western blotting, using an anti-phospho-MAPK antibody and/or an anti-regular MAPK protein antibody.", "The increased phosphorylation may be an elevation in the amount of phosphorylated protein at a single time point or an increase in duration of the phosphorylated protein The methods are effective in diagnosis where the subject lacks clinical manifestations of Alzheimer's disease.", "According to the invention, the methods may further comprise contacting the subject's cells with one or more inhibitors selected from the group consisting of an inhibitor of protein kinase C activity, an inhibitor of PI-3 kinase activity, an inhibitor of C-src protein tyrosine kinase activity, an inhibitor of the IP-3 receptor and an inhibitor of a protein phosphatase.", "Also, by way of characterizing a particularly discriminating embodiment of the invention, the methods may be characterized as having the increased phosphorylation inhibited by contacting the subject's cells with an inhibitor selected from the group consisting of an inhibitor of protein kinase C activity, C-src protein tyrosine kinase activity, PI-3 kinase activity, and the IP-3 receptor.", "In such methods said inhibitor can be selected from the group consisting of BiSM-1, PP1, and 2-aminoethoxydiphenyl borate.", "The invention further provides a method for screening compounds to identify a compound useful for treatment or prevention of Alzheimer's disease comprising: contacting test cells from an AD subject with a compound being screened, before, during, or after the contacting step, stimulating the test cells with an agent that triggers intracellular calcium release via the inositol 1,4,5-trisphosphate (IP3) receptor, measuring the amount of phosphorylation of a MAPK protein in the test cells at one or more time points after stimulating the test cells, comparing the amount of phosphorylation of the MAPK protein in the test cells at the one or more time points with the amount of phosphorylation at the same one or more time points in control cells from an AD subject that are not contacted with the compound.", "The methods may further comprise accepting a compound that inhibits or prevents the increased phosphorylation as a lead compound, and rejecting a compound that does not inhibit or prevent the increased phosphorylation.", "As in all the methods of the invention, the agent may be bradykinin or a bradykinin receptor agonist and the the MAPK protein may be Erk1/2.The methods may comprise measuring the amount of phosphorylation at a single time point after the contacting step.", "A further embodiment provides a method of screening compounds for usefulness as activator compounds in a stimulus-response assay, comprising measuring the effect of the compound on phosphorylation of an indicator protein in AD cells and control cells and selecting a compound that increases phosphorylation of the indicator protein in amount and/or duration in AD cells as compared to control cells.", "Another embodiment of the invention is a diagnostic test kit for Alzheimer's disease comprising anti-phospho-MAPK protein antibody and bradykinin.", "An embodiment provides a method for selecting medication for an Alzheimer's patient comprising selecting a possible therapeutic compound, administering the possible therapeutic compound to the patient, and thereafter, detecting the presence or absence of an abnormally elevated level of a phosphorylated indicator protein in cells of the patient after activating the cells with a compound that stimulates phosphorylation of the indicator protein, the presence of such an elevated level indicating that the possible therapeutic compound is not effective for the patient, and the absence of such a level indicating that the possible therapeutic compound is therapeutic for the patient.", "The method may further comprise treating or preventing Alzheimer's disease in the subject by administering to the subject the compound shown to be therapeutic for the patient.", "The invention provides a method of treating or preventing Alzheimer's disease in a subject comprising administering an effective amount of a medicament that (a) inhibits or prevents abnormally elevated phosphorylation of a MAPK protein in cells of the subject as compared to control cells; and/or (b) inhibits events caused by abnormally elevated phosphorylation of said MAPK protein.", "The medicament may inhibit Erk1/2 phosphorylation, and may be an inhibitor of protein kinase C activity.", "src protein tyrosine kinase activity, or the IP-3 receptor.", "The inhibitor may be selected from the group consisting of BiSM-1, PP 1, and 2ABP.", "In particular, the invention provides a method of diagnosing Alzheimer's disease in a subject, comprising: (a) contacting skin fibroblast cells from the subject and from a non-Alzheimer's control subject with an effective, phosphorylation-stimulating concentration of bradykinin, (b) measuring the amount of phosphorylated Erk1/2 in the subject's cells at one or more time points selected from the group consisting of 2 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes, by Western blotting using an antibody specific for phospho-Erk1/2; (c) measuring the amount of phosphorylated Erk1/2 in cells from a non-Alzheimer's control subject at the same time point or points as in (b) by Western blotting using an antibody specific for phospho-Erk1/2, wherein the amount of phosphorylated Erk1/3 in steps (b) and (c) is normalized to the amount of protein present in said cells; (d) comparing the amount of phosphorylated Erk1/2 in the subject's cells with the amount of phosphorylated Erk1/2 in the control cells at said time points, wherein an increased amount of phosphorylated Erk1/2 in the subject's cells compared to the control cells at one of more of said time points is diagnostic of Alzheimer's disease.", "The method may further comprise contacting the subject's cells with one or more inhibitors selected from the group consisting of the inhibitor of protein kinase C activity, BiSM-1, the inhibitor of C-src protein tyrosine kinase activity, PP1; and the inhibitor of the IP-3 receptor, 2-aminoethoxydiphenyl borate, wherein the bradykinin-induced increase in the amount of phosphorylated Erk1/2 in the subject's cells compared to the control cells is reduced by said inhibitor.", "A further embodiment of the invention provides a method for screening compounds to identify a compound useful for treatment or prevention of Alzheimer's disease comprising: (a) contacting test skin fibroblasts from an AD subject with a compound being screened; (b) contacting control skin fibroblasts from said subject with a control agent for said compound or incubating said control fibroblasts in the absence or either said compound or said control agent; (c) before, during, or after step (a) and (b) stimulating the test and the control fibroblasts with an effective, phosphorylation-stimulating concentration of bradykinin, (d) measuring the amount of phosphorylated Erk1/2 in the test and in the control fibroblasts at one or more time points selected from the group consisting of 2 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes, by Western blotting using an antibody specific for phospho-Erk1/2, wherein the amount of phosphorylated Erk1/3 is normalized to the amount of protein present in said test and control fibroblasts; (e) comparing the amount of phosphorylated Erk1/2 in the test fibroblasts with the amount of phosphorylated Erk1/2 in the control fibroblasts, to determine whether the compound inhibits or prevents bradykinin-induced increase in phosphorylation of Erk1/2 in the test cells compared to the control cells, wherein a compound that inhibits or prevents the increased phosphorylation is identified as useful for the treatment of prevention of Alzheimer's disease.", "A method of the invention is a method of reducing proteolysis of amyloid precursor protein, secretion of amyloid protein β, and/or phosphorylation of tau protein in a human cell, the cell having increased IP3 receptor mediated phosphorylation of MAPK protein compared to a control human cell, comprising contacting the cell with an inhibitor of phosphorylation of MAPK effective to reduce phosphorylation to the level in the control cell The elements of the invention recited herein may be combined or eliminated among the particular embodiments described, as would be apparent to a person of ordinary skill." ], [ "BACKGROUND OF THE INVENTION The invention provides a diagnostic and screening test for Alzheimer's disease (“AD”).", "An example of the test involves detecting abnormally enhanced phosphorylation of extracellular signal-regulated kinase type 1 or 2 (“Erk1/2”) in skin fibroblasts from AD patients after stimulating the cells with agonist such as bradykinin or other agents that stimulate the inositol 1,4,5-trisphosphate (IP3) receptor, in comparison to cells from age-matched controls.", "Enhanced phosphorylation may be measured by Western blot using antibodies specific for the phosphorylated protein or other similar approaches.", "Accumulating evidence indicates that the early pathogenesis of Alzheimer's disease (AD) involves perturbation of intracellular calcium homeostasis and increased levels of oxidative stress that contribute to excitatory toxicity and neuronal death in the AD brain (Putney, 2000; Yoo et al., 2000; Sheehan et al., 1997).", "Studies have reported enhanced elevation of intracellular Ca2+ levels in AD brains as well as in peripheral cells in response to activation of bradykinin receptors and inactivation of a K+ channel (Ito et al., 1994; Etcheberrigaray et al., 1994; Hirashima, et al., 1996; Gibson et al., 1996; Etcheberrigaray et al., 1998).", "Critical proteins such as amyloid precursor protein (APP), presenilin 1 and presenilin 2, mutations of which are associated with the pathogenesis of AD, have been reported to induce dysregulation of both the IP3 receptor (IP3R) and the ryanodine receptor-(RYR-) mediated intracellular Ca2+ homeostasis (Yoo et al., 2000; Leissring et al., 1999; 2000; Mattson et al., 2000; Barrow et al., 2000).", "The alteration in cytosolic Ca2+ concentration is thought to contribute to the pathophysioloy of AD, including increased production of the neurotoxic 42 amino acid β-amyloid peptide (APβ) involved in plaque formation, hyperphosphorylation of tau protein involved in formation of neurfibrillay tangles, and enhanced general vulnerability of neurons to cell death.", "Bradykinin (BK) is a potent vasoactive nonapeptide that is generated in the course of various inflammatory conditions.", "BK binds to and activates specific cell membrane BK receptor(s), thereby triggering a cascade of intracellular events leading to the phosphorylation of proteins known as “mitogen activated protein kinase” (MAPK; see below).", "Phosphorylation of proteins, the addition of a phosphate group to a Ser, Thr or Tyr residue, is mediated by a large number of enzymes known collectively as protein kinases.", "Phosphorylation normally modifies the function of, and usually activates, a protein.", "Homeostasis requires that phosphorylation be a transient process, which is reversed by phosphatase enzymes that dephosphorylate the substrate.", "Any aberration in phosphorylation or dephosphorylation disrupts biochemical pathways and multiple cellular functions.", "Such disruptions may be the basis for certain brain diseases.", "Increased intracellular Ca2+ levels in response to BK is mediated at least by the “type 2” BK receptor (BKb2R), a G-protein-coupled receptor.", "Stimulation of BKb2R by BK activates phospholipase C (PLC) resulting in production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), second messengers involved in regulation of intracellular Ca2+ levels and activation of protein kinase C (PKC).", "The PLC/phospholipid/PKC pathway also interact with the Ras signaling pathway that activates the MAPK pathway.", "MAPK (or MAP kinase) refers to an enzyme family termed “mitogen activated protein kinase,” an important member of which is the “extracellular signal-regulated kinase” type 1 or 2 (“Erk1/2”) (Berridge, 1984; Bassa et al., 1999).", "Erk1/2 receive signals from multiple signal transductional pathways and is part of a pathway that leads to cell proliferation and differentiation by regulation of gene expression through a number of transcriptional factors including cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB).", "Erk1/2 phosphorylates tau protein at multiple Ser/Thr sites including Ser262 and Ser356 (Reynolds et al., 2000), which are in microtubule-binding regions of tau.", "Phosphorylation of Ser262 markedly compromises the ability of tau to assemble and stabilize microtubules (Biemat et al., 1993; Lu et al., 1993).", "Increased oxidative stress, aberrant expression of amyloid precursor protein (APP), and exposure to APβ cause activation of MAPK (McDonald et al., 1998; Ekinci and Shea, 1999; Grant et al., 1999) and enhanced tau phosphorylation (Greenberg et al., 1994).", "Young L T et al., Neurosci Lett, 1988, 94:198-202 studied IP3 receptor binding sites in autopsied brains from 10 subjects with AD and 10 age-matched controls.", "In the parietal cortex and hippocampus, there was a 50-70% loss of [3H]-IP3 binding whereas no significant changes were observed in frontal, occipital and temporal cortices, caudate or amygdala.", "Scatchard analysis confirmed a reduction in receptor density rather than a change in affinity.", "Also, many neurotransmitters, hormones and growth factors act at membrane receptors to stimulate the phosphodiesterase hydrolysis of phosphatidyl-inositol 4,5-bisphosphate (PIP2) generating the comessengers IP3 and diacylglycerol (DAG).", "DAG stimulates PKC while IP3 was initially postulated to activate specific receptors leading to release of intracellular calcium, probably from the endoplasmic reticulum.", "Though earlier reports had detected 32P-IP3 binding to liver and adrenal microsomes and to permeabilized neutrophils and liver cells, Solomon Snyder's group was the first to localize, isolate, analyze and later clone, IP3 receptors.", "Worley P F et al., Nature 1987;325:159-161, demonstrated high affinity, selective binding sites for 3H- and 32P-labelled IP3 in the brain at levels 100-300 times higher than those observed in peripheral tissues.", "These receptors were considered physiologically relevant because the potencies of various myoinositol analogues at the IP3 binding site corresponded to their potencies in releasing calcium from microsomes.", "Brain autoradiograms demonstrated discrete, heterogeneous localization of IP3 receptors.", "In 1988, this group (Supattapone S et al., J Biol Chem, 1988, 263:1530-1534, reported the solubilization, purification to homogeneity, and characterization of an IP3 receptor from rat cerebellum.", "The purified receptor was a globular protein that migrated in electrophoresis as one protein band with an Mr of 260 kDa.", "In a review, Snyder et al.", "(Cell Calcium, 1989, 10:337-342) noted that immunohistochemical studies with antisera to the purified receptor protein localized the receptor to a subdivision of the rough endoplasmic reticulum occurring in synaptic areas and in close association with the nuclear membrane.", "The IP3 receptor protein was selectively phosphorylated by cAMP-dependent protein kinase.", "This phosphorylation decreased 10-fold the potency of IP3 in releasing calcium from brain membranes.", "Ferris C D et al., Proc Natl Acad Sci USA, 1991 88:2232-2235 later studied phosphorylation of IP3 receptors with purified receptor protein reconstituted in liposomes (to remove detergent that can inhibit protein kinases).", "The IP3 receptor was stoichiometrically phosphorylated by protein kinase C (PKC) and CaM kinase II as well as by protein kinase A (PKA).", "IP3 receptors are regulated by phosphorylation catalyzed by the three enzymes which was additive and involved different peptide sequences.", "Phosphorylation by (1) PKC which was stimulated by Ca2+ and DAG, and (2) by CaM kinase II which required Ca2+, provided a means whereby Ca2+ and DAG, formed during inositol phospholipid turnover, regulate IP3 receptors.", "Chadwick C C et al., Proc Natl Acad Sci USA, 1990 87:2132-2136, described the isolation from smooth muscle of an IP3 receptor that was an oligomer of a single polypeptide with a Mr of 224 kDa.", "Furuichi T, et al., FEBS Lett, 1990 267:85-88 examined distribution of IP3 receptor mRNA in mouse tissues.", "The concentration of was greatest in cerebellar tissue.", "Moderate amounts of IP3 receptor mRNA were present in other brain tissue: thymus, heart, lung, liver, spleen, kidney, and uterus.", "Small amounts of IP3 receptor mRNA were observed in skeletal muscle and testicular tissue.", "Based on in situ hybridization, a considerable amount of IP3 receptor mRNA was located in smooth muscle cells, such as those of the arteries, bronchioles, oviduct and uterus.", "Ferris C D et al., J Biol Chem, 1992, 267:7036-7041, demonstrated serine autophosphorylation of the purified and reconstituted IP3 receptor and found serine protein kinase activity of the IP3 receptor toward a specific peptide substrate.", "The investigators concluded that the IP3 receptor protein and the phosphorylating activity reside in the same molecule.", "Ross C A et al.", "(Proc Natl Acad Sci USA, 1992, 89:4265-4269), cloned three IP3R cDNAs, designated IP3R-II, -III, and -IV, from a mouse placenta cDNA library.", "All three displayed strong homology in membrane-spanning domains M7 and M8 to the originally cloned cerebellar IP3R-I, with divergences predominantly in cytoplasmic domains.", "Levels of mRNA for the three additional IP3Rs in general were substantially lower than for IP3R-I, except for the gastrointestinal tract where levels were comparable.", "Cerebellar Purkinje cells expressed at least two and possibly three distinct IP3Rs, suggesting heterogeneity of IP3 action within a single cell.", "Sharp A H, Neuroscience, 1993, 53:927-42, examined in detail the distribution of IP3 receptors in the rat brain and spinal cord using immunohistochemical methods.", "IP3 receptors are present in neuronal cells, fibers and terminals in a wide distribution of areas throughout the CNS, including the olfactory bulb, thalamic nuclei and dorsal hom of the spinal cord, in circumventricular organs and neuroendocrine structures such as the area postrema, choroid plexus, subcommisural organ, pineal gland and pituitary.", "Ca2+ release mediated by the phosphoinositide second messenger system is important in control of diverse physiological processes.", "Studies of IP3 receptors in lymphocytes (T cells) by Snyder's group localized these receptors to the plasma membrane.", "Capping of the T cell receptor-CD3 complex, which is associated with signal transduction, was accompanied by capping of IP3 receptors.", "The IP3 receptor on T cells appears to be responsible for the entry of Ca2+ that initiates proliferative responses (Khan, A A et al., Science, 1992, 257:815-818) Further with regard to IP3, Wilcox R A et al., Trends Pharmacol Sci, 1998, 19:467-475, noted that receptor-mediated activation of PLC to generate IP3 is a ubiquitous signalling pathway in mammalian systems.", "A family of three IP3 receptor subtype monomers form functional tetramers, which act as IP3 effectors, providing a ligand-gated channel that allows Ca2+ ions to move between cellular compartments.", "As IP3 receptors are located principally, although not exclusively, in the endoplasmic reticular membrane, IP3 is considered to be a second messenger that mobilizes Ca2+ from intracellular stores contributing to a variety of physiological and pathophysiological phenomena.", "Patel S et al., Cell Calcium, 1999, 25:247-264, reviewed the molecular properties of IP3 receptors.", "Several Ca2+-binding sites and a Ca2+-calmodulin-binding domain were mapped within the type I IP3 receptor, and studies on purified cerebellar IP3 receptors suggested a second Ca2+-independent calmodulin-binding domain.", "Overexpression of IP3 receptors provided further clues to the regulation of individual IP3 receptor isoforms present within cells, and the role that they play in the generation of IP3-dependent Ca2+ signals.", "IP3 receptors may be involved in cellular processes such as proliferation and apoptosis.", "Abdel-Latif AA.", "Exp Biol Med (Maywood) 2001 March;226(3):153-63 reviewed evidence, both from nonvascular and vascular smooth muscle, for cross talk between the cyclic nucleotides, cAMP and cGMP via their respective protein kinases, and the Ca2+-dependent- and Ca2+-independent-signaling pathways involved in agonist-induced contraction.", "These included the IP3-Ca2+-CaM-myosin light chain kinase (MLCK) pathway and the Ca2+-independent pathways, including PKC, MAP kinase, and Rho-kinase.", "Mikoshiba K et al., Sci STKE 2000 Sep. 26;2000(51):P, described the regulated release of calcium from intracellular stores by the IP3 receptor and the relationship of this release mechanism to calcium influx from the extracellular milieu through store-operated calcium channels.", "They disclosed a model of functional and physical coupling of intracellular and plasma membrane calcium channels.", "Although AD is well known for its severe brain damage and memory loss, pathological changes are manifest elsewhere in the body and can be detected at the cellular level.", "Skin fibroblasts lying in the deep layer of skin reveal characteristic cellular and molecular abnormalities of AD damage.", "Skin fibroblasts are readily obtained and cultured for diagnostic purposes (U.S. Pat.", "No.", "6,107,050, “Diagnostic Test for Alzheimer's Disease,” issued Aug. 22, 2000, which is incorporated herein by reference).", "However, there is a need for simpler, more economical, accurate and reliable methods for diagnosis of Alzheimer's disease.", "It is known e.g.", "from U.S. Pat.", "No.", "6,107,050, Alkon et al., that differential effects of an activator of intracellular Ca2+ release can be measured.", "Both healthy and Alzheimer's cell types exhibit a release of calcium from storage, but Alzheimer's cells exhibit a much greater release.", "Known methods for measuring the release of Ca2+(i) include fluorescent indicators, absorbance indicators or a Ca2+“patch clamp” electrode, and others, and such methods may be used for diagnostic purposes.", "However, there is a tremendous need for more effective techniques for measuring the differential effects of IP3R activators, for diagnostic, research, and clinical purposes.", "SUMMARY OF THE INVENTION The invention provides a method of diagnosing Alzheimer's disease in a patient comprising detecting the presence or absence of an abnormally elevated level of a phosphorylated indicator protein in cells of the patient after activating the cells with a compound that stimulates phosphorylation of the indicator protein, the presence of such an elevated level indicating a positive diagnosis for Alzheimer's disease.", "The diagnostic method comprises measuring a phosphorylation level of an indicator protein in cells of the patient at a predetermined time after stimulating the cells with an activator compound, and determining comparing the stimulis abnormally elevated as compared to a basal phosphorylation level without stimulus.", "The invention provides a method of diagnosing Alzheimer's disease in a subject, said method comprising: (a) measuring a basal level of phosphorylation of an indicator protein in cells from the subject; (b) contacting cells from the subject with an activator compound, the activator compound and indicator protein being selected such that the activator elicits a differential response of activated phosphorylation of the indicator protein in cells of the subject as compared to an activated phosphorylation response in cells from a non-Alzheimer's control subject at a predetermined time after the contacting is initiated; (c) measuring an activated phosphorylation level of the indicator protein in said subject cells at the predetermined time after contacting is initiated; and (d) calculating a ratio of the activated phosphorylation level determined in step (c) to the basal phosphorylation level of step (a); and (e) comparing the calculated ratio of step (d) to previously determined activated phosphorylation ratios measured from known Alzheimer's disease cells and from known non-Alzheimer's disease cells at said predetermined time; wherein if the calculated ratio is not statistically different from the previously determined ratios for said known Alzheimer's disease cells, the diagnosis is positive, and/or if the calculated ratio is not statistically different from the previously determined ratios for said known non-Alzheimer's disease cells, the diagnosis is negative.", "The invention further provides a method of diagnosing Alzheimer's disease in a subject comprising: in cells of the subject, measuring a background phosphorylation level of an indicator calcium signalling pathway protein whose phosphorylation is associated with IP-3R-sensitive Ca2+ elevation in the cells; stimulating cells of the subject by contact with an IP3R agonist that elicits a differential response in the phosphorylation level of the indicator protein in cells of Alzheimer's subjects as compared to the level in a non-Alzheimer's control cell; thereafter, measuring a response phosphorylation level of the indicator protein in the contacted cells, and determining whether the response phosphorylation level of the indicator protein as compared to the background level matches the response level known for a cell from an Alzheimer's subjects or from a healthy control cell.", "The methods may comprise first measuring the background phosphorylation level of the indicator protein in a culture of cells, then adding the IP3R agonist to the culture, and measuring the response phosphorylation level.", "Or the method may comprise measuring the background level in a first aliquot of cells, stimulating a similar aliquot of the cells, and measuring the response level in the aliquot.", "The activator compound may be an IP3-R agonist selected from the group consisting of bradykinin, bombesin, cholecystokinin, thrombin, prostaglandin F2α and vasopressin.", "The cells may be selected from the group consisting of fibroblasts, buccal mucosal cells, neurons, and blood cells.", "According to the invention, a method of diagnosing Alzheimer's disease in a subject comprises (a) obtaining cells from said subject; (b) measuring the basal level of phosphorylation of an indicator protein in said cells; (c) contacting said cells with an activator of phosphorylation of the indicator protein; (d) measuring the phosphorylation level of the indicator protein in said cells at a predetermined time after initiation of the contacting; and (e) calculating a first ratio of the level measured in step (d) to the level measured in step (b) and comparing said first ratio to a previously determined second ratio of said levels obtained at said predetermined time from known Alzheimer's disease cells and a third ratio of said levels obtained at said predetermined time from known non-Alzheimer's disease cells; wherein (i) if the first ratio of step (e) is statistically not different from the previously determined second ratio, the diagnosis is positive, and (ii) if the first ratio of step (e) is not statistically different from the previously determined third ratio, the diagnosis is negative.", "The predetermined time of step (d) may be a time when the difference between the second ratio and the third ratio of step (e) is greatest.", "In the inventive methods, the measuring may comprise an immunoassay of disrupted cells, and the subject's cells may be contacted with an antibody specific for the phosphorylated indicator protein, permitting the antibody to bind to the indicator protein, and detecting the antibody bound to the indicator protein.", "The immunoassay may be a radioimmunoassay, a Western blot assay, an immunofluorescence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay.", "A further inventive method of diagnosing Alzheimer's disease in a subject comprises: (a) incubating cells from said subject with a compound in a diluent, wherein the compound stimulates calcium signaling pathway-mediated phosphorylation of an indicator protein, thereby producing stimulated cells; (b) before, at the same time or after step (a) incubating cells of the same type from the subject with a control compound or with said diluent, thereby producing unstimulated control cells; (c) comparing a level of the phosphorylated indicator protein in the stimulated cells to a level of phosphorylated indicator protein in the unstimulated control cells, wherein an increase in the level of the phosphorylated indicator protein in stimulated cells as compared to the unstimulated cells indicates the presence of Alzheimer's disease.", "The comparing step (c) may include the following steps: (i) contacting a protein sample from said stimulated and/or said unstimulated cells with an antibody which recognizes the phosphorylated indicator protein; and (ii) detecting the binding of said antibody to said indicator protein.", "The method may further comprise contacting a protein sample from said stimulated and/or said unstimulated cells with an antibody which recognizes an unphosphorylated form of said indicator protein, and detecting the binding of said antibody and unphosphorylated indicator protein, and normalizing the level of protein.", "The comparing step may further include the step of obtaining a protein sample from said stimulated and said unstimulated cells.", "A method of diagnosing the presence of Alzheimer's disease in a subject may comprise the steps of: a) stimulating cells from said subject with an activator compound that increases phosphorylation of an indicator protein, and b) comparing the level of unphosphorylated indicator protein and phosphorylated indicator protein in stimulated cells to the level of unphosphorylated indicator protein and phosphorylated indicator protein in unstimulated cells of the same type from said subject, wherein an increase in the relative level of phosphorylated indicator protein in stimulated cells as compared to unstimulated cells indicates the presence of Alzheimer's disease.", "The invention provides a method of diagnosing Alzheimer's disease in a subject, comprising: contacting cells from the subject with an agent that triggers intracellular calcium release via the inositol 1,4,5-trisphosphate (IP3) receptor, measuring the amount of phosphorylation of a MAPK protein in the subject's cells at one or more time points after the contacting step, and comparing the amount of phosphorylation of the MAPK protein in the subject's cells at the one or more time points with the amount of phosphorylation in cells from a non-Alzheimer's control subject at the same time points after contacting the control cells with the agent, wherein increased phosphorylation of the MAPK protein in the subject's cells compared to the control cells is diagnostic of Alzheimer's disease.", "In the methods of the invention, the agent may be bradykinin or a bradykinin receptor agonist, or bombesin, and may be an agonist which induces IP3-mediated Ca2+ release.", "The amount of phosphorylation may be measured at a single time point after the contacting step.", "According to the invention, the measuring may comprise measuring the amount of phosphorylation in a first aliquot of the subject's cells at a first time point after the contacting step, and measuring the amount of phosphorylation in a second aliquot of the subject's cells at a second time point after the contacting step.", "The time points may be about 0.5 minutes or shorter, 1 minute, 2 minutes, 2.5 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, or 1 hour, or longer for some combinations of cell types, activators, and indicator proteins.", "The cells are typically from peripheral tissue, such as skin fibroblasts.", "The measuring step in the inventive methods optionally comprises detecting phosphorylation in a lysate of the subject's cells, in vitro, and may comprise gel electrophoresis, Western blotting, using an anti-phospho-MAPK antibody and/or an anti-regular MAPK protein antibody.", "The increased phosphorylation may be an elevation in the amount of phosphorylated protein at a single time point or an increase in duration of the phosphorylated protein The methods are effective in diagnosis where the subject lacks clinical manifestations of Alzheimer's disease.", "According to the invention, the methods may further comprise contacting the subject's cells with one or more inhibitors selected from the group consisting of an inhibitor of protein kinase C activity, an inhibitor of PI-3 kinase activity, an inhibitor of C-src protein tyrosine kinase activity, an inhibitor of the IP-3 receptor and an inhibitor of a protein phosphatase.", "Also, by way of characterizing a particularly discriminating embodiment of the invention, the methods may be characterized as having the increased phosphorylation inhibited by contacting the subject's cells with an inhibitor selected from the group consisting of an inhibitor of protein kinase C activity, C-src protein tyrosine kinase activity, PI-3 kinase activity, and the IP-3 receptor.", "In such methods said inhibitor can be selected from the group consisting of BiSM-1, PP1, and 2-aminoethoxydiphenyl borate.", "The invention further provides a method for screening compounds to identify a compound useful for treatment or prevention of Alzheimer's disease comprising: contacting test cells from an AD subject with a compound being screened, before, during, or after the contacting step, stimulating the test cells with an agent that triggers intracellular calcium release via the inositol 1,4,5-trisphosphate (IP3) receptor, measuring the amount of phosphorylation of a MAPK protein in the test cells at one or more time points after stimulating the test cells, comparing the amount of phosphorylation of the MAPK protein in the test cells at the one or more time points with the amount of phosphorylation at the same one or more time points in control cells from an AD subject that are not contacted with the compound.", "The methods may further comprise accepting a compound that inhibits or prevents the increased phosphorylation as a lead compound, and rejecting a compound that does not inhibit or prevent the increased phosphorylation.", "As in all the methods of the invention, the agent may be bradykinin or a bradykinin receptor agonist and the the MAPK protein may be Erk1/2.The methods may comprise measuring the amount of phosphorylation at a single time point after the contacting step.", "A further embodiment provides a method of screening compounds for usefulness as activator compounds in a stimulus-response assay, comprising measuring the effect of the compound on phosphorylation of an indicator protein in AD cells and control cells and selecting a compound that increases phosphorylation of the indicator protein in amount and/or duration in AD cells as compared to control cells.", "Another embodiment of the invention is a diagnostic test kit for Alzheimer's disease comprising anti-phospho-MAPK protein antibody and bradykinin.", "An embodiment provides a method for selecting medication for an Alzheimer's patient comprising selecting a possible therapeutic compound, administering the possible therapeutic compound to the patient, and thereafter, detecting the presence or absence of an abnormally elevated level of a phosphorylated indicator protein in cells of the patient after activating the cells with a compound that stimulates phosphorylation of the indicator protein, the presence of such an elevated level indicating that the possible therapeutic compound is not effective for the patient, and the absence of such a level indicating that the possible therapeutic compound is therapeutic for the patient.", "The method may further comprise treating or preventing Alzheimer's disease in the subject by administering to the subject the compound shown to be therapeutic for the patient.", "The invention provides a method of treating or preventing Alzheimer's disease in a subject comprising administering an effective amount of a medicament that (a) inhibits or prevents abnormally elevated phosphorylation of a MAPK protein in cells of the subject as compared to control cells; and/or (b) inhibits events caused by abnormally elevated phosphorylation of said MAPK protein.", "The medicament may inhibit Erk1/2 phosphorylation, and may be an inhibitor of protein kinase C activity.", "src protein tyrosine kinase activity, or the IP-3 receptor.", "The inhibitor may be selected from the group consisting of BiSM-1, PP 1, and 2ABP.", "In particular, the invention provides a method of diagnosing Alzheimer's disease in a subject, comprising: (a) contacting skin fibroblast cells from the subject and from a non-Alzheimer's control subject with an effective, phosphorylation-stimulating concentration of bradykinin, (b) measuring the amount of phosphorylated Erk1/2 in the subject's cells at one or more time points selected from the group consisting of 2 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes, by Western blotting using an antibody specific for phospho-Erk1/2; (c) measuring the amount of phosphorylated Erk1/2 in cells from a non-Alzheimer's control subject at the same time point or points as in (b) by Western blotting using an antibody specific for phospho-Erk1/2, wherein the amount of phosphorylated Erk1/3 in steps (b) and (c) is normalized to the amount of protein present in said cells; (d) comparing the amount of phosphorylated Erk1/2 in the subject's cells with the amount of phosphorylated Erk1/2 in the control cells at said time points, wherein an increased amount of phosphorylated Erk1/2 in the subject's cells compared to the control cells at one of more of said time points is diagnostic of Alzheimer's disease.", "The method may further comprise contacting the subject's cells with one or more inhibitors selected from the group consisting of the inhibitor of protein kinase C activity, BiSM-1, the inhibitor of C-src protein tyrosine kinase activity, PP1; and the inhibitor of the IP-3 receptor, 2-aminoethoxydiphenyl borate, wherein the bradykinin-induced increase in the amount of phosphorylated Erk1/2 in the subject's cells compared to the control cells is reduced by said inhibitor.", "A further embodiment of the invention provides a method for screening compounds to identify a compound useful for treatment or prevention of Alzheimer's disease comprising: (a) contacting test skin fibroblasts from an AD subject with a compound being screened; (b) contacting control skin fibroblasts from said subject with a control agent for said compound or incubating said control fibroblasts in the absence or either said compound or said control agent; (c) before, during, or after step (a) and (b) stimulating the test and the control fibroblasts with an effective, phosphorylation-stimulating concentration of bradykinin, (d) measuring the amount of phosphorylated Erk1/2 in the test and in the control fibroblasts at one or more time points selected from the group consisting of 2 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes, by Western blotting using an antibody specific for phospho-Erk1/2, wherein the amount of phosphorylated Erk1/3 is normalized to the amount of protein present in said test and control fibroblasts; (e) comparing the amount of phosphorylated Erk1/2 in the test fibroblasts with the amount of phosphorylated Erk1/2 in the control fibroblasts, to determine whether the compound inhibits or prevents bradykinin-induced increase in phosphorylation of Erk1/2 in the test cells compared to the control cells, wherein a compound that inhibits or prevents the increased phosphorylation is identified as useful for the treatment of prevention of Alzheimer's disease.", "A method of the invention is a method of reducing proteolysis of amyloid precursor protein, secretion of amyloid protein β, and/or phosphorylation of tau protein in a human cell, the cell having increased IP3 receptor mediated phosphorylation of MAPK protein compared to a control human cell, comprising contacting the cell with an inhibitor of phosphorylation of MAPK effective to reduce phosphorylation to the level in the control cell The elements of the invention recited herein may be combined or eliminated among the particular embodiments described, as would be apparent to a person of ordinary skill.", "BRIEF DESCRIPTION OF THE DRAWINGS FIGS.", "1A1, 1A2 and 1B.", "Time course of the BK-stimulated activation of Erk1/2.AD cells and cells from age-matched normal controls (“AC”) were treated with 10 nM BK for different times and the reactions were terminated as described in the Examples.", "To the control for each cell line was added the same volume of PBS.", "The top images were representative Western blots for activated Erk1/2 (P-Erk1/2) from AD and AC cells.", "The mean density of each sample was normalized to the total amount of protein, which was determined using a anti-“regular MAP kinase” antibody (R-Erk1/2).", "Statistical significance of values from 11 independent cell lines was evaluated by an unpaired Student's t-test.", "Results are summarized and presented in the lower graph.", "FIGS.", "2A and 2B.", "Scatter plot-comparing phosphorylation of Erk1/2 between AD and AC cells after 10 min of BK stimulation (FIG.", "2A).", "Cells from 20 AD patients and 22 age-matched controls were treated with BK and processed as described in the description of FIG.", "1.Activated and the total Erk1/2 were detected by Western blotting with appropriate antibodies.", "Results were processed and analyzed as described for FIG.", "1.FIG.", "2B shows BK-stimulated Erk1/2 phosphorylation from three independent replications for each randomly selected cell line.", "FIGS.", "3A1, 3A2, 3B1 and 3B2.Immunocytochemical staining of activated Erk1/2 in the human fibroblasts.", "Fibroblasts from AD patients and the age-matched controls were cultured on glass cover slips and stimulated with 10 nM BK for 10 min.", "Activated Erk1/2 was detected with an anti-phospho-Erk1/Erk2 antibody.", "The results showed that there were no apparent difference in AC cells (top image) between pre- and after BK treatment.", "In the AD cells, however, highly enhanced immunofluorescent signals (lower right image, 3B2) were seen in the BK-treated cells (10 min).", "FIGS.", "4A1, 4A2, 4B1, 4B2, 4C1, 4C2, 4D1 and 4D2.Effects of different inhibitors on BK-stimulated activation of Erk1/2: Fibroblasts from AD patients and the age-matched controls were respectively preincubated at 37° C. with 50 μM 2APB for 30 min (FIG.", "4A); 5 μM BiSm-1 for 15 min (FIG.", "4B); 10 μM PP1 for 15 min (FIG.", "4C) and 5 μM LY294002 for 15 min (FIG.", "4D).", "A second flask of cells of each cell line was incubated with an identical volume of DMSO vehicle.", "At the end of incubation, cells were treated with 10 nM BK for 5 min before the reaction was terminated.", "The activated and “regular” forms of Erk1/2 were then detected in Western blots with the two antibodies described above.", "The panels at left show representative Western blots and the graphs at right summarize results from 11 cell lines from both AD and AC.", "** p<0.001; * p<0.05.FIGS.", "5A, 5B1, 5B2, 5C1 and 5C2.Changes in phosphorylation of, and amounts of CREB after BK stimulation.", "AC and AD cells were treated with 10 nM BK for 5 or 10 min.", "Phosphorylated CREB (P-CREB) was measured using a anti-phospho-CREB-Ser133 antibody on Western blots.", "The mean densities of P-CREB were normalized against the total amount of CREB measured using an anti-“regular CREB” antibody and subjected to statistic analyses.", "FIG.", "5A shows the BK-induced CREB phosphorylation at 5 and 10 min post-treatment.", "FIG.", "5B shows a representative blot of P-CREB at 10 min after BK treatment.", "FIG.", "5C shows that the total amount of CREB was significantly reduced after 10 min BK treatment.", "FIGS.", "6A1, 6A2, 6B1, 6B2, 6C1 and 6C2.Effects of different inhibitors on BK-stimulated phosphorylation of CREB.", "AC and AD cells were preincubated at 37° C. for 15 min with 5 μM BiSM-1 (FIG.", "6A), 10 μM PP1 (FIG.", "6B), or 5 μM Ly294002 (FIG.", "6C) before incubation with 10 nM BK for 10 min.", "Normalization was performed as with FIGS.", "5A-5C and statistical significance of differences were evaluated by 2-way ANOVA.", "** p<0.0001; * p<0.05.FIGS.", "7A and 7B.", "Distribution of BKb2 receptor in the human skin fibroblasts.", "Fibroblasts were incubated with an anti-BKb2 receptor antibody followed by a fluorescein-labeled secondary antibody.", "The top panel shows the differential interference contrast (DIC) image of the cells, and the lower panel shows the immunofluorescence of the BK b2 receptor in fibroblasts.", "FIG.", "8.The effect of bradykinin on phosphorylation of Erk1/2 on fibroblasts from individuals with Huntington's dementia (HD).", "N=4 and P=0.39, t test.", "DETAILED DESCRIPTION OF THE INVENTION According to the invention, an activator, or agent that stimulates an IP3 mediated intracellular calcium release is one which can be identified by a person of ordinary skill in the art using the methods disclosed herein, based on published information about the IP3 receptor and related pathways, as described in the background.", "An abnormally elevated level or ratio of stimulated/unstimulated phosphorylation levels means one which exceeds a previously determined level for known non-Alzheimer's disease cells at a predetermined time, by an amount that is statistically significantly characteristic of Alzheimer's disease cells and not characteristic of non-Alzheimer's disease cells.", "The term indicator protein, or MAPK protein, generally used interchangeably herein, means a protein which is phosphorylated as part of the calcium signaling pathway, in response to administration of an activator compound, and whose degree of phosphorylation is differentially higher in AD cells than control cells.", "The indicator protein may be a calcium signaling pathway protein whose phosphorylation is associated with IP3R-sensitive Ca2+ elevation in the cells.", "Indicator/MAPK proteins include those which are phosphorylated in response to IP3R mediated calcium release, such as Erk1/2.The activation or stimulation of cells means that the intact cells are contacted with the activator in vitro, typically by adding a solution containing the activator compound, or otherwise as would be known to a person of ordinary skill in the art.", "An activator compound is one that, upon introduction to human cells, stimulates phosphorylation of the indicator protein.", "Activator compounds elicit a differential response in the phosphorylation level of the indicator protein in cells of Alzheimer's subjects as compared to the level in control cells of a non-Alzheimer's subject.", "Activator compounds are effective when delivered in cell culture medium.", "Activator compounds may be referred to as IP3R agonists because they induce an IP3R mediated intracellular Ca2+ elevation, either directly or indirectly.", "The phosphorylation level of an indicator protein is generally determined by measuring the amount of phosphorylated indicator protein and, optionally, of unphosphorylated indicator protein, and normalizing the amount of phosphorylated protein to the total of indicator protein in the sample being analyzed.", "The calculated response phosphorylation level and the basal or background phosphorylation levels are thus not affected by differences in the absolute quantity of the indicator protein at a given time.", "The discriminatory time point, or predetermined time after stimulating the cells with the activator compound is selected to achieve a calibrated statistically significant difference between the phosphorylation level of indicator protein in known AD cells and known control cells.", "The difference may be maximal at the predetermined time but that is not required and depends on other parameters of the test.", "For any given activator compound, there are a finite number of suitable indicator proteins and vice versa.", "According to the invention, the activator compound and indicator protein combination is selected such that the activator elicits a differential response in the phosphorylation level of the indicator protein in cells of Alzheimer's subjects as compared to the level in a healthy control cells at a predetermined time after contacting the cells with the activator.", "A person of ordinary skill may select suitable indicator proteins and activator compounds by determining whether such a differential phosphorylation occurs.", "Having selected a set of indicator proteins and activator compounds, the assay parameters may then be optimized as to the discriminatory time point, suitable concentrations of the protein and activator, suitable antibodies to the phosphoproteins or unphosphorylated proteins, or other detection means, and so on.", "Calculation of a ratio of the activated phosphorylation level to the basal phosphorylation level may of course be reversed.", "That is, for convenience we refer to the activatedibasal ratio being higher than a known level, it is apparent to those skilled in the art that reversing the numerator and denominator in the ratio gives the same result if the viewed from the perspective of the unactivated phosphorylation is considered lower than a known level.", "Thus, where calculation of the ratio is described one way herein it is to be understood to encompass calculating the inverse as is apparent to a person of ordinary skill.", "Also, whereas the calculation of ratios as described herein is beneficial in providing useful comparative numbers, calculation of absolute differences between activated and basal phosphorylation levels, and between test subjects and control subjects, could also be employed and would be effective according to the invention The previously determined ratios for known Alzheimer's disease cells and non-Alzheimer's disease cells at the predetermined or discriminatory time may be written in a chart or set forth in a computer database from which diagnostic results may be determined.", "Accordingly, in practice, a diagnostic test is performed, an activated/basal phosphorylation ratio is calculated, and if the calculated ratio is the same as or greater than the previously determined ratio for known Alzheimer's disease cells, the diagnosis is positive.", "If the calculated ratio is less than the previously determined ratio for known non-Alzheimer's disease cells, the diagnosis is negative.", "The present invention provides a diagnostic test for AD that comprises measurement of the response of a MAP kinase (MAPK) protein to BK stimulation.", "The response in AD cells is compared to the response in cells from age-matched controls (which may include subjects with other diseases, including other diseases associated with dementia).", "In a preferred embodiment, the test is based on the detection of abnormally high phosphorylation of Erk1/Erk2 in skin fibroblasts as diagnostic of the presence of AD (of both familial and non-familial type) after stimulation of the cells with BK.", "Evaluation of phosphorylation may be by Western blotting or other approaches.", "Phosphorylation is a biochemical reaction in which a phosphate group is added to Ser, Thr or Tyr residues of a protein and is catalyzed by protein kinase enzymes.", "Phosphorylation normally modifies the functions of target proteins, typically causing activation.", "As part of the cell's homeostatic mechanisms, phosphorylation is only a transient process which is reversed by other enzyme called phosphatases.", "Any aberration in either side of the reaction (phosphorylation vs. dephosphorylation) can disrupt cellular function.", "These disruptions may be the fundamental underpinnings of various brain diseases.", "According to the invention abnormal phosphorylation activity producing elevated phosphorylation of indicator proteins in response to calcium signaling pathway modulators can be detected in AD patients in a diagnostic analysis.", "The abnormality is an increase or decrease of phosphorylation at a given time relative to the levels in a healthy patient's cells.", "Detection of AD-specific differences in MAPK in any peripheral tissue (i.e., outside the central nervous system) serves as the basis for an economical test for early diagnosis of AD and for screening and identification of therapeutic targets for drug development.", "Thus, this invention provides methods, reagents, and kits for detecting the presence or absence of AD.", "MAPK enzymes play a central role in conveying extracellular signals to the cell nucleus leading to control of gene expression.", "They are also involved in regulating phosphorylation of the microtubule-associated protein tau and generation and secretion of the β amyloid protein, events critical to the pathogenesis of AD.", "According to the invention, abnormally prolonged phosphorylation of the MAPK Erk1/2 occurs in, and is detected in, AD fibroblasts in response to stimulation by BK when compared to age-matched controls.", "Although the present inventors conducted their initial tests with BK as the stimulus, it is to be understood that BK represents but one way to achieve stimulation of cellular IP3 receptors.", "Accordingly, reference herein to BK should be interpreted to encompass other suitable activator or stimulus compounds.", "This stimulation serves as the common step that results in the heightened Erk1/2 phosphorylation characteristic of AD.", "Indeed, it was observed that inhibition of the IP3 receptor totally abolished the BK-stimulated Erk1/2 phosphorylation suggesting that the IP3-sensitive Ca2+ release accounts for this outcome.", "The AD-specific increases in Erk1/2 phosphorylation were also associated with enhanced expression of genes for PKC and MAP kinase kinase (also known as “MAPK/Erk kinase or MEK) isoforms in AD cells, as well as reduced expression in genes for phosphatase 1, 2A, and 2B which dephosphorylate the microtubule-associated protein tau and MAPK.", "The alterations of gene expression in AD cells may change the balance of activities between protein kinases and phosphatases that could contribute to this AD-specific prolonged Erk1/2 activation, as well as to the hyperphosphorylation of tau that forms neurofibrillary tangles in the AD brain.", "Detection of these Erk1/2 abnormal activities in skin fibroblasts reflects similar changes in the brain, and thus is diagnostic of the early pathophysiology of AD.", "Thus, according to the invention, BK stimulation evokes an abnormally prolonged increase in phosphorylation of Erk1/2 in AD fibroblasts, a change that depends upon IP3 receptor-mediated Ca2+ release.", "While the IP3 kinase appears to be involved in the BK-stimulated Erk1/2 phosphorylation in cells from non-AD controls, this phosphorylation of Erk1/2 in the AD cells is IP3 kinase-independent.", "The AD-specific increase in Erk1/2 phosphorylation was subsequent to the IP3-mediated Ca2+ release.", "Protein Kinase C (PKC) and the nonreceptor protein tyrosine kinase (PTK) c-Src are involved in the upstream signaling pathway of the Erk1/2 activation, indicating by the results that both the PKC inhibitor bisindolylmaleimide-1 and the c-Src inhibitor, PP1, completely abolished the BK-stimulated Erk1/2 phosphorylation.", "The PI-3 kinase, LY924002, partially inhibited the BK-stimulated Erk1/2 phosphorylation in the control, nonAD cells, but showed no effect in the AD cells, suggesting the BK-induced Erk1/2 phosphorylation may involve signaling pathways independent of PI-3 kinase.", "Activation of cAMP-responsive element binding protein (CREB), measured as an increase in phosphorylation at Ser-133 was also observed after BK stimulation.", "Similar to Erk1/2 phosphorylation, the BK-induced CREB phosphorylation was completely inhibited by inhibitors of PKC and c-src in both AC and AD cells.", "Only the CREB phosphorylation in the AC but not the AD cells was partially inhibited by IP-3 kinase inhibitor LY-294002.These results suggest a derangement of signaling pathways in response to BK in AD cells leading to abnormally enhanced and prolonged Erk1/2 phosphorylation and activation which, in turn perturb cellular processes such as gene transcription, APP (amyloid precursor protein) processing and tau protein phosphorylation, all of which underlie the pathogenesis of AD.", "The understanding of alterations in these pathways may also help explain the early memory loss that is a hallmark of AD.", "Moreover, detection of AD-specific differences in MAPK in peripheral tissues provides (1) an efficient means for early diagnosis of AD and (2) a biochemical basis for identifying therapeutic targets for drug development.", "In view of the foregoing, and because the same cell may use different combinations of intracellular Ca2+-releasing messengers to encode different external messages, any agent that is capable of stimulating IP3 receptors to cause release of internal stores of calcium, may be used in the test of the present invention an inducer of the increased Erk1/2 phosphorylation characteristic of AD cells.", "Again, BK is but one such agent, and other BK receptor agonists are similarly useful.", "Molecules that are ligands for the IP3 receptor may also be used.", "Because the IP3 receptor is regulated by ATP, calcium, and phosphorylation by protein kinase A, protein kinase C, and Ca2+ caltiodulin-dependent protein kinase 11 (CaM kinase II), agents that modulate the levels or activities of ATP or these kinases can achieve similar effects and can substitute for BK in the present invention.", "One such agent is the 14 amino acid peptide bombesin (Anastasi A. et al., Arch Biochem Biophys, 1972, 148:443-446); Woodruff G N et al., Ann N Y Acad Sci, 1996, 780:223-243, or biologically active analogues or fragments thereof (Rivier J E et al., Biochemistry, 1978, 17:1766-71; Orloff M S et al., Peptides, 1984 5:865-70; Walsh, J H et al., Peptides, 1985, 6 Suppl 3:63-68).", "Another is cholecysokinin.", "For bombesin and cholecytokinin induced calcium, signaling via IP3 and IP3 receptors, see, e.g., Cook S J et al., Biochem J, 1990, 265:617-620 Schulz I et al., Biol Chem, 1999, 380:903-908; Burdakov D, et al., Curr Biol, 2000, 10:993-996 Although the test is exemplified herein using skin fibroblasts, other cells that are as or more convenient to obtain and process may be used in the test of this invention, thus blood cells, preferably lymphocytes or monocytes, are easily prepared from peripheral blood and used in accordance with this invention.", "A person of ordinary skill can calibrate the suitable time points and experimental conditions to obtain a diagnostic method based on such cells, using suitable indicator proteins and activator compounds.", "The present inventors discovered that the activity of an important enzyme, MAPK, meaning mitogen-activated protein kinase (MAP kinase) was stimulated for an abnormally prolonged interval in Alzheimer's disease skin fibroblasts compared to cells from age-matched controls.", "The Examples below show that phosphorylation of Erk1/2 was prolonged in AD fibroblasts upon stimulation with BK.", "IP3 receptor-mediated Ca2+ release, and the activity of PKC and of the nonreceptor protein tyrosine kinase (PTK) c-Src are essential for this Erk1/2 phosphorylation.", "Although an IP3 kinase appears to be involved in BK-stimulated Erk1/2 phosphorylation in AC cells, the same phosphorylation in AD cells is IP3 kinase-independent.", "According to the invention, cells are stimulated with an agent that stimulates the IP3 receptor to release intracellular calcium; examples of such agents are BK and its analogs.", "BK is a potent nonapeptide that binds to and activates specific BK receptor(s), which, in turn triggers a cascade of molecular events inside the cell including stimulation of IP3 receptor and the phosphorylation of MAPK proteins.", "Phosphorylation is a biochemical reaction in which a phosphate group is attached to a protein initiated by an enzyme called protein kinase; it normally modifies functions of target proteins and usually causes activation of a protein.", "Cells need to maintain a balanced system for their functions so phosphorylation is only a transient process, which needs to be reversed by another enzyme, called phosphatase.", "Any aberration in either side of the reaction (phosphorylation vs. dephosphorylation) will break the integrity molecular network and disrupt cellular functions.", "These disruptions may be the fundamental underpinnings of various brain diseases.", "Such long-lasting phosphorylation of MAPK in AD fibroblasts indicates abnormally enhanced activity of this enzyme in those cells.", "This AD-specific effect appears to be related to other abnormal cellular and enzymatic activities.", "The prolonged MAPK phosphorylation was secondary to excessive Ca2+ release mediated by IP3 receptors from a specific store inside the cell.", "Phosphorylation of MAPK was partially dependent on PI3 kinase activity, and a PI3 kinase-dependent mechanism was shown to be involved in phosphorylation of MAPK in AD cells.", "In comparing the gene expression profile in AD cells with the age-matched control cells, the present inventors found that expression of PKC and MEK was increased in AD cells.", "Both PKC and MEK are upstream molecules that catalyze phosphorylation of MAPK.", "The present inventors also found reduced expression of several phosphatases that dephosphorylate MAPK.", "Therefore, the imbalance, ie, combined changes in the amounts and activities of enzymes that regulate MAPK phosphorylation account for the diagnostically-useful difference in AD cells—prolonged MAPK phosphorylation.", "This is the first discovery of abnormally enhanced MAPK activity in AD.", "The MAPK family of enzymes performs crucial steps in cell signaling from the cell membrane to the nucleus.", "They are stimulated by a variety of signals (e.g., mitogens) acting at the cell surface and leading to cell division (mitosis), a process required for growth and development, as well as replacement and/or repair of cells damaged by injury or disease.", "MAPK also plays an important role in transmitting signals in neurons that underlie functions such as learning and memory formation.", "MAPK also regulates secretion of the amyloid peptide βAP and phosphorylation of tau protein in brain cells.", "Accumulation of βAP outside nerve cells and excessive phosphorylation of tau are highly toxic to neurons, leading to plaques and neurofibrillary tangles (NFT), respectively.", "Indeed these are two characteristic pathological features in AD brains.", "According to the present invention, abnormally enhanced MAPK activity contributes to aberrant amyloid processing and tau protein function.", "The present results provide new insights into molecular substrates for normal memory formation in the brain as well as pathophysiology of AD.", "This latter understanding is expected to lead to the identification of new molecular targets for prevention and treatment of the disease.", "More practically for now, the present results have immediate clinical diagnostic significance.", "Because (a) skin fibroblasts manifest the alternations in MAPK activity characteristic of AD, both familial and non-familial, (b) skin fibroblasts are so easily obtained (vs. brain tissue), a, a diagnostic test based on MAPK performed on skin fibroblasts provides an simple and economic test for early diagnosis of AD.", "The present new discoveries are consistent with finding by others that excessive calcium signaling was detectable in BK-stimulated AD fibroblasts (Ito et al., 1994; Gibson et at., 1996; Etcheberrigaray, et al., 1998).", "EXAMPLE I The present study used skin fibroblasts from 20 AD patients with ages ranging from 52-67 years and from 22 age-matched controls.", "Some of the samples were previously banked cells, while others were freshly collected and cultured from human skin.", "Materials and Methods Banked skin fibroblasts from familial (FAD) and nonfamilial (nFAD) AD patients and from the age-matched controls (AC) were purchased from the Coriell Institute for Medical Research.", "The medium was Dulbecco's modified Eagle's medium (DMEM; Gibco BRL) supplemented with fetal bovine serum (FBS; Bio Fluids).", "Bradykinin, diphenylboric acid 2-aminoethyl ester (2ABP), protease and phosphatase inhibitor cocktails were from Sigma; bisindolylmaleimide-1 (BiSM-1) and LY294002 were from Alexis; PP1 was from Dr Anthony Bishop, Princeton University.", "Anti Phospho-Erk1/2, anti-phospho-CREB, and anti-CREB antibodies were from Cell Signaling Technology.", "Anti-Erk1/2 antibody was from Upstate Biotechnology.", "4-20% SDS-mini gels were from Invertrogene Novex.", "Nitrocellulose membranes were from Schleicher & Schuell.", "All the SDS electrophoresis reagents were from BioRad.", "SuperSignal chemiluminescent substrate kit was from Pierce.", "Culture of AC and AD fibroblasts: Banked fibroblasts from Alzheimer's disease patients including both familial (FAD) nonfamilial (nFAD) types, and from the age-matched controls (AC) were maintained and passaged in T-25/T75 flasks with the DMEM+10% FBS.", "Cells were from subjects ranging in age from 52 to 67 years and over 80% of the samples were from males.", "The cells were used during passages 6-17.Processing and culture of fibroblasts from fresh biopsies: The collection and culture of fibroblasts from freshly obtained skin tissue were performed as follows.", "Punch biopsy skin tissues from NFAD patients and the age-matched controls were taken by qualified personnel at Copper Ridge Institute (Sykesville, Md.)", "and Johns Hopkins Hospital (Baltimore, Md.)", "under an approved protocol.", "Samples were placed in 1×PBS and transported in transfer medium to the laboratory for processing.", "Tissue was removed from transfer medium, rinsed with PBS and chopped into 1 mm explants.", "The explants were transferred individually onto the growth surface of vented T-25 flasks with 3 ml of biopsy medium containing 45% FBS and 100 U/ml Penicillin and 100 U/ml streptomycin (Pen/Strep).", "The tissues were cultured at 37° C. for 24 hours before addition of 2 ml of biopsy medium containing 10% FBS.", "The medium was replaced, after 48 hours, with 5 ml of regular culture medium containing 10% FBS and Pen/Strep.", "The cells were then passaged and maintained as above.", "Treatment of fibroblast cells with different pharmacological agents: Fibroblasts were treated with BK, and various inhibitors.", "These include the IP3 receptor inhibitor, diphenylboric acid 2-aminoethyl ester (2ABP, the protein kinase C inhibitor Bisindolylmaleimide I (BiSM-1), C-src protein tyrosine kinase inhibitor PP1, and PI3 kinase inhibitor LY294002.Banked AC and AD fibroblasts were grown to 80-100% confluence before they were “starved” in a serum-free DMEM overnight.", "Cells were treated with 10 nM BK at 37° C. for different intervals to establish a time course for BK effects.", "The same volume of PBS was added to a control flask of cells of each line.", "The reaction was terminated by removing the medium, rapidly rinsing the cells with pre-cooled PBS, pH 7.4, and transferring the flask onto dry ice/ethanol.", "Cells from fresh biopsy tissue were incubated with an optimal concentration of BK, 0.1 nM for 10 min at 37° C. For inhibition studies, cells were preincubated with the following concentration of inhibitor at 37° C. for the intervals indicated: 2ABP (50 μM for 30 min); PP1 (10 μM for 15 min); Ly294002 (5 μM for 15 min); and BiSM-1 (5 μM for 15 min).", "A parallel control flask was incubated with an identical volume of DMSO vehicle.", "At the end of this incubation, the same inhibitor at the indicated concentration was added and followed immediately by BK to a final concentration of 10 nM.", "BK (10 nM) was also added to DMSO controls.", "Basal controls were cells to which neither BK nor inhibitors were added.", "After a 5-min incubation at 37° C., the reaction was terminated as above.", "Cell lysates were prepared from cells of the various groups Flasks were moved from dry ice/ethanol to ice water.", "Each flask received 1 ml of lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH8, 0.5% NP-40, 1% Triton X-100, 1% protease inhibitor cocktail (Sigma), 1% cocktail of serine/threonine (Ser/Thr) phosphatase inhibitor and tyrosine (Tyr) phosphatase inhibitor (Sigma).", "After rocking on an end-to-end shaker in a cold room for 30 min, cells were collected from each flask with a cell scraper.", "Cells were sonicated, centrifuged at 5000 rpm for 5 min, and a sample of the supernatant was subjected to Western blotting.", "Western blotting: Cell lysates were boiled in an equal volume of 2×SDS-sample buffer for 10 min.", "Proteins from each sample were resolved on a 4-20% mini gradient gel and transferred onto a nitrocellulose membrane.", "Phosphorylated Erk1/2 was detected with anti-phospho-Erk1/2 antibody using the SuperSignal ECL detection kit.", "In order to normalize the amount of phosphorylated Erk1/2 against the total amount of Erk1/2, the same membrane blotted with the antibody was stripped with a stripping buffer (62.5 mM Tris-HCl pH6.7, 2% SDS and 100 mM 2-mercaptoethanol) at 60° C. for 45 min.", "After washing with 10 mM PBS pH 7.4 containing 0.01% Tween-20 (3×at 10 min), the membrane was blotted with an anti-non-phospho-Erk1/2 antibody, which allowed calculation of the total amount of Erk1/2 loaded on the gel.", "CREB was detected using analogous methods.", "Data analysis: Signals from phosphorylated and nonphosphorylated Erk1/2 (or CREB) were scanned with a Fujifilm LAS-1000 Plus scanner.", "The mean optical density of each protein band was measured using NIH Image software.", "Values from the phosph-Erk1/2 signals were normalized respectively against the total signal of Erk1/2.After normalization, data from treated cells was converted to a percentage of the basal control and subjected to statistical analyses.", "Immunocytochemistry: Fibroblasts were grown on the surface of glass coverslips coated with 0.02 mg % polylysine.", "After treatment with BK as above, cells were rapidly rinsed with cold PBS, fixed with 4% formaldehyde in PBS at room temperature for 15 min, washed with PBS 3 times 5 min each, and penetrated with 0.1% TritonX-100 in PBS at room temperature for 30 min.", "After incubated with 10% normal horse serum in PBS at room temperature for 30 min, cells were incubated with anti-Phospho-Erk1/2 antibody (1:200) at 4° C. overnight.", "Coverslips were washed with PBS 3 times, and fluorescein-labeled anti-mouse IgG, 1:200 (Vector Lab) was added and allowed to incubate at room temperature for 60 min.", "Following three washes with PBS, and sealing with Vectashield (Vector Lab), immunostaining signals in the cells were observed in a florescence microscope.", "The intensity of the fluorsecent signals was measured with BioRad Quantity One® software (BioRad).", "For localization of BK receptors in the fibroblasts, a monoclonal anti BK B2 antibody was applied to the normal fibroblasts, followed by incubation with CY5-conjugated anti-mouse IgG and the cells examined by fluorescence microscopy.", "Results BK-Induced Activation of Erk1/2 in AC and AD Cells BK elicited marked, but transient phosphorylation of Erk1/2 in human skin fibroblasts from control subjects (FIG.", "1).", "Peak phosphorylation occurred at about 2.5 min following stimulation, after which phosphorylation of Erk1/2 declined, and returned to control levels by 10 min after stimulation.", "At 20 min post-BK treatment, phosphorylation of Erk1/2 was 68% of the control (FIG.", "1).", "In cells from subjects with AD, however, increased phosphorylation levels of Erk1/2 were maintained for a markedly prolonged period (FIG.", "1).", "Differences were statistically significant (Student's t test) at 5 min (p<0.01), with the greatest divergence occurred at 10 min (p<0.0001).", "This difference between AD and AC cells remained significant when measured at 20 min (P=0.002) after BK stimulation.", "FIG.", "2 shows results of a study in which BK-induced Erk1/2 phosphorylation was examined at 10 minutes after stimulation with skin fibroblasts from fresh tissue samples taken from AD patients and age-matched controls as well as from banked cells lines from AD and AC groups.", "Erk1/2 phosphorylation in AD fibroblasts was consistently elevated compared to age-matched controls (FIG.", "2A).", "Similarly, BK-stimulated Erk1/2 phosphorylation in cell lines was reproducibly elevated in AD cell lines in as shown in FIG.", "2B, which illustrates three independent replications (from randomly chosen AC and AD lines).", "When fibroblasts were immunostained with anti-P-Erk1/2 antibody, increased levels of phosphorylated Erk1/2 were observed in AD cells, but not in AC cells, 10 min after -BK treatment (FIG.", "3).", "These enhanced signals were concentrated in the para-nuclear area (FIG.", "3, lower right panel).", "Effects of the IP3 Receptor Inhibitor 2ABP on BK-Induced Erk1/2 Phosphorylation BK stimulates Ca2+ release from IP3-sensitive stores (Cruzblanca et al., 1998; Pascale et al., 1999).", "In the present study, the involvement of Ca2+ release from IP3 receptor in the BK-induced Erk1/2 phosphorylation was examined.", "Addition of 2ABP, a potent membrane-permeable inhibitor of the IP-3R, to cells before BK completely abolished BK-stimulated Erk1/2 phosphorylation—in both AC and AD cells (FIG.", "4A).", "Two-way ANOVA showed that the treatment effects were highly significant (F1,36=187.4, p<0.0001).", "This result proves that phosphorylation of Erk1/2 and the subsequent molecular cascade is downstream of the IP-3R-sensitive Ca2+ elevation in the cells.", "Effect of the PKC Inhibitor BiSM-1 on the BK-Induced Erk1/2 Phosphorylation In this experiment, AC and AD cells were preincubated with the specific PKC inhibitor BiSM-1 before BK.", "As shown in FIG.", "4B, BK-induced phosphorylation of Erk1/2 was again abolished (in both AC and AD cells).", "A two-way ANOVA showed significant treatment effects.", "This result indicates that PKC activity is required for the BK-stimulated activation of Erk1/2 in both AC and AD cells.", "Effect of the C-src Protein Tyrosine Kinase Inhibitor, PP1, on the BK-Induced Erk1/2 Phosphorylation To test whether the non receptor PTK, C-src is involved in activation of Erk1/2 by BK, AC and AD cells were preincubated with 10 μM PP1 prior to BK.", "Similar to 2ABP and BiSM-1, above, PP1 completely inhibited BK-induced Erk1/2 phosphorylation (FIG.", "4C).", "Two-way ANOVA showed significant treatment effects (F1,40=234; p<0.0001).", "This result therefore suggests that C-src PTK activity is also involved in activation of Erk1/2 by BK.", "Effects of PI3 Kinase Inhibitor, LY294002, on BK-Induced Erk1/2 Phosphorylation A study was conducted to test whether PI3 kinase activity is involved in BK-stimulated Erk1/2 activation.", "Fibroblasts were incubated with 5 μM LY294002 prior to BK.", "In contrast to the effects of inhibitors of the IP-3R, PCK and C-src, LY294002 caused a modest, though significant, inhibition of Erk1/2 phosphorylation in AC cells (FIG.", "4D) while it failed to inhibit the BK-induced phosphorylation of Erk1/2 in AD cells (FIG.", "4D).", "A two-way ANOVA showed significant treatment effects (F1,20=136.2; p<0.001) and group effects (F1,20=11.55; p<0.05).", "This result suggests that PI3 kinase activity is not involved in BK-induced Erk1/2 phosphorylation in cells of AD patients.", "Effects of BK on Activation of Cyclic-AMP Response Element Binding Protein (CREB) The effect of BK on activation of CREB, which normally is downstream of Erk1/2, was tested.", "As shown in FIG.", "5, 10 nM BK induced highly significant phosphorylation of CREB in both AC and AD cells.", "The increased phosphorylation was detected as early as 5 min after BK stimulation, and increased further at 10 min (FIG.", "5A).", "A two-way ANOVA showed a significant time effect (F1,28=14.09, p<0.001) but not group effect.", "FIG.", "5B shows similarly elevated CREB phosphorylation 10 min after BK treatment in AC and AD cells.", "The BK treatment also caused a marked reduction in the total amount of CREB protein at 10 min (FIG.", "5C) (p<0.001).", "In summary, there were no significant differences in BK-induced CREB phosphorylation or reduction in total CREB between AC and AD cells.", "Effects of PKC, C-src, and PI3 Kinase Inhibitors on BK-Induced CREB Phosphorylation Treatment of fibroblasts with the PKC inhibitor, BiSM-1, completely abolished the BK-stimulated CREB phosphorylation (FIG.", "6A).", "A two-way ANOVA showed significant treatment effect (F1,30=53.76, p<0.0001), but no group effect.", "Similarly, the c-src inhibitor PP1 inhibited BK-stimulated CREB phosphorylation (FIG.", "6B).", "These results again indicate that both PKC and c-src are involved in the signaling pathway by which BK activates both Erk1/2 and CREB.", "Similar to the effect with Erk1/2, the PI3 kinase inhibitor, LY294002 partially inhibited CREB phosphorylation in AC but did not have any effect on CREB phosphorylation in AD cells (FIG.", "6C).", "A two-way ANOVA showed significant treatment effect (F1,30=36.23; p<0.0001) and group effect (F1,30=4.7; p<0.05).", "Expression of BK B2 Receptor in Fibroblasts To test whether enhanced Erk1/2 and CREB phosphorylation in response to BK was due to an increase in the number of BK receptors expressed on AD cells, a study was performed to measure BK receptor expression in AD and control AC cells by Western blot using a specific antibody for type 2 BK receptor (BKb2R).", "No significant differences in receptor expression were observed.", "By immunofluorescent staining, BK2bR was demonstrated in cytosol, nuclei, and paranuclear areas of fibroblasts (FIG.", "7).", "However, no apparent differences in either intensity or distribution pattern of the BKb2R were observed in AD vs AC cells.", "Discussion of Experimental Results Fibroblasts have served as a useful model for studying AD and other hereditary diseases presumably because these cells, located all over the body, can express genetic abnormalities associated with some diseases of the brain.", "The present study has provided evidence that stimulation of the BK receptor results in abnormal signal transduction by the MAPK pathway in AD fibroblasts.", "BK is one of the most potent endogenous algesic and proinflammatory substances, playing important roles following injury, disease and CNS as well as peripheral inflammation.", "In the present study, BK stimulation enhanced and prolonged Erk1/2 phosphorylation in AD cells in a manner that was dependent on Ca2+ release from IP3 receptor.", "This is consistent with previous reports that elevated IP3-sensitive Ca2+ release was detected in AD skin fibroblasts following BK stimulation (Gibson et al., 1996; Etcheberrigaray et al., 1998).", "However, this prolonged phosphorylation of AD Erk1/2 was not due to increased expression of the BKb2 receptor or Erk1/2, since neither the expression nor concentration of these two proteins was different in AD cells compared to controls.", "It is not possible to rule out specific other changes in the properties or sensitivities of the BK receptor and/or IP3 receptor in AD, in addition to changes in other molecules involved in this signal transduction pathway.", "The activation of the BK2bR stimulates activation of the PLC system, which in turn, together with the elevated intracellular Ca2+, induces PKC activation.", "In addition, some G protein-coupled receptors (“GPCRs”) are known to stimulate c-src PTK activity, which, as a key intermediate in cellular signaling, also may participate in activation of Erk1/2.The present study tested the involvement of both PKC and c-src PTK in BK-stimulated Erk1/2 phosphorylation.", "The fact that this phosphorylation was abolished by a PKC inhibitor, BiSM-1, and a c-src inhibitor, PP1, indicate that Erk1/2 activity is regulated by convergence of multiple upstream protein kinases.", "Like the IP3 receptor inhibitor, 2ABP, both BiSM-1 and PP1 abolished the Erk1/2 phosphorylation similarly in AD and control cells.", "It is unlikely, therefore, that the enhancement of Erk1/2 phosphorylation associated with AD was due to elevated PKC or c-src PTK activity.", "In analyzing the signaling pathway(s) that underlies the AD-specific enhancement of Erk1/2 phosphorylation, the present inventors tested the involvement of PI-3 kinase, another enzyme involved in Erk1/2 activation by GPCRs.", "Based on studies in various systems, activation of PI-3 kinase following GPCR stimulation is dependent on activation of PKC, down stream of Ras and to involve c-src PTK.", "The specific PI-3 kinase inhibitor LY294002 only caused a modest inhibition of the BK-induced Erk1/2 phosphorylation in control fibroblasts but had no effect in AD cells.", "This suggests that, while PI-3 kinase partially contributes to the BK-activated Erk1/2 phosphorylation in normal fibroblasts, Erk1/2 phosphorylation in AD cells appears to be independent of this enzyme.", "A major function of Erk1/2/MAPK is the activation of gene expression by regulation of transcriptional factors.", "Activation of CREB has also been observed in the present study (phosphorylation of Ser133).", "Unlike the phosphorylation of Erk1/2 in control cells, which lasted less than 10 min following BK treatment, highly phosphorylated CREB was present 10 min after BK treatment in both AD and control cells, suggesting a different time course for CREB activation.", "Inhibitors of PKC, c-src and PI3 kinase inhibit phosphorylation of CREB in a manner similar to their inhibition of Erk1/2 phosphorylation, suggesting that CREB activation is regulated by the same signal pathway.", "While PP1 completely inhibited BK-stimulated CREB phosphorylation, it resulted in increased expression of the CREB protein in control cells (FIG.", "6B), presumably reflecting a normal compensatory feedback mechanism.", "This effect of PP1 was not observed in AD cells, however, suggesting that regulation of CREB expression may be impaired in AD.", "The present results provide evidence that AD pathogenesis involves derangement of molecular cascades associated with MAPK as summarized in FIG.", "6.Recent results from others suggest that MAPK is important for brain functions that are related to neuronal plasticity, for example, learning and memory.", "Erk phosphorylates tau protein at multiple Ser/Thr sites, including Ser262 and Ser356 (Reynolds et al., 2000), which are in the microtubule-binding regions of tau.", "Phosphorylation of Ser262 markedly compromises the functional ability of tau to assemble and stabilize microtubules.", "With compromised signaling pathways involving MAPK, cells may respond to extra- and intracellular signals by inducing aberrant expression of numerous other proteins.", "Systemic manifestations of abnormalities in molecular signaling such as enhanced or prolonged MAPK phosphorylation, may reflect aberrations in the CNS, e.g.", "memory loss, that have grave behavioral/cognitive consequences.", "Thus, detection of AD-specific abnormalities in MAPK and its related signaling pathways in cells found at easily accessible peripheral sites, exemplified by skin fibroblasts, provides an efficient and reliable means for early diagnosis of AD as well as for identifying therapeutic targets for drug development.", "EXAMPLE 2 Using skin fibroblasts from individuals with Huntington's dementia (HD), tests were conducted to determine whether the BK-induced prolonged Erk1/2 activity was AD specific.", "FIG.", "8 represents the effect of bradykinin on phosphorylation of Erk1/2 on fibroblasts.", "N=4 and P=0.39, t test.", "The HD cells were treated with 10 nM BK for 10 minutes.", "FIG.", "8 shows that when HD cells were treated with 10 nM BK for 10 minutes, the phosphorylation levels of Erk1/2 were not different from that of the age-matched controls indicating that the BK-induced enhancement of Erk1/2 activity is not present in Huntington's dementia.", "Thus, a MAP kinase assay for Alzheimer's disease according to the invention gives a negative diagnosis for Huntington's dementia, showing that the assay is specific to Alzheimer's.", "In describing preferred embodiments of the present invention, specific terminology is employed for the sake of clarity.", "However, the invention is not intended to be limited to the specific terminology so selected.", "It is to be understood that each specific element includes all technical equivalents, which operate in a similar manner to accomplish a similar purpose.", "The above-described embodiments of the invention may be modified or varied, and elements added or omitted, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings.", "Each reference cited here is incorporated by reference as if each were individually incorporated by reference.", "REFERENCES U.S. provisional patent application 60/312,064, filed Feb. 27, 2001, and the following publications are incorporated herein by reference.", "1.Barrow, P. A. Empson, R. M. Gladwell, S. J. Anderson, C. M. Killick, R., Yu, X., Jefferys, J. G. and Duff, K. (2000) Neurobiol Dis 7, 119-126 (“Barrow et al., 2000”).", "2.Bassa B V, Roh D D, Vaziri N D, Kirschenbaum M A, Kamanna V S (1999) Am J Physiol 277, F328-337 (“Bassa et al., 1999”).", "3.Berridge M J (1984) Biochem J 220, 345-360 (“Berridge, 1984”).", "4.Biemat, J., Gustke, N., Drewes, G., Mandelkow, E. W., and Mandelkow, E. (1993) Neuron 11, 153-163 (“Biernat et al., 1993”).", "5.Cruzblanca H, Koh D S, Hille B.", "(1998) Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons.", "Proc Natl Acad Sci USA 95:7151-7156 (“Cruzblanca et al., 1998”).", "6.Ekinci, F. J. and Shea, T. B.", "(1999) Cell Mol.", "Neurobiol.", "19, 249-260 (“Ekinci and Shea, 1999”).", "7.Etcheberrigaray E, Gibson G E, Alkon D L (1994) Molecular mechanisms of memory and the pathophysiology of Alzheimer's disease.", "Ann N Y Acad Sci 747:245-55 (“Etcheberrigaray et al., 1994”) 8.Etcheberrigaray, R., Hirashima, N., Nee, L., Prince, J., Govoni, S., Racchi, M., Tanzi, R. E. and Alkon, D. I.", "(1998) Neurobiol.", "Diseas.", "5, 37-45 (“Etcheberrigaray, et al., 1998”).", "9.Gibson, G. E., Zhang, H., Toral-Barza, L., Szolosi, S., and Tofel-Grehl, B.", "(1996) Biochim, Biophys Acta 1316, 71-77 (“Gibson et al., 1996”).", "10.Grant S M, Morinville A, Maysinger D, Szyf M, Cuello A C. (1999) Brain Res Mol Brain Res.", "72, 115-20 (“Grant et al., 1999”).", "11.Greenberg S M, Koo E H, Selkoe D J, Qiu W Q, Kosik K S. (1994) Secreted beta-amyloid precursor protein stimulates mitogen-activated protein kinase and enhances tau phosphorylation.", "Proc Natl Acad Sci USA 91, 7104-7108 (“Greenberg et al., 1994”).", "12.Hirashima, N., Etcheberrigaray, R., Bergamashi, S., Racchi, M., Battaini, F., Binetti, G., Govoni, S., Alkon, D. L. (1996) Neurobiol Aging 17, 549-555 (“Hirashima, et al., 1996”).", "cm 13.Ito E, Oka K, Etcheberrigaray R, Nelson T J, McPhie D L, Tofel-Grehl B, Gibson G E, Alkon D L (1994) Internal Ca2+ mobilization is altered in fibroblasts from patients with Alzheimer disease.", "Proc Natl Acad Sci USA 91,534-538 (“Ito et al., 1994”).", "14.Leissring, M. A., Akbari, Y., Fanger, C. M., Cahalan, M. D., Mattson, M. P. and Laferla, F. M. (2000) J Cell Biol 149, 793-798 (“Leissring et al., 2000”).", "15.Leissring, M. A., Parker, I., LaFerla, F. M. (1999) J Biol Chem 274, 32535-32538 (“Leissring et al., 1999”).", "16.Lu, Q., Soria, J. P., and Wood, J. G. (1993) J. Neurosci.", "Res.", "35, 439-444 (“Lu et al., 1993”).", "17.Mattson, M. P., Zhu, H., Yu, J. and Kindy, M. S. (2000) J. Neurosci.", "20, 1358-1364 (“Mattson et al., 2000”).", "18.McDonald D R, Bamberger M E, Combs C K, Landreth G E (1998) J Neurosci 18, 4451-4460 (“McDonald et al., 1998”).", "19.Pascale A, Bhagavan S, Nelson T J, Neve R L, McPhie D L, Etcheberrigaray R. (1999) Enhanced BK-induced calcium responsiveness in PC12 cells expressing the C100 fragment of the amyloid precursor protein.", "Brain Res Mol Brain Res 72:205-2 (“Pascale et al., 1999”).", "20.Putney J. W. Jr. (2000) Neuron 27, 411-412 (“Putney, 2000”).", "21.Reynolds, C. H., Betts, J. C., Blackstock, W. P., Nebreda, A. R. and Anderton, B. H. (2000) J. Neurochem.", "74, 1587-1595 (“Reynolds et al., 2000”).", "22.Sheehan J P, Swerdlow R H, Miller S W, Davis R E, Parks J K, Parker W D, Tuttle J B.", "(1997) J Neurosci 17, 4612-4622 (“Sheehan et al., 1997”).", "23.Yoo, A. S., Cheng, I., Chung, S., Grenfell, T. Z., Lee, H., Pack-Chung, E., Handler, M., Shen, J., Presenilin-mediated modulation of capacitative calcium entry.", "Neuron.", "2000 September;27(3):561-72 (“Yoo et al., 2000”)." ] ]	Patent_10469164
[ [ "Novel Genes Encoding Novel Proteolytic Enzymes", "The invention relates to newly identified gene sequences that encode novel proteases obtainable from Aspergillus niger.", "The invention features the full length gene sequence of the novel genes, their cDNA sequences as well as the full-length functional protein and fragments thereof.", "The invention also relates to methods of using these enzymes in industrial processes and methods of diagnosing fungal infections.", "Also included in the invention are cells transformed with DNA according to the invention and cells wherein a protease according to the invention is genetically modified to enhance or reduce its activity and/or level of expression." ], [ "1.An isolated polynucleotide that encodes a protease wherein said polynucleotide hybridizes to a polynucleotide of SEQ ID NO: 1 to SEQ ID NO: 57 or the complement thereof or of SEQ ID NO: 58 to SEQ ID NO: 114 or the complement thereof under wash conditions of 1×SSC, 0-1% SDS at 50° C. 2.An isolated polynucleotide according to claim 1, which thus hybridizes under stringent conditions.", "3.An isolated polynucleotide according to claim 1 obtainable from a filamentous fungus.", "4.An isolated polynucleotide according to claim 3 obtainable from A. niger.", "5.An isolated polynucleotide encoding a polypeptide having protease activity, said protein comprising an amino acid sequence of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof.", "6.The isolated polynucleotide of claim 5 that encodes a protease or functional portion thereof at least 60% identical to SEQ ID NO: 115 to SEQ ID NO: 171 or to the corresponding functional portion thereof.", "7.", "(canceled) 8.The isolated polynucleotide of claim 1 that has the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 57 or their complements or of SEQ ID NO: 58 to SEQ ID NO: 114 or their complements.", "9.A vector comprising a polynucleotide sequence according to claim 1.10.A vector according to claim 9 wherein said polynucleotide sequence is operatively linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell.", "11.A vector according to claim 10 wherein said suitable host cell is a filamentous fungus.", "12.A method to prepare a protease comprising the steps of culturing a host cell comprising the vector of claim 10 and isolating said protease from said host cell.", "13.An isolated polypeptide having protease activity that has the amino acid sequence of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof.", "14.An isolated polypeptide according to claim 13 obtainable from Aspergillus niger.", "15.An isolated polypeptide having protein activity obtainable by expressing a vector according to claim 10 in a host cell.", "16.A recombinant protease comprising a functional domain of a protease polypeptide, which has an amino acid sequence at least 60% identical to a functional domain of SEQ ID NO: 115 to SEQ ID NO: 171.17.", "(canceled) 18.A recombinant host cell comprising a vector according to claim 10.19.A recombinant host cell expressing a polypeptide according to claim 16.20.A recombinant host cell comprising a polynucleotide encoding a functionally inactivated protease polypeptide.", "21.A recombinant host cell wherein a polynucleotide encoding a protease polypeptide has at least partially been deleted.", "22.A recombinant host cell according to claim 18 wherein said host cell is from an Aspergillus species.", "23.A recombinant host cell functionally deficient in a protease obtainable by a method comprising said steps of: a. in vitro mutagenesis of a polynucleotide according to claim 1, b. transforming a host cell comprising an endogenous gene comprising a polynucleotide sequence hybridisable to said mutagenised polynucleotide obtained in step a), c. selecting and isolating recombinant host cells in which said endogenous gene is replaced by a mutagenised polynucleotide obtained in step a).", "24.Purified antibodies reactive with a polypeptide according to claim 16.25.Fusion protein comprising a polypeptide sequence according to claim 16.26.Method for diagnosing whether an organism is infected with Aspergillus comprising said steps of: a. isolating a biological sample from said organism suspected to be infected with Aspergillus, b. isolating nucleic acid from that sample, c. determining whether said isolated nucleic acid comprises polynucleotides hybridisable to a polynucleotide according to claim 1.27.Method according to claim 26 wherein step c) additionally comprises amplifying said isolated nucleic acid.", "28.Method for diagnosing whether an organism is infected with Aspergillus comprising said steps of: a. isolating a biological sample from said organism suspected to be infected with Aspergillus, b. reacting said biological sample with an antibody according to claim 24, c. determining whether immune complexes are formed." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Proteolytic Enzymes Proteins can be regarded hetero-polymers that consist of amino acid building blocks connected by a peptide bond.", "The repetitive unit in proteins is the central alpha carbon atom with an amino group and a carboxyl group.", "Except for glycine, a so-called amino acid side chain substitutes one of the two remaining alpha carbon hydrogen atoms.", "The amino acid side chain renders the central alpha carbon asymmetric.", "In general, in proteins the L-enantiomer of the amino acid is found.", "The following terms describe the various types of polymerized amino acids.", "Peptides are short chains of amino acid residues with defined sequence.", "Although there is not really a maximum to the number of residues, the term usually indicates a chain which properties are mainly determined by its amino acid composition and which does not have a fixed three-dimensional conformation.", "The term polypeptide is usually used for the longer chains, usually of defined sequence and length and in principle of the appropriate length to fold into a three-dimensional structure.", "Protein is reserved for polypeptides that occur naturally and exhibit a defined three-dimensional structure.", "In case the proteins main function is to catalyze a chemical reaction it usually is called an enzyme.", "Proteases are the enzymes that catalyze the hydrolysis of the peptide bond in (poly)peptides and proteins.", "Under physiological conditions proteases catalyse the hydrolysis of the peptide bond.", "The International Union of Biochemistry and Molecular Biology (1984) has recommended to use the term peptidase for the subset of peptide bond hydrolases (Subclass E.C 3.4.).", "The terms protease and peptide hydrolase are synonymous with peptidase and may also be used here.", "Proteases comprise two classes of enzymes: the endo-peptidases and the exo-peptidases, which cleave peptide bonds at points within the protein and remove amino acids sequentially from either N or C-terminus respectively.", "Proteinase is used as a synonym for endo-peptidase.", "The peptide bond may occur in the context of di-, tri-, tetra-peptides, peptides, polypeptides or proteins.", "In general the amino acid composition of natural peptides and polypeptides comprises 20 different amino acids, which exhibit the L-configuration (except for glycine which does not have a chiral centre).", "However the proteolytic activity of proteases is not limited to peptides that contain only the 20 natural amino acids.", "Peptide bonds between so-called non-natural amino acids can be cleaved too, as well as peptide bonds between modified amino acids or amino acid analogues.", "Some proteases do accept D enantiomers of amino acids at certain positions.", "In general the remarkable stereoselectivity of proteases makes them very useful in the process of chemical resolution.", "Many proteases exhibit interesting side activities such as esterase activity, thiol esterase activity and (de)amidase activity.", "These side activities are usually not limited to amino acids only and might turn out to be very useful in bioconversions in the area of fine chemicals.", "There are a number of reasons why proteases of filamentous fungi, eukaryotic microorganisms, are of particular interest.", "The basic process of hydrolytic cleavage of peptide bonds in proteins appears costly and potentially detrimental to an organism if not properly controlled.", "The desired limits to proteolytic action are achieved through the specificity of proteinases, by compartmentalization of proteases and substrates within the cell, through modification of the substrates allowing recognition by the respective proteases, by regulation via zymogen activation, and the presence or absence of specific inhibitors, as well through the regulation of protease gene expression.", "In fungi, proteases are also involved in other fundamental cellular processes, including intracellular protein turnover, processing, translocation, sporulation, germination and differentiation.", "In fact, Aspergillus nidulans and Neurospora crassa have been used as model organisms for analyzing the molecular basis of a range of physiological and developmental processes.", "Their genetics enable direct access to biochemical and genetical studies, under defined nutrient and cultivation conditions.", "Furthermore, a large group of fungi pathogenic to humans, live-stock and crop, has been isolated and proteolysis has been suggested to play a role in their pathogenicity (host penetration, countering host defense mechanisms and/or nutrition during infection).", "Proteases are also frequently used in laboratory, clinical and industrial processes; both microbial and non-microbial proteases are widely used in the food industry (baking, brewing, cheese manufacturing, meat tenderizing), in tanning industry and in the manufacture of biological detergents (Aunstrup, 1980).", "The commercial interest in exploiting certain filamentous fungi, especially the Aspergilli, as hosts for the production of both homologous and heterologous proteins, has also recently renewed interests in fungal proteases (van Brunt, 1986ab).", "Proteases often cause problems in heterologous expression and homologous overexpression of proteins in fungi.", "In particular, heterologous expression is hampered by the proteolytic degradation of the expressed products by homologous proteases.", "These commercial interests have resulted in detailed studies of proteolytic spectra and construction of protease deficient strains and have improved the knowledge about protease expression and regulation in these organisms.", "Consequently there is a great need to identify and eliminate novel proteases in filamentous fungi.", "Micro-organisms such as for example fungi are particularly useful in the large scale production of proteins.", "In particular when such proteins are secreted into the medium.", "Proteolytic enzymes play a role in these production processes.", "On the one hand particular proteolytic enzymes are in general required for proper processing of the target protein and the metabolic well-being of the production host.", "On the other hand proteolytic degradation may significantly decrease the yield of secreted proteins.", "Poor folding in the secretion pathway may lead to degradation by intracellular proteases.", "This might be a particular problem with producing heterologous proteins.", "The details of the proteolytic processes, which are responsible for the degradation of the proteins that are diverted from the secretory process in fungi are not exactly known.", "In eukaryotes the degradation of cellular proteins is achieved by a proteasome and usually involves ubiquitin labelling of proteins to be degraded.", "In fungi, proteasomal and vacuolar proteases are also likely candidates for the proteolytic degradation of poorly folded secretory proteins.", "The proteolytic degradation is likely cytoplasmic, but endoplamatic reticulum resident proteases cannot be excluded.", "From the aspect of production host strain improvement the proteolytic system may be an interesting target for genetic engineering and production strain improvement.", "Additional copies of protease genes, over-expression of certain proteases, modification of transcriptional control, as well as knock out procedures for deletion of protease genes may provide a more detailed insight in the function a given protease.", "Deletion of protease encoding genes can be a valuable strategy for host strain improvement in order to improve production yield for homologous as well as heterologous proteins.", "Eukaryotic microbial proteases have been reviewed by North (1982).", "More recently, Suarez Rendueles and Wolf (1988) have reviewed the S. cerevisiae proteases and their function.", "Apart from the hydrolytic cleavage of bonds, proteases may also be applied in the formation of bonds.", "Bonds in this aspect comprise not only peptide and amide bonds but also ester bonds.", "Whether a protease catalyses the cleavage or the formation of a particular bond does in the first place depend on the thermodynamics of the reaction.", "An enzyme such as a protease does not affect the equilibrium of the reaction.", "The equilibrium is dependent on the particular conditions under which the reaction occurs.", "Under physiological conditions the thermodynamics of the reactions is in favour of the hydrolysis of the peptide due to the thermodynamically very stable structure of the zwitterionic product.", "By application of physical-chemical principles to influence the equilibrium, or by manipulating the concentrations or the nature of the reactants and products, or by exploiting the kinetic parameters of the enzyme reaction it is possible to apply proteases for the purpose of synthesis of peptide bonds.", "The addition of water miscible organic solvents decreases the extent of ionisation of the carboxyl component, thereby increasing the concentration of substrate available for the reaction.", "Biphasic systems, water mimetics, reverse micelles, anhydrous media, or modified amino and carboxyl groups to invoke precipitation of products are often employed to improve yields.", "When the proteases with the right properties are available the application of proteases for synthesis offers substantial advantages.", "As proteases are stereoselective as well as regio-selective, sensitive groups on the reactants do usually not need protection and reactants do not need to be optically pure.", "As conditions of enzymatic synthesis are mild, racemization and decomposition of labile reactants or products can be prevented.", "Apart from bonds between amino acids, also other compounds exhibiting a primary amino group, a thiol group or a carboxyl group may be linked by properly selected proteases.", "In addition esters, thiol esters and amides may be synthesized by certain proteases.", "Protease have been shown to exhibit regioselectively in the acylation of mono, di- and tri-saccharides, nucleosides, and riboflavin.", "Problems with stability under the sometimes harsh reaction conditions may be prevented by proper formulation.", "Encapsulation and immobilisation do not only stabilise enzymes but also allow easy recovery and separation from the reaction medium.", "Extensive crosslinking, treatment with aldehydes or covering the surface with certain polymers such as dextrans, polyethyleneglycol, polyimines may substantially extend the lifetime of the biocatalyst.", "The Natural Roles of Proteases Traditionally, proteases have been regarded as degrading enzymes, capable of cleaving proteins into small peptides and/or amino acids, and whose role it is to digest nutrient protein or to participate in the turnover of cellular proteins.", "In addition, it has been shown that proteases also play key roles in a wide range of cellular processes, via mechanisms of selective modification by limited proteolysis, and thus can have essential regulatory functions (Holzer and Tschensche 1979; Holzer and Heinrich, 1980).", "The specificity of a proteinase is assumed to be closely related to its physiological function and its mode of expression.", "With respect to the function of a particular protease, its localisation is often very important; for example, a lot of the vacuolar and periplasmic proteases are involved in protein degradation, while many of the membrane-bound proteases are important in protein processing (Suarez Rendueles and Wolf, 1988).", "The different roles of proteases in many cellular processes can be divided into four main functions of proteases: 1) protein degradation, 2) posttranslational processing and (in)activation of specific proteins, 3) morphogenesis, and 4) pathogenesis.", "An obvious role for proteases in organisms which utilise protein as a nutrient source is in the hydrolysis of nutrients.", "In fungi, this would involve the degradation outside the cells by extracellular broad specificity proteases.", "Protein degradation is also important for rapid turnover of cellular proteins and allows the cell to remove abnormal proteins and to adapt their complement of protein to changing physiological conditions.", "Generally, proteases of rather broad specificity should be extremely well-controlled in order to protect the cell from random degradation of other than correct target proteins.", "Contrary to the hydrolysis the synthesis of polypeptides occurs in vivo by an ATP driven process on the ribosome.", "Ultimately the sequence in which the amino acids are linked is dictated by the information derived from the genome.", "This process is known as the transcription.", "Primary translation products are often longer than the final functional products, and after the transcription usually further processing of such precursor proteins by proteases is required.", "Proteases play a key role in the maturation of such precursor proteins to obtain the final functional protein.", "In contrast to the very controlled trimming and reshaping of proteins, proteases can also be very destructive and may completely degrade polypeptides into peptides and amino acids.", "In order to avoid that proteolytic activity is unleashed before it is required, proteases are subject to extensive regulation.", "Many proteases are synthesized as larger precursors known as zymogens, which become activated when required.", "Remarkably this activation always occurs by proteolysis.", "Apart from direct involvement in the processing, selective activation and inactivation of individual proteins are well-known phenomena catalyzed by specific proteases.", "The selectivety of limited proteolysis appears to reside more directly in the proteinase-substrate interaction.", "Specificity may be derived from the proteolytic enzyme which recognizes only specific amino acid target sequences.", "On the other hand, it may also be the result of selective exposure of the ‘processing site’ under certain conditions such as pH, ionic strength or secondary modifications, thus allowing an otherwise non-specific protease to catalyze a highly specific event.", "The activation of vacuolar zymogens by limited proteolysis gives an example of the latter kind.", "Morphogenesis or differentiation can be defined as a regulated series of events leading to changes from one state to another in an organism.", "Although direct relationships between proteases and morphological effects could not be established in many cases, the present evidence suggests a significant involvement of proteases in fungal morphogenesis; apart form the observed extensive protein turnover during differentiation, sporulation and spore germination, proteases are thought to be directly involved in normal processes as hyphal tip branching and septum formation, (Deshpande, 1992).", "Species of Aspergillus , in particular A. fumigatus and A. flavus , have been implicated as the causative agents of a number of diseases in humans and animals called aspergillosis (Bodey and Vartivarian, 1989).", "It has been repeatedly suggested that proteases are involved in virulence of A. fumigatus and A. flavus like there are many studies linking secreted proteases and virulence of bacteria.", "In fact, most human infections due to Aspergillus species are characterised by an extensive degradation of the parenchyma of the lung which is mainly composed of collagen and elastin (Campbell et al., 1994).", "Research has been focussed on the putative role of the secreted proteases in virulence of A. fumigatus and A. flavus which are the main human pathogens and are known to possess elastinolytic and collagenic activities (Kolattukudy et al., 1993).", "These elastinolytic activities were shown to correlate in vitro with infectivity in mice (Kothary et al., 1984).", "Two secreted proteases are known to be produced by A. fumigatus and A. flavus , an alkaline serine protease (ALP) and a neutral metallo protease (MEP).", "In A. fumigatus both the genes encoding these proteases were isolated, characterised and disrupted (Reicherd et al., 1990; Tang et al, 1992, 1993; Jaton-Ogay et al., 1994).", "However, alp mep double mutants showed no differences in pathogenecity when compared with wild type strains.", "Therefore, it must be concluded that the secreted A. fumigatus proteases identified in vitro are not essential factors for the invasion of tissue (Jaton Ogay et al., 1994).", "Although A. fumigatus accounts for only a small proportion of the airborne mould spores, it is the most frequently isolated fungus from lung and sputem (Schmitt et al., 1991).", "Other explanations for the virulence of the fungus could be that the conditions in the bronchia (temperature and nutrients) are favourable for the parasitic growth of A. fumigatus .", "As a consequence, invasive apergillosis could be a circumstancial event, when the host pathogenic defences have been weakened by immunosuppressive treatments or diseases like AIDS.", "Four major classes of proteases are known and are designated by the principal functional groups in their active site: the ‘serine’, the ‘thiol’ or ‘cysteine’, the ‘aspartic’ or ‘carboxyl’ and the ‘metallo’ proteases.", "A detailed state of the art review on these major classes of proteases, minor classes and unclassified proteases can be found in Methods in Enzymology part 244 and 248 (A. J. Barrett ed, 1994 and 1995).", "Specificity of Proteases Apart from the catalytic machinery of proteases another important aspect of proteolytic enzymes is the specificity of proteases.", "The specificity of a protease indicates which substrates the protease is likely to hydrolyze.", "The twenty natural amino acids offer a large number of possibilities to make up peptides.", "Eg with twenty amino acids one can make up already 400 dipeptides and 800 different tripeptide, and so on.", "With longer peptides the number of possibilities will become almost unlimited.", "Certain proteases hydrolyze only particular sequences at a very specific position.", "The interaction of the protease with the peptide substrate may encompass one up to ten amino acid residues of the peptide substrate.", "With large proteinacious substrates there may be even more residues of the substrate that interact with the proteases.", "However this likely involves less specific interactions with protease residues outside the active site binding cleft.", "In general the specific recognition is restricted to the linear peptide, which is bound in the active site of the protease.", "The nomenclature to describe the interaction of a substrate with a protease has been introduced in 1967 by Schechter and Berger (Biochem.", "Biophys.", "Res.", "Corn., 1967, 27, 157-162) and is now widely used in the literature.", "In this system, it is considered that the amino acid residues of the polypeptide substrate bind to so-called sub-sites in the active site.", "By convention, these sub-sites on the protease are called S (for sub-sites) and the corresponding amino acid residues are called P (for peptide).", "The amino acid residues of the N-terminal side of the scissile bond are numbered P3, P2, P1 and those residues of the C-terminal side are numbered P1′, P2′, P3′.", "The P1 or P1′ residues are the amino acid residues located near the scissile bond.", "The substrate residues around the cleavage site can then be numbered up to P8.The corresponding sub-sites on the protease that complement the substrate binding residues are numbered S3, S2, S1, S1′, S2′, S3′, etc, etc.", "The preferences of the sub-sites in the peptide binding site determine the preference of the protease for cleaving certain specific amino acid sequences at a particular spot.", "The amino acid sequence of the substrate should conform with the preferences exhibited by the sub-sites.", "The specificity towards a certain substrate is clearly dependant both on the binding affinity for the substrate and on the velocity at which subsequently the scissile bond is hydrolysed.", "Therefore the specificity of a protease for a certain substrate is usually indicated by its kcat/Km ratio, better known as the specificity constant.", "In this specificity constant kcat represents the turn-over rate and Km is the dissociation constant.", "Apart from amino acid residues involved in catalysis and binding, proteases contain many other essential amino acid residues.", "Some residues are critical in folding, some residues maintain the overall three dimensional architecture of the protease, some residues may be involved in regulation of the proteolytic activity and some residue may target the protease for a particular location.", "Many proteases contain outside the active site one or more binding sites for metal ions.", "These metal ions often play a role in stabilizing the structure.", "In addition secreted eukaryotic microbial proteases may be extensively glycosylated.", "Both N- and O-linked glycosylation occurs.", "Glycosylation may aid protein folding, may increase solubility, prevent aggregation and as such stabilize the mature protein.", "In addition the extent of glycosylation may influence secretion as well as water binding by the protein.", "Regulation of Proteolytic Activity A substantial number of proteases are subject to extensive regulation of the proteolytic activity in order to avoid undesired proteolytic damage.", "To a certain extent this regulation takes place at transcription level.", "For example in fungi the transcription of secreted protease genes appears to be sensitive to external carbon and nitrogen sources, whereas genes encoding intracellular proteases are insensitive.", "The extracellular pH is sensed by fungi and some genes are regulated by pH.", "In this process transcriptional regulator proteins play a crucial role.", "Proteolytic processing of such regulator proteins is often the switch that turns the regulator proteins either on or off.", "Proteases are subject to intra- as well as intermolecular regulation.", "This implies certain amino acids in the proteolytic enzyme molecule that are essential for such regulation.", "Proteases are typically synthesized as larger precursors known as zymogens, which are catalytically inactive.", "Usually the peptide chain extension rendering the precursor protease inactive is located at the amino terminus of the protease.", "The precursor is better known as pro-protein.", "As many of the proteases processed in this way are secreted from the cells they contain in addition a signal sequence (pre sequence) so that the complete precursor is synthesized as a pre-pro-protein.", "Apart from rendering the protease inactive the pro-peptide often is essential for mediating productive folding.", "Examples of proteases include serine proteases (alpha lytic protease, subtilisin, aqualysin, prohormone convertase), thiol proteases (cathepsin L and cruzian), aspartic proteases (proteinase A and cathepsin D) and metalloproteases.", "In addition the pro-peptide might play a role in cellular transport either alone or in conjunction with signal peptides.", "It may facilitate interaction with cellular chaperones or it may facilitate transport over the membrane.", "The size of the extension in the precursor pre-pro-protein may vary substantially, ranging from a short peptide fragment to a polypeptide, which can exist as an autonomous folding unit.", "In particular these larger extensions are often observed to be strong inhibitors of the protease even after cleavage from the protease.", "It was observed that even after cleavage such pro-peptides could assist in proper folding of the proteases.", "As such pro-peptides can be considered to function as molecular chaperones and separate or additional co-expression of such pro-peptides could be advantageous for protease production.", "There is substantial difference in the level of regulation between proteases that are secreted into the medium and proteases that remain intracellular.", "Proteases secreted into the medium are usually after activation no longer subject to control and therefore are usually relatively simple in their molecular architecture consisting of one globular module.", "Intracellular proteases are necessarily subject to continuous control in order to avoid damage to the cells.", "In contrast with zymogens of secreted proteases in more complex regulatory proteases very large polypeptide segments may be inserted between the signal and the zymogen activation domain of the proteolytic module.", "Structure-function studies indicate that such non-protease parts may be involved in interactions with macroscopic structures, membranes, cofactors, substrates, effectors, inhibitors, ions, that regulate activity and activation of the proteolytic module(s) or its (their) zymogens.", "The non-proteolytic modules exhibit remarkable variation in size and structure.", "Many of the modules can exist as such independently from the proteolytic module.", "Therefore such modules can be considered to correspond to independent structural and functional units that are autonomous with respect to folding.", "The value of such a modular organization is that acquisition of new modules can endow the recipient protease with new novel binding specificities and can lead to dramatic changes in its activity, regulation and targeting.", "The principle of modular organized proteolytic enzymes may also be exploited by applying molecular biology tools in order to create novel interactions, regulation, specificity, and/or targeting by shuffling of modules.", "Although in general such additional modules are observed as N or C terminal extension, also large insertions within the exterior loops of the catalytic domain have been observed.", "It is believed that also in this case the principal fold of the protease represents still the essential topology to form a functional proteolytic entity and that the insertion can be regarded as substructure folded onto the surface of the proteolytic module.", "Molecular Structure In principle the modular organization of larger proteins is a general theme in nature.", "In particular within the larger multimodular frameworks typical proteolytic modules show sizes of 100 to 400 amino acids on the average.", "This corresponds with the average size of most of the globular proteolytic enzymes that are secreted into the medium.", "As discussed above polypeptide modules are polypeptide fragments, which can fold and function as independent entities.", "Another term for such modules is domains.", "However domain is used in a broader context than module.", "The term domain as used herein refers usually to a part of the polypeptide chain that depicts in the three-dimensional structure a typical folding topology.", "In a protein domains interact to varying extents, but less extensively than do the structural elements within domains.", "Other terms such as subdomain and folding unit are also used in literature.", "As such it is observed that many proteins that share a particular functionality may share the same domains.", "Such domains can be recognized from the primary structure that may show certain sequence patterns, which are typical for a particular domain.", "Typical examples are the mononucleotide binding fold, cellulose binding domains, helix-turn-helix DNA binding motif, zinc fingers, EF hands, membrane anchors.", "Modules refer to those domains which are expected to be able to fold and function autonomously.", "A person skilled in the art knows how to identify particular domains in a primary structure by applying commonly available computer software to said structure and homologous sequences from other organisms or species.", "Although multimodular or multidomain proteins may appear as a string of beads, assemblies of substantial more complex architecture have been observed.", "In case the various beads reside on the same polypeptide chain the beads are generally called modules or domains.", "When the beads do not reside on one and same polypeptide chain but form assemblies via non-covalent interactions then the term subunit is used to designate the bead.", "Subunits may be transcribed by one and the same gene or by different genes.", "The multi-modular protein may become proteolytically processed after transcription leading to multiple subunits.", "Individual subunits may consist of multiple domains.", "Typically the smaller globular proteins of 100-300 amino acids usually consist only of one domain.", "Molecular Classification of Proteolytic Enzymes In general proteases are classified according to their molecular properties or according to their functional properties.", "The molecular classification is based on the primary structure of the protease.", "The primary structure of a protein represents its amino acid sequence, which can be derived from the nucleotide sequence of the corresponding gene.", "Tracing extensively the similarities in the primary structures may allow for the notice of similarities in catalytic mechanism and other properties, which even may extend to functional properties.", "The term family is used to describe a group of proteases that show evolutionary relationship based on similarity between their primary structures.", "The members of such a family are believed to have arisen by divergent evolution from the same ancestor.", "Within a family further sub-grouping of the primary structures based on more detailed refinement of sequence comparisons results in subfamilies.", "Classification according to three-dimensional fold of the proteases may comprise secondary structure, tertiary structure and quarternary structure.", "In general the classification on secondary structure is limited to content and gross orientation of secondary structure elements.", "Similarities in tertiary structure have led to the recognition of superfamilies or clans.", "A superfamily or a clan is a group of families that are thought to have common ancestry as they show a common 3-dimensional fold.", "In general tertiary structure is more conserved than the primary structure.", "As a consequence similarity of the primary structure does not always reflect similar functional properties.", "In fact functional properties may have diverged substantially resulting in interesting new properties.", "At present quarternary structure has not been applied to classify various proteases.", "This might be due to a certain bias of the structural databases towards simple globular proteases.", "Many proteolytic systems that are subject to activation, regulation, or complex reaction cascades are likely to consist of multiple domains or subunits.", "General themes in the structural organization of such protease systems may lead to new types of classification.", "Classification According to Specificity.", "In absence of sequence information proteases haven been subject to various type of functional classification.", "The classification and naming of enzymes by reference to the reactions which are catalyzed is a general principle in enzyme nomenclature.", "This approach is also the underlying principle of the EC numbering of enzymes ( Enzyme Nomenclature 1992 Academic Press, Orlando).", "Two types of proteases (EC 3.4) can be recognized within Enzyme Nomenclature 1992, those of the exo-peptidases (EC 3.4.11-19) and those of the endo-peptidases (EC 3.4.21-24, 3.4.99).", "Endo-peptidases cleave peptide bonds in the inner regions of the peptide chain, away from the termini.", "Exo-peptidases cleave only residues from the ends of the peptide chain.", "The exo-peptidases acting at the free N-terminus may liberate a single amino acid residue, a dipeptide or a tripeptide and are called respectively amino peptidases (EC 3.4.11), dipeptidyl peptidases (EC 3.4.14) and tripeptidyl peptidase (EC 3.3.14).", "Proteases starting peptide processing from the carboxyl terminus liberating a single amino acid are called carboxy peptidase (EC 3.4.16-18).", "Peptidyl-dipeptidases (EC 3.4.15) remove a dipeptide from the carboxyl terminus.", "Exo- and endo-peptidase in one are the dipeptidases (EC 3.4.13), which cleave specifically only dipeptides in their two amino acid halves.", "Omega peptidases (EC 3.4.19) remove terminal residues that are either substituted, cyclic, or linked by isopeptide bonds Apart from the position where the protease cleaves a peptide chain, for each type of protease a further division is possible based on the nature of the preferred amino acid residues in the substrate.", "In general one can distinguish proteases with broad, medium and narrow specificity.", "Some proteases are simply named after the specific proteins or polypeptides that they hydrolyze, e.g.", "keratinase, collagenase, elastase.", "A narrow specificity may pin down to one particular amino acid or one particular sequence which is removed or which is cleaved respectively.", "When the protease shows a particular preference for one aminoacid in the P1 or P1′ position the name of this amino acid may be a qualifier.", "For example prolyl amino peptidase removes proline from the amino terminus of a peptide (proline is the P1 residue).", "X-Pro or proline is used when the bond on the imino side of the proline is cleaved (proline is P1′ residue), eg proline carboxypeptidase removes proline from the carboxyl terminus.", "Prolyl endopeptidase (or Pro-X) cleaves behind proline while proline endopeptidase (X-Pro) cleaves in front of a proline.", "Amino acid residue in front of the scissile peptide bond refers to the amino acid residue that contributes the carboxyl group to the peptide bond.", "The amino acids residue behind the scissile peptide bond refers to the amino acid residue that contributes the amino group to the peptide bond.", "According to the general convention an amino acid chain runs from amino terminus (the start) to the carboxyl terminus (the end) and is numbered accordingly.", "Endo proteases may also show clear preference for a particular amino acid in the P1 or P1′ position, eg glycyl endopeptidase, peptidyl-lysine endopeptidase, glutamyl endopeptidase.", "In addition proteases may show a preference for a certain group of amino acids that share a certain resemblance.", "Such a group of preferred amino acids may comprise the hydrophobic amino acids, only the bulky hydrophobic amino acids, small hydrophobic, or just small amino acids, large positively charged amino acids, etc, etc.", "Apart from preferences for P1 and P1′ residues also particular preferences or exclusions may exist for residues preferred by other subsites on the protease.", "Such multiple preferences can result in proteases that are very specific for only those sequences that satisfy multiple binding requirements at the same time.", "In general it should be realized that protease are rather promiscuous enzymes.", "Even very specific protease may cleave peptides that do not comply with the generally observed preference of the protease.", "In addition it should be realized that environmental conditions such as pH, temperature, ionic strength, water activity, presence of solvents, presence of competing substrates or inhibitors may influence the preferences of the proteases.", "Environmental condition may not only influence the protease but also influence the way the proteinacious substrate is presented to the protease.", "Classification by Catalytic Mechanism.", "Proteases can be subdivided on the basis of their catalytic mechanism.", "It should be understood that for each catalytic mechanism the above classification based on specificity leads to further subdivision for each type of mechanism.", "Four major classes of proteases are known and are designated by the principal functional group in the active site: the serine proteases (EC 3.4.21 endo peptidase, EC 3.4.16 carboxy peptidase), the thiol or cysteine proteases (EC 3.4.22 endo peptidase, EC 3.4.18 carboxy peptidase), the carboxyl or aspartic proteases (EC 3.4.23 endo peptidase) and metallo proteases (EC 3.4.24 endo peptidase, EC 3.4.18 carboxy peptidase).", "There are characteristic inhibitors of the members of each catalytic type of protease.", "These small inhibitors irreversibly modify an amino acid residue of the protease active site.", "For example, the serine protease are inactivated by Phenyl Methane Sulfonyl Fluoride (PMSF) and Diisopropyl Fluoro Phosphate (DFP), which react with the active Serine whereas the chloromethylketone derivatives react with the Histidine of the catalytic triad.", "Phosphoramidon and 1,10 Phenanthroline typically inhibit metallo proteases.", "Inhibition by Pepstatin generally indicates an aspartic protease.", "E64 inhibits thiol protease specifically.", "Amastatin and Bestatin inhibit various aminopeptidases.", "Substantial variations in susceptibility of the proteases to the inhibitors are observed, even within one catalytic class.", "To a certain extent this might be related to the specificity of the protease.", "In case binding site architecture prevents a mechanism based inhibitor to approach the catalytic site, then such a protease escapes from inhibition and identification of the type of mechanism based on inhibition is prohibited.", "Chymostation for example is a potent inhibitor for serine protease with chymotrypsin like specificity, Elastatinal inhibits elastase like serine proteases and does not react with trypsin or chymostrypsin, 4 amido PMSF (APMSF) inhibits only serine proteases with trypsin like specificity.", "Extensive accounts of the use of inhibitors in the classification of proteases include Barret and Salvesen, Proteinase Inhibitors , Elsevier Amstardam, 1986; Bond and Beynon (eds), Proteolytic Enzymes, A Practical Approach , IRL Press, Oxford, 1989; Methods in Enzymology, eds E. J. Barret, volume 244, 1994 and volume 248, 1995; E. Shaw, Cysteinyl proteinases and their selective inactivation , Adv Enzymol.", "63:271-347 (1990) Classification According to Optimal Performance Conditions.", "The catalytic mechanism of a proteases and the requirement for its conformational integrity determine mainly the conditions under which the protease can be utilized.", "Finding the protease that performs optimal under application conditions is a major challenge.", "Often conditions at which proteases have to perform are not optimal and do represent a compromise between the ideal conditions for a particular application and the conditions which would suit the protease best.", "Apart from the particular properties of the protease it should be realized that also the presentation of a proteinacious substrates is dependant on the conditions, and as such determines also which conditions are most effective for proteolysis.", "Specifications for the enzyme that are relevant for application comprise for example the pH dependence, the temperature dependence, sensitivity for or the dependence of metal ions, ionic strength, salt concentration, solvent compatibility.", "Another factor of major importance is the specific activity of a protease.", "The higher the enzyme's specific activity, the less enzyme is needed for a specific conversion.", "Lower enzyme requirements imply lower costs and lower protein contamination levels.", "The pH is a major parameter that determines protease performance in an application.", "Therefor pH dependence is an important parameter to group proteases.", "The major groups that are recognized are the acid proteases, the neutral proteases, the alkaline proteases and the high alkaline proteases.", "The optimum pH matches only to some extent the proteolytic mechanism, eg aspartic protease show often an optimum at acidic pH, metalloproteases and thiol proteases often perform optimal around neutral pH to slightly alkaline, serine peptidases are mainly active in the alkaline and high alkaline region.", "For each class exceptions are known.", "In addition the overall water activity of the system plays a role.", "The pH optimum of a protease is defined as the pH range where the protease exhibits an optimal hydrolysis rate for the majority of its substrates in a particular environment under particular conditions.", "This range can be narrow, e.g.", "one pH unit, as well as quite broad, 3-4 pH units.", "In general the pH optimum is also dependant on the nature of the proteinacious substrate.", "Both the turnover rate as well as the specificity may vary as a function of pH.", "For a certain efficacy it can be desirable to use the protease far from its pH optimum because production of less desired peptides is avoided.", "Less desired peptides might be for example very short peptides or peptides causing a bitter taste.", "In addition a more narrow specificity can be a reason to choose conditions that deviate from optimal conditions with respect to turnover rate.", "Dependant on the pH the specificity may be narrow, e.g.", "only cleaving the peptide chain in one particular position or before or after one particular amino acid, or broader, e.g.", "cleaving a chain at multiple positions or cleaving before or after more different types of amino acids.", "In fact the pH dependence might be an important tool to regulate the proteolytic activity in an application.", "In case the pH shifts during the process the proteolysis might cease spontaneously without the need for further treatment to inactivate the protease.", "In some cases the proteolysis itself may be the driver of the pH shift.", "Very crucial for application of proteases is their handling and operating stability.", "As protease stability is strongly affected by the working temperature, stability is often also referred to as thermostability.", "In general the stability of a protease indicates how long a protease retains its proteolytic activity under particular conditions.", "Particular conditions may comprise fermentation conditions, conditions during isolation and down stream processing of the enzyme, storage conditions, formulation and operating or application conditions.", "In case particular conditions encompass elevated temperatures stability in general refers to thermostability.", "Apart from the general causes for enzyme inactivation such as chemical modification, unfolding, aggregation etc, main problem with proteases is that they are easy subject to autodegradation.", "Especially for the utilization of proteases the temperature optimum is a relevant criterion to group proteases.", "Although there are different definitions, economically the most useful definition is the temperature or the temperature range in which the protease is most productive in a certain application.", "Protease productivity is a function of both the stability and the turnover rate.", "Where elevated temperature in general will increase the turnover rate, rapid inactivation will counteract the increase in turnover rate and ultimately lead to low productivity.", "The conformational stability of the protease under a given process condition will determine its maximum operating temperature.", "The temperature at which the protease looses it active conformation, often indicated as unfolding or melting point, can be determined according various methods, for example NMR, Circular Dichroism Spectroscopy, Differential Scanning Calorimetry etc etc.", "For protease unfolding is usually accompanied by a tremendous increase in autodegradation rate.", "In applications where low temperatures are required protease may be selected with emphasis on a high intrinsic activity at low to moderate temperature.", "As under such conditions inactivation is relatively slow, under these conditions activity might largely determine productivity.", "In processes where only during a short period protease activity is required, the stability of the protease might be used as a switch to turn the protease off.", "In such case more labile instead of very thermostable protease might be preferred.", "Other environmental parameters which may play a role in selecting the appropriate protease may be its sensitivity to salts.", "The compatibility with metal ions which are found frequently at low concentrations in various natural materials can be crucial for certain applications.", "In particular with metallo proteases certain ions may replace the catalytic metal ion and reduce or even abolish activity completely.", "In some applications metal ions have to be added on purpose in order to prevent the washout of the metal ions coordinated to the protease.", "It is well known that for the sake of enzyme stability and life-time, calcium ions have to be supplied in order to prevent dissociation of protein bound calcium.", "Most microorganisms show a certain tolerance with respect to adapting to changes in the environmental condition.", "As a consequence at least the proteolytic spectrum that the organism is able to produce are likely to show at least similar tolerances.", "Such a proteolyitic spectrum might be covered by many proteases covering together the hole spectrum or by only a few proteases of a broad spectrum.", "Taking into account the whole proteolytic spectrum of a microorganism it can be very important to take the location into account.", "Cellular Localisation and Characterization of Proteolytic Processing and Degradation From an industrial point of view the proteases which are excreted from the cell have specific advantages with respect to producibility at a large scale and stress tolerance as they have to survive without protection of the cell.", "The large group of cellular protease can be further subdivided in soluble and membrane bound.", "Membrane bound may comprise protease at the inside as well the outside of the membrane.", "Intracellular soluble protease may be subdivided further according to specific compartments of the cell where they do occur.", "As the cell shields the proteases to some extent from the environment and because the cell controls the conditions in the cell, intracellular protease might be more sensitive to large environmental changes and their optima might correlate better with the specific intacellualr conditions.", "Knowing the conditions of the cellular department where the protease resides might indicate their preferences.", "Where extracellular protease in general do not require any regulation any more once excreted from the cell, intracellular proteases are often subject to more complicated control and regulation.", "With respect to the function of a particular protease, its localisation is often very important; for example, a lot of the vacuolar and periplasmic proteases are involved in protein degradation, while many of the membrane-bound proteases are important in protein processing (Suarez Rendueles and Wolf, 1988).", "A comprehensive review on the biological properties and evolution of proteases has been published in van den Hombergh: Thesis Landbouwuniversiteit Wageningen: An analysis of the proteolytic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2, which is hereby incorporated by reference herein.", "The Protease Problem An important reason for the interest in microbial proteases are protease related expression problems observed in several expression hosts used in bioprocess industry.", "The increasing use of heterologous hosts for the production of proteins, by recombinant DNA technology, has recently brought this problem into focus, since it seems that heterologous proteins are more prone to proteolysis (Archer et al., 1992; van den Hombergh et al., 1996b).", "In S. cerevisiae , already in the early eighties the protease problem and the involvement of several proteases, thus complicating targetted gene disruption approaches to overcome this problem, was recognised.", "During secretion a protein is exposed to several proteolytic activities residing in the secretory pathway.", "Additionally, in a prototrophic microorganism as Aspergillus secreted proteins can be exposed to several extracellular proteolytic activities The problem of degradation of heterologously expressed proteins is well documented in Aspergillus (van den Hombergh Thesis Landbouwuniversiteit Wageningen: An analysis of the proteolytic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2) and has been reported in the expression of cow prochymosin, human interferon α-2 tPA, GMCSF, IL6, lactoferrin, chicken egg-white lysosyme, porcine pIA2, A. niger pectin lyase B, E. coli enterotoxin B and β-glucoronidase, and Erwinia carotovora pectate lyase 3.The problem of proteolysis may be addressed at several stages in protein production.", "Bioprocess engineers may address the problem of proteolysis by downstream processing at low temperatures, by early separation of product and protease(s) or by use of protease inhibitors.", "These may all lead to successful reduction of the problem.", "However it is certainly not eliminated, because much of the degradation occurs in vivo during the production of the protein.", "In understanding how proteolysis is controlled in the cell, a major question concerns the recognition mechanism by which proteolysis is triggered.", "Into what extent are proteolytically susceptable (heterologous) proteins recognised as aberrant because of misfolding or, if correctly folded, as ‘foreign’, because they do not posses features essential for stability which are specific to the host.", "Various types of stress can cause the overall proteolysis in a cell to increase significantly.", "Factors known to increase rate of proteolysis include nutrient starvation and various other types of stress (i.e.", "elevation of temperature, osmotic stress, toxic substances and expression of certain heterologous proteins).", "To deal with proteolysis-related expression problems in vivo, several approaches have been proven succesfull as will be discussed below.", "However, we have to keep in mind that true ‘non-proteolytic cells’ cannot exist, since proteolysis by intracellular proteases is involved in many essential metabolic and ‘housekeeping’ reactions.", "Reducing proteolysis will therefore always be a process in which the changed genetical background which results in decreased proteolytic has to be analysed for potential secundary effects which could lead to reduced protein production (e.g.", "reduced growth rate or sporulation).", "Disruption of Proteases in Filamentous Fungal Expression Hosts Berka and coworkers (1990) describe the cloning and disruption of the A. awamori pepA gene.", "More recently, three disrupted aspartyl proteases in A. niger have been described.", "Disruptants for both the major extracellular aspartyl proteases and the major vacuolar aspartyl protease were described.", "Double and triple disruptants were generated via recombination and tested for protease spectra and expression and secretion of the A. niger pectin lyase PELB protein, which is very susceptable to proteolytic degradation (van den Hombergh et al., 1995).", "Disruption of pepA and pepB resulted both in reduction of extracellular protease activities, 80% and 6%, respectively.", "In the ΔpepE disruptant also other (vacuolar) protease activities were severely affected caused by inactivating of the proteolytic cascade for other vacuolar proteases.", "Reduced extracellular activities correlated with reduced in vitro degradation of PELB and improved in vivo expression of pelB (van den Hombergh et al., 1996f).", "Protease Deficient (prt) Mutants Filamentous Fungi Several Aspergillus protease deficient mutants have been studied whether protein production is improved.", "Archer and coworkers describe the reduced proteolysis of Hen egg white lysozyme in supernatants of an A. niger double prt mutant generated by Mattern and coworkers (1992) and conclude that although the degradation is not absent, it is significantly reduced.", "Van den Hombergh et al.", "(1995) show that the in vitro degradation of A. niger PELB is reduced in all seven prt complementation groups they have isolated.", "Virtually no degradation is observed in the prtB, prtF and prtG mutants.", "Recently, the expression of the pelB gene was shown to be improved in six complementation groups tested (prtA-F) and highest expression levels were observed in the prtB, prtF and prtG mutants.", "In addition to the single mutants, which contained residual extracellular proteolytic activities varying from 2-80% compared to wild type activity, double mutants were generated both by recombination and by additional rounds of mutagenesis.", "Via this approach several double prt mutants were selected and further characterised, which showed a further reduction of PELB degradation compared to their parental strains.", "Instead of elimination of protease activities via disruption or mutagenesis, reduced proteolysis can also be achieved via down-regulation of the interfering proteolytic activities.", "This may be achieved by genetically altering the promoter or other regulatory sequences of the gene.", "As shown by Fraissinet-Tachet and coworkers (1996) the extracellular proteases in A. niger are all regulated by carbon catabolite repression and nitrogen metabolite repression.", "Nutrient starvation also causes the overall proteolysis rate in a cell to increase stromgly, which makes sense for a cell that lacks nutrients but posses proteins, that under starvation conditions are not needed or needed only in smaller amounts.", "In expression strategies which allow high expression on media containing high glucose and ammonium concentrations reduced proteolysis has been reported.", "Several constitutive glycolytic promoters (gpd and pkiA) are highly expressed under these conditions and can also be used to drive (heterologous) gene expression in continuous fermentations.", "The type of nutrient starvation imposed can influence different proteases to varying extent, which means that the importance of nutrient conditions in a given process depend on the type of proteolysis that is involved.", "Specific proteolysis may therefore be induced by conditions of substrate limitation which are frequently used in many large-scale fermentation processes.", "The protease problem can nowadays be addressed in part by one or more of the above strategies.", "However, the residual proteolytic activity of yet unidentified proteolytic enzymes still constitutes a major problem in the art.", "In order to further reduce the level of unwanted proteolysis, there is a great need in the art to identify novel proteases responsible for degradation of homologously and heterologously expressed proteins.", "This invention provides such novel protease gene sequences encoding novel proteases.", "Once the primary sequence of a novel protease gene is known, one or more of the above recombinant DNA strategies may be employed to produce (knock-out) mutants with reduced proteolytic activity.", "Despite the widespread applications of proteases in a great number of industrial processes, current enzymes also have significant shortcomings with respect to at least one of the following properties.", "When added to animal feed, current proteases are not sufficiently resistant to digestive enzymes present in the gastrointestinal (GI) tract of e.g.", "pigs and poultry.", "With respect to another aspect, the currently available enzymes are not sufficiently resistant to specific (high) temperatures and (high) pressure conditions that are applied during extrusion or pelleting operations.", "Also, the current enzymes are not sufficiently active in a pH range of 3-7, conditions prevailing in many food, beverage products as well as in in the GI tract of most animals.", "According to yet another aspect the specificity of the currently available proteases is very limited which results in the inability of the existing enzymes to degrade or to dissolve certain “protease resistant” proteins thus resulting in low peptide or amino acid yields.", "Moreover proteases with new specificities allow the synthesis of new peptides.", "Yet another drawback of the currently available enzymes is their low specific activity.", "It is therefore clear that for a large number of applications a strong desire exists for proteases that are more resistant to digestive enzymes, high temperature and/or pressure and which exhibit novel specificities regarding their sites of hydrolysis.", "The present invention provides such enzymes." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides for novel polynucleotides encoding novel proteases.", "More in particular, the invention provides for polynucleotides having a nucleotide sequence that hybridises (preferably under highly stringent conditions) to a sequence according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or to a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114.Consequently, the invention provides nucleic acids that are about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114.In a more preferred embodiment the invention provides for such an isolated polynucleotide obtainable from a filamentous fungus, preferably Aspergilli, in particular A. niger is preferred.", "In one embodiment, the invention provides for an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof.", "In a further preferred embodiment, the invention provides an isolated polynucleotide encoding at least one functional domain of a polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof.", "In a preferred embodiment the invention provides a protease gene according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57.In another aspect the invention provides a polynucleotide, preferably a cDNA encoding an A. niger protease selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or variants or fragments of that polypeptide.", "In a preferred embodiment the cDNA has a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 or functional equivalents thereof.", "A genomic clone encoding a polypeptide according to the invention may also be obtained by selecting suitable probes to specifically amplify a genomic region corresponding to any of the sequences according to SEQ ID NO: 1 to SEQ ID NO: 57 or fragments thereof, hybridising that probe under suitable conditions to genomic DNA obtained from a suitable organism, such as Aspergillus , e.g.", "A. niger , amplifying the desired fragment e.g.", "by PCR (polymerase chain reaction) followed by purifying and cloning of the amplified fragment.", "In an even further preferred embodiment, the invention provides for a polynucleotide comprising the coding sequence of the genomic polynucleotides according to the invention, preferred is a polynucleotide sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114.In another preferred embodiment, the invention provides a cDNA obtainable by cloning and expressing a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 into a suitable host organism, such as A. niger.", "A polypeptide according to the invention may also be obtained by cloning and expressing a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 into a suitable host organism, such as A. niger.", "The invention also relates to vectors comprising a polynucleotide sequence according to the invention and primers, probes and fragments that may be used to amplify or detect the DNA according to the invention.", "In a further preferred embodiment, a vector is provided wherein the polynucleotide sequence according to the invention is functionally linked with regulatory sequences suitable for expression of the encoded amino acid sequence in a suitable host cell, such as A. niger or A. oryzea .", "The invention also provides methods for preparing polynucleotides and vectors according to the invention.", "The invention also relates to recombinantly produced host cells that contain heterologous or homologous polynucleotides according to the invention.", "In one embodiment, the invention provides recombinant host cells wherein the expression of a protease according to the invention is significantly reduced or wherein the activity of the protease is reduced or wherein the protease is even inactivated.", "Such recombinants are especially useful for the expression of homologous or heterologous proteins.", "In another embodiment, the invention provides recombinant host cells wherein the expression of a protease according to the invention is significantly increased or wherein the activity of the protease is increased.", "Such recombinants are especially useful for the expression of homologous or heterologous proteins where maturation is seriously hampered in case the required proteolytic cleavage becomes the rate limiting step.", "In another embodiment the invention provides for a recombinantly produced host cell that contains heterologous or homologous DNA according to the invention, preferably DNA encoding proteins bearing signal sequnences and wherein the cell is capable of producing a functional protease according to the invention, preferably a cell capable of over-expressing the protease according to the invention, for example an Aspergillus strain comprising an increased copy number of a gene or cDNA according to the invention.", "In another embodiment the invention provides for a recombinantly produced host cell that contains heterologous or homologous DNA according to the invention and wherein the cell is capable of secreting a functional protease according to the invention, preferably a cell capable of over-expressing and secreting the protease according to the invention, for example an Aspergillus strain comprising an increased copy number of a gene or cDNA according to the invention.", "In yet another aspect of the invention, a purified polypeptide is provided.", "The polypeptides according to the invention include the polypeptides encoded by the polynucleotides according to the invention.", "Especially preferred is a polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof.", "The invention also provides for antibodies reactive with a polypeptide according to the invention.", "These antibodies may be polyclonal, yet especially preferred are monoclonal antibodies.", "Such antibodies are particularly useful for purifying the polypeptides according to the invention.", "Fusion proteins comprising a polypeptide according to the invention are also within the scope of the invention.", "The invention also provides methods of making the polypeptides according to the invention.", "The invention further relates to a method for diagnosing aspergillosis either by detecting the presence of a polypeptide according to the invention or functional equivalents thereof, or by detecting the presence of a DNA according to the invention or fragments or functional equivalents thereof.", "The invention also relates to the use of the protease according to the invention in an industrial process as described herein detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The invention relates to newly identified polynucleotide sequences comprising genes that encode novel proteases isolated from Aspergillus niger.", "The invention features the full length nucleotide sequence of the novel genes, the cDNA sequences comprising the full length coding sequences of the novel proteases as well as the amino acid sequences of the full-length functional proteins and fragments and variants thereof.", "The invention also relates to methods of using these enzymes in industrial processes and methods of diagnosing fungal infections.", "Also included in the invention are cells transformed with a polynucleotide according to the invention and cells wherein a protease according to the invention is genetically modified to enhance or reduce its activity and/or level of expression.", "BACKGROUND OF THE INVENTION Proteolytic Enzymes Proteins can be regarded hetero-polymers that consist of amino acid building blocks connected by a peptide bond.", "The repetitive unit in proteins is the central alpha carbon atom with an amino group and a carboxyl group.", "Except for glycine, a so-called amino acid side chain substitutes one of the two remaining alpha carbon hydrogen atoms.", "The amino acid side chain renders the central alpha carbon asymmetric.", "In general, in proteins the L-enantiomer of the amino acid is found.", "The following terms describe the various types of polymerized amino acids.", "Peptides are short chains of amino acid residues with defined sequence.", "Although there is not really a maximum to the number of residues, the term usually indicates a chain which properties are mainly determined by its amino acid composition and which does not have a fixed three-dimensional conformation.", "The term polypeptide is usually used for the longer chains, usually of defined sequence and length and in principle of the appropriate length to fold into a three-dimensional structure.", "Protein is reserved for polypeptides that occur naturally and exhibit a defined three-dimensional structure.", "In case the proteins main function is to catalyze a chemical reaction it usually is called an enzyme.", "Proteases are the enzymes that catalyze the hydrolysis of the peptide bond in (poly)peptides and proteins.", "Under physiological conditions proteases catalyse the hydrolysis of the peptide bond.", "The International Union of Biochemistry and Molecular Biology (1984) has recommended to use the term peptidase for the subset of peptide bond hydrolases (Subclass E.C 3.4.).", "The terms protease and peptide hydrolase are synonymous with peptidase and may also be used here.", "Proteases comprise two classes of enzymes: the endo-peptidases and the exo-peptidases, which cleave peptide bonds at points within the protein and remove amino acids sequentially from either N or C-terminus respectively.", "Proteinase is used as a synonym for endo-peptidase.", "The peptide bond may occur in the context of di-, tri-, tetra-peptides, peptides, polypeptides or proteins.", "In general the amino acid composition of natural peptides and polypeptides comprises 20 different amino acids, which exhibit the L-configuration (except for glycine which does not have a chiral centre).", "However the proteolytic activity of proteases is not limited to peptides that contain only the 20 natural amino acids.", "Peptide bonds between so-called non-natural amino acids can be cleaved too, as well as peptide bonds between modified amino acids or amino acid analogues.", "Some proteases do accept D enantiomers of amino acids at certain positions.", "In general the remarkable stereoselectivity of proteases makes them very useful in the process of chemical resolution.", "Many proteases exhibit interesting side activities such as esterase activity, thiol esterase activity and (de)amidase activity.", "These side activities are usually not limited to amino acids only and might turn out to be very useful in bioconversions in the area of fine chemicals.", "There are a number of reasons why proteases of filamentous fungi, eukaryotic microorganisms, are of particular interest.", "The basic process of hydrolytic cleavage of peptide bonds in proteins appears costly and potentially detrimental to an organism if not properly controlled.", "The desired limits to proteolytic action are achieved through the specificity of proteinases, by compartmentalization of proteases and substrates within the cell, through modification of the substrates allowing recognition by the respective proteases, by regulation via zymogen activation, and the presence or absence of specific inhibitors, as well through the regulation of protease gene expression.", "In fungi, proteases are also involved in other fundamental cellular processes, including intracellular protein turnover, processing, translocation, sporulation, germination and differentiation.", "In fact, Aspergillus nidulans and Neurospora crassa have been used as model organisms for analyzing the molecular basis of a range of physiological and developmental processes.", "Their genetics enable direct access to biochemical and genetical studies, under defined nutrient and cultivation conditions.", "Furthermore, a large group of fungi pathogenic to humans, live-stock and crop, has been isolated and proteolysis has been suggested to play a role in their pathogenicity (host penetration, countering host defense mechanisms and/or nutrition during infection).", "Proteases are also frequently used in laboratory, clinical and industrial processes; both microbial and non-microbial proteases are widely used in the food industry (baking, brewing, cheese manufacturing, meat tenderizing), in tanning industry and in the manufacture of biological detergents (Aunstrup, 1980).", "The commercial interest in exploiting certain filamentous fungi, especially the Aspergilli, as hosts for the production of both homologous and heterologous proteins, has also recently renewed interests in fungal proteases (van Brunt, 1986ab).", "Proteases often cause problems in heterologous expression and homologous overexpression of proteins in fungi.", "In particular, heterologous expression is hampered by the proteolytic degradation of the expressed products by homologous proteases.", "These commercial interests have resulted in detailed studies of proteolytic spectra and construction of protease deficient strains and have improved the knowledge about protease expression and regulation in these organisms.", "Consequently there is a great need to identify and eliminate novel proteases in filamentous fungi.", "Micro-organisms such as for example fungi are particularly useful in the large scale production of proteins.", "In particular when such proteins are secreted into the medium.", "Proteolytic enzymes play a role in these production processes.", "On the one hand particular proteolytic enzymes are in general required for proper processing of the target protein and the metabolic well-being of the production host.", "On the other hand proteolytic degradation may significantly decrease the yield of secreted proteins.", "Poor folding in the secretion pathway may lead to degradation by intracellular proteases.", "This might be a particular problem with producing heterologous proteins.", "The details of the proteolytic processes, which are responsible for the degradation of the proteins that are diverted from the secretory process in fungi are not exactly known.", "In eukaryotes the degradation of cellular proteins is achieved by a proteasome and usually involves ubiquitin labelling of proteins to be degraded.", "In fungi, proteasomal and vacuolar proteases are also likely candidates for the proteolytic degradation of poorly folded secretory proteins.", "The proteolytic degradation is likely cytoplasmic, but endoplamatic reticulum resident proteases cannot be excluded.", "From the aspect of production host strain improvement the proteolytic system may be an interesting target for genetic engineering and production strain improvement.", "Additional copies of protease genes, over-expression of certain proteases, modification of transcriptional control, as well as knock out procedures for deletion of protease genes may provide a more detailed insight in the function a given protease.", "Deletion of protease encoding genes can be a valuable strategy for host strain improvement in order to improve production yield for homologous as well as heterologous proteins.", "Eukaryotic microbial proteases have been reviewed by North (1982).", "More recently, Suarez Rendueles and Wolf (1988) have reviewed the S. cerevisiae proteases and their function.", "Apart from the hydrolytic cleavage of bonds, proteases may also be applied in the formation of bonds.", "Bonds in this aspect comprise not only peptide and amide bonds but also ester bonds.", "Whether a protease catalyses the cleavage or the formation of a particular bond does in the first place depend on the thermodynamics of the reaction.", "An enzyme such as a protease does not affect the equilibrium of the reaction.", "The equilibrium is dependent on the particular conditions under which the reaction occurs.", "Under physiological conditions the thermodynamics of the reactions is in favour of the hydrolysis of the peptide due to the thermodynamically very stable structure of the zwitterionic product.", "By application of physical-chemical principles to influence the equilibrium, or by manipulating the concentrations or the nature of the reactants and products, or by exploiting the kinetic parameters of the enzyme reaction it is possible to apply proteases for the purpose of synthesis of peptide bonds.", "The addition of water miscible organic solvents decreases the extent of ionisation of the carboxyl component, thereby increasing the concentration of substrate available for the reaction.", "Biphasic systems, water mimetics, reverse micelles, anhydrous media, or modified amino and carboxyl groups to invoke precipitation of products are often employed to improve yields.", "When the proteases with the right properties are available the application of proteases for synthesis offers substantial advantages.", "As proteases are stereoselective as well as regio-selective, sensitive groups on the reactants do usually not need protection and reactants do not need to be optically pure.", "As conditions of enzymatic synthesis are mild, racemization and decomposition of labile reactants or products can be prevented.", "Apart from bonds between amino acids, also other compounds exhibiting a primary amino group, a thiol group or a carboxyl group may be linked by properly selected proteases.", "In addition esters, thiol esters and amides may be synthesized by certain proteases.", "Protease have been shown to exhibit regioselectively in the acylation of mono, di- and tri-saccharides, nucleosides, and riboflavin.", "Problems with stability under the sometimes harsh reaction conditions may be prevented by proper formulation.", "Encapsulation and immobilisation do not only stabilise enzymes but also allow easy recovery and separation from the reaction medium.", "Extensive crosslinking, treatment with aldehydes or covering the surface with certain polymers such as dextrans, polyethyleneglycol, polyimines may substantially extend the lifetime of the biocatalyst.", "The Natural Roles of Proteases Traditionally, proteases have been regarded as degrading enzymes, capable of cleaving proteins into small peptides and/or amino acids, and whose role it is to digest nutrient protein or to participate in the turnover of cellular proteins.", "In addition, it has been shown that proteases also play key roles in a wide range of cellular processes, via mechanisms of selective modification by limited proteolysis, and thus can have essential regulatory functions (Holzer and Tschensche 1979; Holzer and Heinrich, 1980).", "The specificity of a proteinase is assumed to be closely related to its physiological function and its mode of expression.", "With respect to the function of a particular protease, its localisation is often very important; for example, a lot of the vacuolar and periplasmic proteases are involved in protein degradation, while many of the membrane-bound proteases are important in protein processing (Suarez Rendueles and Wolf, 1988).", "The different roles of proteases in many cellular processes can be divided into four main functions of proteases: 1) protein degradation, 2) posttranslational processing and (in)activation of specific proteins, 3) morphogenesis, and 4) pathogenesis.", "An obvious role for proteases in organisms which utilise protein as a nutrient source is in the hydrolysis of nutrients.", "In fungi, this would involve the degradation outside the cells by extracellular broad specificity proteases.", "Protein degradation is also important for rapid turnover of cellular proteins and allows the cell to remove abnormal proteins and to adapt their complement of protein to changing physiological conditions.", "Generally, proteases of rather broad specificity should be extremely well-controlled in order to protect the cell from random degradation of other than correct target proteins.", "Contrary to the hydrolysis the synthesis of polypeptides occurs in vivo by an ATP driven process on the ribosome.", "Ultimately the sequence in which the amino acids are linked is dictated by the information derived from the genome.", "This process is known as the transcription.", "Primary translation products are often longer than the final functional products, and after the transcription usually further processing of such precursor proteins by proteases is required.", "Proteases play a key role in the maturation of such precursor proteins to obtain the final functional protein.", "In contrast to the very controlled trimming and reshaping of proteins, proteases can also be very destructive and may completely degrade polypeptides into peptides and amino acids.", "In order to avoid that proteolytic activity is unleashed before it is required, proteases are subject to extensive regulation.", "Many proteases are synthesized as larger precursors known as zymogens, which become activated when required.", "Remarkably this activation always occurs by proteolysis.", "Apart from direct involvement in the processing, selective activation and inactivation of individual proteins are well-known phenomena catalyzed by specific proteases.", "The selectivety of limited proteolysis appears to reside more directly in the proteinase-substrate interaction.", "Specificity may be derived from the proteolytic enzyme which recognizes only specific amino acid target sequences.", "On the other hand, it may also be the result of selective exposure of the ‘processing site’ under certain conditions such as pH, ionic strength or secondary modifications, thus allowing an otherwise non-specific protease to catalyze a highly specific event.", "The activation of vacuolar zymogens by limited proteolysis gives an example of the latter kind.", "Morphogenesis or differentiation can be defined as a regulated series of events leading to changes from one state to another in an organism.", "Although direct relationships between proteases and morphological effects could not be established in many cases, the present evidence suggests a significant involvement of proteases in fungal morphogenesis; apart form the observed extensive protein turnover during differentiation, sporulation and spore germination, proteases are thought to be directly involved in normal processes as hyphal tip branching and septum formation, (Deshpande, 1992).", "Species of Aspergillus, in particular A. fumigatus and A. flavus, have been implicated as the causative agents of a number of diseases in humans and animals called aspergillosis (Bodey and Vartivarian, 1989).", "It has been repeatedly suggested that proteases are involved in virulence of A. fumigatus and A. flavus like there are many studies linking secreted proteases and virulence of bacteria.", "In fact, most human infections due to Aspergillus species are characterised by an extensive degradation of the parenchyma of the lung which is mainly composed of collagen and elastin (Campbell et al., 1994).", "Research has been focussed on the putative role of the secreted proteases in virulence of A. fumigatus and A. flavus which are the main human pathogens and are known to possess elastinolytic and collagenic activities (Kolattukudy et al., 1993).", "These elastinolytic activities were shown to correlate in vitro with infectivity in mice (Kothary et al., 1984).", "Two secreted proteases are known to be produced by A. fumigatus and A. flavus, an alkaline serine protease (ALP) and a neutral metallo protease (MEP).", "In A. fumigatus both the genes encoding these proteases were isolated, characterised and disrupted (Reicherd et al., 1990; Tang et al, 1992, 1993; Jaton-Ogay et al., 1994).", "However, alp mep double mutants showed no differences in pathogenecity when compared with wild type strains.", "Therefore, it must be concluded that the secreted A. fumigatus proteases identified in vitro are not essential factors for the invasion of tissue (Jaton Ogay et al., 1994).", "Although A. fumigatus accounts for only a small proportion of the airborne mould spores, it is the most frequently isolated fungus from lung and sputem (Schmitt et al., 1991).", "Other explanations for the virulence of the fungus could be that the conditions in the bronchia (temperature and nutrients) are favourable for the parasitic growth of A. fumigatus.", "As a consequence, invasive apergillosis could be a circumstancial event, when the host pathogenic defences have been weakened by immunosuppressive treatments or diseases like AIDS.", "Four major classes of proteases are known and are designated by the principal functional groups in their active site: the ‘serine’, the ‘thiol’ or ‘cysteine’, the ‘aspartic’ or ‘carboxyl’ and the ‘metallo’ proteases.", "A detailed state of the art review on these major classes of proteases, minor classes and unclassified proteases can be found in Methods in Enzymology part 244 and 248 (A. J. Barrett ed, 1994 and 1995).", "Specificity of Proteases Apart from the catalytic machinery of proteases another important aspect of proteolytic enzymes is the specificity of proteases.", "The specificity of a protease indicates which substrates the protease is likely to hydrolyze.", "The twenty natural amino acids offer a large number of possibilities to make up peptides.", "Eg with twenty amino acids one can make up already 400 dipeptides and 800 different tripeptide, and so on.", "With longer peptides the number of possibilities will become almost unlimited.", "Certain proteases hydrolyze only particular sequences at a very specific position.", "The interaction of the protease with the peptide substrate may encompass one up to ten amino acid residues of the peptide substrate.", "With large proteinacious substrates there may be even more residues of the substrate that interact with the proteases.", "However this likely involves less specific interactions with protease residues outside the active site binding cleft.", "In general the specific recognition is restricted to the linear peptide, which is bound in the active site of the protease.", "The nomenclature to describe the interaction of a substrate with a protease has been introduced in 1967 by Schechter and Berger (Biochem.", "Biophys.", "Res.", "Corn., 1967, 27, 157-162) and is now widely used in the literature.", "In this system, it is considered that the amino acid residues of the polypeptide substrate bind to so-called sub-sites in the active site.", "By convention, these sub-sites on the protease are called S (for sub-sites) and the corresponding amino acid residues are called P (for peptide).", "The amino acid residues of the N-terminal side of the scissile bond are numbered P3, P2, P1 and those residues of the C-terminal side are numbered P1′, P2′, P3′.", "The P1 or P1′ residues are the amino acid residues located near the scissile bond.", "The substrate residues around the cleavage site can then be numbered up to P8.The corresponding sub-sites on the protease that complement the substrate binding residues are numbered S3, S2, S1, S1′, S2′, S3′, etc, etc.", "The preferences of the sub-sites in the peptide binding site determine the preference of the protease for cleaving certain specific amino acid sequences at a particular spot.", "The amino acid sequence of the substrate should conform with the preferences exhibited by the sub-sites.", "The specificity towards a certain substrate is clearly dependant both on the binding affinity for the substrate and on the velocity at which subsequently the scissile bond is hydrolysed.", "Therefore the specificity of a protease for a certain substrate is usually indicated by its kcat/Km ratio, better known as the specificity constant.", "In this specificity constant kcat represents the turn-over rate and Km is the dissociation constant.", "Apart from amino acid residues involved in catalysis and binding, proteases contain many other essential amino acid residues.", "Some residues are critical in folding, some residues maintain the overall three dimensional architecture of the protease, some residues may be involved in regulation of the proteolytic activity and some residue may target the protease for a particular location.", "Many proteases contain outside the active site one or more binding sites for metal ions.", "These metal ions often play a role in stabilizing the structure.", "In addition secreted eukaryotic microbial proteases may be extensively glycosylated.", "Both N- and O-linked glycosylation occurs.", "Glycosylation may aid protein folding, may increase solubility, prevent aggregation and as such stabilize the mature protein.", "In addition the extent of glycosylation may influence secretion as well as water binding by the protein.", "Regulation of Proteolytic Activity A substantial number of proteases are subject to extensive regulation of the proteolytic activity in order to avoid undesired proteolytic damage.", "To a certain extent this regulation takes place at transcription level.", "For example in fungi the transcription of secreted protease genes appears to be sensitive to external carbon and nitrogen sources, whereas genes encoding intracellular proteases are insensitive.", "The extracellular pH is sensed by fungi and some genes are regulated by pH.", "In this process transcriptional regulator proteins play a crucial role.", "Proteolytic processing of such regulator proteins is often the switch that turns the regulator proteins either on or off.", "Proteases are subject to intra- as well as intermolecular regulation.", "This implies certain amino acids in the proteolytic enzyme molecule that are essential for such regulation.", "Proteases are typically synthesized as larger precursors known as zymogens, which are catalytically inactive.", "Usually the peptide chain extension rendering the precursor protease inactive is located at the amino terminus of the protease.", "The precursor is better known as pro-protein.", "As many of the proteases processed in this way are secreted from the cells they contain in addition a signal sequence (pre sequence) so that the complete precursor is synthesized as a pre-pro-protein.", "Apart from rendering the protease inactive the pro-peptide often is essential for mediating productive folding.", "Examples of proteases include serine proteases (alpha lytic protease, subtilisin, aqualysin, prohormone convertase), thiol proteases (cathepsin L and cruzian), aspartic proteases (proteinase A and cathepsin D) and metalloproteases.", "In addition the pro-peptide might play a role in cellular transport either alone or in conjunction with signal peptides.", "It may facilitate interaction with cellular chaperones or it may facilitate transport over the membrane.", "The size of the extension in the precursor pre-pro-protein may vary substantially, ranging from a short peptide fragment to a polypeptide, which can exist as an autonomous folding unit.", "In particular these larger extensions are often observed to be strong inhibitors of the protease even after cleavage from the protease.", "It was observed that even after cleavage such pro-peptides could assist in proper folding of the proteases.", "As such pro-peptides can be considered to function as molecular chaperones and separate or additional co-expression of such pro-peptides could be advantageous for protease production.", "There is substantial difference in the level of regulation between proteases that are secreted into the medium and proteases that remain intracellular.", "Proteases secreted into the medium are usually after activation no longer subject to control and therefore are usually relatively simple in their molecular architecture consisting of one globular module.", "Intracellular proteases are necessarily subject to continuous control in order to avoid damage to the cells.", "In contrast with zymogens of secreted proteases in more complex regulatory proteases very large polypeptide segments may be inserted between the signal and the zymogen activation domain of the proteolytic module.", "Structure-function studies indicate that such non-protease parts may be involved in interactions with macroscopic structures, membranes, cofactors, substrates, effectors, inhibitors, ions, that regulate activity and activation of the proteolytic module(s) or its (their) zymogens.", "The non-proteolytic modules exhibit remarkable variation in size and structure.", "Many of the modules can exist as such independently from the proteolytic module.", "Therefore such modules can be considered to correspond to independent structural and functional units that are autonomous with respect to folding.", "The value of such a modular organization is that acquisition of new modules can endow the recipient protease with new novel binding specificities and can lead to dramatic changes in its activity, regulation and targeting.", "The principle of modular organized proteolytic enzymes may also be exploited by applying molecular biology tools in order to create novel interactions, regulation, specificity, and/or targeting by shuffling of modules.", "Although in general such additional modules are observed as N or C terminal extension, also large insertions within the exterior loops of the catalytic domain have been observed.", "It is believed that also in this case the principal fold of the protease represents still the essential topology to form a functional proteolytic entity and that the insertion can be regarded as substructure folded onto the surface of the proteolytic module.", "Molecular Structure In principle the modular organization of larger proteins is a general theme in nature.", "In particular within the larger multimodular frameworks typical proteolytic modules show sizes of 100 to 400 amino acids on the average.", "This corresponds with the average size of most of the globular proteolytic enzymes that are secreted into the medium.", "As discussed above polypeptide modules are polypeptide fragments, which can fold and function as independent entities.", "Another term for such modules is domains.", "However domain is used in a broader context than module.", "The term domain as used herein refers usually to a part of the polypeptide chain that depicts in the three-dimensional structure a typical folding topology.", "In a protein domains interact to varying extents, but less extensively than do the structural elements within domains.", "Other terms such as subdomain and folding unit are also used in literature.", "As such it is observed that many proteins that share a particular functionality may share the same domains.", "Such domains can be recognized from the primary structure that may show certain sequence patterns, which are typical for a particular domain.", "Typical examples are the mononucleotide binding fold, cellulose binding domains, helix-turn-helix DNA binding motif, zinc fingers, EF hands, membrane anchors.", "Modules refer to those domains which are expected to be able to fold and function autonomously.", "A person skilled in the art knows how to identify particular domains in a primary structure by applying commonly available computer software to said structure and homologous sequences from other organisms or species.", "Although multimodular or multidomain proteins may appear as a string of beads, assemblies of substantial more complex architecture have been observed.", "In case the various beads reside on the same polypeptide chain the beads are generally called modules or domains.", "When the beads do not reside on one and same polypeptide chain but form assemblies via non-covalent interactions then the term subunit is used to designate the bead.", "Subunits may be transcribed by one and the same gene or by different genes.", "The multi-modular protein may become proteolytically processed after transcription leading to multiple subunits.", "Individual subunits may consist of multiple domains.", "Typically the smaller globular proteins of 100-300 amino acids usually consist only of one domain.", "Molecular Classification of Proteolytic Enzymes In general proteases are classified according to their molecular properties or according to their functional properties.", "The molecular classification is based on the primary structure of the protease.", "The primary structure of a protein represents its amino acid sequence, which can be derived from the nucleotide sequence of the corresponding gene.", "Tracing extensively the similarities in the primary structures may allow for the notice of similarities in catalytic mechanism and other properties, which even may extend to functional properties.", "The term family is used to describe a group of proteases that show evolutionary relationship based on similarity between their primary structures.", "The members of such a family are believed to have arisen by divergent evolution from the same ancestor.", "Within a family further sub-grouping of the primary structures based on more detailed refinement of sequence comparisons results in subfamilies.", "Classification according to three-dimensional fold of the proteases may comprise secondary structure, tertiary structure and quarternary structure.", "In general the classification on secondary structure is limited to content and gross orientation of secondary structure elements.", "Similarities in tertiary structure have led to the recognition of superfamilies or clans.", "A superfamily or a clan is a group of families that are thought to have common ancestry as they show a common 3-dimensional fold.", "In general tertiary structure is more conserved than the primary structure.", "As a consequence similarity of the primary structure does not always reflect similar functional properties.", "In fact functional properties may have diverged substantially resulting in interesting new properties.", "At present quarternary structure has not been applied to classify various proteases.", "This might be due to a certain bias of the structural databases towards simple globular proteases.", "Many proteolytic systems that are subject to activation, regulation, or complex reaction cascades are likely to consist of multiple domains or subunits.", "General themes in the structural organization of such protease systems may lead to new types of classification.", "Classification According to Specificity.", "In absence of sequence information proteases haven been subject to various type of functional classification.", "The classification and naming of enzymes by reference to the reactions which are catalyzed is a general principle in enzyme nomenclature.", "This approach is also the underlying principle of the EC numbering of enzymes (Enzyme Nomenclature 1992 Academic Press, Orlando).", "Two types of proteases (EC 3.4) can be recognized within Enzyme Nomenclature 1992, those of the exo-peptidases (EC 3.4.11-19) and those of the endo-peptidases (EC 3.4.21-24, 3.4.99).", "Endo-peptidases cleave peptide bonds in the inner regions of the peptide chain, away from the termini.", "Exo-peptidases cleave only residues from the ends of the peptide chain.", "The exo-peptidases acting at the free N-terminus may liberate a single amino acid residue, a dipeptide or a tripeptide and are called respectively amino peptidases (EC 3.4.11), dipeptidyl peptidases (EC 3.4.14) and tripeptidyl peptidase (EC 3.3.14).", "Proteases starting peptide processing from the carboxyl terminus liberating a single amino acid are called carboxy peptidase (EC 3.4.16-18).", "Peptidyl-dipeptidases (EC 3.4.15) remove a dipeptide from the carboxyl terminus.", "Exo- and endo-peptidase in one are the dipeptidases (EC 3.4.13), which cleave specifically only dipeptides in their two amino acid halves.", "Omega peptidases (EC 3.4.19) remove terminal residues that are either substituted, cyclic, or linked by isopeptide bonds Apart from the position where the protease cleaves a peptide chain, for each type of protease a further division is possible based on the nature of the preferred amino acid residues in the substrate.", "In general one can distinguish proteases with broad, medium and narrow specificity.", "Some proteases are simply named after the specific proteins or polypeptides that they hydrolyze, e.g.", "keratinase, collagenase, elastase.", "A narrow specificity may pin down to one particular amino acid or one particular sequence which is removed or which is cleaved respectively.", "When the protease shows a particular preference for one aminoacid in the P1 or P1′ position the name of this amino acid may be a qualifier.", "For example prolyl amino peptidase removes proline from the amino terminus of a peptide (proline is the P1 residue).", "X-Pro or proline is used when the bond on the imino side of the proline is cleaved (proline is P1′ residue), eg proline carboxypeptidase removes proline from the carboxyl terminus.", "Prolyl endopeptidase (or Pro-X) cleaves behind proline while proline endopeptidase (X-Pro) cleaves in front of a proline.", "Amino acid residue in front of the scissile peptide bond refers to the amino acid residue that contributes the carboxyl group to the peptide bond.", "The amino acids residue behind the scissile peptide bond refers to the amino acid residue that contributes the amino group to the peptide bond.", "According to the general convention an amino acid chain runs from amino terminus (the start) to the carboxyl terminus (the end) and is numbered accordingly.", "Endo proteases may also show clear preference for a particular amino acid in the P1 or P1′ position, eg glycyl endopeptidase, peptidyl-lysine endopeptidase, glutamyl endopeptidase.", "In addition proteases may show a preference for a certain group of amino acids that share a certain resemblance.", "Such a group of preferred amino acids may comprise the hydrophobic amino acids, only the bulky hydrophobic amino acids, small hydrophobic, or just small amino acids, large positively charged amino acids, etc, etc.", "Apart from preferences for P1 and P1′ residues also particular preferences or exclusions may exist for residues preferred by other subsites on the protease.", "Such multiple preferences can result in proteases that are very specific for only those sequences that satisfy multiple binding requirements at the same time.", "In general it should be realized that protease are rather promiscuous enzymes.", "Even very specific protease may cleave peptides that do not comply with the generally observed preference of the protease.", "In addition it should be realized that environmental conditions such as pH, temperature, ionic strength, water activity, presence of solvents, presence of competing substrates or inhibitors may influence the preferences of the proteases.", "Environmental condition may not only influence the protease but also influence the way the proteinacious substrate is presented to the protease.", "Classification by Catalytic Mechanism.", "Proteases can be subdivided on the basis of their catalytic mechanism.", "It should be understood that for each catalytic mechanism the above classification based on specificity leads to further subdivision for each type of mechanism.", "Four major classes of proteases are known and are designated by the principal functional group in the active site: the serine proteases (EC 3.4.21 endo peptidase, EC 3.4.16 carboxy peptidase), the thiol or cysteine proteases (EC 3.4.22 endo peptidase, EC 3.4.18 carboxy peptidase), the carboxyl or aspartic proteases (EC 3.4.23 endo peptidase) and metallo proteases (EC 3.4.24 endo peptidase, EC 3.4.18 carboxy peptidase).", "There are characteristic inhibitors of the members of each catalytic type of protease.", "These small inhibitors irreversibly modify an amino acid residue of the protease active site.", "For example, the serine protease are inactivated by Phenyl Methane Sulfonyl Fluoride (PMSF) and Diisopropyl Fluoro Phosphate (DFP), which react with the active Serine whereas the chloromethylketone derivatives react with the Histidine of the catalytic triad.", "Phosphoramidon and 1,10 Phenanthroline typically inhibit metallo proteases.", "Inhibition by Pepstatin generally indicates an aspartic protease.", "E64 inhibits thiol protease specifically.", "Amastatin and Bestatin inhibit various aminopeptidases.", "Substantial variations in susceptibility of the proteases to the inhibitors are observed, even within one catalytic class.", "To a certain extent this might be related to the specificity of the protease.", "In case binding site architecture prevents a mechanism based inhibitor to approach the catalytic site, then such a protease escapes from inhibition and identification of the type of mechanism based on inhibition is prohibited.", "Chymostation for example is a potent inhibitor for serine protease with chymotrypsin like specificity, Elastatinal inhibits elastase like serine proteases and does not react with trypsin or chymostrypsin, 4 amido PMSF (APMSF) inhibits only serine proteases with trypsin like specificity.", "Extensive accounts of the use of inhibitors in the classification of proteases include Barret and Salvesen, Proteinase Inhibitors, Elsevier Amstardam, 1986; Bond and Beynon (eds), Proteolytic Enzymes, A Practical Approach, IRL Press, Oxford, 1989; Methods in Enzymology, eds E. J. Barret, volume 244, 1994 and volume 248, 1995; E. Shaw, Cysteinyl proteinases and their selective inactivation, Adv Enzymol.", "63:271-347 (1990) Classification According to Optimal Performance Conditions.", "The catalytic mechanism of a proteases and the requirement for its conformational integrity determine mainly the conditions under which the protease can be utilized.", "Finding the protease that performs optimal under application conditions is a major challenge.", "Often conditions at which proteases have to perform are not optimal and do represent a compromise between the ideal conditions for a particular application and the conditions which would suit the protease best.", "Apart from the particular properties of the protease it should be realized that also the presentation of a proteinacious substrates is dependant on the conditions, and as such determines also which conditions are most effective for proteolysis.", "Specifications for the enzyme that are relevant for application comprise for example the pH dependence, the temperature dependence, sensitivity for or the dependence of metal ions, ionic strength, salt concentration, solvent compatibility.", "Another factor of major importance is the specific activity of a protease.", "The higher the enzyme's specific activity, the less enzyme is needed for a specific conversion.", "Lower enzyme requirements imply lower costs and lower protein contamination levels.", "The pH is a major parameter that determines protease performance in an application.", "Therefor pH dependence is an important parameter to group proteases.", "The major groups that are recognized are the acid proteases, the neutral proteases, the alkaline proteases and the high alkaline proteases.", "The optimum pH matches only to some extent the proteolytic mechanism, eg aspartic protease show often an optimum at acidic pH, metalloproteases and thiol proteases often perform optimal around neutral pH to slightly alkaline, serine peptidases are mainly active in the alkaline and high alkaline region.", "For each class exceptions are known.", "In addition the overall water activity of the system plays a role.", "The pH optimum of a protease is defined as the pH range where the protease exhibits an optimal hydrolysis rate for the majority of its substrates in a particular environment under particular conditions.", "This range can be narrow, e.g.", "one pH unit, as well as quite broad, 3-4 pH units.", "In general the pH optimum is also dependant on the nature of the proteinacious substrate.", "Both the turnover rate as well as the specificity may vary as a function of pH.", "For a certain efficacy it can be desirable to use the protease far from its pH optimum because production of less desired peptides is avoided.", "Less desired peptides might be for example very short peptides or peptides causing a bitter taste.", "In addition a more narrow specificity can be a reason to choose conditions that deviate from optimal conditions with respect to turnover rate.", "Dependant on the pH the specificity may be narrow, e.g.", "only cleaving the peptide chain in one particular position or before or after one particular amino acid, or broader, e.g.", "cleaving a chain at multiple positions or cleaving before or after more different types of amino acids.", "In fact the pH dependence might be an important tool to regulate the proteolytic activity in an application.", "In case the pH shifts during the process the proteolysis might cease spontaneously without the need for further treatment to inactivate the protease.", "In some cases the proteolysis itself may be the driver of the pH shift.", "Very crucial for application of proteases is their handling and operating stability.", "As protease stability is strongly affected by the working temperature, stability is often also referred to as thermostability.", "In general the stability of a protease indicates how long a protease retains its proteolytic activity under particular conditions.", "Particular conditions may comprise fermentation conditions, conditions during isolation and down stream processing of the enzyme, storage conditions, formulation and operating or application conditions.", "In case particular conditions encompass elevated temperatures stability in general refers to thermostability.", "Apart from the general causes for enzyme inactivation such as chemical modification, unfolding, aggregation etc, main problem with proteases is that they are easy subject to autodegradation.", "Especially for the utilization of proteases the temperature optimum is a relevant criterion to group proteases.", "Although there are different definitions, economically the most useful definition is the temperature or the temperature range in which the protease is most productive in a certain application.", "Protease productivity is a function of both the stability and the turnover rate.", "Where elevated temperature in general will increase the turnover rate, rapid inactivation will counteract the increase in turnover rate and ultimately lead to low productivity.", "The conformational stability of the protease under a given process condition will determine its maximum operating temperature.", "The temperature at which the protease looses it active conformation, often indicated as unfolding or melting point, can be determined according various methods, for example NMR, Circular Dichroism Spectroscopy, Differential Scanning Calorimetry etc etc.", "For protease unfolding is usually accompanied by a tremendous increase in autodegradation rate.", "In applications where low temperatures are required protease may be selected with emphasis on a high intrinsic activity at low to moderate temperature.", "As under such conditions inactivation is relatively slow, under these conditions activity might largely determine productivity.", "In processes where only during a short period protease activity is required, the stability of the protease might be used as a switch to turn the protease off.", "In such case more labile instead of very thermostable protease might be preferred.", "Other environmental parameters which may play a role in selecting the appropriate protease may be its sensitivity to salts.", "The compatibility with metal ions which are found frequently at low concentrations in various natural materials can be crucial for certain applications.", "In particular with metallo proteases certain ions may replace the catalytic metal ion and reduce or even abolish activity completely.", "In some applications metal ions have to be added on purpose in order to prevent the washout of the metal ions coordinated to the protease.", "It is well known that for the sake of enzyme stability and life-time, calcium ions have to be supplied in order to prevent dissociation of protein bound calcium.", "Most microorganisms show a certain tolerance with respect to adapting to changes in the environmental condition.", "As a consequence at least the proteolytic spectrum that the organism is able to produce are likely to show at least similar tolerances.", "Such a proteolyitic spectrum might be covered by many proteases covering together the hole spectrum or by only a few proteases of a broad spectrum.", "Taking into account the whole proteolytic spectrum of a microorganism it can be very important to take the location into account.", "Cellular Localisation and Characterization of Proteolytic Processing and Degradation From an industrial point of view the proteases which are excreted from the cell have specific advantages with respect to producibility at a large scale and stress tolerance as they have to survive without protection of the cell.", "The large group of cellular protease can be further subdivided in soluble and membrane bound.", "Membrane bound may comprise protease at the inside as well the outside of the membrane.", "Intracellular soluble protease may be subdivided further according to specific compartments of the cell where they do occur.", "As the cell shields the proteases to some extent from the environment and because the cell controls the conditions in the cell, intracellular protease might be more sensitive to large environmental changes and their optima might correlate better with the specific intacellualr conditions.", "Knowing the conditions of the cellular department where the protease resides might indicate their preferences.", "Where extracellular protease in general do not require any regulation any more once excreted from the cell, intracellular proteases are often subject to more complicated control and regulation.", "With respect to the function of a particular protease, its localisation is often very important; for example, a lot of the vacuolar and periplasmic proteases are involved in protein degradation, while many of the membrane-bound proteases are important in protein processing (Suarez Rendueles and Wolf, 1988).", "A comprehensive review on the biological properties and evolution of proteases has been published in van den Hombergh: Thesis Landbouwuniversiteit Wageningen: An analysis of the proteolytic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2, which is hereby incorporated by reference herein.", "The Protease Problem An important reason for the interest in microbial proteases are protease related expression problems observed in several expression hosts used in bioprocess industry.", "The increasing use of heterologous hosts for the production of proteins, by recombinant DNA technology, has recently brought this problem into focus, since it seems that heterologous proteins are more prone to proteolysis (Archer et al., 1992; van den Hombergh et al., 1996b).", "In S. cerevisiae, already in the early eighties the protease problem and the involvement of several proteases, thus complicating targetted gene disruption approaches to overcome this problem, was recognised.", "During secretion a protein is exposed to several proteolytic activities residing in the secretory pathway.", "Additionally, in a prototrophic microorganism as Aspergillus secreted proteins can be exposed to several extracellular proteolytic activities The problem of degradation of heterologously expressed proteins is well documented in Aspergillus (van den Hombergh Thesis Landbouwuniversiteit Wageningen: An analysis of the proteolytic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2) and has been reported in the expression of cow prochymosin, human interferon α-2 tPA, GMCSF, IL6, lactoferrin, chicken egg-white lysosyme, porcine pIA2, A. niger pectin lyase B, E. coli enterotoxin B and β-glucoronidase, and Erwinia carotovora pectate lyase 3.The problem of proteolysis may be addressed at several stages in protein production.", "Bioprocess engineers may address the problem of proteolysis by downstream processing at low temperatures, by early separation of product and protease(s) or by use of protease inhibitors.", "These may all lead to successful reduction of the problem.", "However it is certainly not eliminated, because much of the degradation occurs in vivo during the production of the protein.", "In understanding how proteolysis is controlled in the cell, a major question concerns the recognition mechanism by which proteolysis is triggered.", "Into what extent are proteolytically susceptable (heterologous) proteins recognised as aberrant because of misfolding or, if correctly folded, as ‘foreign’, because they do not posses features essential for stability which are specific to the host.", "Various types of stress can cause the overall proteolysis in a cell to increase significantly.", "Factors known to increase rate of proteolysis include nutrient starvation and various other types of stress (i.e.", "elevation of temperature, osmotic stress, toxic substances and expression of certain heterologous proteins).", "To deal with proteolysis-related expression problems in vivo, several approaches have been proven succesfull as will be discussed below.", "However, we have to keep in mind that true ‘non-proteolytic cells’ cannot exist, since proteolysis by intracellular proteases is involved in many essential metabolic and ‘housekeeping’ reactions.", "Reducing proteolysis will therefore always be a process in which the changed genetical background which results in decreased proteolytic has to be analysed for potential secundary effects which could lead to reduced protein production (e.g.", "reduced growth rate or sporulation).", "Disruption of Proteases in Filamentous Fungal Expression Hosts Berka and coworkers (1990) describe the cloning and disruption of the A. awamori pepA gene.", "More recently, three disrupted aspartyl proteases in A. niger have been described.", "Disruptants for both the major extracellular aspartyl proteases and the major vacuolar aspartyl protease were described.", "Double and triple disruptants were generated via recombination and tested for protease spectra and expression and secretion of the A. niger pectin lyase PELB protein, which is very susceptable to proteolytic degradation (van den Hombergh et al., 1995).", "Disruption of pepA and pepB resulted both in reduction of extracellular protease activities, 80% and 6%, respectively.", "In the ΔpepE disruptant also other (vacuolar) protease activities were severely affected caused by inactivating of the proteolytic cascade for other vacuolar proteases.", "Reduced extracellular activities correlated with reduced in vitro degradation of PELB and improved in vivo expression of pelB (van den Hombergh et al., 1996f).", "Protease Deficient (prt) Mutants Filamentous Fungi Several Aspergillus protease deficient mutants have been studied whether protein production is improved.", "Archer and coworkers describe the reduced proteolysis of Hen egg white lysozyme in supernatants of an A. niger double prt mutant generated by Mattern and coworkers (1992) and conclude that although the degradation is not absent, it is significantly reduced.", "Van den Hombergh et al.", "(1995) show that the in vitro degradation of A. niger PELB is reduced in all seven prt complementation groups they have isolated.", "Virtually no degradation is observed in the prtB, prtF and prtG mutants.", "Recently, the expression of the pelB gene was shown to be improved in six complementation groups tested (prtA-F) and highest expression levels were observed in the prtB, prtF and prtG mutants.", "In addition to the single mutants, which contained residual extracellular proteolytic activities varying from 2-80% compared to wild type activity, double mutants were generated both by recombination and by additional rounds of mutagenesis.", "Via this approach several double prt mutants were selected and further characterised, which showed a further reduction of PELB degradation compared to their parental strains.", "Instead of elimination of protease activities via disruption or mutagenesis, reduced proteolysis can also be achieved via down-regulation of the interfering proteolytic activities.", "This may be achieved by genetically altering the promoter or other regulatory sequences of the gene.", "As shown by Fraissinet-Tachet and coworkers (1996) the extracellular proteases in A. niger are all regulated by carbon catabolite repression and nitrogen metabolite repression.", "Nutrient starvation also causes the overall proteolysis rate in a cell to increase stromgly, which makes sense for a cell that lacks nutrients but posses proteins, that under starvation conditions are not needed or needed only in smaller amounts.", "In expression strategies which allow high expression on media containing high glucose and ammonium concentrations reduced proteolysis has been reported.", "Several constitutive glycolytic promoters (gpd and pkiA) are highly expressed under these conditions and can also be used to drive (heterologous) gene expression in continuous fermentations.", "The type of nutrient starvation imposed can influence different proteases to varying extent, which means that the importance of nutrient conditions in a given process depend on the type of proteolysis that is involved.", "Specific proteolysis may therefore be induced by conditions of substrate limitation which are frequently used in many large-scale fermentation processes.", "The protease problem can nowadays be addressed in part by one or more of the above strategies.", "However, the residual proteolytic activity of yet unidentified proteolytic enzymes still constitutes a major problem in the art.", "In order to further reduce the level of unwanted proteolysis, there is a great need in the art to identify novel proteases responsible for degradation of homologously and heterologously expressed proteins.", "This invention provides such novel protease gene sequences encoding novel proteases.", "Once the primary sequence of a novel protease gene is known, one or more of the above recombinant DNA strategies may be employed to produce (knock-out) mutants with reduced proteolytic activity.", "Despite the widespread applications of proteases in a great number of industrial processes, current enzymes also have significant shortcomings with respect to at least one of the following properties.", "When added to animal feed, current proteases are not sufficiently resistant to digestive enzymes present in the gastrointestinal (GI) tract of e.g.", "pigs and poultry.", "With respect to another aspect, the currently available enzymes are not sufficiently resistant to specific (high) temperatures and (high) pressure conditions that are applied during extrusion or pelleting operations.", "Also, the current enzymes are not sufficiently active in a pH range of 3-7, conditions prevailing in many food, beverage products as well as in in the GI tract of most animals.", "According to yet another aspect the specificity of the currently available proteases is very limited which results in the inability of the existing enzymes to degrade or to dissolve certain “protease resistant” proteins thus resulting in low peptide or amino acid yields.", "Moreover proteases with new specificities allow the synthesis of new peptides.", "Yet another drawback of the currently available enzymes is their low specific activity.", "It is therefore clear that for a large number of applications a strong desire exists for proteases that are more resistant to digestive enzymes, high temperature and/or pressure and which exhibit novel specificities regarding their sites of hydrolysis.", "The present invention provides such enzymes.", "OBJECT OF THE INVENTION It is an object of the invention to provide novel polynucleotides encoding novel proteases.", "A further object is to provide naturally and recombinantly produced proteases as well as recombinant strains producing these.", "Such strains may also be used to produce classical fermentation products faster or with higher yields.", "Yet another object of the invention is to provide a filamentous fungus strain defective in producing a protease according to the invention.", "Such strains may be used for a more efficient production of heterologous or homologous proteins.", "Also antibodies and fusion polypeptides are part of the invention as well as methods of making and using the polynucleotides and polypeptides according to the invention.", "SUMMARY OF THE INVENTION The invention provides for novel polynucleotides encoding novel proteases.", "More in particular, the invention provides for polynucleotides having a nucleotide sequence that hybridises (preferably under highly stringent conditions) to a sequence according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or to a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114.Consequently, the invention provides nucleic acids that are about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114.In a more preferred embodiment the invention provides for such an isolated polynucleotide obtainable from a filamentous fungus, preferably Aspergilli, in particular A. niger is preferred.", "In one embodiment, the invention provides for an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof.", "In a further preferred embodiment, the invention provides an isolated polynucleotide encoding at least one functional domain of a polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof.", "In a preferred embodiment the invention provides a protease gene according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57.In another aspect the invention provides a polynucleotide, preferably a cDNA encoding an A. niger protease selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or variants or fragments of that polypeptide.", "In a preferred embodiment the cDNA has a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 or functional equivalents thereof.", "A genomic clone encoding a polypeptide according to the invention may also be obtained by selecting suitable probes to specifically amplify a genomic region corresponding to any of the sequences according to SEQ ID NO: 1 to SEQ ID NO: 57 or fragments thereof, hybridising that probe under suitable conditions to genomic DNA obtained from a suitable organism, such as Aspergillus, e.g.", "A. niger, amplifying the desired fragment e.g.", "by PCR (polymerase chain reaction) followed by purifying and cloning of the amplified fragment.", "In an even further preferred embodiment, the invention provides for a polynucleotide comprising the coding sequence of the genomic polynucleotides according to the invention, preferred is a polynucleotide sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114.In another preferred embodiment, the invention provides a cDNA obtainable by cloning and expressing a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 into a suitable host organism, such as A. niger.", "A polypeptide according to the invention may also be obtained by cloning and expressing a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 into a suitable host organism, such as A. niger.", "The invention also relates to vectors comprising a polynucleotide sequence according to the invention and primers, probes and fragments that may be used to amplify or detect the DNA according to the invention.", "In a further preferred embodiment, a vector is provided wherein the polynucleotide sequence according to the invention is functionally linked with regulatory sequences suitable for expression of the encoded amino acid sequence in a suitable host cell, such as A. niger or A. oryzea.", "The invention also provides methods for preparing polynucleotides and vectors according to the invention.", "The invention also relates to recombinantly produced host cells that contain heterologous or homologous polynucleotides according to the invention.", "In one embodiment, the invention provides recombinant host cells wherein the expression of a protease according to the invention is significantly reduced or wherein the activity of the protease is reduced or wherein the protease is even inactivated.", "Such recombinants are especially useful for the expression of homologous or heterologous proteins.", "In another embodiment, the invention provides recombinant host cells wherein the expression of a protease according to the invention is significantly increased or wherein the activity of the protease is increased.", "Such recombinants are especially useful for the expression of homologous or heterologous proteins where maturation is seriously hampered in case the required proteolytic cleavage becomes the rate limiting step.", "In another embodiment the invention provides for a recombinantly produced host cell that contains heterologous or homologous DNA according to the invention, preferably DNA encoding proteins bearing signal sequnences and wherein the cell is capable of producing a functional protease according to the invention, preferably a cell capable of over-expressing the protease according to the invention, for example an Aspergillus strain comprising an increased copy number of a gene or cDNA according to the invention.", "In another embodiment the invention provides for a recombinantly produced host cell that contains heterologous or homologous DNA according to the invention and wherein the cell is capable of secreting a functional protease according to the invention, preferably a cell capable of over-expressing and secreting the protease according to the invention, for example an Aspergillus strain comprising an increased copy number of a gene or cDNA according to the invention.", "In yet another aspect of the invention, a purified polypeptide is provided.", "The polypeptides according to the invention include the polypeptides encoded by the polynucleotides according to the invention.", "Especially preferred is a polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof.", "The invention also provides for antibodies reactive with a polypeptide according to the invention.", "These antibodies may be polyclonal, yet especially preferred are monoclonal antibodies.", "Such antibodies are particularly useful for purifying the polypeptides according to the invention.", "Fusion proteins comprising a polypeptide according to the invention are also within the scope of the invention.", "The invention also provides methods of making the polypeptides according to the invention.", "The invention further relates to a method for diagnosing aspergillosis either by detecting the presence of a polypeptide according to the invention or functional equivalents thereof, or by detecting the presence of a DNA according to the invention or fragments or functional equivalents thereof.", "The invention also relates to the use of the protease according to the invention in an industrial process as described herein DETAILED DESCRIPTION OF THE INVENTION Polynucleotides The present invention provides polynucleotides encoding proteases having an amino acid sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof.", "The sequence of these genes was determined by sequencing a genomic clone obtained from Aspergillus niger.", "The invention provides polynucleotide sequences comprising the gene encoding these proteases as well as their complete cDNA sequence and its coding sequence.", "Accordingly, the invention relates to an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 or functional equivalents thereof.", "More in particular, the invention relates to an isolated polynucleotide hybridisable under stringent conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 preferably under highly stringent conditions.", "Advantageously, such polynucleotides may be obtained from filamentous fungi, in particular from Aspergillus niger.", "More specifically, the invention relates to an isolated polynucleotide having a nucleotide sequence according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114.The invention also relates to an isolated polynucleotide encoding at least one functional domain of a polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof.", "As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which may be isolated from chromosomal DNA, which include an open reading frame encoding a protein, e.g.", "an A. niger protease.", "A gene may include coding sequences, non-coding sequences, introns and regulatory sequences.", "Moreover, a gene refers to an isolated nucleic acid molecule as defined herein.", "A nucleic acid molecule of the present invention, such as a nucleic acid molecule having the nucleotide sequence of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 or a functional equivalent thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.", "For example, using all or portion of the nucleic acid sequence of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or the nucleotide sequence of a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 as a hybridization probe, nucleic acid molecules according to the invention can be isolated using standard hybridization and cloning techniques (e. g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.", "2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).", "Moreover, a nucleic acid molecule encompassing all or a portion of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information contained in a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114.A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.", "The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.", "Furthermore, oligonucleotides corresponding to or hybridisable to nucleotide sequences according to the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.", "In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114.The sequence of a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 corresponds to the coding region of the A. niger protease cDNA.", "This cDNA comprises sequences encoding the A. niger protease polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171.In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 or a functional equivalent of these nucleotide sequences.", "A nucleic acid molecule which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleotide sequence thereby forming a stable duplex.", "One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a functional equivalent thereof such as a biologically active fragment or domain, as well as nucleic acid molecules sufficient for use as hybridisation probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.", "An “isolated polynucleotide” or “isolated nucleic acid” is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived.", "Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5′ non-coding (e.g., promotor) sequences that are immediately contiguous to the coding sequence.", "The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences.", "It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).", "Moreover, an “isolated nucleic acid fragment” is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.", "As used herein, the terms “polynucleotide” or “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.", "The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.", "The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides).", "Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.", "Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a protease nucleic acid molecule, e.g., the coding strand of a protease nucleic acid molecule.", "Also included within the scope of the invention are the complement strands of the nucleic acid molecules described herein.", "Sequencing Errors The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.", "The specific sequences disclosed herein can be readily used to isolate the complete gene from filamentous fungi, in particular A. niger which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.", "Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above.", "Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors.", "Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.", "The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.", "As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.", "The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.", "Nucleic Acid Fragments, Probes and Primers A nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence shown in a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114, for example a fragment which can be used as a probe or primer or a fragment encoding a portion of a protease protein.", "The nucleotide sequence determined from the cloning of the protease gene and cDNA allows for the generation of probes and primers designed for use in identifying and/or cloning other protease family members, as well as protease homologues from other species.", "The probe/primer typically comprises substantially purified oligonucleotide which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, preferably about 22 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequence shown in a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 or of a functional equivalent thereof.", "Probes based on the protease nucleotide sequences can be used to detect transcripts or genomic protease sequences encoding the same or homologous proteins for instance in other organisms.", "In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.", "Such probes can also be used as part of a diagnostic test kit for identifying cells which express a protease protein.", "Identity & Homology The terms “homology” or “percent identity” are used interchangeably herein.", "For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).", "The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.", "When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.", "The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions (i.e.", "overlapping positions)×100).", "Preferably, the two sequences are the same length.", "The skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences.", "For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.", "In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol.", "Biol.", "(48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.", "In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.In another embodiment, the percent identity two amino acid or nucleotide sequence is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989) which has been incorporated into the ALIGN program (version 2.0) (available at http://vega/igh.cnrs.fr/bin/align-guess.cgi), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences.", "Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.", "(1990) J. Mol.", "Biol.", "215:403-10.BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to protease nucleic acid molecules of the invention.", "BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protease protein molecules of the invention.", "To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res.", "25(17):3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.", "See http://www.ncbi.nlm.nih.gov.", "Hybridisation As used herein, the term “hybridizing” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 50%, at least about 60%, at least about 70%, more preferably at least about 80%, even more preferably at least about 85% to 90%, more preferably at least 95% homologous to each other typically remain hybridized to each other.", "A preferred, non-limiting example of such hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 1×SSC, 0.1% SDS at 50° C., preferably at 55° C., preferably at 60° C. and even more preferably at 65° C. Highly stringent conditions include, for example, hybridizing at 68° C. in 5×SSC/5× Denhardt's solution/1.0% SDS and washing in 0.2×SSC/0.1% SDS at room temperature.", "Alternatively washing may be performed at 42° C. The skilled artisan will know which conditions to apply for stringent and highly stringent hybridisation conditions.", "Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al.", "(eds.", "), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).", "Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3′ terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule contain a poly (A) stretch or the complement thereof (e.g., practically any double-standed cDNA clone).", "Obtaining Full Length DNA from Other Organisms In a typical approach, cDNA libraries constructed from other organisms, e.g.", "filamentous fungi, in particular from the species Aspergillus can be screened.", "For example, Aspergillus strains can be screened for homologous protease polynucleotides by Northern blot analysis.", "Upon detection of transcripts homologous to polynucleotides according to the invention, cDNA libraries can be constructed from RNA isolated from the appropriate strain, utilizing standard techniques well known to those of skill in the art.", "Alternatively, a total genomic DNA library can be screened using a probe hybridisable to a protease polynucleotide according to the invention.", "Homologous gene sequences can be isolated, for example, by performing PCR using two oligonucleotide primers or two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.", "The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a polynucleotide according to the invention.", "The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new protease nucleic acid sequence, or a functional equivalent thereof.", "The PCR fragment can then be used to isolate a full length cDNA clone by a variety of known methods.", "For example, the amplified fragment can be labeled and used to screen a bacteriophage or cosmid cDNA library.", "Alternatively, the labeled fragment can be used to screen a genomic library.", "PCR technology also can be used to isolate full length cDNA sequences from other organisms.", "For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source.", "A reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5′ end of the amplified fragment for the priming of first strand synthesis.", "The resulting RNA/DNA hybrid can then be “tailed” (e.g., with guanines) using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed (e.g., with a poly-C primer).", "Thus, cDNA sequences upstream of the amplified fragment can easily be isolated.", "For a review of useful cloning strategies, see e.g., Sambrook et al., supra; and Ausubel et al., supra.", "Vectors Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a protease protein or a functional equivalent thereof.", "As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.", "One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.", "Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.", "Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).", "Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.", "Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked.", "Such vectors are referred to herein as “expression vectors”.", "In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.", "The terms “plasmid” and “vector” can be used interchangeably herein as the plasmid is the most commonly used form of vector.", "However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.", "The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.", "Within a recombinant expression vector, “operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).", "The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal).", "Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).", "Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g.", "tissue-specific regulatory sequences).", "It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.", "The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g.", "protease proteins, mutant forms of protease proteins, fragments, variants or functional equivalents thereof, fusion proteins, etc.).", "The recombinant expression vectors of the invention can be designed for expression of protease proteins in prokaryotic or eukaryotic cells.", "For example, protease proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells.", "Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).", "Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and 17 polymerase.", "Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.", "The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.", "Other suitable promoters will be known to the skilled person.", "In a specific embodiment, promoters are preferred that are capable of directing a high expression level of proteases in filamentous fungi.", "Such promoters are known in the art.", "The expression constructs may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.", "The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.", "Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.", "As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-percipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipid mediated transfection or electroporation.", "Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al.", "(Molecular Cloning: A Laboratory Manual, 2nd, ed.", "Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.", "For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome.", "In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.", "Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methatrexate.", "Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a protease protein or can be introduced on a separate vector.", "Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g.", "cells that have incorporated the selectable marker gene will survive, while the other cells die).", "Expression of proteins in prokaryotes is often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins.", "Fusion vectors add a number of amino acids to a protein encoded therein, e.g.", "to the amino terminus of the recombinant protein.", "Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.", "Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.", "Such enzymes, and their cognate recognation sequences, include Factor Xa, thrombin and enterokinase.", "As indicated, the expression vectors will preferably contain selectable markers.", "Such markers include dihydrofolate reductase or neomycin resistance for eukarotic cell culture and tetracyline or ampicilling resistance for culturing in E. coli and other bacteria.", "Representative examples of appropriate host include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma; and plant cells.", "Appropriate culture mediums and conditions for the above-described host cells are known in the art.", "Among vectors preferred for use in bacteria are pQE70, pQE60 and PQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.", "Among preferred eukaryotic vectors are PWLNEO, pSV2CAT, pOG44, pZT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.", "Other suitable vectors will be readily apparent to the skilled artisan.", "Among known bacterial promotors for use in the present invention include E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (“RSV”), and metallothionein promoters, such as the mouse metallothionein-I promoter.", "Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector.", "Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.", "Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.", "For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretation signal may be incorporated into the expressed polypeptide.", "The signals may be endogenous to the polypeptide or they may be heterologous signals.", "The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.", "Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.", "Also, peptide moieties may be added to the polypeptide to facilitate purification.", "Polypeptides According to the Invention The invention provides an isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171, an amino acid sequence obtainable by expressing a polynucleotide according to the invention or in a preferred embodiment of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 in an appropriate host, as well as an amino acid sequence obtainable by expressing a polynucleotide sequences selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 in an appropriate host.", "Also, a peptide or polypeptide comprising a functional equivalent of the above polypeptides is comprised within the present invention.", "The above polypeptides are collectively comprised in the term “polypeptides according to the invention” The terms “peptide” and “oligopeptide” are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages.", "The word “polypeptide” is used herein for chains containing more than seven amino acid residues.", "All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus.", "The one-letter code of amino acids used herein is commonly known in the art and can be found in Sambrook, et al.", "(Molecular Cloning: A Laboratory Manual, 2nd, ed.", "Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) By “isolated” polypeptide or protein is intended a polypeptide or protein removed from its native environment.", "For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).", "The protease according to the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.", "For analytical purposes most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.", "Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.", "Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.", "In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.", "Moreover, a protein according to the invention may be a precursor protein such as a zymogen, a hybrid protein, a protein obtained as a pro sequence or pre-pro sequence, or any other type of immature form.", "Protein Fragments The invention also features biologically active fragments of the polypeptides according to the invention.", "Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protease protein (e.g., the amino acid sequence of a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171), which include fewer amino acids than the full length protein, and exhibit at least one biological activity of the corresponding full-length protein.", "Typically, biologically active fragments comprise a domain or motif with at least one activity of the protease protein.", "A biologically active fragment of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.", "Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the invention.", "The invention also features nucleic acid fragments which encode the above biologically active fragments of the protease protein.", "Fusion Proteins The proteins of the present invention or functional equivalents thereof, e.g., biologically active portions thereof, can be operatively linked to a non-protease polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.", "As used herein, a protease “chimeric protein” or “fusion protein” comprises a protease polypeptide operatively linked to a non-protease polypeptide.", "A “protease polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a polypeptide sequence according to the invention, whereas a “non-protease polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to a protein according to the invention, e.g., a protein which is different from the protease protein and which is derived from the same or a different organism.", "Within a protease fusion protein the protease polypeptide can correspond to all or a portion of a protein according to the invention.", "In a preferred embodiment, a protease fusion protein comprises at least one biologically active fragment of a protein according to the invention.", "In another preferred embodiment, a protease fusion protein comprises at least two biologically active portions of a protein according to the invention.", "Within the fusion protein, the term “operatively linked” is intended to indicate that the protease polypeptide and the non-protease polypeptide are fused in-frame to each other.", "The non-protease polypeptide can be fused to the N-terminus or C-terminus of the protease polypeptide.", "For example, in one embodiment, the fusion protein is a GST-protease fusion protein in which the protease sequences are fused to the C-terminus of the GST sequences.", "Such fusion proteins can facilitate the purification of recombinant protease.", "In another embodiment, the fusion protein is a protease protein containing a heterologous signal sequence at its N-terminus.", "In certain host cells (e.g., mammalian and Yeast host cells), expression and/or secretion of protease can be increased through use of a hetereologous signal sequence.", "In another example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).", "Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, Calif.).", "In yet another example, useful prokarytic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).", "A signal sequence can be used to facilitate secretion and isolation of a protein or polypeptide of the invention.", "Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events.", "Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway.", "The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved.", "The protein can then be readily purified from the extracellular medium by art recognized methods.", "Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.", "Thus, for instance, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused polypeptide.", "In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag provided in a pOE vector (Qiagen, Inc.), among others, many of which are commercially available.", "As described in Gentz et al, Proc.", "Natl.", "Acad.", "Sci.", "USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purificaton of the fusion protein.", "The HA tag is another peptide useful for purification which corresponds to an epitope derived of influenza hemaglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984), for instance.", "Preferably, a protease chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.", "For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.", "In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.", "Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds.", "Ausubel et al.", "John Wiley & Sons: 1992).", "Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide).", "A protease-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protease protein.", "Functional Equivalents The terms “functional equivalents” and “functional variants” are used interchangeably herein.", "Functional equivalents of a DNA according to the invention are isolated DNA fragments that encode a polypeptide that exhibits a particular function of an A. niger protease as defined herein.", "A functional equivalent of a polypeptide according to the invention is a polypeptide that exhibits at least one function of an A. niger protease as defined herein.", "Functional protein or polypeptide equivalents may contain only conservative substitutions of one or more amino acids of a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or substitutions, insertions or deletions of non-essential amino acids.", "Accordingly, a non-essential amino acid is a residue that can be altered in a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 without substantially altering the biological function.", "For example, amino acid residues that are conserved among the protease proteins of the present invention, are predicted to be particularly unamenable to alteration.", "Furthermore, amino acids conserved among the protease proteins according to the present invention and other proteases are not likely to be amenable to alteration.", "The term “conservative substitution” is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain.", "These families are known in the art and include amino acids with basic side chains (e.g.", "lysine, arginine and hystidine), acidic side chains (e.g.", "aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).", "Functional nucleic acid equivalents may typically contain silent mutations or mutations that do not alter the biological function of encoded polypeptide.", "Accordingly, the invention provides nucleic acid molecules encoding protease proteins that contain changes in amino acid residues that are not essential for a particular biological activity.", "Such protease proteins differ in amino acid sequence from a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 yet retain at least one biological activity.", "In one embodiment the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises a substantially homologous amino acid sequence of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171.For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., Science 247:1306-1310 (1990) wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change.", "The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.", "The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selects or screens to identify sequences that maintain functionality.", "As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions.", "The authors further indicate which changes are likely to be permissive at a certain position of the protein.", "For example, most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved.", "Other such phenotypically silent substitutions are described in Bowie et al, supra, and the references cited therein.", "An isolated nucleic acid molecule encoding a protease protein homologous to the protein selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 can be created by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequences according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded protein.", "Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.", "The term “functional equivalents” also encompasses orthologues of the A. niger protease protein.", "Orthologues of the A. niger protease protein are proteins that can be isolated from other strains or species and possess a similar or identical biological activity.", "Such orthologues can readily be identified as comprising an amino acid sequence that is substantially homologous to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO:171.As defined herein, the term “substantially homologous” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second amino acid or nucleotide sequence such that the first and the second amino acid or nucleotide sequences have a common domain.", "For example, amino acid or nucleotide sequences which contain a common domain having about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or more are defined herein as sufficiently identical.", "Also, nucleic acids encoding other protease family members, which thus have a nucleotide sequence that differs from a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114, are within the scope of the invention.", "Moreover, nucleic acids encoding protease proteins from different species which thus have a nucleotide sequence which differs from a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 are within the scope of the invention.", "Nucleic acid molecules corresponding to variants (e.g.", "natural allelic variants) and homologues of the protease DNA of the invention can be isolated based on their homology to the protease nucleic acids disclosed herein using the cDNAs disclosed herein or a suitable fragment thereof, as a hybridisation probe according to standard hybridisation techniques preferably under highly stringent hybridisation conditions.", "In addition to naturally occurring allelic variants of the protease sequence, the skilled person will recognise that changes can be introduced by mutation into the nucleotide sequences of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 thereby leading to changes in the amino acid sequence of the protease protein without substantially altering the function of the protease protein.", "In another aspect of the invention, improved protease proteins are provided.", "Improved protease proteins are proteins wherein at least one biological activity is improved.", "Such proteins may be obtained by randomly introducing mutations along all or part of the protease coding sequence, such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity.", "For instance, the art provides for standard assays for measuring the enzymatic activity of proteases and thus improved proteins may easily be selected.", "In a preferred embodiment the protease protein has an amino acid sequence according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171.In another embodiment, the protease polypeptide is substantially homologous to the amino acid sequence according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 and retains at least one biological activity of a polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171, yet differs in amino acid sequence due to natural variation or mutagenesis as described above.", "In a further preferred embodiment, the protease protein has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridising to a nucleic acid according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114, preferably under highly stringent hybridisation conditions.", "Accordingly, the protease protein is a protein which comprises an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 and retains at least one functional activity of the polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171.Functional equivalents of a protein according to the invention can also be identified e.g.", "by screening combinatorial libraries of mutants, e.g.", "truncation mutants, of the protein of the invention for protease activity.", "In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level.", "A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).", "There are a variety of methods that can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence.", "Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al.", "(1984) Annu.", "Rev.", "Biochem.", "53:323; Itakura et al.", "(1984) Science 198:1056; Ike et al.", "(1983) Nucleic Acid Res.", "11:477).", "In addition, libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants.", "For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector.", "By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.", "Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations of truncation, and for screening cDNA libraries for gene products having a selected property.", "The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.", "Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan (1992) Proc.", "Natl.", "Acad.", "Sci.", "USA 89:7811-7815; Delgrave et al.", "(1993) Protein Engineering 6(3):327-331).", "In addition to the protease gene sequence shown in a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57, it will be apparent for the person skilled in the art that DNA sequence polymorphisms that may lead to changes in the amino acid sequence of the protease protein may exist within a given population.", "Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation.", "Allelic variants may also include functional equivalents.", "Fragments of a polynucleotide according to the invention may also comprise polynucleotides not encoding functional polypeptides.", "Such polynucleotides may function as probes or primers for a PCR reaction.", "Such polynucleotides may also be useful when it is desired to abolish the functional activity of a protease in a particular organism (knock-out mutants).", "Nucleic acids according to the invention irrespective of whether they encode functional or non-functional polypeptides, can be used as hybridization probes or polymerase chain reaction (PCR) primers.", "Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having a protease activity include, inter alia, (1) isolating the gene encoding the protease protein, or allelic variants thereof from a cDNA library e.g.", "from other organisms than A. niger; (2) in situ hybridization (e.g.", "FISH) to metaphase chromosomal spreads to provide precise chromosomal location of the protease gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis for detecting expression of protease mRNA in specific tissues and/or cells and 4) probes and primers that can be used as a diagnostic tool to analyse the presence of a nucleic acid hybridisable to the protease probe in a given biological (e.g.", "tissue) sample.", "Also encompassed by the invention is a method of obtaining a functional equivalent of a protease gene or cDNA.", "Such a method entails obtaining a labelled probe that includes an isolated nucleic acid which encodes all or a portion of the sequence according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or a variant thereof; screening a nucleic acid fragment library with the labelled probe under conditions that allow hybridisation of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes, and preparing a full-length gene sequence from the nucleic acid fragments in any labelled duplex to obtain a gene related to the protease gene.", "In one embodiment, a protease nucleic acid of the invention is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to a nucleic acid sequence shown in a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57, a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 or the complement thereof.", "In another preferred embodiment a protease polypeptide of the invention is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the amino acid sequence shown in a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171.Host Cells In another embodiment, the invention features cells, e.g., transformed host cells or recombinant host cells that contain a nucleic acid encompassed by the invention.", "A “transformed cell” or “recombinant cell” is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention.", "Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are cells from filamentous fungi, in particular Aspergillus niger.", "A host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion.", "Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.", "Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products.", "Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed.", "To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.", "Such host cells are well known in the art.", "Host cells also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and choroid plexus cell lines.", "If desired, the polypeptides according to the invention can be produced by a stably-transfected cell line.", "A number of vectors suitable for stable transfection of mammalian cells are available to the public, methods for constructing such cell lines are also publicly known, e.g., in Ausubel et al.", "(supra).", "Antibodies The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind protease proteins according to the invention.", "As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protease protein.", "Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl.", "Med.", "24:316-325 (1983)).", "Thus, these fragments are preferred.", "The antibodies of the present invention may be prepared by any of a variety of methods.", "For example, cells expressing the protease protein or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.", "In a preferred method, a preparation of protease protein is prepared and purified to render it substantially free of natural contaminants.", "Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.", "In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protease protein binding fragments thereof).", "Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur.", "J. Immunol.", "6:511 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp.", "563-681 (1981)).", "In general, such procedures involve immunizing an animal (preferably a mouse) with a protease protein antigen or, with a protease protein expressing cell.", "The splenocytes of such mice are extracted and fused with a suitable myeloma cell line.", "Any suitable myeloma cell line may be employed in accordance with the present inventoin; however, it is preferably to employ the parent myeloma cell line (SP2O), available from the American Type Culture Collection, Rockville, Md.", "After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al.", "(Gastro-enterology 80.225-232 (1981)).", "The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the protease protein antigen.", "In general, the polypeptides can be coupled to a carrier protein, such as KLH, as described in Ausubel et al., supra, mixed with an adjuvant, and injected into a host mammal.", "In particular, various host animals can be immunized by injection of a polypeptide of interest.", "Examples of suitable host animals include rabbits, mice, guinea pigs, and rats.", "Various adjuvants can be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), adjuvant mineral gels such as aluminum hydroxide, surface actve substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, BCG (bacille Calmette-Guerin) and Corynebacterium parvum.", "Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.", "Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.", "The hybridomas producing the mAbs of this invention can be cultivated in vitro or in vivo.", "Once produced, polyclonal or monoclonal antibodies are tested for specific recognition of an protease polypeptide or functional equivalent thereof in an immunoassay, such as a Western blot or immunoprecipitation analysis using standard techniques, e.g., as described in Ausubel et al., supra.", "Antibodies that specifically bind to protease proteins or functional equivalents thereof are useful in the invention.", "For example, such antibodies can be used in an immunoassay to detect protease in pathogenic or non-pathogenic strains of Aspergillus (e.g., in Aspergillus extracts).", "Preferably, antibodies of the invention are produced using fragments of the protease polypeptides that appear likely to be antigenic, by criteria such as high frequency of charged residues.", "For example, such fragments may be generated by standard techniques of PCR, and then cloned into the pGEX expression vector (Ausubel et al., supra).", "Fusion proteins may then be expressed in E. coli and purified using a glutathione agarose affinity matrix as described in Ausubel, et al., supra.", "If desired, several (e.g., two or three) fusions can be generated for each protein, and each fusion can be injected into at least two rabbits.", "Antisera can be raised by injections in a series, typically including at least three booster injections.", "Typically, the antisera are checked for their ability to immunoprecipitate a recombinant protease polypeptide or functional equivalents thereof whereas unrelated proteins may serve as a control for the specificity of the immune reaction.", "Alternatively, techniques decribed for the production of single chain antibodies (U.S. Pat.", "Nos.", "4,946,778 and 4,704,692) can be adapted to produce single chain antibodies against a protease polypeptide or functional equivalents thereof.", "Kits for generating and screening phage display libraries are commercially available e.g.", "from Pharmacia.", "Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat.", "No.", "5,223,409; PCT Publication No.", "WO 92/18619; PCT Publication No.", "WO 91/17271; PCT Publication No.", "WO 20791; PCT Publication No.", "WO 92/20791; PCT Publication No.", "WO 92/15679; PCT Publication No.", "WO 93/01288; PCT Publication No.", "WO 92/01047; PCT Publication No.", "WO 92/09690; PCT Publication No.", "WO 90/02809; Fuchs et al.", "(1991) Bio/Technology 9:1370-1372; Hay et al.", "(1992) Hum.", "Antibod.", "Hybridomas 3:81-85; Huse et al.", "(1989) Science 246;1275-1281; Griffiths et al.", "(1993) EMBO J.", "12:725-734.Polyclonal and monoclonal antibodies that specifically bind protease polypeptides of functional equivalents thereof can be used, for example, to detect expression of a protease gene or a functional equivalent thereof e.g.", "in another strain of Aspergillus.", "For example, protease polypeptide can be readily detected in conventional immunoassays of Aspergillus cells or extracts.", "Examples of suitable assays include, without limitation, Western blotting, ELISAs, radioimmune assays, and the like.", "By “specifically binds” is meant that an antibody recognizes and binds a particular antigen, e.g., a protease polypeptide, but does not substantially recognize and bind other unrelated molecules in a sample.", "Antibodies can be purified, for example, by affinity chromatography methods in which the polypeptide antigen is immobilized on a resin.", "An antibody directed against a polypeptide of the invention (e.g., monoclonal antibody) can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation.", "Moreover, such an antibody can be used to detect the protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide.", "The antibodies can also be used diagnostically to monitor protein levels in cells or tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen or in the diagnosis of Aspergillosis.", "Detection can be facilitated by coupling the antibody to a detectable substance.", "Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.", "Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125I, 131I, 35S or 3H.", "Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions.", "Hydrophobicity plots of the proteins of the invention can be used to identify hydrophilic regions.", "The antigenic peptide of a protein of the invention comprises at least 7 (preferably 10, 15, 20, or 30) contiguous amino acid residues of the amino acid sequense of a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.", "Preferred epitopes encompassed by the antigenic peptide are regions of protease that are located on the surface of the protein, e.g., hydrophilic regions, hydrophobic regions, alpha regions, beta regions, coil regions, turn regions and flexible regions.", "Immunoassays Qualitative or quantitative determination of a polypeptide according to the present invention in a biological sample can occur using any art-known method.", "Antibody-based techniques provide special advantages for assaying specific polypeptide levels in a biological sample.", "In these, the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies.", "As a result, an immunocomplex is obtained.", "Accordingly, the invention provides a method for diagnosing whether a certain organism is infected with Aspergillus comprising the steps of: Isolating a biological sample from said organism suspected to be infected with Aspergillus, reacting said biological sample with an antibody according to the invention, determining whether immune complexes are formed.", "Tissues can also be extracted, e.g., with urea and neutral detergent, for the liberation of protein for Western-blot or dot/slot assay.", "This technique can also be applied to body fluids.", "Other antibody-based methods useful for detecting protease gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).", "For example, protease-specific monoclonal antibodies can be used both as an immunoabsorbent and as an enzyme-labeled probe to detect and quantify the protease protein.", "The amount of protease protein present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.", "In another ELISA assay, two distinct specific monoclonal antibodies can be used to detect protease protein in a biological fluid.", "In this assay, one of the antibodies is used as the immuno-absorbent and the other as the enzyme-labeled probe.", "The above techniques may be conducted essentially as a “one-step” or “two-step” assay.", "The “one-step” assay involves contacting protease protein with immobilized antibody and, without washing, contacting the mixture with the labeled antibody.", "The “two-step” assay involves washing before contacting the mixture with the labeled antibody.", "Other conventional methods may also be employed as suitable.", "It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample.", "Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate.", "Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labelled antibody/substrate reaction.", "Besides enzymes, other suitable labels include radioisotopes, such as iodine (125I, 121I), carbon (14C), sulphur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.", "Specific binding of a test compound to a protease polypeptide can be detected, for example, in vitro by reversibly or irreversibly immobilizing the protease polypeptide on a substrate, e.g., the surface of a well of a 96-well polystyrene microtitre plate.", "Methods for immobilizing polypeptides and other small molecules are well known in the art.", "For example, the microtitre plates can be coated with a protease polypeptide by adding the polypeptide in a solution (typically, at a concentration of 0.05 to 1 mg/ml in a volume of 1-100 ul) to each well, and incubating the plates at room temperature to 37° C. for 0.1 to 36 hours.", "Polypeptides that are not bound to the plate can be removed by shaking the excess solution from the plate, and then washing the plate (once or repeatedly) with water or a buffer.", "Typically, the polypeptide is contained in water or a buffer.", "The plate is then washed with a buffer that lacks the bound polypeptide.", "To block the free protein-binding sites on the plates, the plates are blocked with a protein that is unrelated to the bound polypeptide.", "For example, 300 ul of bovine serum albumin (BSA) at a concentration of 2 mg/ml in Tris-HCl is suitable.", "Suitable substrates include those substrates that contain a defined cross-linking chemistry (e.g., plastic substrates, such as polystyrene, styrene, or polypropylene substrates from Corning Costar Corp. (Cambridge, Mass.", "), for example).", "If desired, a beaded particle, e.g., beaded agarose or beaded sepharose, can be used as the substrate.", "Binding of the test compound to the polypeptides according to the invention can be detected by any of a variety of art known methods.", "For example, a specific antibody can be used in an immunoassay.", "If desired, the antibody can be labeled (e.g., fluorescently or with a radioisotope) and detected directly (see, e.g., West and McMahon, J.", "Cell Biol.", "74:264, 1977).", "Alternatively, a second antibody can be used for detection (e.g., a labeled antibody that binds the Fc portion of an anti-AN97 antibody).", "In an alternative detection method, the protease polypeptide is labeled, and the label is detected (e.g., by labeling a protease polypeptide with a radioisotope, fluorophore, chromophore, or the like).", "In still another method, the protease polypeptide is produced as a fusion protein with a protein that can be detected optically, e.g., green fluorescent protein (which can be detected under UV light).", "In an alternative method, the protease polypeptide can be covalently attached to or fused with an enzyme having a detectable enzymatic activity, such as horse radish peroxidase, alkaline phosphatase, a-galactosidase, or glucose oxidase.", "Genes encoding all of these enzymes have been cloned and are readily available for use by those of skill in the art.", "If desired, the fusion protein can include an antigen, and such an antigen can be detected and measured with a polyclonal or monoclonal antibody using conventional methods.", "Suitable antigens include enzymes (e.g., horse radish peroxidase, alkaline phosphatase, and a-galactosidase) and non-enzymatic polypeptides (e.g., serum proteins, such as BSA and globulins, and milk proteins, such as caseins).", "Epitopes, Antigens and Immunogens.", "In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.", "The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.", "An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.", "These immunogenic epitopes are believed to be confined to a few loci on the molecule.", "On the other hand, a region of a protein molecule to which an antibody can bind is defined as an “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes.", "See, for instance, Geysen, H. M. et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 81:3998-4002 (1984).", "As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein.", "See, for instance, Sutcliffe, J. G. et al., Science 219:660-666 (1984).", "Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.", "Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer, soluble peptides, especially those containing proline residues, usually are effective.", "Sutcliffe et al., supra ?.", "For instance, 18 of 20 peptides designed according to these guidelines, containing 8-39 residues covering 75% of the sequence of the influenza virus hemagglutinin HAI polypeptide chain, induced antibodies that reacted with the HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.", "Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention.", "Thus, a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with an antigen epitope-bearing peptide generally secrete antibody reactive with the native protein.", "Sutcliffe et al., supra, at 663.The antibodies raised by antigenic epitope bearing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes posttranslation processing.", "The peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about 9 amino acids) can bind and displace the larger peptides in immunoprecipitation assays.", "See, for instance, Wilson, I.", "A. et al., Cell 37:767-778 at 777 (1984).", "The anti-peptide antibodies of the invention also are useful for purification of the mimicked protein, for instance, by adsorption chromatography using methods well known in the art.", "Antigenic epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.", "However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein.", "Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.", "The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention.", "For instance, a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies.", "Epitope-bearing peptides also may be synthesized using known methods of chemical synthesis.", "For instance, Houghten has described a simple method for synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing single amino acid variants of a segment of the HAI polypeptide which were prepared and characterized (by ELISA-type binding studies) in less than four weeks.", "Houghten, R. A., Proc.", "Natl.", "Acad.", "Sci.", "USA 82:5131-5135 (1985).", "This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in U.S. Pat.", "No.", "4,631,211 to Houghten et al.", "(1986).", "In this procedure the individual resins for the solid-phase synthesis of various peptides are contained in separate solvent-permeable packets, enabling the optimal use of the many identical repetitive steps involved in solid-phase methods.", "A manual procedure allows 500-1000 or more syntheses to be conducted simultaneously.", "Houghten et al., supra, at 5134.Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art.", "See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 82:910-914; and Bittle, F. J. et al., J. Gen. Virol.", "66:2347-2354 (1985).", "Generally, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid.", "For instance, peptides containing cysteine may be coupled to carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde.", "Animals such as rabbits, rats and mice are immunized with either free or carrier coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ug peptide or carrier protein and Freund's adjuvant.", "Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.", "The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.", "Immunogenic epitope-bearing peptides of the invention, i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art.", "For instance, Geysen et al., 1984, supra, discloses a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay.", "Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support.", "In this manner a peptide bearing an immunogenic epitope of a desired protein may be identified routinely by one of ordinary skill in the art.", "For instance, the immunologically important epitope in the coat protein of foot-and-mouth disease virus was located by Geysen et al.", "with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein.", "Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope were synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined.", "Thus, peptide analogs of the epitope-bearing peptides of the invention can be made routinely by this method.", "U.S. Pat.", "No.", "4,708,781 to Geysen (1987) further describes this method of identifying a peptide bearing an immunogenic epitope of a desired protein.", "Further still, U.S. Pat.", "No.", "5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a “mimotope”) which is complementary to a particular paratope (antigen binding site) of an antibody of interest.", "More generally, U.S. Pat.", "No.", "4,433,092 to Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest.", "Similarly, U.S. Pat.", "No.", "5,480,971 to Houghten, R. A. et al.", "(1996) on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest.", "Thus, non-peptide analogs of the epitope-bearing peptides of the invention also can be made routinely by these methods.", "Removal or Reduction of Protease Activity The present invention also relates to methods for producing a mutant cell of a parent cell, which comprises disrupting or deleting a nucleic acid sequence encoding the protease or a control sequence thereof, which results in the mutant cell producing less of the protease than the parent cell.", "The construction of strains which have reduced protease activity may be conveniently accomplished by modification or inactivation of a nucleic acid sequence necessary for expression of the protease activity in the cell.", "The nucleic acid sequence to be modified or inactivated may be, for example, a nucleic acid sequence encoding the protease or a part thereof essential for exhibiting protease activity, or the nucleic acid sequence may have a regulatory function required for the expression of the protease from the coding sequence of the nucleic acid sequence.", "An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i.e., a part which is sufficient for affecting expression of the protease.", "Other control sequences for possible modification include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a signal sequence, and a termination site.", "Modification or inactivation of the nucleic acid sequence may be performed by subjecting the cell to mutagenesis and selecting for cells in which the protease producing capability has been reduced or eliminated.", "The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis.", "Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.", "Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.", "When such agents are used, the mutagenesis is typically performed by incubating the cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for cells exhibiting reduced or no expression of protease activity.", "Modification or inactivation of production of a protease of the present invention may be accomplished by introduction, substitution, or removal of one or more nucleotides in the nucleic acid sequence encoding the protease or a regulatory element required for the transcription or translation thereof.", "For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change of the open reading frame.", "Such modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art.", "Although, in principle, the modification may be performed in vivo, i.e., directly on the cell expressing the nucleic acid sequence to be modified, it is preferred that the modification be performed in vitro as exemplified below.", "An example of a convenient way to inactivate or reduce production by a host cell of choice is based on techniques of gene replacement or gene interruption.", "For example, in the gene interruption method, a nucleic acid sequence corresponding to the endogenous gene or gene fragment of interest is mutagenized in vitro to produce a defective nucleic acid sequence which is then transformed into the host cell to produce a defective gene.", "By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene or gene fragment.", "It may be desirable that the defective gene or gene fragment also encodes a marker which may be used for selection of transformants in which the gene encoding the protease has been modified or destroyed.", "Alternatively, modification or inactivation of the nucleic acid sequence encoding a protease of the present invention may be performed by established anti-sense techniques using a nucleotide sequence complementary to the protease encoding sequence.", "More specifically, production of the protease by a cell may be reduced or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence encoding the protease which may be transcribed in the cell and is capable of hybridizing to the protease mRNA produced in the cell.", "Under conditions allowing the complementary antisense nucleotide sequence to hybridize to the protease mRNA, the amount of protease translated is thus reduced or eliminated.", "It is preferred that the cell to be modified in accordance with the methods of the present invention is of microbial origin, for example, a fungal strain which is suitable for the production of desired protein products, either homologous or heterologous to the cell.", "The present invention further relates to a mutant cell of a parent cell which comprises a disruption or deletion of a nucleic acid sequence encoding the protease or a control sequence thereof, which results in the mutant cell producing less of the protease than the parent cell.", "The protease-deficient mutant cells so created are particularly useful as host cells for the expression of homologous and/or heterologous polypeptides.", "Therefore, the present invention further relates to methods for producing a homologous or heterologous polypeptide comprising (a) culturing the mutant cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.", "In the present context, the term “heterologous polypeptides” is defined herein as polypeptides which are not native to the host cell, a native protein in which modifications have been made to alter the native sequence, or a native protein whose expression is quantitatively altered as a result of a manipulation of the host cell by recombinant DNA techniques.", "The methods of the present invention for producing an essentially protease-free product is of particular interest in the production of eukaryotic polypeptides, in particular fungal proteins such as enzymes.", "The protease-deficient cells may also be used to express heterologous proteins of interest for the food industry, or of pharmaceutical interest.", "Use of Proteases in Industrial Processes The invention also relates to the use of the protease according to the invention in a selected number of industrial and pharmaceutical processes.", "Despite the long term experience obtained with these processes, the protease according to the invention features a number of significant advantages over the enzymes currently used.", "Depending on the specific application, these advantages can include aspects like lower production costs, higher specificity towards the substrate, less antigenic, less undesirable side activities, higher yields when produced in a suitable microorganism, more suitable pH and temperature ranges, better tastes of the final product as well as food grade and kosher aspects.", "In large scale industrial applications aimed at food or feed production, proteolytic enzymes are commonly used to improve aspects like protein solubility, extraction yields, viscosity or taste, texture, nutritional value, minimalisation of antigenicity or antinutrional factors, colour or functionality as well as processing aspects like filterablity of the proteinaceous raw material.", "In these applications the proteinaceous raw material can be of animal or vegetable origin and examples include vegetable proteins such as soy protein, wheat gluten, rape seed protein, pea protein, alfalfa protein, sunflower protein, fabaceous bean protein, cotton or sesame seed protein, maize protein, barley protein, sorghum protein, potato protein, rice protein, coffee proteins, and animal derived protein such as milk protein (e.g.", "casein, whey protein), egg white, fish protein, meat protein including gelatin, collagen, blood protein (e.g.", "haemoglobin), hair, feathers and fish meal.", "An important aspect of the proteases according to the invention is that they cover a whole range of pH and temperature optima which are ideally suited for a variety of applications.", "For example many large scale processes benefit from relatively high processing temperatures of 50 degrees C. or higher to control the risks of microbial infections.", "Several proteases according to the invention comply with this demand but at the same time exhibit no extreme heat stabilities so that they resist attempts to inactivate the enzyme by an additional heat treatment.", "The latter feature allows production routes that yield final products free of residual proteolytic activity.", "Similarly many feed and food products have slightly acidic pH values so that for their processing proteases with acidic or near neutral pH optima are preferred.", "A protease according to the invention complies with this requirement as well.", "The specificity of endoproteases is usually defined in terms of preferential cleavages of bonds between the carboxyl of the amino acid residue in position P1 and the amino group of the residue in position P1′ respectively.", "The preference may be conditioned predominantly either by P1 (e.g.", "positively charged residues in substrates for trypsin), by P1′ (e.g.", "hydrophobic residues in cleavages by thermolysin) or by both P1 and P2 (e.g.", "specific cleavages between two positively charged residues by adrenal medulla serine endoprotease).", "In some cases more distant residues may determine the cleavage preference, e.g.", "P2 for streptococcal peptidase A.", "Some residues are known to influence cleavages negatively; it is well known that bonds with proline in position P1′ are resistant to the action of many proteases.", "Most endoproteases cleave preferentially either in a hydrophobic environment or in the proximity of negatively charged residues.", "For example, industrially available endoproteases like chymotrypsin (obtained from bovine pancreas) or subtilisin, neutral metallo endoprotease or thermolysin (all obtained from Bacillus species) tend to favour cleavage “behind” hydrophobic amino acids like -Phe, -Leu and -Tyr.", "Other industrially available endoproteases are trypsin (obtained from bovine pancreas) preferring cleavage behind -Arg and -Lys and papain (a complex mixture of various enzymes including proteases obtained from papaya fruits) preferring cleavage behind -Arg.", "In contrast, peptide bonds formed by small sized residues such as Ala, Gly, Ser, Thre as well as IIe and Pro are poor substrates (Keil, B et al.", "; Protein Seq Data Anal (1993) 5; 401-407).", "This situation has a profound implications for the pharmaceutical, the food and beverages, the agro and even the chemical industry.", "A protease according to the invention exhibits uncommon cleavage preferences.", "The exopeptidases act only near the ends of polypeptide chains.", "Those acting at a free N-terminus liberate a single amino acid residue (socalled aminopeptidases) or a dipeptide or a tripeptide (socalled dipeptidyl-peptidases and tripeptidyl-peptidases) Those acting at a free C-terminus liberate a single residue (socalled carboxypeptidases) or a dipeptide (socalled peptidyl-dipeptidases) The carboxypeptidases are allocated to three groups on the basis of catalytic mechanism i.e.", "serine-type carboxypeptidases, metallocarboxypeptidases and cystein-type carboxypeptidases.", "Other exopeptidases are specific for dipeptides (socalled dipeptidases) or are able to cleave peptide linkages other than those of alpha-carboxyl or alpha-amino groups (socalled omega peptidases).", "Examples of such new omega peptidases are the pyroglutamyl-peptidase and the acylaminoacyl-peptidase as identified in the present invention (see Table 1, genes 18 and 45 respectively).", "Typical examples of industrial application which depend on the use of pure endoproteases and in which the protease according to the invention can be expected to deliver a superior performance include the processing of materials of vegetable or animal origin.", "These processing steps can be-aimed at modifying a large array of characteristics of either the crude material or the (partially) purified protein fraction.", "For example, these processing steps can be aimed at maximising product solubilities, filterabilities, separabilities, protein extraction yields and digestibilities or minimising toxicities, off-tastes and viscosities.", "Furthermore the treatment can be directed at altering physico-chemical characteristics of the crude material or the purified (or partially purified) protein.", "These advantages apply not only if the endoprotease according to the invention is applied as a processing aid in industrial applications but also if applied as an active enzyme component in animal feed.", "Specifically the endoprotease according to the invention can be applied as bread improver in the bakery industry, e.g.", "to retard the staling of bread or to diminishing the viscosity of doughs.", "Or the endoprotease can be used in the beer and wine industry to prevent or to minimise the formation of undesirable protein hazes.", "Alternatively it can be used in the beer industry to optimise the protein extraction yields of cereals used in the preparation of the wort.", "Furthermore, it can also be advantageously used in the dairy industry as a milk clotting agent with superior characteristics or to optimise the texturising, foaming or setting characteristics of various milk components.", "Another application in the dairy industry is the use of the new protease in the preparation of Enzyme Modified Cheeses (EMC's).", "Moreover, various proteinaceous substrates can be subjected to an endoprotease according to the invention, usually in combination with other proteolytic enzymes to obtain hydrolysates for medical or non-medical applications.", "Here the endoprotease according to the invention is surprisingly effective in achieving a complete hydrolysis of the proteinaceous substrate so that even protease resistant parts are fully hydrolysed, the endoprotease is also surprisingly active in minimising the allergenicity of the final hydrolysate or in suppressing the formation of bitter off-tastes.", "More specifically the endoprotease according to the invention is characterised by its preference for cleaving proteins at unusual peptide bonds, especially with the small size amino acid residues of Ala, Gly, Ser and Thr, or the residues lie and Pro in either the P1 or the P1′ position (Keil, B et al.", "; Protein Seq Data Anal (1993) 5; 401-407).", "As the result those fractions of the proteinaceous starting materials that resist hydrolysis upon using prior art endoproteases, can be dissolved and hydrolysed using the endoprotease according to the invention.", "Non limiting examples of such protease resistant fractions include socalled extensins in plant materials and collagen, gelatin but also specific milk components in material of animal origen.", "Various feedstuffs such as e.g.", "soybeans contain trypsin inhibitors.", "These proteins inhibit trypsin activity in the GI-tract of e.g.", "pigs and poultry.", "This trypsin inhibiting activity results in sub-optimal protein digestibility in these animals resulting in increased waste production and poor economics.", "This problem may partly be overcome by toasting soybeans at high temperatures.", "Two different types of trypsin inhibitors have been identified in soybeans, i.e.", "the Bowman-Birk type trypsin inhibitors and the Kunitz type trypsin inhibitors.", "This invention now provides an alternative way to degrade trypsin inhibiting activity over toasting, in that it provides a cysteine proteases (EC 3.4.22, table 1) capable of cleaving at Leucine176-Aspartate177 peptide bond near the carboxyl-terminus of the Kunitz type trypsin inhibitor (as reviewed by Wilson (1988) in CRC Critical Reviews in Biotechnology 8 (3): 197-216).", "This results in inactivation of this trypsin inhibitor in soybean.", "It was surprisingly found that the cysteine proteases secreted by the fungus Aspergillus niger fulfilled these criteria far better than similar enzymes derived from other organisms.", "Proteases are also widely used in the art of cheese-making.", "In the production of cheese it is necessary to coagulate the cheese milk to be able to separate the cheese matters e.g.", "casein from the whey.", "Several milk coagulating enzymes, also referred to as coagulants, have been described and include (bovine) chymosin, bovine pepsin, porcine pepsin as well as microbial enzymes like Rhizomucor miehei protease, Rhizomucor pusillus protease and Cryptonectria parasitica protease.", "Chymosin can be obtained from calf stomachs but can also be produced microbially by for example Kluyveromyces lactis.", "All these enzymes are characterized by having specificity for the peptide bond between residue 105 (phenylalanine) and residue 106 (methionine) or the bond adjacent to that in K-casein.", "This means that by employing these enzymes in cheese making, the K-casein is split at the junction between para-K-casein and the macro-peptide moiety called glycomacropeptide (GMP) carrying the negative charges.", "When this occurs the macropeptide diffuses into the whey, its stabilizing effect on the solubility of the casein micelles is lost, and the casein micelles can start to aggregate once sufficient kappa-casein has been hydrolyzed.", "For further elaboration on the enzymatic coagulation of milk (e.g.", "D. G. Dalgleish in Advanced Dairy Chemistry vol.", "1 ed by P. F. Fox, Elsevier, London, 1992.The currently available coagulants allow for a rather high yield of cheese, however, it should be realised that due to the enormous volumes of cheese produced, an increased yield in the order of magnitude of tenths of percent points may constitute a substantial economical advantage.", "Consequently there is a great need in the art for coagulants with an (even slightly) improved yield.", "Coagulants are characterized by their high substrate specificity, which is, however, dependent on pH and temperature.", "In a typical cheese making process the pH will change from the initial pH 6.3 to lower pH values in the range of 4.5-5.5, the end-value depends on the conditions used during the cheese production process.", "Some coagulants are more sensitive to pH changes than others.", "The Rhizomucor pusillus protease for example is more sensitive to pH changes than chymosin.", "Besides pH, also other parameters like temperature and water content may affect the protease specificity.", "It is well known that most coagulants show a changing substrate specificity with changing pH, resulting in altered proteolytic activity in later stages of the cheese making process.", "It is also well known that coagulants differ in the extent of casein proteolysis; they may also show differences in the peptide patterns produced during proteolysis.", "These are relevant factors during cheese ripening and may affect cheese properties like taste, flavor and texture.", "In some cases coagulants give rise to undesired effects like the formation of bitter tasting peptides or off-taste.", "In addition, changes in proteolytic specificity may lead to a reduction in yield.", "Pepsin, a well known component in many bovine chymosin preparations, is an example of a protease that gives rise to lower yields and taste effects as compared to pure chymosin.", "There is still a need for coagulants with give rise to new, improved cheese texture and taste.", "Such new coagulants result in the accelerated development of taste and texture profiles related to cheese aging, therewith providing a substantial economical benefit.", "It is well known that free amino acids are very important in taste and flavour generation.", "Especially the amino acids leucine, phenylalanine, methionine and valine play an important role in the generation of typical cheese taste and flavor components.", "The free amino acids are converted via fermentation by micro organisms that are added during the cheese manufacturing process into the actual flavor and taste generating compounds like methanediol, dimethyldisulphide, methylpropanoic acid and methylpropanal.", "Exo-peptidases play an important role in the generation of free amino acids.", "They can only be effective, however, when they are combined with an endo-protease of appropriate specificity.", "Appropriate combinations of exo- and endo-peptidases can be used in cheese making, resulting in the manufacture of cheeses with new and improved taste profiles.", "The enzymes according to the invention may be used to hydrolyze proteinaceous materials of animal origin such as whole milk, skim milk, casein, whey protein or mixtures of casein and whey protein.", "Such mixtures of casein and whey protein may be used, for example, in ratios similar to those found in human milk.", "Furthermore, the enzyme mixture according to the invention may be used to hydrolyze proteinaceous materials of plant origin such as, for example, wheat gluten malted or unmalted barley or other cereals used for making beer, soy milk, concentrates or isolates thereof, maize protein concentrates and isolates thereof, and rice proteins.", "Within the area of large scale industrial processes, some applications rely on the use of endoproteases only whereas in other applications combinations of endoproteases with exoproteases are essential.", "Typical examples which depend on the use of pure endoproteases and in which the protease according to the invention can deliver a superior performance include applications like the processing of soy or peas or cereals proteins aimed at minimising viscosities or optimising foaming or other physics-chemical characteristics, bread improvers in the bakery industry also aimed at diminishing the viscosity of doughs, processing aids in the beer and wine industry aimed at the prevention of protein hazes or optimising the extraction yields of cereals, feed additives in the bio industry aimed at enhancing intestinal absorption or modulating microbial activities in the gut, processing aids in the dairy industry aimed at optimising the clotting, foaming or setting characteristics of various milk components.", "Moreover, v For specific market segments proteins derived from milk or soy or collagen are exposed to proteases to produce socalled protein hydrolysates.", "Although the main outlets for these protein hydrolysates are infant formula and food products for hospitalised persons, products intended for persons with non-medical needs, such as athletes or people on a slimming diet form a rapidly growing segment.", "In all of these applications protein hydrolysates offer attractive advantages such as lowered allergenicities, facilitated gastrointestinal uptake, less chemical deterioration of desirable amino acids like glutamine and cystein and finally, absence of proteinaceous precipitations in acid beverages during prolonged storage periods.", "All these advantages can be combined if the hydrolysate is offered as a mixture of di- and tripeptides.", "However, currently all commercially available hydrolysates are produced by combining several endoproteases.", "The latter approach implies a non-uniform and incomplete degradation of the protein.", "To obtain the desired mixture of di- and tripeptides, a hydrolysis process involving a combination of various di- and tripeptidylpeptidases would be ideal.", "Unfortunately, only few of these enzymes from food grade and industrially acceptable microorganisms are known, let alone industrially available.", "According to the invention several of highly useful di- and tripeptidylpeptidases are economically obtainable in a relatively pure state.", "Preferred are those di- or tripeptidylpeptidases that exhibit a low selectivity towards the substrate to be cleaved, i.e.", "exhibit minimal amino acid residue cleavage preferences only.", "Preferred are combinations of those di- or tripeptidylpeptidases that hydrolyse high percentage of the naturally occurring peptide bonds.", "Despite this high activity to naturally occurring peptide bonds, a total hydrolysis to free amino acids is prevented by the nature of the di- and tripeptidylpeptidases.", "Also preferred are those di- or tripeptidylpeptidases that are optimally active between pH 4 to 8 and exhibit adequate temperature stability.", "Adequate temperature stability implies that at least 40%, preferably at least 60%, more preferably between 70 and 100% of the initial hydrolytic activity survives after heating the enzyme together with the substrate for 1 hour at 50 degrees C. Although the process towards an efficiebnt production of mixtures di-or tripeptides or di- and tripeptides hinges on the availability of the enzymes according to the invention, the first enzyme incubation with the proteinaceous substrate will usually be an endoprotease.", "Preferably an endoprotease with a broad spectrum endopeptidase suited for the situation, e.g.", "subtilisin (Delvolase from DSM), neutral metallo protease (Neutrase from NOVO) or thermolysin (Thermoase from Daiwa Kasei) for the near neutral conditions and pepsin or aspergillopepsin (e.g.", "Sumizyme AP from Shin Nihon, Japan) for the acidic conditions.", "Aim of this first digestion is to improve the solubility, to reduce the viscosity and to reduce the heat setting characteristics of the water/protein mixture.", "Furthermore this pretreatment with an endonuclease is essential to create enough starting points for the di- and tripeptidylpeptidases hereby accellerating the proces of di- or tripeptide formation.", "Optionally a protease intended for debittering of the hydrolysate can be included in this stage of the process or later, together with the di-or tripeptidylpeptidases.", "Main aim of the latter hydrolysates is to minimize the allergenicity of the product or to facilitate gastrointestinal uptake.", "In the production of such hydrolysates the use of dipeptidyl- and tripeptidyl-peptidases is of special importance as these s offer an efficient way for producing hydrolysates.", "Other applications in these food and feed industries totally rely upon combinations of one or more endoprotease(s) with one or more exoprotease(s).", "Such combinations of an endoprotease with an exoprotease are typically used in industries to improve aspects like taste and colour of the final product.", "The reason for this is that the development of taste and colour is largely dependent upon the presence of free amino acids.", "Free amino acids can not only be obtained by exoproteases such as carboxypeptidases and aminopeptidases but also by peptidyl-dipeptidases.", "If combined with endoproteases or even dipeptidyl-or tripeptidyl-peptidases, carboxypeptidases, aminopeptidases and peptidyl-dipeptidases can create larger quantities of free amino acids in less time.", "However, in all of these processes an uncontrolled release of amino acids or even non-proteinaceous components should be avoided to minimise undesirable side reactions.", "Though free amino acids as such, can elicit a number of taste impressions, these taste impressions are very basic (bitter, sweet, sour and “umami”) and the amino acid concentration required for perceiving these tastes are high.", "Despite these high threshold values, free amino acids are able to create major sensory effects at much lower concentration ranges through a number of flavour enhancing mechanisms.", "One of these mechanism involves the combination of free amino acids with sugars in so-called Maillard reactions.", "Compared with free amino acids, with these Maillard products overwhelmingly complex flavour and odour systems can develop with threshold values that are several orders of magnitude lower than those recorded for the free amino acids.", "Maillard products are formed at elevated temperatures usually during cooking, baking or roasting when preparing food or feed products.", "During these treatments both colour and a large array of aromas develop.", "In these reactions amino groups react with reducing compounds as a first step and ultimately leading to a whole family of reaction pathways.", "In foods or feeds the amino compounds involved are predominantly free amino acids which are released from the proteinaceous raw material by various proteases and the required reducing compounds primarily represent reducing sugars.", "The implication is that during the processsing of the raw material undesired release of free amino acids and sugars should be avoided to minimise off tastes that could be generated during subsequent heating steps as e.g.", "during spray drying or sterilisation.", "The latter notion emphasises once more the benefits of superior purity and low in-use costs of the enzyme according to the invention.", "Apart from Maillard reactions, amino acids can also undergo important chemical transitions at ambient temperatures.", "The latter type of transitions are enzyme dependent and are quite common in fermented foods such as beer, yogurt, cheese ripening and meat and wine maturation processes.", "In these fermentation processes, free amino acids are liberated from the raw materials used by the proteases added or by proteolytic enzyme activity from the raw material or the microbial starters used.", "During the maturation phase microbial metabolic activity then converts the free amino acids into derivatives with increased sensoric properties.", "For example, L-leucine, L-isoleucine and L-valine lead to the formation of valuable fusel alcohols like amylalcohols and isobutanol in beer fermentation.", "Similarly cheese volatiles such as methanethiol and dimethyldisulphide have been traced back to the occurrence of methionine in cheese as well as methylpropanoic acid and methylpropanal to valine.", "Finally the free amino acid glutamate and can create strong savoury enhancing effects because of its synergy with the breakdown products of RNA, so-called 5′-ribonucleotides.", "If combined with proper concentrations of 5′-ribonucleotides such as 5′-IMP and 5′-GMP, the detection threshold of the umami taste generated by glutamate is known to be lowered by almost two orders of magnitude.", "In order to obtain pronounced and precise taste effects in all of these processes, the proteinaceous substrates should be hydrolysed using a combination of an endo- and an exoprotease, wherein at least one of the endo or exoprotease, preferably both the endo- and exoprotease, are pure and preferably selective towards a specific set of amino acid(s) or preferentially release the preferred amino acid(s).", "So preferred proteases are characterised by a high selectivity towards the amino acid sequences that can be cleaved which notion makes the enzyme category in Aspergillus known as “maturases” of particular importance.", "Apart from the food and feed industries, proteases are also commonly applied by the chemical, pharmaceutical, diagnostic and personal care industries.", "In the personal care industry proteases are used to create peptides which are added to a variety of products to improve aspects like skin feel, gloss or protection.", "Moreover there is a new tendency towards direct topical application of the protease.", "Very similar to the enzyme use in the leather industry, the prime aim in the latter application is to clean, dehair and soften the skin.", "In the chemical and pharmaceutical industry proteases are being developed as valuable tools in producing costly ingredients or intermediates.", "In these industries proteases are not only used because of their hydrolytic capacity but also because of their capacity to synthesise peptides from natural or non-natural amino acids.", "The latter option is clearly demonstrated by the possibility to synthesize aspartame from its amino acid based building blocks by using an endoprotease like thermolysin.", "Unlike the situation in the food and feed industry, the stereo- and regioselectivity of proteases are also considered important assets although unusual reaction conditions may be needed to accomplish the desired chemical transformation.", "Typical examples of the application of proteases in this industry include the use of endoproteases, aminopeptidases as well as carboxypeptidases in the production of various intermediates for drugs like insulin, antibiotics, renin and ACE-inhibitors An overview of such uses is presented in Industrial Biotransformations, A. Liese, K. Seelbach, C. Wandrey, Wiley-VCH; ISBN 3-527-30094-5.In view of the desired specificities, stereo- and regioselectivities, the absence of side activities and resistance to unusual reaction conditions such as high solvent concentrations, the improved performance of the protease according to the invention offers substantial advantages.", "From a pharmaceutical point of view the role of proteases is illustrated by a substantial number of references in Martindale's, “The Extra Pharmacopoeia” (Pharmaceutical Press, London, UK).", "Moreover the important role of very specific proteases in regulating all kinds of biological processes is illustrated by the fact that many hormones become active only after the processing of an, mostly inactive, precursor molecule by such a very specific protease.", "Inhibitors active towards certain categories of such specific proteases have been implicated in the development of all kinds of new drugs.", "Therefore new and effective inhibitors for protease may now be identified using the sequences provided herein.", "The entire disclosure of each document cited herein is hereby incorporated by reference TABLE 1 SEQ ID number Gene cDNA Protein Function of encoded protein EC number 1 58 115 Pepsin A3 EC3.4.23.1 2 59 116 Metalloprotease EC3.4.24.56 3 60 117 acylaminoacyl-peptidase EC3.4.19.1 4 61 118 Tripeptidylaminopeptidase EC3.4.14.- 5 62 119 serine carboxypeptidase EC3.4.16.6 6 63 120 Serine endoprotease EC3.4.21.- 7 64 121 Carboxypeptidase Y EC3.4.16.5 8 65 122 aspergillopepsin II - hom EC3.4.23.19 9 66 123 Tripeptidyl peptidase EC3.4.14.9 10 67 124 Tripeptidyl peptidase EC3.4.14.9 11 68 125 aspergillopepsin II - hom EC3.4.23.19 12 69 126 Tripeptidyl peptidase EC3.4.14.9 13 70 127 Metalloprotease EC3.4.24.- 14 71 128 aspergillopepsin I EC3.4.23.18 15 72 129 Pepsinogen E EC3.4.23.25 16 73 130 aspergillopepsin I - hom EC3.4.23.18 17 74 131 aspergillopepsin II EC3.4.23.19 18 75 132 Pyro-Glu peptidase EC3.4.19.3 19 76 133 dipeptidyl peptidase EC3.4.14.2 20 77 134 Secr.", "aminopeptidase EC3.4.11.10 21 78 135 alkaline D-peptidase EC3.4.16.4 22 79 136 Carboxypeptidase EC3.4.16.1 23 80 137 Carboxypeptidase EC3.4.16.1 24 81 138 Carboxypeptidase-II EC3.4.16.1 25 82 139 aspartic proteinase EC3.4.23.- 26 83 140 Tripeptidyl peptidase EC3.4.14.9 27 84 141 Carboxypeptidase EC3.4.16.1 28 85 142 cysteine proteinase EC3.4.22.- 29 86 143 Metallocarboxypeptidase EC3.4.17.- 30 87 144 Subtilisin hom.", "EC3.4.21.62 31 88 145 Carboxypeptidase Y EC3.4.16.5 32 89 146 Metalloprotease EC3.4.24.- 33 90 147 Carboxypeptidase Y EC3.4.16.5 34 91 148 Metalloprotease EC3.4.24.- 35 92 149 Tripeptidyl peptidase EC3.4.14.9 36 93 150 Aspartic protease EC3.4.23.24 37 94 151 Aspartic protease EC3.4.23.24 38 95 152 Pepsin A3 EC3.4.23.1 39 96 153 Aspartic protease EC3.4.23.24 40 97 154 Aspartic protease EC3.4.23.24 41 98 155 Kex EC3.4.21.61 42 99 156 Serine protease EC3.4.21.- 43 100 157 Glutamyl endoprotease EC3.4.21.82 44 101 158 aspergillopepsin II - hom EC3.4.23.19 45 102 159 acylaminoacyl-peptidase EC3.4.19.1 46 103 160 Tripeptidylaminopeptidase EC3.4.14.- 47 104 161 serine carboxypeptidase EC3.4.16.6 48 105 162 Gly-X carboxypeptidase EC3.4.17.4 49 106 163 aspartic proteinase EC3.4.23.- 50 107 164 Tripeptidyl peptidase EC3.4.14.9 51 108 165 Carboxypeptidase-I EC3.4.16.1 52 109 166 serine carboxypeptidase EC3.4.16.6 53 110 167 serine carboxypeptidase EC3.4.16.6 54 111 168 Secr.", "aminopeptidase EC3.4.11.10 55 112 169 Prolyl endopeptidase EC3.4.21.26 56 113 170 aspergillopepsin I - hom EC3.4.23.18 57 114 171 Aminopeptidase EC3.4.11.- EXAMPLES Example 1 Assaying Proteolytic Activity and Specificity Protease specificity may be explored by using various peptide substrates.", "Synthetic substrates are widely used to detect proteolytic enzymes in screening, in fermentation, during isolation, to assay enzyme activity, to determine enzyme concentrations, to investigate specificity and to explore interaction with inhibitors.", "Peptide p-nitroanilides are preferably used to assay protease activity as the activity can be followed continuously and therefore allow for kinetic measurement.", "The cleavage of peptide p-nitroanilides can be followed by measuring the increase in adsorption at 410 nm upon release of the 4-nitroanilide.", "Paranitroanilide substrates are generally used for serine and cysteine proteases.", "In addition peptide thioesters and 7-amino-p-methylcoumarin peptide derivates are used.", "Peptide thioesters are very sensitive substrates for serine and metalloproteases that exhibit relatively high turnover rate since the thioester bond is easier to cleave than the amide bond.", "Cleavage of thiolesters may be followed with a thiol reagent such 4,4-dithiopyridine (324 nm) or 5,5-dithiobis 2-nitrobenzoic acid (405 nm).", "The same increased turnover rate is usually observed for the cleavage of ester bonds relative to amide bond.", "The most well known substrates to assay the esterase activity of proteases are p-nitrophenol derivates.", "The release of p-nitrophenol can be monitored at different wavelength dependent on the pH that is used, eg around neutral pH a wavelength of 340 nm is used while above pH 9 monitoring is done around 405 nm.", "In addition the hydrolysis of esters can also be followed by titration using pH-stat equipment.", "In case of qualitative measurement of esterase activity pH sensitive dyes can be applied.", "As an alternative, peptides may be attached to a fluorescent leaving group.", "Proteolysis is accompanied by an increase in fluorescence when monitored at the appropriate wavelengths.", "Peptidyl 2-naphtylamides and peptidyl 4-methyl-7-coumarylamides are commonly used.", "The release of for example 7-amino-4 methylcoumarin is measured using an excitation wavelength of 350 nm and an emission wavelength of 460 nm.", "The use of 7-amino-4 trifluoromethylcoumarin has the advantage of the leaving group being both chromogenic (absorbtion 380 nm) as well as flourogenic (excitation 400 nm, emission 505 nm).", "When it is essential that at both sides of the scissile bond an amino acid is present, the introduction of a group that quenches the fluorescence might be useful.", "The general characteristics of such substrates is that the peptide sequence separates a fluorescent donor group from an acceptor group that acts as a quencher of of fluorescence.", "Cleavage of a peptide bond between the quenching group and the fluorophore will lead to substantial increase in fluorescence.", "Several donor-acceptor pairs have been reported, including o-aminobenzoic acid (Abz) as the donor and 2,4 dinitrophenyl (Dnp) as the acceptor, 5-[(2′aminoethyl)-amino]naphtalenesulfonic acid (EDANS) as the donor and 4-[[4′-(dimethylamino]phenyl]azo]-benzoic acid (DABCYL) as the acceptor.", "The Abz/EDDnp represents a very convenient donor-aceptor pair since after total hydrolysis, the fluorescence increases by a factor 7 to 100 and the absorption spectrum of EDDnp does not change with pH.", "Moreover, the peptide sequence may contain up to 10 residues without loss of the quenching effect.", "As the size of the connecting peptides increases, the position of the scissile bond may become less specific.", "Therefore in addition to establishing whether proteolysis occurred, additional analysis of the products may be required.", "This may be done by analysing and separating the produced peptides by HPLC and determining the the amino acid sequence of the fragments.", "In addition the peptide composition of the digest may be directly analysed by using combined HPLC/mass-spectroscopy technique.", "Apart from using peptides of a defined sequence also synthetic peptide libraries can be used to study protease specificity.", "Peptides are synthesised by solid phase synthesis in random or semi-random fashion.", "E.g.", "Meldal et al.", "(PNAS USA 91,3314,1994) report the preparation of a family of protease substrates by starting with H-Lys(Abz)-resin, extending the resin with peptides to a length of six amino acids, and finally coupling Tyr(NO2) to the peptides.", "Each resin bead has a unique sequence and on treatment with the proteases the most susceptible becomes fluorescent as the Tyr(NO2) containing peptide is released.", "Sequence analysis of the peptides on the susceptible will give information on the specificity of the protease.", "Protease activity is usually expressed in units.", "Generally the international standard unit (IU) is defined as the amount of enzyme, which under defined conditions transfers one micromole of substrate per minute.", "Specifically with proteases the IU would relate to the hydrolysis of one micromole peptide bond per minute.", "However in the case of protease units deviations of the international definition are more rule than exception.", "Where with the model peptides, which are cleaved specifically at one bond the calculation of IU's is strait-forward, for proteinacious substrates where the protease can cleave at various positions to a various degree many deviating unit definition are used.", "Apart from a definition of the unit used, any hydrolysis experiment requires an adequate description of the conditions under which the units are measured.", "Such conditions comprise e.g.", "the substrate concentration, the enzyme-substrate ratio, the pH and temperature.", "Typical assays for determining the specific activity of a proteases comprise a proteinacious substrate such as for example denaturated hemoglobin, insulin or casein.", "The polypeptide substrate is digested by a protease at fixed conditions during a fixed time interval.", "Undigested and large polypeptides are precipitated with TCA and TCA soluble product is determined by measuring absorbance at 220 or 280 nm, or by titrating the soluble peptides with folin reagent, ninhydrin, fluro 2,4, dinitrobenzene/dansylchloride, TNBS method or fluorescein.", "Instead of labeling the product after hydrolysis, also polypeptide substrates may be used which are already labeled by specific dyes or fluorophores such as for example fluorescein.", "In addition standard methods of amino acid analysis may be applied using standard laboratory analyzers.", "In order to hget insight in the size distribution of the peptides generated by a protease, gel chromatography experiments may be performed.", "In addition to this HPLC using reverse phase techniques is applied in order to get better resolution of the peptide patterns generated by the protease.", "The course of the hydrolysis of proteinacious substrates is usually expressed in the degree of hydrolysis or DH.", "In case pH-stat is used to follow the course of hydrolysis, DH can be derived from the base consumption during hydrolysis (Enzymatic Hydrolysis of Food Protein, J. Adler-Nissen, 1986, Elsevier Apilied Science Publishers LTD).", "The DH is related to various useful functional properties of the hydrolysate such as solubility, emulsifying capacity, foaming and foam stability, whipping expansion, organoleptic quality.", "In addition taste is an important aspect of food grade hydrolysates.", "Bitterness can be a major problem in protein hydrolysates.", "Termination of the hydrolysis reaction may be done by changing the pH, heat inactivation, denaruring agents such as SDS, acetonitril etc.", "Polypeptides shown in Tabel 1 were expressed and at least partially purified according to standard procedures known in the art.", "They were analysed according to al least one of the methods described above and found to have the activities listed in Table 1.Example 2 Direct Determination of the kcat/Km Ratio for Protease Substrates.", "Synthetic substrates can be used to monitor the enzymatic activity during purification, to determine enzyme concentration, to determine inhibition constants or to investigate the substrate specificity.", "Determination of the kcat/Km ratio gives a measurement of the substrate specificity.", "It allows to compare the specificity of different substrates for a same enzyme or the comparison of hydrolysis rates with different enzymes cleaving the same substrate.", "This ratio has a unit of a second order rate constant and is then expressed as 1/(concentration.time).", "Substrates having a kcat/Km ratio in the range 10.5-10.6 M−1.sec−1 are considered to be very good substrates i.e good affinity and rapid turn-over.", "However, some substrates may be very specific with kcat/Km values in the 10.4 M−1.sec−1 range.", "The kcat/Km ratio may be calculated after determination of individual parameters.", "In that case, Km and Vm may be obtained from various linear plots (e.g Hanes or Cornish-Bowden method) or by a non-linear regression method.", "Knowing that Vm=kcat.", "Et (where Et is the final active enzyme concentration then kcat=Vm/Et.", "Determination of the kcat/Km ratio by the previous method may be prevented when product or substrate inhibition occur, or when Substrate precipitates at high concentration.", "It is however possible to obtain an accurate value of the kcat/Km ratio working under first-order conditions i.e at a substrate concentration far below the estimated Km.", "In these conditions, the Michaelis-Menten equation: v=(Vm.S)/(Km+S) becomes: v=(Vm.S)/Km since S<<Km or v=(Vm/Km).S=kobs.", "S=−dS/dt which integrates as InS=−kobs.t+InSo where So is the starting substrate concentration and S the substrate concentration at a given time.", "The velocity is proportionnal to the substrate concentration.", "In other words, the substrate hydrolysis obeys a first order process with kobs as the first-order rate constant.", "kobs=Vm/Km=(kcat.Et)/Km since Vm=kcat.Et A continuously recording of the substrate hydrolysis will allow the graphical determination of kobs from the InS vs time graph.", "The kcat/Km ratio is simply inferred from kobs providing the active enzyme concentration is known: kcat/Km=kobs/Et Assay method: Use a starting substrate concentration far below the estimated Km and a low enzyme concentration to allow the substrate hydrolysis to be recorded.", "You will obtain a first-order curve for the product generation: After total hydrolysis of the substrate, the absorbance (or fluorescence units) of the product will allow the accurate determination of So, since Pt=So.", "kobs is determined from the slope of the InS vs time graph or alternatively using a fitting software (Enzfitter, SigmaPlot .", ".", ".", ").", "NB: Do not forget to calculate the substrate concentration for any given time from the product concentration (S=So−P) since plotting P vs time would not provide the correct kobs (dP/dt=kobs.S does not integrate in the same way).", "Alternatively, one can measure successive t½ (half-time) from the product apparition curve since in a first order process: t½=In2/kobs=0.693/kobs then kobs=0.693/t½ Using this method allows to check that you have a true first order decay (identical values for the successive t½).", "Example 3 Inactivating Protease Genes in Aspergillus The most conveniant way of inactivating protease genes in the genome of Aspergillus is the technique of gene replacement (also called “one step gene disruption”).", "The basics of this technique have been described by Rothstein R J in Meth.", "Enzymol.", "101, p202, 1983.Essentially the technique is based on homologous recombination of transformed DNA fragments with the genomic DNA of a fungal cell.", "Via double crossover the gene to be inactivated is (partly) replaced by the DNA fragment with which the cell is transformed.", "Preverably the transformed DNA fragment contains a selectable marker gene for Aspergillus niger.", "Basically the manipulation of DNA and generation of a inactivation construct are done using general molecular biological techniques.", "First, genomic DNA is isolated from the Aspergillus niger strain that is later on used for the inactivation of the protease gene.", "Genomic DNA of A. niger can be isolated by any of the techniques described, e.g.", "by the method described by de Graaff et al.", "(1988) Curr.", "Genet.", "13, 315-321, and known to the person skilled in the art.", "This genomic DNA is used as template for amplification of the flanking regions of the protease gene by using the polymerase chain reaction (PCR; Sambrook et al.", "(1989) Molecular cloning, a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York).", "With flanking regions is meant here the non-coding regions upstream and downstream of the protease gene that will be inactivated.", "Preferably the flanking regions should each be more than 1.0 kb in length.", "Two single stranded DNA oligonucleotides are used for the priming of the PCR amplification of each flanking region.", "For the 5′-flanking region, one primer is homologous to a DNA sequence upstream of the start of the coding sequence of the protease gene.", "Preferably the homologous region is located more than 1.0 kb upstream of the translation start site.", "The second primer is homologous to the complementary and inverse DNA sequence located immediately upstream of the coding sequence of the protease gene.", "For the 3′-flanking region, one primer is homologous to the DNA sequence immediately downstream of the coding sequence of the protease gene.", "The second primer is homologous to a complementary and inverse DNA sequence located preferably more than 1.0 kb downstream of the coding sequence of the protease gene.", "The DNA sequence included in all primers and homologous to the A. niger genome should be minimally 15 nucleotides in length, preferably more than 18 nucleotides in length.", "Most conveniently, all primers should contain a DNA sequence coding for the recognition site of suitable restriction enzymes upstream of the sequence that is homologous to the A. niger genome.", "These extra recognition sites facilitate the cloning process.", "Both primers and the genomic DNA of A. niger are used in a PCR reaction under conditions known to those skilled in the art.", "The annealing temperature of the primers can be calculated from the part of the DNA sequence that is homologous to the A. niger genome.", "Both fragments containing the 5′-flanking region and the 3′-flanking region are cloned into a vector that can be propagated in E. coli using general molecular biological techniques.", "A gene that can be used as selection marker in Aspergillus niger is then cloned in between the two flanking regions.", "Most conveniantly the marker gene is under control of a promoter that comes to expression in A. niger, preferably an endogenous A. niger promoter.", "The orientation of the insertion of the marker gene is preferably in the same direction as the original protease gene.", "The final inactivation fragment contains the 5′-flanking region, a selection marker gene preferably under control of a A. niger endogenous promoter, and the 3′-flanking region, all in this direction and orientation.", "DNA of the final construct is cloned into a vector that can be propagated in E. coli.", "The inactivation construct is digested with suitable restriction enzymes to remove the E. coli vector sequences and the inactivation fragment is isolated using standard techniques (Sambrook et al.", "(1989) Molecular cloning, a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York).", "Finally Aspergillus niger is transformed with the inactivation fragment using a method described in literature, e.g.", "by the method described by Kusters-van Someren et al.", "(1991) Curr.", "Genet.", "20, 293-299.Transformed cells are selected by plating the transformation mixture on agar plates that are selective for growth of Aspergillus niger strains that do express the marker gene.", "After purification of the transformed Aspergillus strains by replica plating, a representative number of strains is analysed by Southern blotting using standard methods (Sambrook et al.", "(1989) Molecular cloning, a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York).", "Therefore, genomic DNA of mycelium of transformed strains is isolated and digested with suitable restriction enzymes.", "Restriction fragments are separated using agarose gel electrophoresis, blotted to nitrocellulose membranes and probed with a labeled fragment of the marker gene.", "Hybridization and washing is under stringent conditions.", "Strains that contain labeled restriction fragments of the correct length are considered correct.", "Using this method A. niger strains can be selected with an inactivated protease gene of choice.", "Example 4 Isolating Proteases by Ion Exchange Chromatography Small quanties of the protease encoded by the nucleotide sequence as provided herein are obtained by constructing an expression plasmid containing the relevant DNA sequence, transforming an A. niger strain with this plasmid and growing the A. niger strain in a suitable medium.", "After collecting the broth free of contaminating cells, the protease sought can be purified.", "To isolate the protease as encoded by the provided nucleotide sequence in an essentially pure form several strategies can be followed.", "All of these strategies have been adequately described in the relevant scientific literature (see for example the Protein Purification Handbook, 18-1132-29 Edition AA as published by Amersham Pharmacia Biotech, Uppsala, Sweden).", "A procedure which is applicable to purify proteases from complex mixtures is provided hereunder.", "Essential is that a suitable assay is available that is selective towards the enzyme characteristics sought.", "For proteases typically a chromogenic, synthetic peptide substrate is used as described in Example 1.Such peptide substrates can be selective towards endoproteases, carboxypeptidases, aminopeptidases or omegapeptidases.", "In Example 11 the selectivity towards a specific tripeptidylpeptidase is described.", "By choosing the right amino acid residues in the relevant synthetic peptide, proteases with the desired specificity can be selected.", "First it should be determined whether the protease is excreted into the medium, depending on the expression system chosen to produce the protease, it may be excreted or contained in the cell.", "If the protease is excreted into the fermentation medium, the producing cells or fragments of these cells have to be removed by centrifugation or filtration and the resulting clear or clarified medium is the starting point for further purification.", "In those cases in which the protease sought is not excreted, the producing cells have to be disrupted to enable purification of the protease.", "In such cases the collected cell mass is best ground with an abrasive, milled with beads, ultrasonicated or subjected to a French press or a Manton-Gaulin homogeniser and then filtered or centrifuged.", "In case the protease is hydrophobic or membrane bound, the addition of a non-ionic detergent to solubilise the protease before the filtering or centrifugation step may be necessary.", "After the clarification step, a three phase purification strategy can be applied to obtain the unknown proteases in an essentially pure state.", "In all or some of these three phases addition of a detergent may be necessary.", "In the first or capture phase the target protease is isolated, partly purified and concentrated.", "During the subsequent intermediate purification phase most of the bulk impurities are removed and in the final polishing phase trace amounts of remaining impurities of larger amounts of closely related substances are removed and the enzyme is dissolved in the desired buffer.", "Depending upon the nature and physical properties of the protease at hand, a person skilled in the art is capable of optimising the three phases using slightly modified versions of the different protein binding materials and apply these under somewhat changed conditions.", "However, in all cases a selective analytical assay is indispensible as it will enable the continuous monotoring of the increasingly purified proteolytic activity.", "Analytical assays suitable for the purpose include the use of chromogenic peptide substrates as has been mentioned before.", "In the first capturing phase of the purification a strong ion exchange resin of the anionic type is preferably used to apply the clarified and desalted enzyme containing medium.", "To guarantee binding of the desired proteolytic activity to the resin, three or four different pH values of medium and resin are tested under low conductivity conditions.", "In these tests the resin is always equilibrated with a buffer of the same pH value and conductivity as the enzyme containing medium.", "The medium is then applied to the column under pH conditions which has been shown to allow adequate binding of the protease to the resin i.e.", "none of the desired enzymatic activity can be traced back in the run-through medium.", "Subsequently the desired enzymatic activity is eluted from the ion exchange resin using a continuous salt gradient which starts with the resin equilibration buffer and ends with this buffer to which 1 molliter of NaCl has been added.", "Eluted fractions containing the desired activity according to the assay are pooled and then prepared for an additional purification step.", "This additional purification step depends on the purity of the desired enzyme in the pooled fraction: if almost pure, an additional gel filtration step will proof to be adequate; if not almost pure, chromatography over a hydrophobic interaction resin is applied followed by a gel filtration step.", "Chromatography over a hydrophobic interaction resin is carried out by first increasing the salt content of the pooled fraction obtained from the ion exchange resin to 4 mol/liter of NaCl and by removing any precipitate formed.", "If the resulting clear fraction does not contain the desired activity, this activity is obviously present in the precipitate and and can be recovered in an essentially pure state.", "If the resulting clear fraction still exhibits the desired activity in the assay, then the liquid is applied as such to a phenyl sepharose resin (Pharmacia) equilibrated in this high salt buffer with an identical pH and conductivity.", "If the desired enzymatic activity binds to the phenyl sepharose resin, the activity is eluted with a continuous gradient of decreasing salt content followed by a salt free wash and, if nesessary, with a chaotropic agent.", "Like before those fractions from the gradient that exhibit activity in the assay are pooled and finally subjected to a gel filtration step.", "If the desired enzymatic activity does not bind to the phenyl sepharose resin, many of the contaminants will, so the desired proteolytic activity as present in the void volume of the column requires only an additional ultrafiltration step to obtain the activity in a more concentrated form before applying it to the gel filtration column.", "The gel filtration column does not only remove trace contaminations but also brings the enzyme in the buffer which is required by subsequent use.", "Although this method is generally applicable for the isolation and purification of proteases according to the invention, a more specific isolation technique is described in Example 4.In that Example the isolation of an Aspergillus protease is described by using immobilised bacitracin, a peptide antibiotic known for its selective interaction with various types of proteases.", "Example 5 Isolating Proteases by Affinity Chromatography An alternative method for purifying small quantities of protease is by affinity chromatography.", "To obtain the protease in a purified form, a 100 milliliter culture is grown in a well aerated shake flask.", "After centrifugation to remove any non-soluble matter, the supernatant is applied to a 40 milliliter bacitracin-Sepharose column equilibrated with 0.05 mol/litre sodium acetate pH 5.0.Proteases bound to the column are eluted using the acetate buffer supplemented with 1 mol/litre of NaCl and 10% (v/v) isopropanol (J. Appl.", "Biochem., 1983 pp420-428).", "Active fractions are collected, dialysed against distilled water and applied on a 20 milliliter bacitracin-Sepharose column, again equilibrated with acetate buffer.", "As before, elution is carried out using the acetate buffer supplemented with NaCl and isopropanol.", "Active fractions, i.e.", "fractions displaying the activities sought, are collected, dialysed against a 5 millimol/litre acetate buffer pH 5.0 and then concentrated by means of ultrafiltration with a Amicon PM-10 membrane.", "To obtain the protease in an essentially pure state, the concentrated liquid is chromatographed over a Superdex 75 column equilibrated with the 0.05 mol/litre sodium acetate buffer pH 5.0 and supplemented with 0.5 mol/litre NaCl.", "Further experiments carried out with the purified enzyme on PAGE may confirm if the molecular weight is in line with what can be expected on the basis of the available sequence data.", "Final confirmation can be obtained by carrying out a partial, N-terminal amino acid analysis.", "Example 6 Properties of a Novel Cysteine Protease from A. niger.", "In this Example Aspergillus gene nr 28 was cloned and overexpressed in A. niger as described before.", "The enzyme obtained was purified according to procedures described in Example 4 and used to destroy trypsin inhibiting activity from soybeans under various conditions.", "As reference materials papain and bromelain were used.", "Bromelain was obtained from Sigma, papain was obtained from DSM Food Specialties Business Unit Beverage Ingredients, PO Box 1, 2600 MA Delft, the Netherlands.", "Trypsin inhibition was measured according to the method of Kakade, M. L., Rackis, J. J., McGhee, J. E. and Puski, G. (1974): J. Cereal Chemistry 51: 376-382.Degradation of the substrate N-benzoyl-L-arghinine-p-nitroaniline to N-benzoyl-L-arginine and p-nitroaniline was taken as a measure of trypsin activity.", "Trypsin was obtained from British Drug Houses Ltd and was derived from cow's pancreas containing more than 0.54 Anson Units per gram of product.", "The Kunitz inhibitor for soybeans was also obtained from Sigma.", "The trypsin inhibitor was pre-incubated at a concentration of 2 mg/ml with the above mentioned cysteine protease enzymes at pH 3 in 50 mM Na-acetate buffer prior to measuring trypsin inhibition.", "Enzymes were added at a ratio of enzyme protein to trypsin inhibitor of 1:100 (w/w).", "Albumin served as a negative control for the enzymes.", "Remaining trypsin activity was measured after incubation during 3 hours at 37° C. Results are shown in Table 2.TABLE 2 Effects of various cysteine proteases on the enzymatic inactivation of the Kunitz trypsin inhibitor from soybeans.", "2 3 4 5 Remaining Remaining TI Remaining TI Remaining TI 1 TI activity after activity after activity after Enzyme activity pepsin heat treatment heat treatment tested (%) treatment at 75° C. at 90° C. Papain 25 55 78 95 Bromelain 30 62 86 99 A. niger 26 26 28 35 Albumin 100 100 100 100 (control) TI: Trypsin Inhibitor activity Experiments were repeated in the presence of pepsin during the pre-incubation of cysteine proteases with the trypsin inhibitor.", "Pepsin was added at final concentration of 1.3 mg/ml.", "Results are shown in column 3.Another series of experiments were conducted to check for heat stability.", "The cysteine proteases were incubated at 75 and 90° C. during 5 minutes prior to the addition of these enzymes to the pre-incubation with the trypsin inhibitors.", "Results are shown in columns 4 and 5.These results clearly demonstrate the superior activity of these novel cysteine proteases from Aspergillus niger over currently available cysteine proteases for the inactivation of trypsin inhibitors in animal feed.", "Example 7 Exo-Peptidases Promoting Cheese Ripening and Cheese Taste.", "The amino-peptidases encoded by genes nr 20 and 54 (see Table 1) were overexpressed in A. niger according to methods described earlier.", "Purification of these enzymes was carried out according to procedures as described in Example 4.The activity of the purified enzyme samples was determined at pH7.2 in an aqueous phosphate buffer (50 mM) containing the para-nitro anilide derivative of a number of hydrophobic amino acids (3 mM) as the substrate.", "The conversion of the substate by the amino peptidase was determined by monitoring the change in optical density at 400 nm as a result of substrate conversion, using a solution not contaning the enzyme as the reference.", "Activity (A) was calculated as the change in OD per minute and expressed as e.g.", "Phe-AP, Leu-AP or Val-AP units, depending on the substrate used.", "Normal cheese milk was inoculated with starter culture of the Delvo-tec™ DX 31 range (DSM Food Specialities Delft, The Netherlands) to obtain a Gouda-type cheese and coagulating was executed with an average dosis of coagulant (50 IMCU per liter of cheese milk).", "In addition, 25 Phe-units of each exo-protease was added to two experimental cheeses whereas the control did not contain either one of the exo-proteases.", "Cheese making parameters were used conform the procedure applied for semi-hard cheese for both cheeses.", "A difference was noted in terms of flavor and aroma development between the experimental cheeses and control cheese to such an extent that the experimental cheeses has obtained most of its organoleptical properties after three (3) weeks whereas the control cheese has obtained a similar qualification after six (6) weeks.", "The level of free amino acids after three weeks was shown to be twice as high in the experimental cheeses; after six weeks of ripening the levels were comparable again.", "Amino acid analysis was carried out according to the Picotag method of Waters (Milford Mass., USA).", "These data suggests that the product is ready for sale three weeks earlier without decreasing the keeping quality of the cheese.", "The organoleptic character of the experimental cheeses differed from the control to the extent that the bland cheese flavor with a slight tendency to bitterness of the control cheese was overcome in the experimental cheese in the presence of the amino-peptidase.", "The texture of the cheeses was found to be somewhat smoother as well.", "Example 8 Novel Specificity of a Protease Encoded by Gene 55 As explained earlier, certain proteins can resist enzymatic hydrolysis as the result of specific amino acid compositions or specific tertiary structures.", "In such cases the quantity of peptides that can be solubilised from protease resistant proteins can be dramatically improved by using proteases exhibiting novel specificities.", "Beta-casein is a protein with very limited tertiary structure but with an extraordinary high level of proline residues.", "Many proteases have difficulties in cleaving proline containing sequences so that the hydrolysis of beta-casein with commonly available proteases yields a hydrolysate that is relatively rich in large, protease-resistant peptides.", "The latter resistant peptides can attribute to a number of undesirable properties of the hydrolysate.", "For example, it is well known that these larger peptides have a relatively strong effect on allergenicity and bitterness.", "Moreover, these peptides withstand a further degradation into free amino acids so that in certain processes the occurrence of these large, protease resistant peptides are synonymous with yield losses.", "Therefore, the availability and use of proteases that are capable of cleaving the protease-resistant parts of the proteins, translate into serious technical and economical benefits.", "Beta-casein represents one of the major casein fractions of bovine milk.", "The protein has been well characterised in terms of its amino acid sequence and is commercially available in an almost pure form.", "As such, beta-casein offers an excellent test substrate for studying the relationship between enzyme cleavage sites and the length of various peptides formed during enzyme hydrolysis.", "This Example demonstrates that despite the broad spectrum cleavage character of the endoprotease subtilisin, the addition of a very specific enzyme like a prolyl endopeptidase as encoded by gene 55 (see Table 1) has a major impact on the size of the beta-casein fragments formed.", "Beta-casein from bovine milk (lyophilised, essentially salt-free powder) with a minimum 90% beta-casein was obtained from Sigma.", "Subtilisin from B. licheniformis (Delvolase®, 560 000 DU per gram) was obtained from DSM Food Specialities (Seclin, France).", "The proline-specific endoprotease as encoded by gene 55 was overexpressed in A. niger and purified using procedures described in Example 4.Beta-casein powder was dissolved at a concentration of 10% (w/w) together with 0.1% (w/w) Delvolase™ powder in a 0.1 mol/liter phosphate buffer pH7.0.After an incubation of 24 hours at 45° C. in a shaking waterbath, the reaction was stopped by heating the solution for 15 minutes at 90° C. To one half of the solution (1 ml containing 100 milligrams of beta-casein) 100 microliter of the proline-specific protease was added and the reaction was continued for another 24 hours at 45° C. After another heat shock at 90° C., samples of both the Delvolase™ and the Delvolase™+proline-specific endoprotease treated beta-casein material were analysed by LC/MS equipment to study the precise peptide size distributions in the two samples.", "LC/MS Analysis HPLC using an ion trap mass spectrometer (Thermoquest™, Breda, the Netherlands) coupled to a P4000 pump (Thermoquest™, Breda, the Netherlands) was used in characterising the enzymatic protein hydrolysates produced by the inventive enzyme mixture.", "The peptides formed were separated using a PEPMAP C18 300A (MIC-15-03-C18-PM, LC Packings, Amsterdam, The Netherlands) column in combination with a gradient of 0.1% formic acid in Milli Q water (Millipore, Bedford, Mass., USA; Solution A) and 0.1% formic acid in acetonitrile (Solution B) for elution.", "The gradient started at 100% of Solution A and increased to 70% of solution B in 45 minutes and was kept at the latter ratio for another 5 minutes.", "The injection volume used was 50 microliters, the flow rate was 50 microliter per minute and the column temperature was maintained at 30° C. The protein concentration of the injected sample was approx.", "50 micrograms/milliliter.", "Detailed information on the individual peptides was obtained by using the “scan dependent” MS/MS algorithm which is a characteristic algorithm for an ion trap mass spectrometer.", "Full scan analysis was followed by zoom scan analysis for the determination of the charge state of the most intense ion in the full scan mass range.", "Subsequent MS/MS analysis of the latter ion resulted in partial peptide sequence information, which could be used for database searching using the SEQUEST application from Xcalibur Bioworks (Thermoquest™, Breda, The Netherlands).", "Databanks used were extracted from the OWL.fasta databank, available at the NCBI (National Centre for Biotechnology informatics), containing the proteins of interest for the application used.", "By using this technique as a screening method only peptides with a mass ranging from approx.", "400 to 2000 Daltons were considered suitable for further analysis by MS sequencing.", "Angiotensin (M=1295.6) was used to tune for optimal sensitivity in MS mode and for optimal fragmentation in MS/MS mode, performing constant infusion of 60 μg/ml, resulting in mainly doubly and triply charged species in MS mode, and an optimal collision energy of about 35% in MS/MS mode.", "In the sample digested with Delvolase alone, the LC/MS/MS analysis identified 40 peptides covering various parts of the beta-casein molecule.", "Together these peptides accounted for 79% of the total beta-casein sequence.", "Different retention times of the peptides on the C18 column could be traced back to peptide lengths ranging from 2 to 23 amino acid residues.", "Together <15% of the peptides found were smaller than 6 amino acids.", "The sample digested with Delvolase™ and the proline-specific protease also generated a large number ofl identifiable peptides from beta-casein.", "Together these peptides covered >50% of the total beta-casein protein sequence.", "In this sample the peptide size distribution was remarkably homogeneous, as the peptides ranged in length only between 2 and 6 residues.", "The results show that in the hydrolysate made with the proline-specific protease contain a large fraction of di-, tri-, up to 6 AA peptides, showing the distinct beneficial effect of the co-incubation with an endoprotease featuring an unusual specificity.", "It is also clear from these experiments that the endoprotease according to gene 55 encodes an endoprotease that cleaves the peptide chain at the carboxy terminus of the proline residue.", "Example 9 The Selective Release of Specic Amino Acids to Promote Flavour Formation.", "Free amino acids like leucine and phenylalanine have not only been implicated in Maillard reactions but also as precursor for desirable aromas in various food fermentations.", "To promote the formation of such aromas in food fermentations or during the heating, roasting or baking phase of food, it would be advantageous to incorporate into these products a protein hydrolysate that contains relatively high levels of these specific amino acids in a free form.", "In this Example we describe the production of yeast extracts selectively enriched t in leucine and phenylalanine.", "This enrichment is obtained by combining an endoprotease with a cleavage preference for a selected set of amino acid residues with an exoprotease favouring the release of a similar set of amino acid residues.", "The preference of the endoprotease should match with the preference of the exoprotease used.", "For example we have established that the aminopeptidases encoded by genes 20 and 54 (see Table 1) feature a definite preference for releasing leucine and phenylalanine residues which matches with the cleavage preferences of thermolysin.", "The carboxypeptidases encoded by genes 23 and 24 have a preference for releasing arginine and lysine residues which matches the cleavage preferences of trypsin.", "Carboxypeptidase encoded by gene 5 features a highly unusual preference for releasing glycine which could be combined with certain endoproteases present in papaine.", "The carboxypeptidase encoded by gene 51 is capable of removing glutamate residues which matches the glutamate specific protease encoded by gene 43.The endoprotease thermolysin (commercially available as Thermoase)C 180 from Daiwa Kasei KK (Osaka, Japan) is known to cleave peptide bonds at the amino terminal side of bulky, hydrophobic amino acids like Leu and Phe.", "To liberate the thus exposed amino acids from the newly formed peptides, we used the amino-peptidases encoded by genes nr 20 and 54 (see Table 1).", "These genes were overexpressed in A. niger according to methods described earlier and purification of these enzymes was carried out according to procedures as described in Example 4.To release as much leucine and phenylalanine as possible without concomitant release of undesired amino acids with this combination of enzymes, it is evident that the conditions used during enzymatic hydrolysis should be carefully selected.", "Moreover, the yeasts own endogeneous (and probably a specific) proteases have to be inactivated.", "After a number of test incubations, a protocol was worked out that leads to a surprisingly selective and effective release of leucine and phenylalanine from the yeast proteins using these two new enzymes.", "To inactivate the yeasts endogeneous proteases, the yeast suspension was kept for 5 minutes at 95 degrees C. Then the suspension was quickly cooled down to the required temperature and the pH was adjusted to 7.0 using 4N NaOH.", "The yeast, the thermolysin and one of the aminopeptidases were all incubated simultaneously under the following conditions.", "After the heat shock, the pH of the 2000 milliliters yeast suspension was adjusted to 7.0 after which 680 milligrams of Thermoase were added and, after stirring, the purified aminopeptidase.", "The mixture was incubated with stirring at 50 degrees C. for 3 hours and centrifuged.", "To stop all enzymatic activities the pH of the supernatant was adjusted to 4 and subjected to another heat treatment of 45 minutes at 95 degrees C. After another centrifugation a sample for amino acid analysis was obtained from the supernatant.", "Precipitated or non-dissolved matter was removed by centrifugation for 15 minutes at 3500 rpm in an Hereaus Megafuge 2.0 R centrifuge.", "Supernatant was removed and kept frozen at −20° C. Samples of the supernatant, were analysed for amino acid content according to the Picotag method of Waters (Milford Mass., USA) immediately after thawing.", "In the amino acid analysis Trp and Cys values were omitted And Asp and Asn values were summed as one value.", "According to the data obtained, in the resulting hydrolysate the ratio between alanine and leucine (21.3:11.7) was 1:0.5 Commercially available yeast hydrolysates typically exhibit alanine versus leucine ratio's of 1:0.3.In a second experiment a yeast extract was prepared that was enriched in free glutamate.", "To achieve this, use was made of an endoprotease exhibiting a preference for cleaving at the C-terminal end of glutamate residues (encoded by gene nr 43 in Table 1) and a carboxypeptidase (encoded by gene nr 51 in Table 1) capable of removing these glutamate residues thus exposed.", "The endoprotease encoded by gene nr 43 and the carboxypeptidase encoded by gene 51 (see Table 1) were overexpressed in A. niger according to methods described earlier.", "Purification of these enzymes was carried out according to procedures as described in Example 4.The essential role of free glutamate in a number of aroma forming processes is well documented and MSG, the sodium salt of glutamic acid, is recognized as the single most important taste enhancing component.", "In this Example the pH of the 200 ml heat shocked yeast suspension is adjusted to 8.0, then the purified enzyme product encoded by gene 43 is added and the mixture was incubated for 4 hours at 50 degrees C. Then the pH was lowered to 5.0 and the suspension was centrifuged.", "To 100 milliliters of supernatant the purified gene product of gene 51 is added.", "Incubation with this carboxypeptidase took place for 30 minutes at 50 degrees C. with continuous pH adjustments.", "After stopping the enzyme incubation by a heat treatment of 5 minutes by 95 degrees C., the material was again centrifuged (see above) and a sample was obtained for amino acid analysis.", "According to the amino acid data obtained (see above), in the resulting hydrolysate the ratio between alanine and glutamate (30.0:48.7) was 1:1.6.Commercially available yeast hydrolysates typically exhibit alanine versus glutamate ratio's of 1:1.Example 10 Flavour Evaluation of Yeast Hydrolysates Enriched in Specific Amino Acids.", "To prove that a protein hydrolysate enriched in specific amino acids according to the invention can generate specific aroma's, a number of experiments were carried out with the yeast hydrolysates described in an earlier Example.", "To that end larger portions of these hydrolysates were prepared and lyophilised.", "The performance of the resulting powders were compared with the performance of a commercially availble yeast extract (Gistex LS, obtainable from DSM Food Specialties, Delft, The Netherlands) in a standardised mixture under several reaction conditions.", "The standardised mixture consisted of one of the hydrolysates, base mixture and water.", "The base mixture contained 22 grams of Maxarome Plus Powder (a specialised yeast extract with a high content of natural nucleotides, also obtainable from DSM Food Specialties), 29.2 grams of glucose, 9 grams of REFEL-F fat (hydrogenated soy oil, obtainable from Barentz, Hoofddorp, The Netherlands) and 0.2 grams of calcium stearoyl lactylate (emulsifyer, obtainable from Abitec, Northampton, UK) thoroughly mixed in a mortar.", "All standardised mixtures contained 5 grams of yeast hydrolysate powder (i.e.", "either the leucine or the glumate enriched material or the commercial yeast extract), 3 grams of the base mixture and 3 grams of water.", "After thorough mixing, these three slurries were subjected to different heating regimes i.e.", "either 65 minutes at 90-95 degrees C. in a reaction vial (liquid reaction) or dried at 20 millibar at 120 degrees C. in a vacuum oven (vacuum roast reaction) or heated in an open reaction vial at 120 degrees C. for 10 minutes after the dissipation of all water (roast reaction).", "After the heat treatment all three products Shad assumed colours ranging from dark brown to almost black.", "In case of the vacuum roast reaction only the light coloured top layers were used.", "Taste evaluation of the heated products was carried out by grinding the blackened cakes into fine powders and dissolving these powders to a concentration of 2% (w/w) in water containing 0.6% (w/w) NaCl.", "The observations of the taste panel are specified in Table 3.TABLE 3 Reference Leucine Glutamate Liquid Bouillon, slightly Cold tea, slightly More bouillon, meaty, roast flowery, yeasty yeasty Vacuum Burnt, fried Astringent, beans, Burnt, bouillon, yeasty roast potatoes yeasty Roast Dark roast, Less roast, flowery, Roast, more bouillon, bouillon, umami umami more umami Example 11 Non-Allergenic Whey Protein Hydrolysates Formed with Tripeptidylpeptidases.", "The dipeptidylpeptidases encoded by the genes 19 and 55 as well as the tripeptidylpeptidases encoded by the genes 4, 9, 10, 12, 26, 35, 46, and 50 (see Table 1) may be overproduced as described and may be purified according to the methods provided in Example 4.After purification the pH optimum and the temperature stability of each individual enzyme may be established by any of the methods available and known by the skilled person.", "Furthermore, the specificity of each individual enzyme may be determined using the methods outlined in Example 1.The selectivity exhibited by tripeptidylpeptidases is illustrated in the following experiment.", "The enzyme encoded by gene 12 was overproduced in an Aspergillus niger host cell and purified by procedures described in Example 4.The enzyme thus obtained was incubated at pH 5 and 50 degrees C. with different synthetic chromogenic substrates i.e.", "Ala-Ala-Phe-pNA and Ala-Phe-pNA (both from Bachem, Switserland).", "The incubation with the Ala-Ala-Phe-pNA substrate led to a significant increase of the absorbance at 410 nm whereas the incubation with Ala-Phe-pNA did not.", "This observation clearly demonstrates that tripeptidylpeptidases cleave off tripeptides and do not exhibit aminopeptidase activity that can lead to an undesirable increase of free amino acids.", "Moreover, the enzyme encoded by gene 12 shows favourable enzyme stability characteristics as shown in the following experiment.", "Four samples of the enzyme were incubated at pH 5 for one hour at 0, 40, 50 and 60 degrees C. respectively.", "Then each enzyme sample was incubated with the above mentioned Ala-Ala-Phe-pNA substrate in a citrate buffer at pH5 and the residual activity in each individual sample was determined by measuring the increase in absorbance at 410 nm.", "With the 0 degrees C. sample showing 100% activity, the 40 degrees sample showed 96% residual activity, the 50 degrees sample 92% residual activity and the 60 degrees sample 88% residual activity.", "In a typical process aimed at producing a hydrolysate with a high proportion of tripeptides, whey protein (WPC 75) may be dissolved/suspended in a concentration of 100 grams of protein/liter, in an aqueous medium having a pH of 8.5.The first enzyme incubation is with the broad spectrum endoprotease subtilisin (Delvolase®, 560 000 DU per gram from DSM).", "After a predigestion of the whey with this enzyme in a concentration of 0.5% enzyme concentrate per gram of protein for 2 hours at 60 degrees C., the mixture is heat-treated to inactivate the endoprotease used.", "Then the temperature is adjusted to 50 degrees C. and the tripeptidylpeptidase is added and the whole mixture is incubated until the desired level of tripeptides is reached.", "Further processing steps of the hydrolysate thus obtained depend on the specific application but may incorporate microfiltration or centrfugation followed by evaporation and spray drying." ] ]	Patent_10469204
[ [ "Compound", "This invention relates to new polypeptide compound represented by the following general formula (I): wherein R1, R2, R3, R4, R5 and R6 are as defined in the description or a salt thereof which has antimicrobial activities (especially, antifungal activities), inhibitory activity on β-1,3-glucan synthase, to process for preparation thereof, to a pharmaceutical composition comprising the same, and to a method for prophylactic and/or therapeutic treatment of infectious diseases including Pneumocystis carinii infection (e.g.", "Pneumocystis carinii pneumonia) in a human being or an animal." ], [ "1.A polypeptide compound of the following general formula (I): wherein R1 is acyl group, R2 is hydrogen or acyl group, R3 is lower alkyl which has one or more hydroxy or protected hydroxy, R4 is hydrogen or hydroxy, R5 is hydrogen, hydroxy, lower alkoxy or hydroxy sulfonyloxy, and R6 is hydroxy or acyloxy, or a salt thereof: 2.A compound of claim 1, wherein R1 is phenyl(lower)alkenoyl substituted with one or more suitable substituent(s), benzoyl substituted with one or more suitable substituent(s) or naphthoyl substituted with one or more suitable substituent(s), R2 is hydrogen, R3 is lower alkyl which has one or more hydroxy, R4 is hydrogen or hydroxy, R5 is hydroxy or hydroxysulfonyloxy and R6 is hydroxy.", "3.A compound of claim 2, wherein R1 is phenyl(lower)alkenoyl substituted with one or more suitable substituent(s), benzoyl substituted with one ore more suitable substituent(s) or naphthoyl substituted with one or more suitable substituent (s), R2 is hydrogen, R3 is lower alkyl which has two hydroxy, R4 is hydrogen or hydroxy; R5 is hydroxy or hydroxysulfonyloxy; and R6 is hydroxy.", "4.A compound of claim 3, wherein R1 is naphthoyl substituted with higher alkoxy, naphthoyl substituted with lower alkoxy(higher)alkoxy, naphthoyl substituted with higher alkyl, phenyl(lower)alkenoyl substituted with lower alkoxy, benzoyl substituted with a suitable substituent selected from the group consisting of phenyl substituted with a suitable substituent selected from the group consisting of lower alkoxy, higher alkoxy and higher alkyl, thiadiazolyl substituted with phenyl which has a suitable substituent selected from the group consisting of piperazinyl substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, piperazinyl substituted with lower alkoxy(higher)alkyl, piperazinyl substituted with tetrahydropyran, piperazinyl substituted with dioxaspiro(higher)alkyl which may have lower alkyl, piperazinyl substituted with lower alkyl having pyridyl, piperidyl substituted with lower alkoxy and chlorophenyl, piperidyl substituted with lower alkoxy, piperidyl substituted with lower alkoxy having cyclo(lower)alkyl, piperidyl substituted with lower alkoxy(higher)alkoxy, dioxaazaspiro(higher)alkyl, tetrahydropyrazolopyridyl substituted with phenyl, cyclo(lower)alkyloxy, piperidyloxy substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, piperidyloxy substituted with lower alkoxy(higher)alkyl, piperidyloxy substituted with phenyl which may have lower alkoxy, piperidyl substituted with lower alkoxy higher alkyl, and piperidyl substituted with lower alkoxy(lower)alkoxy, thiadiazolyl substituted with pyridyl having piperidyl substituted with phenyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy(lower)alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy and cyclo(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with phenyl which may have lower alkoxy, imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, imidazothiadiazolyl substituted with phenyl having tetrahydropyridyl substituted with cyclo(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperidyl substituted with lower alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperazinyl substituted with lower alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy(higher)alkyl, imidazothiazolyl substituted with phenyl having lower alkoxy(lower)alkoxy, phenyl substituted with piperazinyl having phenyl substituted with lower alkoxy, phenyl substituted with piperazinyl having phenyl substituted with piperidyloxy having lower alkoxy(lower)alkyl, phenyl substituted with diazabicyclo(higher)alkyl having cyclo(lower)alkyl, phenyl substituted with hexahydrodiazepinyl having cyclo(lower)alkyl, phenyl substituted with piperidyl having phenyl, phenyl substituted with piperazinyl having phenyl substituted with piperazinyl having lower alkoxy(lower)alkyl, piperazinyl substituted with thiadiazolyl having phenyl substituted with lower alkoxy(higher)alkoxy, thiazolyl substituted with phenyl having lower alkoxy, oxadiazolyl substituted with phenyl having higher alkoxy, oxadiazolyl substituted with phenyl having phenyl substituted with lower alkoxy, oxadiazolyl substituted with phenyl having piperazinyl substituted with cyclo(lower)alkyl having lower alkyl, pyrazolyl substituted with phenyl having phenyl, and pyrazolyl substituted with phenyl having lower alkoxy, R2 is hydrogen, R3 is lower alkyl which has two hydroxy, R4 is hydrogen or hydroxy; R5 is hydroxy or hydroxysulfonyloxy; and R6 is hydroxy.", "5.A process for preparing a polypeptide compound (I) of claim 1, or a salt thereof, which comprises, 1) reacting a compound (II) of the formula: wherein R1, R4, R5 and R6 are defined in claim 1, or its reactive derivative at the amino group or a salt thereof, with a compound (III) of the formula: R3═O (III) wherein R3 is defined in claim 1, or its reactive derivative or a salt thereof, to give a compound (Ia) of the formula: wherein R1, R3, R4, R5 and R6 are defined above, or a salt thereof, or ii) reacting a compound (Ia) of the formula: wherein R1, R3, R4, R5 and R6 are defined in claim 1, or its reactive derivative at the amino group or a salt thereof, with a compound (IV) of the formula: Ra2—OH (IV) wherein R1, Ra2 is acyl group, or its reactive derivative at the carboxy group or a salt thereof, to give a compound (Ib) of the formula: wherein R1, Ra2, R3, R4, R5 and R6 are defined above, or a salt thereof, or iii) subjecting a compound (Ib) of the formula: wherein R1, R3, R4, R5 and R6 are defined in claim 1, Ra2 is acyl group, or a salt thereof, to elimination reaction of the acyl group, to give a compound (Ia) of the formula: wherein R1, R3, R4, R5 and R6 are defined above, or a salt thereof, or iv) reacting a compound (Ic) of the formula: wherein R2, R3, R4, R5 and R6 are defined in claim 1, or its reactive derivative at the amino group or a salt thereof, with a compound (V) of the formula: Ra1—OH (V) wherein Ra1 is acyl group, or its reactive derivative at the carboxy group or a salt thereof, to give a compound (Id) of the formula: wherein R2, R3, R4, R5 and R6 are defined in claim 1, Ra1 is defined above, or a salt thereof.", "6.A pharmaceutical composition which comprises, as an active ingredient, a compound of claim 1 or a pharmaceutically acceptable salt thereof in admixture with pharmaceutically acceptable carriers or excipients.", "7.Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament.", "8.A compound of claim 1 or a pharmaceutically acceptable salt thereof for use as a medicament.", "9.A method for the prophylactic and/or therapeutic treatment of infectious diseases caused by pathogenic microorganisms, which comprises administering a compound of claim 1 or a pharmaceutically acceptable salt thereof to a human being or an animal.", "10.A commercial package comprising the pharmaceutical composition of claim 7 and a written matter associated therewith, wherein the written matter states that the pharmaceutical composition can or should be used for preventing or treating infections disease.", "11.An article of manufacture, comprising packaging material and the compound (I) identified in claim 1 contained within said packaging material, wherein said the compound (I) is therapeutically effective for preventing or treating infectious diseases, and wherein said packaging material comprises a label or a written material which indicates that said compound (I) can or should be used for preventing or treating infectious diseases." ], [ "<SOH> BACKGROUND ART <EOH>In U.S. Pat.", "Nos.", "5,376,634, 5,569,646, WO 96/11210 and WO 99/40108, there are disclosed the polypeptide compound and a pharmaceutically acceptable salt thereof, which have antimicrobial activities (especially antifungal activity)." ], [ "TECHNICAL FIELD The present invention relates to new polypeptide compounds and salts thereof which are useful as a medicament.", "BACKGROUND ART In U.S. Pat.", "Nos.", "5,376,634, 5,569,646, WO 96/11210 and WO 99/40108, there are disclosed the polypeptide compound and a pharmaceutically acceptable salt thereof, which have antimicrobial activities (especially antifungal activity).", "DISCLOSURE OF INVENTION The present invention relates to new polypeptide compound and a salt thereof.", "More particularly, it relates to new polypeptide compound and a salt thereof, which have antimicrobial activities [especially, antifungal activities, in which the fungi may include Aspergillus, Cryptococcus, Candida, Mucor, Actinomyces, Histoplasma, Dermatophyte, Malassezia, Fusarium and the like.", "], inhibitory activity on β-1,3-glucan synthase, and further which are expected to be useful for the prophylactic and/or therapeutic treatment of Pneumocystis carinii infection (e.g.", "Pneumocystis carinii pneumonia) in a human being or an animal, to a process for preparation thereof, to a pharmaceutical composition comprising the same, and to a method for the prophylactic and/or therapeutic treatment of infectious disease including Pneumocystis carinii infection (e.g.", "Pneumocystis carinii pneumonia) in a human being or an animal.", "The object polypeptide compounds of the present invention are new and can be represented by the following general formula (I): wherein R1 is acyl group, R2 is hydrogen or acyl group, R3 is lower alkyl which has one or more hydroxy or protected hydroxy, R4 is hydrogen or hydroxy, R5 is hydrogen, hydroxy, lower alkoxy or hydroxysulfonyloxy, and R6 is hydroxy or acyloxy, or a salt thereof.", "The new polypeptide compound (I) or a salt thereof can be prepared by the process as illustrated in the following reaction schemes.", "wherein R1, R2, R3, R4, R5 and R6 are defined above, Ra1 is acyl group, and Ra2 is acyl group.", "Suitable salt of the new polypeptide compound (I) is a pharmaceutically acceptable and conventional non-toxic salt, and may include a salt with a base or an acid addition salt such as a salt with an inorganic base, for example, an alkali metal salt (e.g., sodium salt, potassium salt, etc.", "), an alkaline earth metal salt (e.g., calcium salt, magnesium salt, etc.", "), an ammonium salt; a salt with an organic base, for example, an organic amine salt (e.g., triethylamine salt, diisopropylethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N′-dibenzylethylenediamine salt, 4-dimethylaminopyridine salt, etc.", "); an inorganic acid addition salt (e.g., hydrochloride hydrobromide, sulfate, phosphate, etc.", "); an organic carboxylic sulfonic acid addition salt (e.g., formate, acetate, trifluoroacetate, maleate, tartrate, fumarate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.", "); a salt with a basic or acidic amino acid (e.g., arginine, aspartic acid, glutamic acid, etc.).", "Suitable examples and illustration of the various definitions in the above and subsequent descriptions of the present specification, which the present invention intends to include within the scope thereof, are explained in detail as follows: The term “lower” is used to intend a group having 1 to 6 carbon atom(s), unless otherwise provided.", "Suitable example of “one or more” may be the number of 1 to 6, in which the preferred one may be the number of 1 to 3, and the most preferred one may be the number of 1 or 2.Suitable example of “halogen” may be fluorine, chlorine, bromine, iodine and the like.", "Suitable example of “lower alkoxy” may include straight or branched one such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, tert-pentyloxy, neo-pentyloxy, hexyloxy, isohexyloxy and the like.", "Suitable example of “higher alkoxy” may include straight or branched one such as heptyloxy, octyloxy, 3,5-dimethyloctyloxy, 3,7-dimethyloctyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, hexadecyloxy, heptadecyloxy, octadecyloxy, nonadecyloxy, icosyloxy, and the like.", "Suitable example of “lower alkyl” may include straight or branched one having 1 to 6 carbon atom(s), such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, tert-pentyl, neo-pentyl, hexyl, isohexyl and the like.", "Suitable example of “higher alkyl” may include straight or branched one such as heptyl, octyl, 3,5-dimethyloctyl, 3,7-dimethyloctyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, and the like.", "Suitable example of “aryl” and “ar” moiety may include phenyl which may have lower alkyl (e.g., phenyl, mesityl, xylyl, tolyl, etc.", "), naphthyl, anthryl, indanyl, fluorenyl, and the like, and this “aryl” and “ar” moiety may have one or more halogen.", "Suitable example of “aroyl” may include benzoyl, toluoyl, naphthoyl, anthrylcarbonyl, and the like.", "Suitable example of “heterocyclic group” may include unsaturated 3 to 8-membered (more preferably 5 or 6-membered) heteromonocyclic group containing 1 to 4 nitrogen atom(s), for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g.", "; 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.", "), tetrazolyl (e.g.", "1H-tetrazolyl, 2H-tetrazolyl, etc.", "), etc.", "; saturated 3 to 8-membered (more preferably 5 or 6-membered) heteromonocyclic group containing 1 to 4 nitrogen atom(s), for example, pyrrolidinyl, imidazolidinyl, piperidyl, piperazinyl, azetidinyl, etc.", "; unsaturated condensed heterocyclic group containing 1 to 4 nitrogen atom(s), for example, indolyl, isoindolyl, indolinyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, etc.", "; unsaturated 3 to 8-membered (more preferably 5 or 6-membered) heteromonocyclic group containing 1 or 2 oxygen atom(s) and 1 to 3 nitrogen atom(s), for example, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.", "), etc.", "; saturated 3 to 8-membered (more preferably 5 or 6-membered) heteromonocyclic group containing 1 or 2 oxygen atom(s) and 1 to 3 nitrogen atom(s), for example, morpholinyl, sydnonyl, morpholino, etc.", "; unsaturated condensed heterocyclic group containing 1 or 2 oxygen atom(s) and 1 to 3 nitrogen atom(s), for example, benzoxazolyl, benzoxadiazolyl, etc.", "; unsaturated 3 to 8-membered (more preferably 5 or 6-membered) heteromonocyclic group containing 1 or 2 sulfur atom(s) and 1 to 3 nitrogen atom(s), for example, thiazolyl, isothiazolyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.", "), dihydrothiazinyl, etc.", "; saturated 3 to 8-membered (more preferably 5 or 6-membered) heteromonocyclic group containing 1 or 2 sulfur atom(s) and 1 to 3 nitrogen atom(s), for example thiazolidinyl, thiomorpholinyl, thiomorpholino, etc.", "; unsaturated 3 to 8-membered (more preferably 5 or 6-membered) heteromonocyclic group containing 1 or 2 sulfur atom(s), for example, thienyl, dihydrodithiinyl, dihydrodithionyl, etc.", "; unsaturated condensed heterocyclic group containing 1 or 2 sulfur atom(s) and 1 to 3 nitrogen atom(s), for example, benzothiazolyl, benzothiadiazolyl, imidazothiadiazolyl, etc.", "; unsaturated 3 to 8-membered (more preferably 5 or 6-membered) heteromonocyclic group containing an oxygen atom, for example, furyl etc.", "; saturated 3 to 8-membered (more preferably 5 or 6-membered) heteromonocyclic group containing 1 or 2 oxygen atom(s), for example, tetrahydrofuran, tetrahydropyran, dioxacyclopentane, dioxacyclohexane, etc.", "; unsaturated 3 to 8-membered (more preferably 5 or 6-membered) heteromonocyclic group containing an oxygen atom and 1 or 2 sulfur atom(s), for example, dihydrooxathiinyl, etc.", "; unsaturated condensed heterocyclic group containing 1 or 2 sulfur atom(s), for example benzothienyl, benzodithiinyl, etc.", "; unsaturated condensed heterocyclic group containing an oxygen atom and 1 or 2 sulfur atom(s), for example, benzoxathiinyl, etc.", "; and the like, and this “heterocyclic group” may have one or more suitable substituent(s) selected from the group consisting of lower alkyl, oxo, cyclo(lower)alkyl, hydroxy(lower)alkyl, carboxy(lower)alkanoyl which may have amino and heterocycliccarbonyl.", "Suitable example of “cyclo(lower)alkyl” may include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, and this “cyclo(lower)alkyl” may have one or more lower alkyl.", "Suitable example of “cyclo(lower)alkyloxy” may include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.", "Suitable example of “acyl group” may include aliphatic acyl, aromatic acyl, arylaliphatic acyl and heterocyclic-aliphatic acyl derived from carboxylic acid, carbonic acid, carbamic acid, sulfonic acid, and the like.", "Suitable example of said “acyl group” may be illustrated as follows.", "Carboxy; carbamoyl; mono or di(lower)alkylcarbamoyl (e.g., methylcarbamoyl, dimethylcarbamoyl, ethylcarbamoyl, diethylcarbamoyl, etc.)", "Aliphatic acyl such as lower or higher alkanoyl (e.g., formyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl, icosanoyl, etc.", "); lower or higher alkoxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, t-pentyloxycarbonyl, heptyloxycarbonyl, etc.", "); lower alkenyloxycarbonyl (e.g., vinyloxycarbonyl, propenyloxycarbonyl, allyloxycarbonyl, butenyloxycarbonyl, butedienyloxycarbonyl, pentenyloxycarbonyl, hexenyloxycarbonyl, etc.", "); lower or higher alkylsulfonyl (e.g., methylsulfonyl, ethylsulfonyl, etc.", "); lower or higher alkoxysulfonyl (e.g., methoxysulfonyl, ethoxysulfonyl, etc.", "); or the like; Aromatic acyl such as aroyl (e.g., benzoyl, toluoyl, naphthoyl, etc.", "); ar(lower)alkanoyl [e.g., phenyl(C1-C6)alkanoyl (e.g., phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutanoyl, phenylpentanoyl, phenylhexanoyl, etc.", "), naphthyl(C1-C6)alkanoyl (e.g., naphthylacetyl, naphthylpropanoyl, naphthylbutanoyl, etc.", "), etc.", "]; ar(lower)alkenoyl [e.g., phenyl(C3-C6)alkenoyl (e.g., phenylpropenoyl, phenylbutenoyl, phenylacryloyl, phenylmethacryloyl, phenylpentanoyl, phenylhexenoyl, etc.", "), naphthyl(C3-C6)alkenoyl (e.g., naphthylpropenoyl, naphthylbutenoyl, etc.", "), etc.]", "substituted with one or more suitable substituent(s); ar(lower)alkoxycarbonyl [e.g., phenyl(C1-C6)alkoxycarbonyl (e.g., benzyloxycarbonyl, etc.", "), fluorenyl(C1-C6)alkoxy-carbonyl (e.g., fluorenylmethyloxycarbonyl, etc.", "), etc.", "]; aryloxycarbonyl (e.g., phenoxycarbonyl, naphthyloxycarbonyl, etc.", "); aryloxy(lower)alkanoyl (e.g., phenoxyacetyl, phenoxypropionyl, etc.", "); arylcarbamoyl (e.g., phenylcarbamoyl, etc.", "); arylthiocarbamoyl (e.g., phenylthiocarbamoyl, etc.", "); arylglyoxyloyl (e.g., phenylglyoxyloyl, naphthylglyoxyloyl, etc.", "); arylsulfonyl which may have 1 to 4 lower alkyl (e.g., phenylsulfonyl, p-tolylsulfonyl, etc.", "); aroyl (e.g., benzoyl, naphthoyl, etc.)", "substituted with one or more suitable substituent(s); or the like; Heterocyclic acyl such as heterocycliccarbonyl; heterocyclic(lower)alkanoyl (e.g., heterocyclicacetyl, heterocyclicpropanoyl, heterocyclicbutanoyl, heterocyclicpentanoyl, heterocyclichexanoyl, etc.", "); heterocyclic(lower)alkenoyl (e.g., heterocyclicpropenoyl, heterocyclicbutenoyl, heterocyclicpentenoyl, heterocyclichexenoyl, etc.", "); heterocyclicglyoxyloyl; or the like; in which suitable “heterocyclic” moiety in the terms “heterocycliccarbonyl”, “heterocyclic(lower)alkanoyl”, “heterocyclic(lower)alkenoyl” and “heterocyclicglyoxyloyl” can be referred to aforementioned “heterocyclic” moiety.", "Suitable example of “acyl group” of R1 can be referred to aforementioned “acyl group”, in which the preferred one may be lower alkoxy carbonyl, higher alkanoyl, phenyl(lower)alkenoyl substituted with one or more suitable substituent(s), benzoyl substituted with one or more suitable substituent(s) and naphthoyl substituted with one or more suitable substituent(s).", "Suitable example of “suitable substituent(s)” in the term of “phenyl(lower)alkenoyl substituted with one or more suitable substituent(s)”, “benzoyl substituted with one or more suitable substituent(s)” or “naphthoyl substituted with one or more suitable substituent(s)” may be higher alkoxy, lower alkoxy(higher)alkoxy, higher alkyl, phenyl substituted with a suitable substituent selected from the group consisting of lower alkoxy, higher alkoxy and higher alkyl, thiadiazolyl substituted with phenyl which has a suitable substituent selected from the group consisting of piperazinyl substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, piperazinyl substituted with lower alkoxy(higher)alkyl, piperazinyl substituted with tetrahydropyran, piperazinyl substituted with dioxaspiro(higher)alkyl which may have lower alkyl, piperazinyl substituted with lower alkyl having pyridyl, piperidyl substituted with lower alkoxy and chlorophenyl, piperidyl substituted with lower alkoxy, piperidyl substituted with lower alkoxy having cyclo(lower)alkyl, piperidyl substituted with lower alkoxy(higher)alkoxy, dioxaazaspiro(higher)alkyl, tetrahydropyrazolopyridyl substituted with phenyl, cyclo(lower)alkyloxy, piperidyloxy substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, piperidyloxy substituted with lower alkoxy(higher)alkyl, piperidyloxy substituted with phenyl which may have lower alkoxy, piperidyl substituted with lower alkoxy higher alkyl, and piperidyl substituted with lower alkoxy(lower)alkoxy, thiadiazolyl substituted with pyridyl having piperidyl substituted with phenyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy(lower)alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy and cyclo(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with phenyl which may have lower alkoxy, imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, imidazothiadiazolyl substituted with phenyl having tetrahydropyridyl substituted with cyclo(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperidyl substituted with lower alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperazinyl substituted with lower alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy(higher)alkyl, imidazothiazolyl substituted with phenyl having lower alkoxy(lower)alkoxy, phenyl substituted with piperazinyl having phenyl substituted with lower alkoxy, phenyl substituted with piperazinyl having phenyl substituted with piperidyloxy having lower alkoxy(lower)alkyl, phenyl substituted with diazabicyclo(higher)alkyl having cyclo(lower)alkyl, phenyl substituted with hexahydrodiazepinyl having cyclo(lower)alkyl, phenyl substituted with piperidyl having phenyl, phenyl substituted with piperazinyl having phenyl substituted with piperazinyl having lower alkoxy(lower)alkyl, piperazinyl substituted with thiadiazolyl having phenyl substituted with lower alkoxy(higher)alkoxy, thiazolyl substituted with phenyl having lower alkoxy, oxadiazolyl substituted with phenyl having higher alkoxy, oxadiazolyl substituted with phenyl having phenyl substituted with lower alkoxy, oxadiazolyl substituted with phenyl having piperazinyl substituted with cyclo(lower)alkyl having lower alkyl, pyrazolyl substituted with phenyl having phenyl, or pyrazolyl substituted with phenyl having lower alkoxy, in which the preferred one may be heptyloxy, methoxyoctyloxy, heptyl, phenyl substituted with a substituent selected from the group consisting of butoxy, pentyloxy, nonyloxy and heptyl, thiadiazolyl substituted with phenyl which has a substituent selected from the group consisting of piperazinyl substituted with cyclohexyl having methyl, piperazinyl substituted with cyclopentyl, piperazinyl substituted with cycloheptyl, piperazinyl substituted with cyclohexyl having methoxyhexyloxy, piperazinyl substituted with methoxyheptyl, piperazinyl substituted with tetrahydropyran, piperazinyl substituted with dioxaspirodecan which may have dimethyl, piperazinyl substituted with methyl having pyridyl, piperidyl substituted with methoxy and chlorophenyl, piperidyl substituted with 4-methylpentyloxy, piperidyl substituted with butoxy, piperidyl substituted with pentyloxy, piperidyl substituted with methoxy having cyclohexyl, piperidyl substituted with methoxyheptyloxy, dioxaazospirodecan, tetrahydropyrazolopyridyl substituted with phenyl, cyclohexyloxy, piperidyloxy substituted with cyclohexyl which may have methoxyhexyloxy, piperidyloxy substituted with methoxyoctyl, piperidyloxy substituted with phenyl which may have methoxy, piperidyl substituted with methoxyheptyl, and piperidyl substituted with methoxyhexyloxy, thiadiazolyl substituted with pyridyl having piperidyl substituted with phenyl, imidazothiadiazolyl substituted with phenyl having methoxypentyloxymethyl, imidazothiadiazolyl substituted with phenyl having methoxy and cyclohexyl, imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with phenyl which may have methoxy, imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with cyclohexyl which may have methoxyhexyloxy, imidazothiadiazolyl substituted with phenyl having tetrahydropyridyl substituted with cyclohexyl, imidazothiadiazolyl substituted with phenyl having piperidyl substituted with methoxyhexyl, imidazothiadiazolyl substituted with phenyl having piperazinyl substituted with methoxypentyl, imidazothiadiazolyl substituted with phenyl having piperazinyl substituted with methoxyhexyl, imidazothiadiazolyl substituted with phenyl having methoxyheptyl, imidazothiazolyl substituted with phenyl having methoxypentyloxy, phenyl substituted with piperazinyl having phenyl substituted with methoxy, phenyl substituted with piperazinyl having phenyl substituted with piperidyloxy having methoxyhexyl, phenyl substituted with diazabicycloheptyl having cyclohexyl, phenyl substituted with hexahydrodiazepinyl having cyclohexyl, phenyl substituted with piperidyl having phenyl, phenyl substituted with piperazinyl having phenyl substituted with piperazinyl having methoxyhexyl, piperazinyl substituted with thiadiazolyl having phenyl substituted with methoxyheptyloxy, thiazolyl substituted with phenyl having pentyloxy, oxadiazolyl substituted with phenyl having octyloxy, oxadiazolyl substituted with phenyl having phenyl substituted with propoxy, oxadiazolyl substituted with phenyl having piperazinyl substituted with cyclohexyl having methyl, pyrazolyl substituted with phenyl having phenyl, or pyrazolyl substituted with phenyl having hexyloxy.", "The more suitable example of “acyl group” may be naphthoyl substituted with heptyloxy, naphthoyl substituted with methoxyoctyloxy, naphthoyl substituted with heptyl, phenylacryloyl substituted with phenyl substituted with a substituent selected from the group consisting of butoxy and pentyloxy, benzoyl substituted with phenyl substituted with a substituent selected from the group consisting of nonyloxy and heptyl, benzoyl substituted with thiadiazolyl substituted with phenyl which has a substituent selected from the group consisting of piperazinyl substituted with cyclohexyl having methyl, piperazinyl substituted with cyclopentyl, piperazinyl substituted with cycloheptyl, piperazinyl substituted with cyclohexyl having methoxyhexyloxy, piperazinyl substituted with methoxyheptyl, piperazinyl substituted with tetrahydropyran, piperazinyl substituted with dioxaspirodecan which may have dimethyl, piperazinyl substituted with methyl having pyridyl, piperidyl substituted with methoxy and chlorophenyl, piperidyl substituted with 4-methylpentyloxy, piperidyl substituted with butoxy, piperidyl substituted with pentyloxy, piperidyl substituted with methoxy having cyclohexyl, piperidyl substituted with methoxyheptyloxy, dioxaazospirodecan, tetrahydropyrazolopyridyl substituted with phenyl, cyclohexyloxy, piperidyloxy substituted with cyclohexyl which may have methoxyhexyloxy, piperidyloxy substituted with methoxyoctyl, piperidyloxy substituted with phenyl which may have methoxy, piperidyl substituted with methoxyheptyl, and piperidyl substituted with methoxyhexyloxy, benzoyl substituted with thiadiazolyl substituted with pyridyl having piperidyl substituted with phenyl, benzoyl substituted with imidazothiadiazolyl substituted with phenyl having methoxypentyloxymethyl, benzoyl substituted with imidazothiadiazolyl substituted with phenyl having methoxy and cyclohexyl, benzoyl substituted with imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with phenyl which may have methoxy, benzoyl substituted with imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with cyclohexyl which may have methoxyhexyloxy, benzoyl substituted with has imidazothiadiazolyl substituted with phenyl having tetrahydropyridyl substituted with cyclohexyl, benzoyl substituted with imidazothiadiazolyl substituted with phenyl having piperidyl substituted with methoxyhexyl, benzoyl substituted with imidazothiadiazolyl substituted with phenyl having piperazinyl substituted with methoxypentyl, benzoyl substituted with imidazothiadiazolyl substituted with phenyl having piperazinyl substituted with methoxyhexyl, benzoyl substituted with imidazothiadiazolyl substituted with phenyl having methoxyheptyl, benzoyl substituted with imidazothiazolyl substituted with phenyl having methoxypentyloxy, benzoyl substituted with phenyl substituted with piperazinyl having phenyl substituted with methoxy, benzoyl substituted with phenyl substituted with piperazinyl having phenyl substituted with piperidyloxy having methoxyhexyl, benzoyl substituted with phenyl substituted with diazabicycloheptyl having cyclohexyl, benzoyl substituted with phenyl substituted with hexahydrodiazepinyl having cyclohexyl, benzoyl substituted with phenyl substituted with piperidyl having phenyl, benzoyl substituted with phenyl substituted with piperazinyl having phenyl substituted with piperazinyl having methoxyhexyl, benzoyl substituted with piperazinyl substituted with thiadiazolyl having phenyl substituted with methoxyheptyloxy, benzoyl substituted with thiazolyl substituted with phenyl having pentyloxy, benzoyl substituted with oxadiazolyl substituted with phenyl having octyloxy, benzoyl substituted with oxadiazolyl substituted with phenyl having phenyl substituted with propoxy, benzoyl substituted with oxadiazolyl substituted with phenyl having piperazinyl substituted with cyclohexyl having methyl, benzoyl substituted with pyrazolyl substituted with phenyl having phenyl, or benzoyl substituted with pyrazolyl substituted with phenyl having hexyloxy.", "Suitable example of “lower alkyl” in the term of “lower alkyl which has one or more hydroxy or protected hydroxy” can be referred to aforementioned “lower alkyl”, in which the preferred one may be methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.", "Suitable example of “hydroxy protective group” in the term of “protected hydroxy” may include acyl (e.g., lower alkanoyl, etc.)", "as mentioned above, phenyl(lower)alkyl which may have one or more suitable substituent(s) (e.g., benzyl, 4-methoxybenzyl, trityl, etc.", "), tri-substituted silyl [e.g., tri(lower)alkylsilyl(e.g., trimethylsilyl, t-butyldimethylsilyl, etc.", "), etc.", "], tetrahydropyranyl and the like.", "Suitable example of “lower alkyl which has one or more hydroxy or protected hydroxy” may be dihydroxypropyl, dihydroxyisopropyl, trihydroxybutyl, tetrahydroxypentyl, pentahydroxyhexyl and diacetyloxyisopropyl.", "Suitable example of “acyl group” of R2 can be referred to aforementioned “acyl group”, in which the preferred one may be “amino protective group” mentioned below, and the most preferred one may be acetyl, 2-acetyloxypropionyl, methylsulfonyl, 2,5-diaminopentanoyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, allyloxycarbonyl, tert-butoxycarbonyl and (5-methyl-2-oxo-1,3-dioxol-4-yl)methoxycarbonyl.", "Suitable example of “amino protective group” may be included in aforementioned “acyl group”, a conventional protective group such as ar(lower)alkoxycarbonyl and lower alkoxycarbonyl, in which the preferred one may be phenyl-(C1-C4)alkoxycarbonyl and fluorenyl(C1-C4) alkoxycarbonyl and (C1-C4)alkoxycarbonyl, and the most preferred one may be benzyloxycarbonyl, fluorenylmethoxycarbonyl and tert-butoxycarbonyl.", "Suitable example of “acyl” moiety of “acyloxy” can be referred to aforementioned “acyl group”, in which the preferred one may be lower alkenyloxycarbonyl, and the most preferred one may be allyloxycarbonyl.", "Suitable example of “acyloxy” may be lower alkenyloxycarbonyloxy, and the more preferred one may be allyloxycarbonyloxy.", "Particularly, the preferred examples of the cyclic polypeptide compound (I) of the present invention are as follows: the compound (I), wherein R1 is phenyl(lower)alkenoyl substituted with one or more suitable substituent(s), benzoyl substituted with one or more suitable substituent(s) or naphthoyl substituted with one or more suitable substituent (s), R2 is hydrogen, R3 is lower alkyl which has one or more hydroxy, R4 is hydrogen or hydroxy; R5 is hydroxy or hydroxysulfonyloxy; and R6 is hydroxy.", "And, more preferred one may be the compound (I) wherein R1 is naphthoyl substituted with higher alkoxy, naphthoyl substituted with lower alkoxy(higher)alkoxy, naphthoyl substituted with higher alkyl, phenyl(lower)alkenoyl substituted with phenyl substituted with lower alkoxy, benzoyl substituted with a suitable substituent selected from the group consisting of phenyl substituted with a suitable substituent selected from the group consisting of lower alkoxy, higher alkoxy and higher alkyl, thiadiazolyl substituted with phenyl which has a suitable substituent selected from the group consisting of piperazinyl substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, piperazinyl substituted with lower alkoxy(higher)alkyl, piperazinyl substituted with tetrahydropyran, piperazinyl substituted with dioxaspiro(higher)alkyl which may have lower alkyl, piperazinyl substituted with lower alkyl having pyridyl, piperidyl substituted with lower alkoxy and chlorophenyl, piperidyl substituted with lower alkoxy, piperidyl substituted with lower alkoxy having cyclo(lower)alkyl, piperidyl substituted with lower alkoxy(higher)alkoxy, dioxaazaspiro(higher)alkyl, tetrahydropyrazolopyridyl substituted with phenyl, cyclo(lower)alkyloxy, piperidyloxy substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, piperidyloxy substituted with lower alkoxy(higher)alkyl, piperidyloxy substituted with phenyl which may have lower alkoxy, piperidyl substituted with lower alkoxy higher alkyl, and piperidyl substituted with lower alkoxy(lower)alkoxy, thiadiazolyl substituted with pyridyl having piperidyl substituted with phenyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy(lower)alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy and cyclo(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with phenyl which may have lower alkoxy, imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, imidazothiadiazolyl substituted with phenyl having tetrahydropyridyl substituted with cyclo(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperidyl substituted with lower alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperazinyl substituted with lower alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy(higher)alkyl, imidazothiazolyl substituted with phenyl having lower alkoxy(lower)alkoxy, phenyl substituted with piperazinyl having phenyl substituted with lower alkoxy, phenyl substituted with piperazinyl having phenyl substituted with piperidyloxy having lower alkoxy(lower)alkyl, phenyl substituted with diazabicyclo(higher)alkyl having cyclo(lower)alkyl, phenyl substituted with hexahydrodiazepinyl having cyclo(lower)alkyl, phenyl substituted with piperidyl having phenyl, phenyl substituted with piperazinyl having phenyl substituted with piperazinyl having lower alkoxy(lower)alkyl, piperazinyl substituted with thiadiazolyl having phenyl substituted with lower alkoxy(higher)alkoxy, thiazolyl substituted with phenyl having lower alkoxy, oxadiazolyl substituted with phenyl having higher alkoxy, oxadiazolyl substituted with phenyl having phenyl substituted with lower alkoxy, oxadiazolyl substituted with phenyl having piperazinyl substituted with cyclo(lower)alkyl having lower alkyl, pyrazolyl substituted with phenyl having phenyl, and pyrazolyl substituted with phenyl having lower alkoxy, R2 is hydrogen, R3 is lower alkyl which has two hydroxy, R4 is hydrogen or hydroxy; R5 is hydroxy or hydroxysulfonyloxy; and R6 is hydroxy.", "The processes for preparing the polypeptide compound (I) of the present invention are explained in detail in the following.", "Process 1 The object compound (Ia) or a salt thereof can be prepared by reacting the compound (II) or its reactive derivative at the amino group or a salt thereof with the compound (III) of the formula: R3═O (III) or its reactive derivative, or a salt thereof.", "Suitable reactive derivative of the compound (III) may include an acid halide, an acid anhydride, an activated ester, and the like.", "The suitable example may be an acid chloride; acid azide; a mixed acid anhydride with an acid such as substituted phosphoric acid (e.g., dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid, dibenzylphosphoric acid, halogenated phosphoric acid, etc.", "), dialkylphosphorous acid, sulfurous acid, thiosulfuric acid, alkanesulfonic acid (e.g., methanesulfonic acid, ethanesulfonic acid, etc.", "), sulfuric acid, alkylcarbonic acid, aliphatic carboxylic acid (e.g., pivalic acid, pentanoic acid, isopentanoic acid, 2-ethylbutyric acid, trichloroacetic acid, etc.", "); aromatic carboxylic acid (e.g., benzoic acid, etc.", "); a symmetrical acid anydride; an activated amide with imidazole, 4-substitutd imidazole, dimethylpyrazole, triazole or tetrazole; an activated ester (e.g., cyanomethyl, ester methoxymethyl ester, vinyl ester, propargyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl ester, pentachlorophenyl ester, mesylphenyl ester, phenylazophenyl ester, phenylthioester, p-nitrophenyl thioester, p-cresyl thioester, carboxymethyl thioester, pyranyl ester, pyridyl ester, piperidyl ester, 8-quinolyl thioester, etc.", "); an ester with a N-hydroxy compound (e.g., N,N-dimethylhydroxylamine, 1-hydroxy-2-(1H)-pyridone, N-hydroxysuccinimide, N-hydroxybenzotriazole, N-hydroxyphthalimide, 1-hydroxy-6-chloro-1H-benzotriazole, etc.", "); and the like.", "These reactive derivatives can optionally be selected from them according to the kind of the compound (III) to be used.", "The reaction is usually carried out in a conventional solvent such as water, acetone, dioxane, acetonitrile, chloroform, methylene chloride, ethylene chloride, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, pyridine or any other organic solvent which do not adversely affect the reaction, or the mixture thereof.", "When the compound (III) is used in free acid form or its salt form in the reaction, the reaction is preferably carried out in the presence of a conventional condensing agent such as N,N′-dicyclohexylcarbodiimide; N-cyclohexyl-N′-morpholinoethylcarbodiimide); N-cyclohexyl-N′-(4-diethylaminocyclohexyl)carbodiimide; N,N′-diisopropylcarboxiimide; N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide; N,N-carbonyl-bis(2-methylimidazole); pentamethyleneketene-N-cyclohexylimine; diphenylketene-N-cyclohexylimine, ethoxyacetylene; 1-alkoxy-1-chloroethylene; trialkyl phosphite; isopropyl polyphosphate; phosphorous oxychloride (phosphoryl chloride); phosphorous trichloride; thionyl chloride; oxalyl chloride; triphenylphosphite; 2-ethyl-7-hydroxybenzisoxazolium salt; 2-ethyl-S-(m-sulfophenyl) isoxazolium hydroxide intra-molecular salt; 1-(p-chlorobenzenesulfonyloxy)-6-chloro-1H-benzotriazole; so-called Vilsmeier reagent prepared by the reaction of N,N-dimethylformamide with thionyl chloride, phosgene, phosphorous oxychloride, etc.", "; or the like.", "The reaction may also be carried out in the presence of an organic or inorganic base such as an alkali metal bicarbonate, tri(lower)alkylamine (e.g., triethylamine, diisopropylethylamine, etc.", "), pyridine, di(lower)alkylaminopyridine (e.g., 4-dimethylaminopyridine, etc.)", "N-(lower)alkylmorphorine, N,N-di(lower)alkylbenzylamine, or the like.", "The reaction temperature is not critical, and the reaction is usually carried out under cooling to heating.", "Process 2 The object compound (Ib) or a salt thereof can be prepared by reacting the compound (Ia) or its reactive derivative at the amino group or a salt thereof with the compound (IV) of the formula: Ra2—OH (IV) (wherein Ra2 is acyl group) or its reactive derivative at the carboxy group or a salt thereof.", "Suitable reactive derivative of the compound (IV) may include an acid halide, an acid anhydride, an activated ester, and the like.", "The suitable example may be an acid chloride; acid azide; a mixed acid anhydride with an acid such as substituted phosphoric acid (e.g., dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid, dibenzylphosphoric acid, halogenated phosphoric acid, etc.", "), dialkylphosphorous acid, sulfurous acid, thiosulfuric acid, alkanesulfonic acid (e.g., methanesulfonic acid, ethanesulfonic acid, etc.", "), sulfuric acid, alkylcarbonic acid, aliphatic carboxylic acid (e.g., pivalic acid, pentanoic acid, isopentanoic acid, 2-ethylbutyric acid, trichloroacetic acid, etc.", "); aromatic carboxylic acid (e.g., benzoic acid, etc.", "); a symmetrical acid anydride; an activated amide with imidazole, 4-substitutd imidazole, dimethylpyrazole, triazole or tetrazole; an activated ester (e.g., cyanomethyl, ester methoxymethyl ester, vinyl ester, propargyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl ester, pentachlorophenyl ester, mesylphenyl ester, phenylazophenyl ester, phenylthioester, p-nitrophenyl thioester, p-cresyl thioester, carboxymethyl thioester, pyranyl ester, pyridyl ester, piperidyl ester, 8-quinolyl thioester, etc.", "); an ester with a N-hydroxy compound (e.g., N,N-dimethylhydroxylamine, 1-hydroxy-2-(1H)-pyridone, N-hydroxysuccinimide, N-hydroxybenzotriazole, N-hydroxyphthalimide, 1-hydroxy-6-chloro-1H-benzotriazole, etc.", "); and the like.", "These reactive derivatives can optionally be selected from them according to the kind of the compound (IV) to be used.", "The reaction is usually carried out in a conventional solvent such as water, acetone, dioxane, acetonitrile, chloroform, methylene chloride, ethylene chloride, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, pyridine or any other organic solvent which do not adversely affect the reaction, or the mixture thereof.", "When the compound (IV) is used in free acid form or its salt form in the reaction, the reaction is preferably carried out in the presence of a conventional condensing agent such as N,N′-dicyclohexylcarbodiimide; N-cyclohexyl-N′-morpholinoethylcarbodiimide); N-cyclohexyl-N′-(4-diethylaminocyclohexyl)carbodiimide; N,N′-diisopropylcarboxiimide; N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide; N,N-carbonyl-bis(2-methylimidazole); pentamethyleneketene-N-cyclohexylimine; diphenylketene-N-cyclohexylimine, ethoxyacetylene; 1-alkoxy-1-chloroethylene; trialkyl phosphite; isopropyl polyphosphate; phosphorous oxychloride (phosphoryl chloride); phosphorous trichloride; thionyl chloride; oxalyl chloride; triphenylphosphite; 2-ethyl-7-hydroxybenzisoxazolium salt; 2-ethyl-5-(m-sulfophenyl) isoxazolium hydroxide intra-molecular salt; 1-(p-chlorobenzenesulfonyloxy)-6-chloro-1H-benzotriazole; so-called Vilsmeier reagent prepared by the reaction of N,N-dimethylformamide with thionyl chloride, phosgene, phosphorous oxychloride, etc.", "; or the like.", "The reaction may also be carried out in the presence of an organic or inorganic base such as an alkali metal bicarbonate, tri(lower)alkylamine (e.g., triethylamine, diisopropylethylamine, etc.", "), pyridine, di(lower)alkylaminopyridine (e.g., 4-dimethylaminopyridine, etc.)", "N-(lower)alkylmorphorine, N,N-di(lower)alkylbenzylamine, or the like.", "The reaction temperature is not critical, and the reaction is usually carried out under cooling to heating.", "Process 3 The object compound (Ia) or a salt thereof can be prepared by subjecting a compound (Ib) or a salt thereof to elimination reaction of the acyl group.", "This reaction is carried out in accordance with a conventional method such as hydrolysis, reduction or the like.", "The hydrolysis is preferably carried out in the presence of a base or an acid including Lewis acid.", "Suitable base may include an inorganic base and an organic base such as an alkali metal [e.g.", "sodium, potassium, etc.", "], an alkaline earth metal [e.g.", "magnesium, calcium, etc.", "], the hydroxide or carbonate or bicarbonate thereof, trialkylamine [e.g.", "trimethylamine, triethylamine, etc.", "], picoline, 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, or the like.", "Suitable acid may include an organic acid [e.g.", "formic acid, acetic acid, propionic acid, trichloroacetic acid, trifluoroacetic acid, etc.]", "and an inorganic acid [e.g.", "hydrochloric acid, hydrobromic acid, sulfuric acid, hydrogen chloride, hydrogen bromide, etc.].", "The elimination using Lewis acid such as trihaloacetic acid [e.g.", "trichloroacetic acid, trifluoroacetic acid, etc.]", "or the like is preferably carried out in the presence of cation trapping agents [e.g.", "anisole, phenol, etc.).", "The reaction is usually carried out in a solvent such as water, an alcohol [e.g.", "methanol, ethanol, etc.", "], methylene chloride, tetrahydrofuran, a mixture thereof or any other solvent which does not adversely influence the reaction.", "A liquid base or acid can be also used as the solvent.", "The reaction temperature is not critical and the reaction is usually carried out under cooling to warming.", "The reduction method applicable for the elimination reaction may include chemical reduction and catalytic reduction.", "Suitable reducing agents to be used in chemical reduction are a combination of metal [e.g.", "tin, zinc, iron, etc.]", "or metallic compound [e.g.", "chromium chloride, chromium acetate, etc.]", "and an organic or inorganic acid [e.g.", "formic acid, acetic acid, propionic acid, trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid, hydrobromic acid, etc.].", "Suitable catalysts to be used in catalytic reduction are conventional ones such as platinum catalysts [e.g.", "platinum plate, spongy platinum, platinum black, colloidal platinum, platinum oxide, platinum wire, etc.", "], palladium catalysts [e.g.", "spongy palladium, palladium black, palladium oxide, palladium on carbon, colloidal palladium, palladium on barium, sulfate, palladium on barium carbonate, etc.", "], nickel catalysts [e.g.", "reduced nickel, nickel oxide, Raney nickel, etc.", "], cobalt catalysts [e.g.", "reduced cobalt, Raney cobalt, etc], iron catalysts [e.g.", "reduced iron, Raney iron, etc], copper catalysts [e.g.", "reduced copper, Raney copper, Ullman copper, etc.]", "and the like.", "The reduction is usually carried out in a conventional solvent which does not adversely influence the reaction such as water, methanol, ethanol, propanol, N,N-dimethylformamide, or a mixture thereof.", "Additionally, in case that the above-mentioned acids to be used in chemical reduction are in liquid, they can also be used as a solvent.", "Further, a suitable solvent to be used in catalytic reduction may be the above-mentioned solvent, and other conventional solvent such as diethyl ether, dioxane, tetrahydrofuran, etc., or a mixture thereof.", "The reaction temperature of this reduction is not critical and the reaction is usually carried out under cooling to warming.", "Process 4 The object compound (Id) or a salt thereof can be prepared by reacting the compound (Ic) or its reactive derivative at the amino group or a salt thereof with the compound (V) of the formula: Ra1—OH (V) (wherein Ra1 is acyl group) or its reactive derivative at the carboxy group or a salt thereof.", "Suitable reactive derivative at the carboxy group of the compound (V) may include an acid halide, an acid anhydride, an activated amide, an activated ester, and the like.", "Suitable examples of the reactive derivatives may be an acid chloride; an acid azide; a mixed acid anhydride with an acid such as substituted phosphoric acid (e.g., dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid, dibenzylphosphoric acid, halogenated phosphoric acid, etc.", "], dialkylphosphorous acid, sulfurous acid, thiosulfuric acid, sulfuric acid, sulfonic acid [e.g., methanesulfonic acid, etc.", "], aliphatic carboxylic acid [e.g., acetic acid, propionic acid, butyric acid, isobutyric acid, pivaric acid, pentanoic acid, isopentanoic acid, 2-ethylbutyric acid trichloroacetic acid, etc.", "]; or aromatic carboxylic acid [e.g., benzoic acid, etc.", "]; a symmetrical acid, anhydride; an activated amide with imidazole, 4-substituted imidazole, dimethylpyrazole, triazole, tetrazole or 1-hydroxy-1H-benzotriazole; or an activated ester [e.g., cyanomethyl ester, methoxymethyl ester, vinyl ester, propargyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl ester, pentachloropentyl ester, mesylphenyl ester, phenylazophenyl ester, phenyl thioester, p-nitrophenyl thioester, p-cresyl thioester, carboxymethyl thioester, pyranyl ester, pyridyl ester, piperidyl ester, 8-quinolyl thioester, etc.", "], or an ester with a N-hydroxy compound [e.g.", "N,N-dimethylhydroxylamine, 1-hydroxy-2-(1H)-pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxy-1H-benzotriazole, etc.", "], and the like.", "These reactive derivatives can optionally be selected from them according to the kind of the compound (V) to be used.", "Suitable salts of the compound (V) and its reactive derivative can be referred to the ones as exemplified for the polypeptide compound (I).", "The reaction is usually carried out in a conventional solvent such as water, alcohol [e.g., methanol, ethanol, etc.", "], acetone, dioxane, acetonitrile, chloroform, methylene chloride, ethylene chloride, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, pyridine or any other organic solvent which does not adversely influence the reaction.", "These conventional solvent may also be used in a mixture with water.", "In this reaction, when the compound (V) is used in a free acid form or its salt form, the reaction is preferably carried out in the presence of a conventional condensing agent such as N,N-dicyclohexylcarbodiimide; N-cyclohexyl-N′-morpholinoethylcarbodiimide; N-cyclohexyl-N′-(4-diethylaminocyclohexyl)carbodiimide; N,N′-diethylcarbodiimide; N,N′-diisopropylcarbodiimide; N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide; N,N-carbonylbis-(2-methylimidazole); pentamethyleneketene-N-cyclohexylimine; diphenylketene-N-cyclohexylimine, ethoxyacetylene; 1-alkoxy-2-chloroethylene; trialkyl phosphite; ethyl polyphosphate; isopropyl polyphosphate; phosphorus oxychloride (phosphoryl chloride); phosphorus trichloride; thionyl chloride; oxalyl chloride; lower alkyl haloformate [e.g., ethyl chloroformate, isopropyl chloroformate, etc.", "]; triphenylphosphine; 2-ethyl-7-hydroxybenzisoxazolium salt; 2-ethyl-5-(m-sulfophenyl)isoxazolium hydroxide intramolecular salt; 1-(p-chlorobenzenesulfonyloxy)-6-chloro-1H-benzotriazole; so-called Vilsmeier reagent prepared by the reaction of N,N-dimethylformamide with thionyl chloride, phosgene, trichloromethyl chloroformate, phosphorous oxychloride, methanesulfonyl chloride, etc.", "; or the like.", "The reaction may also be carried out in the presence of an inorganic or organic base such as an alkali metal carbonate, alkali metal bicarbonate, tri(lower)alkylamine (e.g., triethylamine, diisopropylethylamine, etc.", "), pyridine, di(lower)alkylaminopyridine (e.g., 4-dimethylaminopyridine, etc.", "), N-(lower)alkylmorpholine, N,N-di(lower)alkylbenzylamine, or the like.", "The reaction temperature is not critical, and the reaction is usually carried out under cooling to warming.", "The compounds obtained by the above Processes 1 to 4 can be isolated and purified by a conventional method such as pulverization, recrystallization, column-chromatography, high-performance liquid chromatography (HPLC), reprecipitation, desalting resin column chromatography, or the like.", "The compounds obtained by the above Processes 1 to 4 may be obtained as its solvate (e.g., hydrate, ethanolate, etc.", "), and its solvate (e.g., hydrate, ethanolate, etc.)", "is included within the scope of the present invention.", "It is to be noted that each of the polypeptide compound (I) may include one or more stereoisomer such as optical isomer(s) and geometrical isomer(s) due to asymmetric carbon atom(s) and double bond(s) and all such isomers and the mixture thereof are included within the scope of the present invention.", "The polypeptide compound (I) or a salt thereof may include solvated compound [e.g., hydrate, ethanolate, etc.].", "The polypeptide compound (I) or a salt thereof may include both its crystal form and non-crystal form.", "It should be understood that the polypeptide compound (I) of the present invention may include the prodrug form.", "The patent applications and publications cited herein are incorporated by reference.", "In order to show the usefulness of the polypeptide compound (I) of the present invention, the biological data of the representative compound is explained in the following.", "Biological Property of the Polypeptide Compound (I) of the Present Invention Test (Antimicrobial Activity): In vitro antimicrobial activity of the object compound of Examples 41, 46, 53 and 56 disclosed later was determined by MICs in mouse serum as described below.", "Test Method: The MICS in mouse serum were determined by the microdilution method using ICR mouse serum buffered with 20 mM HEPES buffer (pH 7.3) as a test medium.", "Inoculum suspension of 106 cells/ml were prepared by a hemocytometric procedure and diluted to obtain an inoculum size of approximately 1.0×103 cells/ml.", "Microplates were incubated at 37° C. for 24 hours in 5% CO2.The MICS were defined as the lowest concentrations at which no visible growth was observed.", "Test Result: MIC (μg/ml) Test organism Test compound Candida albicans FP-633 The object compound of <0.3 Example 41 The object compound of <0.3 Example 46 The object compound of <0.3 Example 53 The object compound of <0.3 Example 56 From the test result, it is realized that the polypeptide compound (I) of the present invention has an antimicrobial activity (especially, antifungal activity).", "In more details, the polypeptide compound (I) of the present invention have an antifungal activity, particularly against the following fungi.", "Acremonium; Absidia (e.g., Absidia corymbifera, etc); Aspergillus (e.g., Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus versicolor, etc); Blastomyces (e.g., Blastomyces dermatitidis, etc); Candida (e.g., Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida tropicalis, candida utilis, etc.", "); Cladosporium (e.g., Cladosporium trichloides, etc); Coccidioides (e.g., Coccidioides immitis, etc); Cryptococcus (e.g., Cryptococcus neoformans, etc); Cunninghamella (e.g., Cunninghamella elegans, etc); Dermatophyte; Exophiala (e.g., Exophiala dermatitidis, Exophiala spinifera, etc); Epidermophyton (e.g., Epidermophyton floccosum, etc); Fonsecaea (e.g., Fonsecaea pedrosoi, etc); Fusarium (e.g., Fusarium solani, etc); Geotrichum (e.g., Geotrichum candiddum, etc); Histoplasma (e.g., Histoplasma capsulatum var.", "capsulatum, etc).", "Malassezia (e.g., Malassezia furfur, etc); Microsporum (e.g., Microsporum canis, Microsporum gypseum, etc); Mucor; Paracoccidioides (e.g., Paracoccidioides brasiliensis, etc); Penicillium (e.g., Penicillium marneffei, etc); Phialophora; Pneumocystis (e.g., Pneumocystis carinii, etc); Pseudallescheria (e.g., Pseudallescheria boydii, etc); Rhizopus (e.g., Rhizopus microsporus var.", "rhizopodiformis, Rhizopus oryzae, etc); Saccharomyces (e.g., Saccharomyces cerevisiae, etc); Scopulariopsis; Sporothrix (e.g., Sporothrix schenckii, etc); Trichophyton (e.g., Trichophyton mentagrophytes, Trichophyton rubrum, etc); Trichosporon (e.g., Trichosporon asahii, Trichosporon cutaneuin, etc).", "The above fungi are well-known to cause various infection diseases in skin, eye, hair, nail, oral mucosa, gastrointestinal tract, bronchus, lung, endocardium, brain, meninges, urinary organ, vaginal protion, oral cavity, ophthalmus, systemic, kidney, bronchus, heart, external auditory canal, bone, nasal cavity, paranasal cavity, spleen, liver, hypodermal tissue, lymph doct, gastrointestine, articulation, muscle, tendon, interstitial plasma cell in lung, blood, and so on.", "Therefore, the polypeptide compound (I) of the present invention are useful for preventing and treating various infectious diseases, such as dermatophytosis (e.g., trichophytosis, etc), pityriasis versicolor, candidiasis, cryptococcosis, geotrichosis, trichosporosis, aspergillosis, penicilliosis, fusariosis, zygomycosis, sporotrichosis, chromomycosis, coccidioidomycosis, histoplasmosis, blastomycosis, paracoccidioidomycosis, pseudallescheriosis, mycetoma, mycotic keratitis, otomycosis, pneumocystosis, fungemia, and so on.", "The combination use of azoles such as fluconazole, voriconazole, itraconazole, ketoconazole, miconazole, ER 30346 and SCH 56592; polyenes such as amphotericin B, nystatin, liposamal and lipid forms thereof such as Abelcet, AmBisome, and Amphocil; purine or pyrimidine nucleotide inhibitors such as flucytosine; or polyxins such as nikkomycines, in particular nikkomycine Z or nikkomycine X; other chitin inhibitors; elongation factor inhibitors such as sordarin and analogs thereof; mannan inhibitors such as predamycin, bactericidal/permeability-inducing (BPI) protein products such as XMP.97 or XMP.127; or complex carbohydrate antifungal agents such as CAN-296; or the combination use of immunosuppressant such as tacrolimus with the polypeptide compound (I) or a salt thereof is effective against above infectious diseases.", "The pharmaceutical composition of the present invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the polypeptide compound (I) or a pharmaceutically acceptable salt thereof, as an active ingredient in admixture with an organic or inorganic carrier or excipient which is suitable for rectal; pulmonary (nasal or buccal inhalation); ocular; external (topical); oral administration; parenteral (including subcutaneous, intravenous and intramuscular) administrations; insufflation (including aerosols from metered dose inhalator); nebulizer; or dry powder inhalator.", "The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers in a solid form such as granules, tablets, dragees, pellets, troches, capsules, or suppositories; creams; ointments; aerosols; powders for insufflation; in a liquid form such as solutions, emulsions, or suspensions for injection; ingestion; eye drops; and any other form suitable for use.", "And, if necessary, there may be included in the above preparation auxiliary substance such as stabilizing, thickening, wetting, emulsifying and coloring agents; perfumes or buffer; or any other commonly may be used as additives.", "The polypeptide compound (I) or a pharmaceutically acceptable salt thereof is/are included in the pharmaceutical composition in an amount sufficient to produce the desired antimicrobial effect upon the process or condition of diseases.", "For applying the composition to humans, it is preferable to apply it by intravenous, intramuscular, pulmonary, oral administration, eye drop administration or insufflation.", "While the dosage of therapeutically effective amount of the polypeptide compound (I) varies from and also depends upon the age and condition of each individual patient to be treated, in the case of intravenous administration, a daily dose of 0.01-400 mg of the polypeptide compound (I) per kg weight of human being in the case of intramuscular administration, a daily dose of 0.1-20 mg of the polypeptide compound (I) per kg weight of human being, in case of oral administration, a daily dose of 0.5-50 mg of the polypeptide compound (I) per kg weight of human being is generally given for treating or preventing infectious diseases.", "Especially in case of the treatment of prevention of Pneumocystis carinii infection, the followings are to be noted.", "For administration by inhalation, the compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation form pressurized as powders which may be formulated and the powder compositions may be inhaled with the aid of an insufflation powder inhaler device.", "The preferred delivery system for inhalation is a metered dose inhalation aerosol, which may be formulated as a suspension or solution of compound in suitable propellants such as fluorocarbons or hydrocarbons.", "Because of desirability to directly treat lung and bronchi, aerosol administration is a preferred method of administration.", "Insufflation is also a desirable method, especially where infection may have spread to ears and other body cavities.", "Alternatively, parenteral administration may be employed using drip intravenous administration.", "For administration by intravenous administration, the preferred pharmaceutical composition is the lyophilized form containing the polypeptide compound (I) or its pharmaceutically acceptable salt.", "The amount of the polypeptide compound (I) or its pharmaceutically acceptable salt contained in the composition for a single unit dosage of the present invention is 0.1 to 400 mg, more preferably 1 to 200 mg, still more preferably 5 to 100 mg, specifically 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 mg.", "The present invention further provides the following ones.", "An article of manufacture, comprising packaging material and the compound (I) identified in the above contained within said packaging material, wherein said the compound (I) is therapeutically effective for preventing or treating infectious diseases caused by pathogenic microorganism, and wherein said packaging material comprises a label or a written material which indicates that said compound (I) can or should be used for preventing or treating infectious diseases caused by pathogenic microorganism.", "A commercial package comprising the pharmaceutical composition containing the compound (I) identified in the above and a written matter associated therewith, wherein the written matter states that the compound (I) can or should be used for preventing or treating infectious diseases caused by pathogenic microorganism.", "The following Preparations and Examples are given for the purpose of illustrating the present invention in more detail.", "Preparation 1 To a solution of cyclohexanone (706 mg) and tert-butyl 1,4-diazepane-1-carboxylate (1.2 g) in a mixed solvent of methanol (20 ml), tetrahydrofuran (15 ml) and acetic acid (1.03 ml) was added sodium cyanoborohydride (452 mg).", "The mixture was stirred at room temperature for 5 hours.", "The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate solution.", "To the reaction mixture was added ethyl acetate.", "The organic layer was taken and dried over magnesium sulfate.", "The magnesium sulfate was filtered off, and the filtrate was concentrated under reduced pressure to give tert-butyl 4-cyclohexylhexahydro-1H-1,4-diazepine-1-carboxylate (1.763 g).", "NMR (CDCl3, δ): 1.46 (9H, s), 0.9-2.2 (12H, m), 2.3-2.55 (1H, m), 2.6-2.8 (4H, m), 2.35-2.55 (4H, m) MASS (m/z): 283 (M++H) Preparation 2 To a solution of 8-(1-hydroxycyclohexyl)-1,4-dioxaspiro[4.5]decan-8-ol (2.75 g) and iodomethane (2.67 ml) in N,N-dimethylformamide (28 ml) was added sodium hydride (60% dispersion in mineral oil) (1.29 g) at 0° C. The solution was stirred for 30 minutes at 0° C. and at room temperature for 26 hours.", "The reaction mixture was added to a mixture of water and ether.", "The organic layer was washed with brine and dried over magnesium sulfate.", "The magnesium sulfate was filtered off, and the filtrate was concentrated under reduced pressure.", "The residue was purified by silica gel chromatography (4:1 hexane-ethyl acetate elution) to give 8-methoxy-8-(1-methoxycyclohexyl)-1,4-dioxaspiro[4.5]decane (2.398 g).", "NMR (CDCl3, δ): 1.0-2.0 (18H, m), 3.43 (3H, s), 3.44 (3H, s), 3.9-4.0 (4H, m) MASS (m/z): 307 (M++23) Preparation 3 A solution of 4-hexyloxybromobenzene (14.3 g) in tetrahydrofuran (200 ml at −60° C. under a nitrogen atmosphere was treated with 1.52M n-butyl lithium in hexane solution (43.9 ml) dropwise over 10 minutes, then stirred for 2 hours at the same temperature.", "Tri-isopropyl borate (12.55 g) in tetrahydrofuran (15 ml) was added dropwise over 30 minutes and after 1 hour at −60° C. the cooling bath removed and the temperature warmed to room temperature over 2 hours.", "Excess 1N-hydrochloric acid was added and the mixture was stirred for 30 minutes then extracted with ethyl acetate.", "The organic layer was washed with water (×5), saturated sodium chloride solution (×1), dried over magnesium sulfate, evaporated and the crude product triturated with hexane to afford 4-hexyloxybenzene boronic acid (6.3 g) as a white solid.", "NMR (CDCl3, δ): 0.89-0.95 (3H, m), 1.35-1.55 (6H, m), 1.76-1.86 (2H, m), 4.04 (2H, t, J=6.5 Hz), 7.00 (2H, d, J=8.5 Hz), 8.15 (2H, d, J=8.5 Hz) Preparation 4 A solution of methyl 4-[2-[4-[4-(4-methylcyclohexyl)-1-piperazinyl]benzoyl]hydrazinocarbonyl]benzoate (2.2 g) in phosphorous oxychloride (25 ml) was heated at reflux for 6 hours then cooled, poured into water, adjusted to pH 7 with sodium hydroxide solution (1N), filtered and the precipitate was washed thoroughly with water and dried to afford methyl 4-[5-[4-[4-(4-methylcyclohexyl)-1-piperazinyl]phenyl]-1,3,4-oxadiazol-2-yl]benzoate (1.93 g) as an off-white solid.", "NMR (CDCl3, δ): 0.98 (3H, d, J=7 Hz), 1.4-2.0 (9H, m), 2.5-2.8 (1H, m), 2.8-3.2 (4H, m), 3.5-3.8 (4H, m), 3.97 (3H, s), 6.99 (2H, d, J=8.8 Hz), 8.03 (2H, d, J=8.8 Hz), 8.20 (4H, s) API-ES(+) MASS: 461.4 (MH+) Preparation 5 To a solution of 1,4-dioxaspiro[4.5]decan-8-ol (3.0 g) in methanol (30 ml) was portionwise added sodium borohydride (1.45 g) with stirring at ambient temperature and the mixture was stirred at the same temperature for 1 hour.", "The reaction mixture was concentrated in vacuo and chromatographed on silica gel (150 ml) eluting with a mixture of n-hexane and ethyl acetate (2:1 v/v).", "The fractions containing the desired compound were collected and evaporated under reduced pressure to give 1,4-dioxaspiro[4.5]decan-8-ol (3.43 g).", "NMR (CDCl3, δ): 1.45-1.95 (8H, m), 3.70-3.85 (1H, m), 3.95 (4H, s) APCI MASS (m/z): 159 (M++H) Preparation 6 To a solution of tert-butyl 4-oxo-1-piperidinecarboxylate (7.0 g) in THF (35 ml) was dropwise added lithium diisopropylamine mono(tetrahydrofuran) (1.5M solution cyclohexane) (25.8 ml) at −70° C. and stirred at the same temperature for 20 minutes.", "To the solution was dropwise added a solution of N-phenyltrifluoromethanesulfonimide (13.43 g) in THF (35 ml) at −70° C. and the mixture was warmed up to 0° C. and stirred at 0° C. for 3 hours.", "The reaction mixture was concentrated in vacuo.", "The resulting residue was dissolved in dichloromethane (50 ml).", "The solution was subjected to column chromatography on Florisil (100-200 mesh) (400 ml) eluting with a mixture of hexane and ethyl acetate (9:1 v/v).", "The first fractions containing the object compound were collected and evaporated under reduced pressure to give the crude vinyl triflate.", "The residue was dissolved in a mixture of dimethoxyethane (250 ml) and aqueous sodium carbonate (Na2CO3 10.4 g in water (50 ml)).", "To this solution were added 4-(methoxycarbonyl)phenylboric acid (8.85 g), lithium chloride (3.20 g) and tetrakis(triphenylphosphine)palladium (2.02 g) at room temperature and the mixture was refluxed for 2 hours.", "To the reaction mixture was added ethyl acetate (200 ml) and the solution was washed in turn with water (60 ml) and aqueous sodium chloride (60 ml), dried over magnesium sulfate and evaporated in vacuo.", "The resulting residue was chromatographed on silica gel (200 g) eluting with a mixture of hexane and ethyl acetate (5:1 v/v).", "The fractions containing the object compound were collected and evaporated under reduced pressure to give tert-butyl 4-[4-(methoxycarbonyl)phenyl]-3,6-dihydro-1(2H)-pyridinecarboxylate (4.19 g) NMR (CDCl3, δ): 1.49 (9H, s), 2.50-2.65 (2H, m), 3.65 (2H, t, J=5.69 Hz), 3.95 (3H, s), 4.05-4.30 (2H, m), 6.16 (1H, br s), 7.43 (2H, J=8.52 Hz), 8.00 (2H, J=8.56 Hz) ESI MASS (Positive)(m/z): 340.2 (M++Na) Preparation 7 A mixture of tert-butyl 4-[4-(methoxycarbonyl)phenyl]-3,6-dihydro-1(2H)-pyridinecarboxylate (3.68 g) and 10% palladium on carbon (50% wet) (1.8 g) in methanol (40 ml) and tetrahydrofuran (40 ml) was stirred for 5 hours at room temperature under hydrogen atmosphere.", "After removal of insoluble solids, the filtrate was concentrated in vacuo and the residue was purified by column chromatography on silica gel eluting with a mixture of toluene and ethyl acetate (20:1→10:1).", "The eluted fractions containing the desired product were collected and evaporated in vacuo to give tert-butyl 4-[4-(methoxycarbonyl)phenyl]-1-piperidinecarboxylate (2.98 g).", "IR (Nujol): 1724, 1705, 1421, 1269, 1228, 1159, 1122, 1012 cm−1 NMR (DMSO-d6, δ): 1.3-1.6 (2H, m), 1.42 (9H, s), 1.7-1.9 (2H, m), 2.6-2.9 (3H, m), 3.84 (3H, s), 4.0-4.2 (2H, m), 7.3-7.5 (2H, m), 7.8-8.0 (2H, m) ESI MASS (Positive): 342.3 (M++Na) Preparation 8 A mixture of ethyl 4-(4-oxo-1-piperidyl)benzoate (12 g) and dimethylformamide dimethylacetal (12.7 g) was heated at 110° C. for 6 hours, cooled, diluted with hexane and the resulting precipitate was collected by filtration and washed with hexane to afford ethyl 4-[(3E)-3-(dimethylaminomethylene)-4-oxo-1-piperidyl]benzoate as a light yellow powder (10.5 g).", "NMR (CDCl3, δ): 1.37 (3H, t, J-7.1 Hz), 2.59 (2H, t, J=6.2 Hz), 3.16 (6H, s), 3.65 (2H, t, J=6.2 Hz), 4.32 (2H, q, J=7.1 Hz), 4.52 (2H, s), 6.77 (2H, d, J=9.1 Hz), 7.55 (1H, s), 7.93 (2H, d, J=9.1 Hz) Preparation 9 A solution of ethyl 4-[(3E)-3-(dimethylaminomethylene)-4-oxo-1-piperidyl]benzoate (2 g) and phenylhydrazine (787 mg) in ethanol (20 ml) was heated at reflux.", "After 2 hours, the reaction mixture was evaporated and dried in vacuo to give an amorphous solid.", "This solid was dissolved in Ethanol (20 ml), and treated with hydrazine hydrate then refluxed for 40 hours, cooled then extracted with ethyl acetate then washed with water, dried over magnesium sulfate and the crude solid was triturated with ethyl acetate-hexane to give 4-(2-phenyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)benzohydrazide as a yellow powder (1.3 g).", "NMR (DMSO-d6, δ): 2.80-3.05 (2H, m), 3.60-3.80 (2H, m), 4.36 (2H, s), 4.36-4.45 (2H, m), 7.04 (2H, d, J=9 Hz), 7.25-7.79 (8H, m), 9.47 (1H, s) APCI-MASS: 334.13 (M++H) Preparation 10 To a solution of 4-bromo cyanobenzene (3.0 g) in THF (30 ml) was added triisopropoxyborate (5.32 ml) at −70° C. with stirring and then dropwise added n-butyllithium (1.6M solution in hexane) (13.4 ml) at the same temperature.", "The mixture was stirred at −60˜−70° C. for 1 hour.", "The reaction mixture was poured into 2N HCl (25 ml) and extracted twice with ethyl acetate (100 ml), washed successively with water and saturated aqueous sodium chloride, dried over magnesium sulfate and evaporated in vacuo.", "The resulting residue was triturated with diisopropyl ether (50 ml).", "The resulting precipitates were collected by filtration and dried in vacuo to give 4-cyanophenylboric acid (1.90 g).", "NMR (DMSO-d6, δ): 7.79 (2H, d, J=8.09 Hz), 7.94 (2H, d, J=8.10 Hz) Preparation 11 To a solution of tert-butyl 4-(trifluoromethylsulfonyloxy)-3,6-dihydro-1(2H)-pyridinecarboxylate (8.33 g) in a mixture of dimethoxyethane (160 ml) and aqueous sodium carbonate (Na2CO3 10.4 g in water (50 ml)).", "To a solution were added 4-cyanophenylboric acid (5.16 g), lithium chloride (2.28 g) and tetrakis(triphenylphosphine)palladium (1.44 g) at room temperature and the mixture was refluxed for 2 hours and then cooled on ice bath.", "The reaction mixture was evaporated in vacuo and dissolved in a mixture of dichloromethane (200 ml), 2N aqueous sodium carbonate (100 ml) and conc.", "ammonium hydroxide (10 ml).", "The organic layer was separated and the aqueous layer was extracted with dichloromethane (100 ml).", "The extracts were washed with saturated aqueous sodium chloride, dried over magnesium sulfate and evaporated in vacuo.", "The resulting residue was chromatographed on silica gel (200 g) eluting with a mixture of hexane and ethyl acetate (5:1 v/v).", "The fractions containing the object compound were collected and evaporated under reduced pressure to give tert-butyl 4-(4-cyanophenyl)-3,6-dihydro-1(2H)pyridinecarboxylate (4.19 g).", "NMR (CDCl3, δ): 1.49 (9H, s), 2.45-2.60 (2H, m), 3.65 (2H, t, J=5.6 Hz), 4.11 (2H, q, J=2.81 Hz), 6.18 (1H, br s), 7.46 (2H, d, J=8.41 Hz), 7.62 (2H, J=8.39 Hz) ESI MASS (Positive)(m/z): 307.2 (M++Na) Preparation 12 To a solution of 4-(ethoxycarbonyl)piperidine (10.0 g) in THF (100 ml) were added triethylamine (11.5 ml) and di-tert-butyl dicarbonate (14.6 g) with stirring at ambient temperature and the mixture was stirred at the same temperature for 3 hours.", "The reaction mixture was concentrated in vacuo.", "The resulting residue was dissolved in ethyl acetate (200 ml) and the solution was washed successively with 1N hydrochloride, saturated aqueous sodium chloride, saturated aqueous sodium hydrogen carbonate and saturated aqueous sodium chloride dried over magnesium sulfate and evaporated in vacuo.", "The residue was chromatographed on silica gel (400 ml) eluting with a mixture of n-hexane and ethyl acetate (5:1 v/v).", "The fractions containing the desired compound were collected and evaporated under reduced pressure to give tert-butyl 4-(ethoxycarbonyl)-1-piperidinecarboxylate (16.09 g).", "This compound was immediately used as the starting compound for the next step.", "Preparation 13 To a solution of lithium aluminum hydride (1.33 g) in THF (60 ml) was dropwise added a solution of tert-butyl 4-(ethoxycarbonyl)-1-piperidinecarboxylate (6.00 g) in THF (30 ml) with stirring at 0˜−20° C. and the mixture was stirred at the same temperature for 1 hour.", "To a reaction mixture were added sodium fluoride (5.87 g) and then dropwise added water (1.90 ml) with stirring.", "After 10 minutes, the mixture was filtrated by celite and evaporated in vacuo.", "The resulting residue was chromatographed on silica gel (250 ml) eluting with a mixture of dichloromethane and methanol (9:1 v/v).", "The fractions containing the desired compound were collected and evaporated under reduced pressure to give tert-butyl 4(hydroxymethyl)-1-piperidinecarboxylate (4.13 g).", "NMR (CDCl3, δ): 1.00-1.30 (2H, m), 1.46 (9H, s), 1.50-1.80 (4H, m), 2.55-2.85 (2H, m), 3.40-3.60 (2H, m), 4.00-4.25 (2H, m) APCI MASS (Positive)(m/z): 238.4 (M++Na) Preparation 14 To a solution of oxalylchloride (2.4 g) in dichloromethane (30 ml) was dropwise added the solution of dimethylsulfoxide (2 g) in dichloromethane (10 ml) at −10° C. with stirring.", "The mixture was stirred at −5-10° C. for 0.5 hour.", "To a reaction mixture was dropwise added the solution of 4-(5-methoxypentyloxy)phenethylalcohol (3.0 g) in dichloromethane (40 ml) at −60° C. with stirring.", "The mixture was stirred at −60° C. for an hour and then dropwise added the triethylamine (8 g) at −60° C. with stirring.", "The mixture was stirred at −60° C. for an hour and stirred at room temperature for 1.5 hours.", "The reaction mixture was poured into ice-water and extracted with dichloromethane.", "The dichloromethane layer was washed with water and dried over magnesium sulfate.", "The magnesium sulfate was filtered off and the filtrate was concentrated under reduced pressure to give oil.", "The oil was subjected to column chromatography on silica gel (silica gel 60F254, Merck) and eluted a mixture of ethyl acetate and n-hexane (1:4).", "The fraction containing the object compound were combined and concentrated under reduced pressure to give 4-(5-methoxypentyloxy)phenylacetaldehyde (1.0 g).", "NMR (CDCl3, δ): 1.40-1.90 (6H, m), 3.34 (3H, s), 3.40 (2H, t, J=6.2 Hz), 3.62 (2H, d, J=2.4 Hz), 3.96 (2H, t, J=6.4 Hz), 6.88 (2H, d, J=8.7 Hz), 7.11 (2H, d, J=8.7 Hz), 9.72 (1H, br s) API-ES MASS (Negative): 249 (M++Na), 236 (M), 235 (M−−1) Preparation 15 To a solution of 4-(5-methoxypentyloxy)-phenylacetaldehyde (0.47 g) in dichloromethane (5 ml) was dropwise added the solution of bromine (0.35 g) in dichloromethane (1 ml) at −10° C. with stirring.", "The mixture was stirred at room temperature for 0.5 hour and stirred at reflux for 40 minutes.", "The reaction mixture was concentrated under nitrogen gas at 40° C. and added the thiourea (0.15 g) and ethanol (10 ml) to the residue.", "The mixture was refluxed for 6 hours with stirring.", "The reaction mixture was concentrated under reduced pressure and added the water to the residue.", "The solution was adjusted to pH 8.5 using the sodium bicarbonate and extracted with the mixture was ethyl acetate and tetrahydrofuran (1:1).", "The organic layer was washed with saturated sodium chloride aqueous solution and dried over magnesium sulfate.", "The magnesium sulfate was filtered off and the filtrate was concentrated under reduced pressure to give oily.", "The oil was subjected to column chromatography on silica gel (silica gel 60F254, Merck) and eluted the mixture of chloroform and methanol (10:1).", "The fractions containing the objective compound was combined and concentrated under reduced pressure to give 2-amino-5-(5-methoxypentyloxyphenyl)thiazole (0.32 g).", "NMR (DMSO-d6, δ): 1.40-1.90 (6H, m), 3.22 (3H, s), 3.20-3.40 (2H, m), 4.03 (2H, t, J=6.3 Hz), 6.80-7.40 (4H m), 7.10 (2H, s), 7.63 (1H, s) MASS (m/z): 371 (M++Br), 293 (M++H) Preparation 16 N,N-diisopropylamine (26.2 ml) is added dropwise to a solution of butyllithium (107.5 ml:1.6M in hexane) in tetrahydrofuran (300 ml) under nitrogen atmosphere and cooled in an ice bath at 0-5° C. After maintaining the solution at 0-5° C. for an additional 30 minutes, cyclohexanecarboxylic acid (10 g) is added at once.", "The cooling bath is removed and the reaction solution is allowed to stir at room temperature for 4 hours.", "A solution of 1,4-dioxaspiro[4.5]decan-8-one (12.2 g) in tetrahydrofuran is added at once.", "After stirred at room temperature for 14 hours, the mixture is poured into ice water, washed once with diethyl ether, acidified to pH 1 with conc.", "HCl, and extracted with chloroform-methanol (9:1).", "The extracts are dried over magnesium sulfate, filtered, and concentrated in vacuo.", "The residue is triturated with a solvent mixture consisting of ethyl acetate (50 ml), diethyl ether (250 ml) and hexane (250 ml), collected by filtration, and dried to give 1′-(8-hydroxy-1,4-dioxaspiro[4.5]dec-8-yl)cyclohexanecarboxylic acid (13.068 g).", "NMR (CDCl3, δ): 0.8-2.4 (22H, m), 3.8-4.1 (4H, m) MASS (m/z): 283 (M+−H) Preparation 17 Dimethylformamide dineopentylacetal was added at once to a stirred slurry of 1′-(8-hydroxy-1,4-dioxaspiro[4.5]dec-8-yl)cyclohexanecarboxylic acid (10 g) in acetonitrile at room temperature.", "The mixture was stirred at room temperature for 1 hour and then was heated for 21 hours at gentle reflux.", "The mixture was cooled, diluted with diethyl ether, washed with ice water, brain, and the organic layer was dried over magnesium sulfate.", "The solution is filtered and concentrated in vacuo.", "The residue was purified by silica gel chromatography (10:1 hexane-ethyl acetate elution) to give 8-cyclohexylidene-1,4-dioxaspiro[4.5]decane (7.19 g).", "NMR (CDCl3, δ): 1.4-1.7 (10H, m), 2.1-2.4 (8H, m), 3.97 (4H, s) MASS (m/z): 222.80 (M++H) Preparation 18 To a stirred solution of 8-cyclohexylidene-1,4-dioxaspiro[4.5]decane (5 g) in dichloromethane (200 ml) was added-dropwise the oxidant solution KMnO4 (5.33 g), triethylbenzylammonium chloride (7.68 g) and dichloromethane (400 ml) at such a rate that the temperature was maintained at 0-3° C. under cooling with ice bath.", "After addition was completed, stirring was continued until permanganate ion was completely consumed.", "The homogeneous dark brown solution was treated with 3% sodium hydrogen carbonate solution (300 ml) at room temperature for 18 hours.", "The organic layer was washed with brine and dried over magnesium sulfate.", "The magnesium sulfate was filtered off, and the filtrate was concentrated under reduced pressure.", "The residue was purified by silica gel chromatography (3:1-1:1 hexane-ethyl acetate elution) to give 8-(1-hydroxycyclohexyl)-1,4-dioxaspiro[4.5]decan-8-ol (2.805 g).", "NMR (CDCl3, δ): 1.0-2.1 (20H, m), 3.85-4.0 (4H, m) MASS (m/z): 279 (M++23) Preparation 19 To a solution of 1-hydroxy-4-methylcyclohexane (13.5 g) and triethylamine (21.4 ml) in ethyl acetate (135 ml) was added dropwise with stirring methanesulfonyl chloride (20 ml) at 0° C. The mixture was then stirred for 25 hours at 0° C. The reaction mixture was added to a mixture of 1 mol/l hydrochloric acid and ethyl acetate.", "The organic layer was washed with water, sodium hydrogen carbonate solution and brain.", "The organic layer was taken and dried over magnesium sulfate.", "The magnesium sulfate was filtered off, and the filtrate was concentrated under reduced pressure to give cis-4-methylcyclohexyl methanesulfonate (22.87 g).", "NMR (CDCl3, δ): 0.93 (3H, d, J-6.5 Hz), 1.2-1.75 (7H, m), 1.95-2.15 (2H, m), 3.01 (3H, s), 4.9-5.0 (1H) MASS (m/z): 215 (M++23) The following compound was obtained according to a similar manner to that of Preparation 19.Preparation 20 Trans-4-Methylcyclohexyl methanesulfonate NMR (CDCl3, δ): 0.90 (3H, d, J=6.5 Hz), 0.95-1.2 (2H, m), 1.2-1.9 (5H, m), 2.05-2.25 (2H, m), 3.00 (3H, s), 5.0-5.7 (1H, m) MASS (m/z): 215 (M++23) Preparation 21 A solution of piperazine (8.96 g) in methanol was heated at 120° C. Since the solvent was disappeared, to the solution was added 4-methylcyclohexyl methanesulfonate (5 g).", "The solution was mixed for 4 hours at 120° C. The reaction mixture was purified by silica gel chromatography (5:1 dichloromethane-methanol elution) to give 1-(cis-4-methylcyclohexyl)piperazine (1.2 g) NMR (CDCl3, δ): 0.93 (3H, d, J=7.0 Hz), 1.4-2.0 (10H, m), 2.1-2.3 (1H, m), 2.45-2.65 (4H, m), 2.9-3.0 (4H, m) MASS (m/z): 183 (M++H) The following compound was obtained according to a similar manner to that of Preparation 21.Preparation 22 1-(trans-4-Methylcyclohexyl)piperazine NMR (CDCl3, δ): 0.8-1.4 (8H, m), 1.65-2.0 (5H, m), 2.05-2.3 (1H, m), 2.45-2.6 (4H, m), 2.8-3.0 (4H, m) MASS (m/z): 183 (M++H) Preparation 23 Sodium hydride, 60% dispersion in mineral oil (950 mg) was added portionwise to a solution of methyl 4-hydroxybenzoate (3 g) in N,N-dimethylformamide (15 ml) at ambient temperature.", "The mixture was stirred at 60° C. for 2 hours.", "The mixture was added portionwise to 1,7-dibromoheptane (10.1 ml) in N,N-dimethylformamide (15 ml) at ambient temperature, and stirred for 16 hours.", "The reaction mixture was diluted with a mixture of ethyl acetate and water, and the organic layer was separated, washed with water and brine, dried, and evaporated under reduced pressure.", "The residue was chromatographed on a column of silica gel eluting with a mixture of n-hexane and ethyl acetate (20:1) to give methyl 4-(7-bromoheptyloxy)benzoate (4.19 g).", "IR (KBr): 2942.8, 1710.6, 1606.6, 1251.6 cm−1 NMR (CDCl3, δ): 1.37-1.56 (6H, m), 1.74-1.91 (4H, m), 3.42 (2H, t, J=6.8 Hz), 3.88 (3H, s), 4.01 (2H, t, J=6.4 Hz), 6.86-6.93 (2H, m), 7.94-8.02 (2H, m) ESI MASS (Positive)(m/z): 351.1 (M++Na) The following compounds [Preparation 24 and 25] were obtained according to a similar manner to that of Preparation 23.Preparation 24 8-(6-Bromohexyloxy)-1,4-dioxaspiro[4.5]decane NMR (CDCl3, δ): 1.30-1.95 (16H, m), 3.25-3.45 (5H, m), 3.94 (4H, s) ESI MASS (Positive)(m/z): 343.2 (M++Na) Preparation 25 4-[4-(7-Bromohexyloxy)piperidin-1-yl]benzoate NMR (CDCl3, δ): 1.36 (3H, t, J=7.1 Hz), 1.22-1.50 (6H, m), 1.50-2.12 (8H, m), 2.98-3.19 (2H, m), 3.32-3.57 (5H, m), 3.57-3.75 (2H, m), 4.32 (2H, q, J=7.1 Hz), 6.86 (2H, d, J=9.0 Hz), 7.91 (2H, d, J=8.9 Hz) MASS (m/z): 426, 428 (M++H, M++3) Preparation 26 A mixture of 4-(4-piperidyloxy)benzonitrile (1.5 g), iodobenzene (1 ml), palladium(II) acetate (83 mg), racemic-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (0.23 g) and cesium carbonate (4.8 g) was stirred for 14.5 hours at 90° C. under nitrogen atmosphere.", "After being cooled to room temperature, the reaction mixture was poured into a mixture of ethyl acetate and water.", "The organic layer was successively washed with water and brine and dried over magnesium sulfate.", "The solvent was evaporated in vacuo and the residue was purified by column chromatography on silica gel eluting with a mixture of hexane and ethyl acetate (5:1).", "The eluted fractions containing the desired product were collected and evaporated in vacuo to give 4-(1-phenyl-4-piperidyloxy)benzonitrile (1.26 g).", "IR (KBr): 2218, 1599, 1500, 1298, 1254, 1228, 1171, 1120, 1034, 837, 754, 689 cm−1 NMR (DMSO-d6, δ): 1.6-1.9 (2H, m), 2.0-2.2 (2H, m), 3.0-3.2 (2H, m), 3.4-3.6 (2H, m), 4.6-4.8 (1H, m), 6.76 (1H, t, J=7.2 Hz), 6.96 (2H, d, J=7.9 Hz), 7.1-7.3 (4H, m), 7.76 (2H, d, J=8.9 Hz) ESI MASS (Positive): 301.2 (M++H) The following compounds [Preparation 27 to 29] were obtained according to a similar manner to that of Preparation 26.Preparation 27 Ethyl 4-(1-phenyl-4-piperidyloxy)benzoate IR (Nujol): 1705, 1603, 1493, 1277, 1254, 1173, 1103, 1034 cm−1 NMR (DMSO-d6, δ): 1.30 (3H, t, J-7.1 Hz), 1.6-1.9 (2H, m), 2.0-2.2 (2%, m), 3.0-3.2 (2H, m), 3.4-3.6 (2H, m), 4.28 (2H, q, J=7.1 Hz), 4.6-4.8 (1H, m), 6.7-7.3 (7H, m), 7.90 (2H, d, J=8.8 Hz) ESI MASS (Positive): 326.3 (M+−H) Preparation 28 Ethyl 4-[1-(4-methoxyphenyl)-4-piperidyloxy]benzoate IR (Nujol): 1701, 1508, 1252, 1171, 1103, 1034 cm−1 NMR (DMSO-d6, δ): 1.30 (3H, t, J=7.1 Hz), 1.7-1.9 (2H, m), 2.0-2.2 (2H, m), 2.8-3.0 (2H, m), 3.3-3.5 (2H, m), 3.68 (3H, s), 4.27 (2H, d, J=7.1 Hz), 4.6-4.7 (1H, m), 6.8-7.0 (4H, m), 7.09 (2H, d, J=8.8 Hz), 7.90 (2H, d, J=8.8 Hz) ESI MASS (Positive): 356.3 (M++H) Preparation 29 4-[1-(4-Methoxyphenyl)-4-piperidyloxy]benzonitrile IR (Nujol): 2222, 1510, 1257 cm−1 NMR (DMSO-d6, δ): 1.6-1.9 (2H, m), 2.0-2.2 (2H, m), 2.8-3.0 (2H, m), 3.3-3.4 (2H, m), 3.68 (3H, s), 4.6-4.8 (1H, m), 6.7-7.0 (4H, m), 7.1-7.2 (2H, m), 7.7-7.8 (2H, m) ESI MASS (Positive): 331.2 (M++Na) Preparation 30 To a mixture of 4-hydroxybenzonitrile (10.7 g), tert-butyl 4-hydroxy-1-piperidinecarboxylate (27.1 g) and triphenylphosphine (35.4 g) in tetrahydrofuran (250 ml) was added diethyl azodicarboxylate (21.3 ml) at room temperature under nitrogen atmosphere.", "After stirring for 6 hours at room temperature under nitrogen atmosphere, the solvent was evaporated in vacuo.", "Then to the residue was added ethyl ether and insoluble solids were filtered off.", "The filtrate was evaporated in vacuo and the residue was purified by column chromatography on silica gel eluting with a mixture of hexane and ethyl acetate (10:1-+5:1).", "The eluted fractions containing the desired product were collected and evaporated in vacuo to give tert-butyl 4-(4-cyanophenoxy)-1-piperidinecarboxylate (26.7 g).", "NMR (DMSO-d6, δ): 1.3-1.6 (11H, m), 1.8-2.0 (2H, m), 3.0-3.3 (2H, m), 3.6-3.8 (2H, m), 4.6-4.8 (1H, m), 7.15 (2H, d, J=8.8 Hz), 7.76 (2H, d, J=8.8 Hz) ESI MASS (Positive): 325.2 (M++Na) The following compounds [Preparation 31 and 32] were obtained according to a similar manner to that of Preparation 30.Preparation 31 Tert-Butyl 4-4-(ethoxycarbonylphenoxy)-1-piperidinecarboxylate IR (Nujol): 2981, 2935, 2875, 1699, 1687, 1601, 1417, 1367, 1313, 1277, 1254, 1236, 1163, 1105, 1038 cm−1 NMR (DMSO-d6, δ): 1.30 (3H, t, J=7.1 Hz), 1.41 (9H, s), 1.4-1.6 (2H, m), 1.8-2.0 (2H, m), 3.1-3.3 (2H, m), 3.6-3.8 (2H, m), 4.27 (2H, q, J=7.1 Hz), 4.6-4.8 (1H, m), 7.08 (2H, d, J=8.9 Hz), 7.89 (2H, d, J=8.8 Hz) ESI MASS (Positive): 372.3 (M++H) Preparation 32 Tert-Butyl 4-(4-bromophenoxy)-1-piperidinecarboxylate NMR (CDCl3, δ): 1.47 (9H, s), 1.6-2.0 (4H, m), 3.2-3.4 (2H, m), 3.6-3.8 (2H, m), 4.3-4.5 (1H, m), 6.78 (2H, d, J=6.8 Hz), 7.36 (2H, d, J=6.8 Hz) Preparation 33 To a solution of 4-pentyloxy-1-(tert-butoxycarbonyloxy)piperidine (6 g) in ethyl acetate (30 ml) was added dropwise 4N hydrogen chloride in ethyl acetate (28 ml) at 0-10° C., and stirred at ambient temperature for 2 hours.", "The reaction mixture was evaporated under reduced pressure to give 4-pentyloxy piperidine (3.87 g).", "IR (KBr): 3488.6, 2944.8, 1591.0, 1091.5 cm−1 NMR (CDCl3, δ): 0.90 (3H, t, J=6.6 Hz), 1.27-2.16 (11H, m), 3.04-3.59 (7H, m) ESI MASS (Positive)(m/z): 172.07 (M++H) The following compounds [Preparation 34 and 35] were obtained according to a similar manner to that of Preparation 33.Preparation 34 4-Butoxy piperidine hydrochloride salt IR (KBr): 2966.0, 1589.1, 1110.8 cm−1 NMR (CDCl3, δ): 0.92 (3H, t, J=7.2 Hz), 1.23-1.61 (4H, m), 1.91-2.16 (5H, m), 2.15-3.63 (7H, m), 9.36 (1H, br s) ESI MASS (Positive)(m/z): 157.93 (M++H) (free) Preparation 35 1-(4-Bromophenyl)piperazine NMR (CDCl3, δ): 2.95-3.2 (8H, m), 6.7-6.9 (2H, m), 7.3-7.5 (2H, m) MASS (m/z): 241, 243 (M++H) Preparation 36 To a mixture of tert-butyl 4-(4-cyanophenoxy)-1-piperidinecarboxylate (2.3 g) and anisole (4.2 ml) in dichloromethane (23 ml) was added portionwise trifluoroacetic acid (12 ml) under ice-cooling.", "After stirring for 8 hours under ice-cooling, the solvent was evaporated in vacuo.", "The residue was poured into a mixture of ethyl acetate and water and the solution was adjusted to pH 10 with potassium carbonate.", "Then the organic layer was concentrated in vacuo and the residue was pulverized from isopropyl ether to give 4-(4-piperidyloxy)benzonitrile (1.55 g).", "NMR (DMSO-d6, δ): 1.5-1.8 (2H, m), 1.9-2.1 (2H, m), 2.8-3.0 (2H, m), 3.0-3.2 (2H, m), 4.6-4.8 (1H, m), 7.1-7.2 (2H, m), 7.7-7.8 (2H, m) ESI MASS (Positive): 203.2 (M++H) The following compounds [Preparation 37 to 43] were obtained according to a similar manner to that of Preparation 36.Preparation 37 Ethyl 4-(4-piperidyloxy)benzoate NMR (DMSO-d6, δ): 1.30 (3H, t, J=7.1 Hz), 1.4-1.6 (2H, m), 1.9-2.1 (2H, m), 2.6-2.8 (2H, m), 2.9-3.1 (2H, m), 3.89 (1H, br s), 4.27 (2H, q, J=7.1 Hz), 4.4-4.7 (1H, m), 7.0-7.1 (2H, m), 7.8-8.0 (2H, m) ESI MASS (Positive): 250.2 (M++H) Preparation 38 Methyl 4-(4-piperidyl)benzoate IR (Nujol): 1709, 1277, 1107 cm−1 NMR (DMSO-d6, δ): 1.4-1.8 (4H, m), 2.6-2.8 (3H, m), 3.0-3.1 (2H, m), 3.84 (3H, s), 7.38 (2H, d, J=8.3 Hz), 7.90 (2H, d, J=8.3 Hz) ESI MASS (Positive): 220.4 (M++H) Preparation 39 4-4-(Methoxybutyloxymethyl)piperidine trifluoroacetate This compound was immediately used as the starting compound for the next step.", "Preparation 40 4-(1,2,3,6-Tetrahydro-4-pyridyl)benzonitrile IR (Neat): 2226, 1651, 1603, 1558, 1541, 1506, 1419 cm−1 NMR (DMSO-d6, δ): 2.3-2.4 (2H, m), 2.8-3.0 (2H, m), 3.3-3.4 (2H, m), 6.4-6.5 (1H, m), 7.5-7.7 (2H, m), 7.7-7.9 (2H, m), 7.98 (1H, s) ESI MASS (Positive): 185.2 (M++H) Preparation 41 4-(5-Methoxypentyloxymethyl)piperidine trifluoroacetate This compound was immediately used as the starting compound for the next step.", "Preparation 42 4-(4-Methylpentyloxy)piperidine trifluoroacetate This compound was used in the next reaction without further purification.", "Preparation 43 4-(Cyclohexylmethoxy)piperidine trifluoroacetate This compound was used in the next reaction without further purification.", "Preparation 44 To a solution of tert-butyl 4-(4-bromophenyl)-1-piperazinecarboxylate (1.1 g) in dichloromethane (11 ml) was added dropwise with stirring trifluoroacetic acid (5 ml) at 0° C. The mixture was then stirred for 2 hours at room temperature.", "Then the solvent was evaporated and the reaction mixture was added to a mixture of ethyl acetate and tetrahydrofuran.", "The organic layer was washed with sodium hydrogen carbonate solution and sodium chloride solution.", "The organic layer was taken and dried over magnesium sulfate.", "The magnesium sulfate was filtered off, and the filtrate was concentrated under reduced pressure to give 1-(4-bromophenyl)piperazine (710 mg).", "NMR (CDCl3, δ): 2.95-3.2 (8H, m), 6.7-6.9 (2H, m), 7.3-7.5 (2H, m) MASS (m/z): 241, 243 (M++H) The following compounds [Preparation 45 to 47] were obtained according to a similar manner to that of Preparation 44.Preparation 45 1-Cyclohexylhexahydro-1H-1,4-diazepine NMR (CDCl3, δ): 0.8-2.0 (12H, m), 2.3-2.65 (2H, m), 2.7-3.1 (8H, m) MASS (m/z): 183 (M++H) Preparation 46 2-Cyclohexyl-2,5-diazabicyclo[2.2.1]heptane NMR (CDCl3, δ): 1.0-3.0 (16H, m), 3.1-3.9 (4H, m) MASS (m/z): 181 (M++H) Preparation 47 4-(4-Bromophenoxy)piperidine NMR (CDCl3, δ): 2.0-2.3 (5H, m), 3.1-3.5 (4H, m), 4.55-4.7 (1H, m), 6.79 (2H, d, J=8.9 Hz), 7.41 (2H, d, J=8.9 Hz) MASS (m/z): 256, 258 (M++H) Preparation 48 A solution of 4-fluorobenzonitrile (1.89 g), 4-4-(methoxybutyloxymethyl)piperidine trifluoroacetate (3.6 g) and potassium carbonate (4.73 g) in DMSO (40 ml) was stirred at 140-150° C. for 4 hours.", "The reaction mixture was poured into water (150 ml) and extracted twice with ethyl acetate (80 ml).", "The extracts were collected, washed with saturated aqueous sodium chloride, dried over magnesium sulfate and evaporated in vacuo.", "The resulting residue was chromatographed on silica gel (200 ml) eluting with a mixture of n-hexane and ethyl acetate (2:1 v/v).", "The fractions containing the desired compound were collected and evaporated under reduced pressure to give 4-(4-methoxybutyloxymethyl)-1-(4-cyanophenyl)piperidine (2.76 g).", "NMR (CDCl3, δ): 1.20-1.45 (2H, s), 1.55-1.75 (4H, m), 1.75-1.90 (3H, m), 2.86 (2H, dt, J=2.37, 12.5 Hz), 3.28 (2H, d, J=6.03 Hz), 3.34 (3H, s), 3.35-3.50 (4H m), 3.70-3.90 (2H, m), 5.70 (2H, br s), 6.90 (2H, d, J=8.96 Hz), 7.64 (2H, d, J=8.86 Hz) APCI MASS (Positive) (m/z): 377.3 (M++H) The following compounds [Preparation 49 to 52] were obtained according to a similar manner to that of Preparation 48.Preparation 49 Ethyl 4-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)benzoate NMR (CDCl3, δ): 1.37 (3H, t, J=7.1 Hz), 1.77-1.83 (4H, m), 3.45-3.51 (4H, m), 4.00 (4H, s), 4.32 (2H, q, J=7.1 Hz), 6.88 (2H, d, J=7 Hz), 7.90 (2H, d, J=7 Hz) APCI MASS: 292.13 (M++H) Preparation 50 4-[4-(5-Methoxypentyloxymethyl)-1-piperidyl]benzonitrile NMR (CDCl3, δ): 1.15-1.50 (4H, m), 1.50-1.70 (4H, m), 1.75-1.90 (3H, m), 2.70-2.95 (2H, m), 3.28 (2H, d, J=6.01 Hz), 3.33 (3H, s), 3.35 (2H, d, J=6.60 Hz), 3.43 (2H, d, J=6.41 Hz), 3.70-3.90 (2H, m), 6.85 (2H, d, J=9.06 Hz), 7.46 (2H, d, J=9.00 Hz) ESI MASS (Positive)(m/z): 339.3 (M++Na) Preparation 51 Tert-Butyl 4-(4-bromophenyl)-1-piperazinecarboxylate NMR (CDCl3, δ): 1.48 (9H, s), 3.05-3.15 (4H, m), 3.5-3.6 (4H, m), 6.79 (2H, d, J=9.0 Hz), 7.35 (2H, d, J=9.0 Hz) MASS (m/z): 340, 342 (M++H) Preparation 52 Ethyl 4-(4-butoxypiperidin-1-yl)benzoate IR (KBr): 2954.4, 1695.1, 1240.0, 1112.7 cm−1 NMR (CDCl3, δ): 0.93 (3H, t, J=7.2 Hz), 1.29-2.03 (11H, m), 3.04-3.16 (2H, m), 3.44-3.55 (3H, m), 3.60-3.72 (2H, m), 4.32 (2H, q, J=7.1 Hz), 6.84-6.90 (2H, m), 7.87-7.94 (2H, m) ESI MASS (Positive)(m/z): 306.20 (M++H) Preparation 53 A solution of 8-(6-methoxyhexyloxy)-1,4-dioxaspiro[4.5]decane (1.55 g) in a mixture of THF (16 ml) and 3N hydrochloric acid (5.7 ml) was stirred at ambient temperature for 5 hours.", "The reaction mixture was then concentrated in vacuo.", "The resulting residue was dissolved in ethyl acetate (50 ml) and saturated aqueous sodium hydrogen carbonate (10 ml).", "The solution was washed with saturated aqueous sodium chloride, dried over magnesium sulfate and evaporated in vacuo to give 4-(6-methoxyhexyloxy)cyclohexanone (1.24 g).", "NMR (CDCl3, δ): 1.30-1.40 (4H, m), 1.45-1.65 (4H, m), 1.80-2.35 (6H, m), 2.40-2.65 (2H, m), 3.33 (3H, s), 3.37 (2H, t, J=6.58 Hz), 3.49 (2H, t, J=6.38 Hz), 3.60-3.75 (1H, m) ESI MASS (Positive)(m/z): 25.13 (M++Na) The following compounds [Preparation 54 and 55] were obtained according to a similar manner to that of Preparation 53.Preparation 54 Ethyl 4-(4-oxo-1-piperidyl)benzoate NMR (CDCl3, δ): 1.38 (3H, t, J=7.1 Hz), 2.57 (4H, t, J=6.1 Hz), 3.75 (4H, t, J=6 Hz), 4.34 (2H, q, J=7.1 Hz), 6.91 (2H, d, J=8.9 Hz), 7.97 (2H, d, J=8.9 Hz) APCI MASS: 248.2 (M++H) Preparation 55 1,1′-Dimethoxy-1,1′-bi(cyclohexyl)-4-one NMR (CDCl3, δ): 1.0-2.0 (12H, m), 2.1-2.4 (4H, m), 2.4 2.7 (2H, m), 3.44 (3H, s), 3.52 (3H, s) MASS (m/z): 263 (M++23) Preparation 56 Sodium hydride, 60% dispersion in mineral oil (3.1 g) was added slowly to a solution of 1-(tert-butoxycarbonyloxy)piperidin-4-ol (12 g) in N,N-dimethylformamide (60 ml) at ambient temperature.", "The mixture was stirred at 60° C. for 1.5 hours.", "To the reaction mixture was added dropwise 1-iodobutane (8.82 ml) at ambient temperature, and stirred for 19 hours.", "The reaction mixture was poured into water (400 ml), and extracted with ethyl acetate.", "The extract was washed with brine and dried, and evaporated under reduced pressure.", "The residue was chromatographed on a column of silica gel eluting with a mixture of n-hexane and ethyl acetate (4:1) to give 4-butoxy-1-(tert-butoxycarbonyloxy)piperidine (3.70 g).", "IR (KBr): 2956.3, 1702.8, 1689.3, 1174.4 cm−1 NMR (CDCl3, δ): 0.92 (3H, t, J=7.2 Hz), 1.26-1.86 (17H, m), 3.01-3.82 (7H, m) ESI MASS (Positive)(m/z): 157.93 (M+−tBoc) The following compound was obtained according to a similar manner to that of Preparation 56.Preparation 57 4-Pentyloxy-1-(tert-butoxycarbonyloxy)piperidine IR (KBr): 2933.2, 1693.2, 1105.0 cm−1 NMR (CDCl3, δ): 0.90 (3H, t, J=6.6 Hz), 1.28-1.86 (19H, m), 3.00-3.82 (7H, m) ESI MASS (Positive)(m/z): 172.00 (M+−tBoc+1) Preparation 58 To a solution of 4-(4-butoxypiperidin-1-yl)benzohydrazide (2.55 g) and pyridine (2.61 ml) in tetrahydrofuran (76.5 ml) was added dropwise phenylchloroformate (1.82 g) with stirring under ice-cooling, and the mixture was stirred at the ambient temperature for 3.5 hours.", "The reaction mixture was added water (770 ml) and the resulting precipitate collected, and dried to give methyl 4-[2-[4-(4-butoxypiperidin-1-yl)benzoyl]hydrazinocarbonyl]-benzoate (3.74 g).", "IR (KBr): 3263.0, 2954.4, 1724.0, 1278.6, 1108.9 cm−1 NMR (CDCl3, δ): 0.93 (3H, t, J=7.2 Hz), 1.30-1.93 (8H, m), 3.04-3.68 (7H, m), 3.94 (3H, s), 6.82-6.87 (2H, m), 7.22-7.77 (2H, m), 7.89-7.93 (2H, m), 8.04-8.08 (2H, m), 9.46 (1H, d, J=5.1 Hz), 9.98 (1H, d, J=5.5 Hz) ESI MASS (Positive)(m/z): 454.33 (M++H) Preparation 59 Ethyl 4-[4-[2-[4-(7-methoxyheptyloxy)benzoyl]-hydrazinocarbonyl]-1-piperazinyl]benzoate (1.25 g) IR (KBr): 3280.3, 2979.5, 1706.7, 1648.3, 1110.8 cm−1 NMR (CDCl3, δ): 1.26-1.72 (13H, m), 3.21 (3H, s), 3.26-3.60 (10H, m), 4.02 (2H, t, J=6.4 Hz), 4.25 (2H, q, J=7.1 Hz), 6.98-7.04 (4H, m), 7.78-7.87 (4H, m), 8.70 (1H, br s), 9.95 (1H, br s) ESI MASS (Positive) (m/z): 540.87 (M)+ Preparation 60 A solution of tert-butyl 4-(hydroxymethyl)-1-piperidinecarboxylate (4.12 g) in DMF (21 ml) was sodium hydride (60% in oil) (0.995 g) at ambient temperature with stirring and the mixture was stirred at 60° C. for 2 hours.", "The reaction mixture was dropwise added to a solution of 1,5-dibromoheptane (17.6 ml) in DMF (35 ml) with stirring at ambient temperature and stirred at the same temperature for 5 hours.", "The reaction mixture was poured into water (200 ml) and extracted twice with a mixture of ethyl acetate (80 ml) and n-hexane (40 ml).", "The extracts were washed with saturated aqueous sodium chloride, dried over magnesium sulfate and evaporated in vacuo.", "The resulting residue was chromatographed on silica gel (200 ml) eluting with a mixture of n-hexane and ethyl acetate (5:1 v/v).", "The fractions containing the desired compound were collected and evaporated under reduced pressure to give tert-butyl 4-(4-bromopentyloxymethyl)-1-piperidinecarboxylate (1.89 g).", "NMR (CDCl3, δ): 1.00-1.30 (6H, m), 1.45 (9H, s), 1.46-1.80 (7H, m), 1.80-1.96 (2H, m), 2.55-2.80 (2H, m), 3.24 (2H, d, J=6.05 Hz), 3.35-3.45 (4H, m), 4.00-4.20 (2H, m) APCI MASS (Positive)(m/z): 388.2, 386.2 (M++Na) The following compounds [Preparation 61 and 62] were obtained according to a similar manner to that of Preparation 60.Preparation 61 1-Bromo-4-(hexyloxy)benzene NMR (CDCl3, δ): 0.87-0.93 (3H, m), 1.28-1.55 (6H, m), 1.69-1.83 (2H, m), 3.91 (2H, t, J=6.5 Hz), 6.73-6.80 (2H, m), 7.31-7.39 (2H, m) EI MASS (m/z): 256, 258 (M+, Br isotopes) Preparation 62 1-(6-Bromohexyl)piperazine NMR (DMSO-d6, δ): 1.20-1.50 (14H, m), 1.65-1.90 (2H, m), 2.10-2.30 (4H, m), 3.20-3.35 (4H, m), 3.50 (2H, t, J=4.0 Hz) MASS: 351 (M+), 349 (M) Preparation 63 To a mixture of cesium carbonate (2.53 g), palladium(II) acetate (62.3 mg) and 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (259 mg) in toluene (11 ml) was successively added methyl 4′-trifluoromethylsulfonyloxy-[1,1′-biphenyl]-4-carboxylate (2.00 g) and 4-phenylpiperidine (1.07 g) in stream of nitrogen.", "The mixture was stirred at ambient temperature for 30 minutes and at 110° C. for further 18 hours.", "After cooling to room temperature, water and acetonitrile were added to the reaction mixture.", "The resulting precipitate was collected by filtration and washed with water and acetonitrile and dried to give methyl 4′-(4-phenyl-1-piperidyl)-1,1′-biphenyl-4-carboxylate (393 mg).", "NMR (CDCl3, δ): 1.8-2.1 (4H, m), 2.6-3.0 (3H, m), 3.8-4.0 (5H, m), 7.06 (2H, d, J=8.9 Hz), 7.15-7.4 (5H, m), 7.5-7.7 (4H, m), 8.0-8.15 (2H, m) MASS (m/z): 372 (M++H) The following compounds [Preparation 64 to 71] were obtained according to a similar manner to that of Preparation 63.Preparation 64 Methyl 4′-(4-cyclohexylhexahydro-1H-1,4-diazepin-1-yl)-1,1′-biphenyl-4-carboxylate NMR (CDCl3, δ): 0.7-2.3 (12H, m), 2.6-3.2 (5H, m), 3.5-3.8 (4H, m), 3.93 (3H, s), 6.77 (2H, d, J=8.9 Hz), 7.54 (2H, d, J=8.9 Hz), 7.61 (2H, d, J=8.4 Hz), 8.05 (2H, d, J=8.4 Hz) MASS (m/z): 393 (M++H) Preparation 65 Tert-Butyl 5-cyclohexyl-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate NMR (CDCl3, δ): 1.0-1.4 (5H, m), 1.47 (9H, s), 1.5-2.6 (9H, m), 3.05-3.2 (2H, m), 3.4-3.65 (1H, m), 3.75 (1H, s), 4.15-4.4 (1H, m) MASS (m/z): 281 (M++H) Preparation 66 Methyl 4′-(5-cyclohexyl-2,5-diazabicyclo[2.2.1]hept-2-yl)-1,1′-biphenyl-4-carboxylate NMR (CDCl3, δ): 1.0-1.35 (5H, m), 1.4-2.3 (8H, m), 2.45-2.6 (1H, m), 3.25-3.5 (3H, m), 3.8-4.0 (4H, m), 4.27 (1H, s), 6.64 (2H, d, J=8.7 Hz), 7.54 (2H, d, J=8.7 Hz), 7.62 (2H, d, J=8.4 Hz), 7.05 (2H, d, J=8.4 Hz) MASS (m/z): 391 (M++H) Preparation 67 Methyl 4′-[4-(cis-4-methylcyclohexyl)-1-piperazinyl]-1,1′-biphenyl-4-carboxylate NMR (CDCl3, δ): 0.95 (3H, d, J=6.9 Hz), 1.4-1.85 (9H, m), 2.1-2.3 (1H, m), 2.65-2.8 (4H, m), 3.2-3.35 (4H, m), 3.93 (3H, s), 7.00 (2H, d, J=8.8 Hz), 7.56 (2H, d, J=8.8 Hz), 7.62 (2H, d, J=8.5 Hz), 8.06 (2H, d, J=8.5 Hz) MASS (m/z): 393 (M++H) Preparation 68 Methyl 4′-[4-(trans-4-methylcyclohexyl)-1-piperazinyl]-1,1′-biphenyl-4-carboxylate NMR (CDCl3, δ): 0.8-1.45 (8H, m), 1.7-2.05 (4H, m), 2.2-2.4 (1H, m), 2.7-2.8 (4H, m), 3.2-3.35 (4H, m), 3.92 (3H, s), 6.99 (2H, d, J=8.8 Hz), 7.55 (2H, d, J-8.8 Hz), 7.62 (2H, d, J=8.4 Hz), 8.06 (2H, d, J=8.4 Hz) MASS (m/z): 393 (M++H) Preparation 69 Methyl 4′-[4-4-[4-(6-methoxyhexyl)-1-piperazinyl]phenyl-1-piperazinyl]-1,1′-biphenyl-4-carboxylate NMR (CDCl3, δ): 1.2-2.7 (8H, m), 2.3-2.7 (5H, m), 3.05-3.5 (14H, m), 3.33 (3H, s), 3.93 (3H, s), 6.8-7.1 (6H, m), 7.5-7.7 (4H, m), 8.07 (2H, d, J=8.4 Hz) MASS (m/z): 571 (M++H) Preparation 70 Methyl 4′-[4-4-[1-(6-methoxyhexyl)-4-piperidyloxy]phenyl-1-piperazinyl]-1,1′-biphenyl-4-carboxylate NMR (CDCl3, δ): 1.2-2.6 (16H, m), 2.7-2.9 (2H, m), 3.2-3.5 (13H, m), 3.93 (3H, s), 4.15-4.35 (1H, m), 6.8-7.0 (4H, m), 7.05 (2H, d, J=8.9 Hz), 7.5-7.7 (4H, m), 8.07 (2H, d, J=5 Hz) MASS (m/z): 586 (M++H) Preparation 71 Methyl 4′-[4-4-[4-(6-methoxyhexyl)-1-piperazinyl]phenyl-1-piperazinyl]-1,1′-biphenyl-4-carboxylate NMR (CDCl3, δ): 1.2-2.7 (8H, m), 2.3-2.7 (5H, m), 3.05-3.5 (14H, m), 3.33 (3H, s), 3.93 (3H, s), 6.8-7.1 (6H, m), 7.5-7.7 (4H, m), 8.07 (2H, d, J=8.4 Hz) MASS (m/z): 571 (M++H) Preparation 72 To a stirred solution of 4-methylphenyl-p-toluenesulfonate (1.88 g) in DMF (20 ml) was added NaH (60% oil suspension; 405 mg) slowly at 0° C., and the suspension was stirred for 1 hour with warming to room temperature and for 1 hour at 65° C. 4-Hydroxy-1-tert-butoxycarbonyloxypiperidine (2 g) in DMF (10 ml) was added dropwise to the above solution and stirring was continued for 2 hours at this temperature.", "Water (10 ml) was added to the solution, and the whole was extracted with EtOAc, and the extract was washed with water and brine and dried over MgSO4.Usual work up followed by flash chromatography (SiO2; EtOAc:hexane=1:5) gave tert-butyl 4-(4-methylpentyloxy)-1-piperidinecarboxylate (2.09 g).", "NMR (CDCl3, δ): 0.88 (6H, d, J=6.6 Hz), 1.1-1.3 (3H, m), 1.4-1.7 (4H, m), 1.45 (9H, s), 1.7-1.9 (2H, m), 3.0-3.2 (2H, m), 3.3-3.5 (3H, m), 3.7-3.9 (2H, m) (+) APCI MASS (Positive): 186.20 (M+−Boc+H) The following compounds [Preparation 73 and 74] were obtained according to a similar manner to that of Preparation 72.Preparation 73 Tert-Butyl 4-(methoxybutyloxymethyl)-1-piperidinecarboxylate NMR (CDCl3, δ): 1.45 (9H, s), 1.55-1.75 (4H, m), 2.60-2.80 (2H, m), 3.25 (2H, d, J=6.05 Hz), 3.28 (3H, s), 3.33-3.68 (4H, m), 4.00-4.10 (2H, m) Preparation 74 Tert-Butyl 4-(cyclohexylmethoxy)-1-piperidinecarboxylate NMR (CDCl3, δ): 0.8-1.9 (15H, m), 1.45 (9H, s), 3.0-3.2 (2H, m), 3.19 (2H, d, J=9.1 Hz), 3.3-3.5 (1H, m), 3.6-3.9 (2H, m) (+)APCI MASS (Positive): 198.33 (M++Boc+H) Preparation 75 A mixture of tert-butyl 1-piperazinecarboxylate (1.6 g), 5-methoxypentyl 4-methylbenzenesulfonate (2.8 g) and potassium carbonate (1.4 g) in dimethylformamide (16 ml) was stirred for 20.5 hours at room temperature.", "The reaction mixture was poured into a mixture of ethyl acetate and water.", "The organic layer was successively washed with water and brine and dried over magnesium sulfate.", "The solvent was evaporated in vacuo and the residue was purified by column chromatography on silica gel eluting with a mixture of dichloromethane and methanol (20:1).", "The eluted fractions containing the desired product were collected and evaporated in vacuo to give tert-butyl 4-(5-methoxypentyl)-1piperazinecarboxylate (1.73 g).", "NMR (CDCl3, δ): 1.3-1.7 (15H, m), 2.3-2.4 (6H, m), 3.3-3.5 (9H, m) (+)APCI MASS: 286.80 (M++H) The following compounds [Preparation 76 to 79] were obtained according to a similar manner to that of Preparation 75.Preparation 76 Ethyl 4-[4-(7-methoxyheptyl)-1-piperazinyl]benzoate NMR (CDCl3, δ): 1.3-1.7 (13H, m), 2.38 (2H, t, J=8.0 Hz), 2.58 (4H, t, J=5.0 Hz), 3.3-3.4 (9H, m), 4.33 (2H, q, J=7.2 Hz), 6.86 (2H, d, J=9.0 Hz), 7.92 (2H, d, J=9.0 Hz) (+) APCI MASS (Positive): 363.33 (M++H) Preparation 77 1-(4-Bromophenyl)-4-(6-methoxyhexyl)piperazine NMR (CDCl3, δ): 1.25-1.7 (8H, m), 2.38 (2H, t, J=7.6 Hz), 2.5-2.65 (4H, m), 3.1-3.2 (4H, m), 3.33 (3H, s), 3.37 (2H, t, J=6.5 Hz), 6.7-6.85 (2H, m), 7.25-7.4 (2H, m) MASS (m/z): 355, 357 (M++H) Preparation 78 4-(4-Bromophenoxy)-1-(6-methoxyhexyl)piperidine NMR (CDCl3, δ): 1.2-2.45 (16H, m), 2.65-2.8 (2H, m), 3.25-3.45 (5H, m), 4.2-4.35 (1H, m), 6.7-6.85 (2H, m), 7.3-7.4 (2H, m) MASS (m/z): 370, 372 (M++H) Preparation 79 1-(4-Bromophenyl)-4-(6-methoxyhexyl)piperazine NMR (CDCl3, δ): 1.25-1.7 (8H, m), 2.38 (2H, t, J=7.6 Hz), 2.5-2.65 (4H, m), 3.1-3.2 (4H, m), 3.33 (3H, s), 3.37 (2H, t, J=6.5 Hz), 6.7-6.85 (2H, m), 7.25-7.4 (2H, m) MASS (m/z): 355, 357 (M++H) Preparation 80 A solution of 4-hexyloxybenzeneboronic acid (1.15 g), 4-iodopyrazole (500 mg), pyridine (409.5 mg) and 4A° molecular sieves (powdered) (1.9 g) in methylene chloride (25 ml) was treated with anhydrous cuprous acetate (Cu(OAc)2) and stirred 3 days at room temperature under an air atmosphere.", "The mixture was filtered then diluted with ethyl acetate, washed with saturated sodium chloride solution (×1), saturated sodium hydrogen carbonate solution (×1), saturated sodium chloride solution (×2), dried over magnesium sulfate and evaporated to afford a crude product that was purified by silica gel chromatography, eluting with 20:1 hexane-ethyl acetate to afford 1-(4-hexyloxyphenyl)-4-iodo-1H-pyrazole (1 g) as a white solid.", "NMR (CDCl3, δ): 0.8-1.0 (3H, m), 1.2-1.5 (6H, m), 1.73-1.83 (2H, m), 3.98 (2H, t, J=6.5 Hz), 6.95 (2H, d, J=9 Hz), 7.51 (2H, d, J=9 Hz), 7.68 (1H, s), 7.85 (1H, s) MASS (m/z): 371 (MH+) The following compounds [Preparation 81 to 88] were obtained according to a similar manner to that of Preparation 80.Preparation 81 Ethyl 4-[4-(1,1′-biphenyl-4-yl)-1H-pyrazol-1-yl]benzoate NMR (CDCl3, δ): 1.43 (3H, t, J=7.1 Hz), 4.41 (2H, q, J=7.1 Hz), 7.32-7.50 (3H, m), 7.65 (4H, s), 7.61-7.71 (2H, m), 7.83 (2H, d, J=8.8 Hz), 8.08 (1H, s), 8.17 (2H, d, J=8.8 Hz), 8.27 (1H, s) MASS (m/z): 369 (MH+) Preparation 82 Ethyl 4-[4-(4-hexyloxyphenyl)-1H-pyrazol-1-yl]benzoate NMR (CDCl3, δ): 0.90-0.95 (3H, m), 1.20-1.57 (9H, m), 1.73-1.84 (2H, m), 3.99 (2H, t, J=6.5 Hz), 4.40 (2H, q, J=7.2 Hz), 6.94 (2H, d, J=8.6 Hz), 7.47 (2H, d, J=8.7 Hz), 7.80 (2H, d, J=7.2 Hz), 7.97 (1H, s), 8.15 (1H, s), 8.16 (2H, d, J-8.5 Hz) MASS (m/z): 393 (MH+) Preparation 83 Methyl 4-[1-4-(hexyloxyphenyl)-1H-pyrazol-4-yl]benzoate NMR (CDCl3, δ): 0.89-0.95 (3H, m), 1.2-1.5 (6H, m), 1.74-1.84 (2H, m), 3.93 (3H, s), 4.00 (2H, t, J=6.5 Hz), 6.98 (2H, d, J=9 Hz), 7.59-7.63 (4H, m), 8.01 (1H, s), 8.06 (2H, d, J-8.4 Hz), 8.14 (1H, s) MASS (m/z): 379 (MH+) Preparation 84 Methyl 4-[1-(8-methoxyoctyl)-4-piperidyloxy]benzoate IR (Neat): 2927, 2856, 1720, 1605, 1508, 1458, 1437, 1309, 1282, 1244, 1169, 1117, 1103, 1043 cm−1 NMR (DMSO-d6, δ): 1.2-1.7 (14H, m), 1.9-2.1 (2H, m), 2.1-2.3 (4H, m), 2.6-2.8 (2H, m), 3.21 (3H, s), 3.29 (2H, t, J=6.4 Hz), 3.81 (3H, s), 4.4-4.6 (1H, m), 7.04 (2H, d, J=8.9 Hz), 7.88 (2H, d, J=8.8 Hz) ESI MASS (Positive): 378.3 (M++H) Preparation 85 Methyl 4-[1-(7-methoxyheptyl)-4-piperidyl]benzoate IR (Nujol): 1728, 1279, 1109 cm−1 NMR (DMSO-d6, δ): 1.2-1.8 (14H, m), 1.9-2.1 (2H, m), 2.2-2.4 (2H, m), 2.4-2.7 (1H, m), 2.9-3.1 (2H, m), 3.21 (3H, s), 3.2-3.4 (2H, m), 3.83 (3H, s), 7.40 (2H, d, J=8.3 Hz), 7.86 (2H, d, J=8.3 Hz) ESI MASS (Positive): 348.3 (M++H) Preparation 86 6-Methoxy-1-hexanol NMR (CDCl3, δ): 1.3-1.5 (4H, m), 1.5-1.7 (5H, m), 3.33 (3H, s), 3.3-3.5 (2H, m), 3.5-3.7 (2H, m) Preparation 87 Tert-Butyl 4-(7-methoxyheptyl)-1-piperazinecarboxylate NMR (CDCl3, δ): 1.2-1.6 (19H, m), 2.3-2.4 (6H, m), 3.33 (3H, s), 3.3-3.5 (6H, m) ESI MASS (Positive): 315.5 (M++H) Preparation 88 5-Methoxy-1-pentanol NMR (CDCl3, δ): 1.3-1.7 (6H, m), 3.3-3.5 (5H, m), 3.6-3.7 (2H, m) Preparation 89 To a solution of 4-methoxybutanol (10 g) in a mixture of dichloromethane (100 ml), triethylamine (17.4 ml) and pyridine (20 ml) was added p-toluenesulfonyl chloride (20.1 g) and the mixture was stirred at ambient temperature overnight.", "The reaction mixture was concentrated in vacuo and dissolved in ethyl acetate (200 ml).", "The solution was washed in turn with 1N hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate and saturated aqueous sodium chloride, dried over magnesium sulfate and evaporated in vacuo.", "The resulting residue was chromatographed on silica gel (200 ml) eluting with a mixture of n-hexane and ethyl acetate (2:1 v/v).", "The fractions containing the desired compound were collected and evaporated under reduced pressure to give 4-methoxybutyl-4-methylbenzenesulfonate (8.34 g).", "NMR (CDCl3, δ): 1.45-1.80 (5H, m), 2.45 (3H, s), 3.30 (3H, s), 3.36 (2H, t, J=6.10 Hz), 3.36 (2H, t, J=6.30 Hz), 7.34 (2H, d, J=8.21 Hz), 7.79 (2H, s, J=8.33 Hz) ESI MASS (Positive)(m/z): 281.2(M++Na) The following compounds [Preparation 90 to 93] were obtained according to a similar manner to that of Preparation 89.Preparation 90 6-Methoxyhexyl 4-methylbenzenesulfonate NMR (CDCl3, δ): 1.2-1.8 (8H, m), 2.45 (3H, s), 3.31 (3H, s), 3.2-3.4 (2H, m), 4.02 (2H, t, J=6.4 Hz), 7.34 (2H, d, J=8.3 Hz), 7.79 (2H, d, J=8.3 Hz) ESI MASS (Positive): 309.3 (M++Na) Preparation 91 5-Methoxypentyl 4-methylbenzenesulfonate NMR (CDCl3, δ): 1.3-1.8 (6H, m), 2.45 (3H, s), 3.3-3.4 (5H, m), 4.02 (2H, t, J=6.4 Hz), 7.3-7.4 (2H, m), 7.7-7.9 (2H, m) ESI MASS (Positive): 295.2 (M++H) Preparation 92 4-Methylpentyl 4-methylbenzenesulfonate NMR (CDCl3, δ): 0.84 (6H, d, J=6.6 Hz), 1.1-1.3 (2H, m), 1.4-1.7 (3H, m), 2.45 (3H, s), 4.01 (2H, t, J=6.6 Hz), 7.34 (2H, d, J=8.0 Hz), 7.7-7.9 (2H, m) ESI MASS (Positive): 279.3 (M++Na) Preparation 93 Cyclohexylmethyl 4-methylbenzenesulfonate NMR (CDCl3, δ): 0.7-1.8 (11H, m), 2.45 (3H, s), 3.81 (2H, d, J=6.0 Hz), 7.34 (2H, d, J=8.0 Hz), 7.7-7.8 (2H, m) ESI MASS (Positive): 291.3 (M++Na) Preparation 94 To a solution of tert-butyl 4-cyclohexyl-4-methoxy-1-piperidinecarboxylate (3.0 g) in a mixture of dichloromethane (60 ml) and anisole (7.67 ml) was dropwise added trifluoroacetic acid (15.5 ml) with stirring under ice-cooling.", "The mixture was stirred at ambient temperature for 1 hour and then concentrated in vacuo.", "The resulting residue was azeotropically distilled three times with toluene (50 ml) and dried in vacuo.", "The obtained residue was dissolved in DMSO (30 ml).", "To the solution were added 4-fluorobenzonitrile (1.47 g) and potassium carbonate (4.18 g) and the mixture was stirred at 140° C. for 4 hours.", "The reaction mixture was poured into water (100 ml) and extracted twice with ethyl acetate (100 ml).", "The extracts were collected, washed with saturated aqueous sodium chloride, dried over magnesium sulfate and evaporated in vacuo.", "The resulting residue was chromatographed on silica gel (200 ml) eluting with a mixture of n-hexane and ethyl acetate (8:1 v/v).", "The fractions containing the desired compound were collected and evaporated under reduced pressure to give 4-(4-cyclohexyl-4-methoxy-1-piperidyl)benzonitrile (3.08 g).", "NMR (CDCl3, δ): 0.85-1.45 (5H, m), 1.50-1.85 (10H, m), 3.00-3.20 (2H, m), 3.16 (3H, s), 3.50-3.70 (2H, m), 6.85 (2H, d, J=90.10 Hz), 7.46 (2H, d, J=9.08 Hz) ESI MASS (Positive)(m/z): 619.5 (2M++Na), 321.3 (M++Na) The following compounds [Preparation 95 to 97] were obtained according to a similar manner to that of Preparation 94.Preparation 95 4-(4-Cyanophenyl)-1-(6-methoxyhexyl)piperazine IR (KBr): 3560, 3392, 2935, 2856, 2212, 1603, 1516 cm−1 NMR (CDCl3, δ): 1.20-1.40 (4H, m), 1.40-1.70 (6H, m), 2.30-2.60 (2H, m), 2.50-2.60 (4H, m), 3.30-3.50 (4H, m), 3.33 (3H, s), 6.85 (2H, d, J=9.0 Hz), 7.48 (2H, d, J=9.0 Hz) MASS (m/z): 302 (M++H) Preparation 96 4-[4-(7-Methoxyheptyl)-1-piperazinyl]benzonitrile IR (KBr): 2929, 2856, 2212, 1603, 1518, 1452, 1389, 1248, 1180, 1132, 1095, 924, 833 cm−1 NMR (DMSO-d6, δ): 1.2-1.6 (10H, m), 2.2-2.6 (6H, m), 3.20 (3H, s), 3.2-3.4 (6H, m), 7.01 (2H, d, J=9.0 Hz), 7.57 (2H, d, J=9.0 Hz) (+) APCI MASS: 316.07 (M++H) Preparation 97 4-[4-(5-Methoxypentyl)-1-piperazinyl]benzonitrile IR (KBr): 2935, 2212, 1603, 1514, 1452, 1387, 1363, 1250, 1180, 1111, 947, 922, 825 cm−1 NMR (DMSO-d6, δ): 1.2-1.6 (6H, m), 2.2-2.5 (6H, m), 3.21 (3H, s), 3.3-3.4 (6H, m), 7.01 (2H, d, J=9.0 Hz), 7.57 (2H, d, J=9.0 Hz) (+) APCI MASS (m/z): 288.13 (M++H) Preparation 98 A solution of tert-butyl 4-(4-bromopentyloxymethyl)-1-piperidinecarboxylate (1.88 g) in a mixture of 28% sodium hydroxide in methanol (10.5 ml) and methanol (10 ml) was refluxed for 2 hours.", "The reaction mixture was concentrated in vacuo and was chromatographed on silica gel (200 ml) eluting with a mixture of n-hexane and ethyl acetate (4:1 v/v).", "The fractions containing the desired compound were collected and evaporated under reduced pressure to give tert-butyl 4-(5-methoxypentyloxymethyl)-1-piperidinecarboxylate (1.53 g).", "NMR (CDCl3, δ): 1.10-1.25 (2H, m), 1.30-1.45 (2H, m), 1.45 (9H, s), 1.50-1.80 (7H, m), 2.55-2.80 (2H, m), 3.24 (2H, d, J=6.07 Hz), 3.33 (3H, s), 3.34-3.45 (4H, m), 4.00-4.20 (2H, m) ESI MASS (Positive)(m/z): 338.4 (M++Na) The following compounds [Preparation 99 to 102] were obtained according to a similar manner to that of Preparation 98.Preparation 99 4-(7-Methoxyheptyloxy)benzoic acid NMR (DMSO-d6, δ): 1.32-1.75 (10H, m), 3.30 (2H, t, J=6.4 Hz), 4.02 (2H, t, J=6.5 Hz), 6.99 (2H, d, J=6.4 Hz), 7.87 (2H, d, J=8.8 Hz) ESI MASS (Negative)(m/z): 265.4 (M+−H) Preparation 100 8-(6-Methoxyhexyloxy)-1,4-dioxaspiro[4.5]decane NMR (CDCl3, δ): 1.25-1.90 (16H, m), 3.33 (3H, s), 3.35-3.45 (5H, m), 3.92 (4H, s) ESI MASS (Positive)(m/z): 295.4 (M++Na) Preparation 101 4-tert-Butoxycarbonyl-1-(6-methoxyhexyl)piperazine NMR (CDCl3, δ): 1.20-1.40 (4H, m), 1.45-1.70 (12H, m), 2.20-2.40 (6H, m), 3.33 (3H, s), 3.35-3.50 (6H, m) API-ES MASS (Positive): 323 (M++Na), 301 (M++H) Preparation 102 Methyl 4-[4-(7-methoxyheptyloxy)piperidin-1-yl]benzoate NMR (CDCl3, δ): 1.25-1.48 (6H, m), 1.48-1.78 (6H, m), 1.88-2.06 (2H, m), 3.00-3.20 (2H, m), 3.32 (3H, s), 3.27-3.54 (5H, m), 3.58-3.75 (2H, m), 3.86 (3H, s), 6.87 (2H, d, J=8.9 Hz), 7.90 (2H, d, J=9.1 Hz) MASS (m/z): 364 (M++H) Preparation 103 A mixture of ethyl 4-fluorobenzoate (7.88 g), 4-iodopyrazole (10 g) and potassium carbonate (7.11 g) in N,N-dimethylformamide (50 ml) was heated at 100° C. for 6 hours then cooled to room temperature.", "The mixture was diluted with ethyl acetate then washed with water (×5), brine, dried over magnesium sulfate, filtered and evaporated to give a crude product that was recrystallized from acetone-hexane to afford ethyl 4-(4-iodo-1H-pyrazol-1-yl)benzoate (2 crops, 6.5 g+4.6 g) as a light yellow solid.", "NMR (CDCl3, δ): 1.41 (3H, t, J=7.1 Hz), 4.40 (2H, q, J=7.1 Hz), 7.73 (2H, d, J=8.5 Hz), 7.75 (1H, s), 8.05 (1H, s), 8.14 (2H, d, J=8.5 Hz) MASS (m/z): 343 (MH+) The following compounds [Preparation 104 to 111] were obtained according to a similar manner to that of Preparation 103.Preparation 104 Ethyl 4-[1-(8-bromooctyl)-4-piperidyloxy)benzoate IR (Neat): 2931, 1713, 1605, 1508, 1277, 1252, 1169, 1105, 1043 cm−1 NMR (DMSO-d6, δ): 1.2-1.9 (17H, m), 1.9-2.1 (2H, m), 2.1-2.5 (2H, m), 2.6-2.9 (2H, m), 3.2-3.6 (2H, m), 3.53 (2H, t, J=6.7 Hz), 4.27 (2H, q, J=7.1 Hz), 4.4-4.6 (1H, m), 7.05 (2H, d, J=8.9 Hz), 7.88 (2H, d, J=8.8 Hz) ESI MASS (Positive): 440.2, 442.2 (M++H) Preparation 105 Methyl 4-[1-(7-bromoheptyl)-4-piperidyl]benzoate IR (Neat): 2933, 1720, 1281, 1111 cm−1 NMR (DMSO-d6, δ): 1.2-2.1 (16H, m), 2.2-2.4 (2H, m), 2.5-2.7 (1H, m), 2.9-3.1 (2H, m), 3.53 (2H, t, J=6.7 Hz), 3.83 (3H, s), 7.40 (2H, d, J=8.3 Hz), 7.89 (2H, d, J=8.3 Hz) ESI MASS (Positive): 396.3, 398.3 (M++H) Preparation 106 4-[1-(6-Methoxyhexyl)-4-piperidyl]benzonitrile IR (Neat): 2937, 2858, 2227, 1608, 1504, 1466, 1450, 1379, 1119 cm−1 NMR (DMSO-d6, δ): 1.2-1.8 (12H, m), 1.8-2.1 (2H, m), 2.5-2.7 (1H, m), 2.9-3.0 (2H, m), 3.21 (3H, s), 3.2-3.4 (2H, m), 7.46 (2H, d, J=8.3 Hz), 7.75 (2H, d, J=8.3 Hz) ESI MASS (Positive): 301.4 (M++H) Preparation 107 Tert-Butyl 4-(7-bromoheptyl)-1-piperazinecarboxylate NMR (CDCl3, δ): 1.2-1.6 (19H, m), 1.7-1.9 (2H, m), 2.3-2.4 (4H, m), 3.3-3.5 (6H, m) (+) APCI MASS: 362.60, 364.53 (M++H) Preparation 108 Ethyl 4-(4-pentyloxypiperidin-1-yl)benzoate IR (KBr): 2952.5, 1695.1, 1369.2, 1110.8 cm−1 NMR (CDCl3, δ): 0.90 (3H, t, J=6.8 Hz), 1.29-2.01 (13H, m), 3.04-3.72 (7H, m), 4.32 (2H, q, J=7.1 Hz), 6.84-6.89 (2H, m), 7.87-7.93 (2H, m) ESI MASS (Positive)(m/z): 320.40 (M++H) Preparation 109 Tert-Butyl 4-(4-bromophenyl)-1-piperazinecarboxylate NMR (CDCl3, δ): 1.48 (9H, s), 3.05-3.15 (4H, m), 3.5-3.6 (4H, m), 6.79 (2H, d, J=9.0 Hz), 7.35 (2H, d, J=9.0 Hz) MASS (m/z): 340, 342 (M++H) Preparation 110 Ethyl 4-[4-(4-methylpentyloxy)-1-piperidyl]benzoate NMR (CDCl3, δ): 0.89 (6H, d, J=6.6 Hz), 1.1-1.2 (3H, m), 1.36 (3H, t, J=7.1 Hz), 1.4-1.8 (4H, m), 1.8-2.1 (2H, m), 3.0-3.2 (2H, m), 3.4-3.6 (3H, m), 3.6-3.8 (2H, m), 4.32 (2H, q, J=7.1 Hz), 6.8-6.9 (2H, m), 7.8-8.0 (2H, m) (+) APCI MASS (Positive): 334.40 (M++H) Preparation 111 Ethyl 4-[4-(cyclohexylmethoxy)-1-piperidyl]benzoate NMR (CDCl3, δ): 0.8-1.4 (6H, m), 1.36 (3H, t, J=7.2 Hz), 1.4-2.1 (9H, m), 3.0-3.2 (2H, m), 3.26 (2H, d, J=6.4 Hz), 3.3-3.5 (1H, m), 3.5-3.8 (2H, m), 4.32 (2H, q, J=7.2 Hz), 6.8-6.9 (2H, m), 7.8-8.0 (2H, m) (+) APCI MASS (Positive): 346.27(M++H) Preparation 112 To a mixture of 4-(1,2,3,6-tetrahydro-4-pyridyl)benzonitrile (1.0 g), cyclohexanone (1.1 ml) and acetic acid (0.93 ml) in methanol (10 ml) was added sodium cyanoborohydride (0.41 g).", "After stirring for 22 hours at room temperature, the solvent was evaporated in vacuo.", "The residue was poured into a mixture of ethyl acetate and water.", "The solution was adjusted to pH 10 with potassium carbonate.", "The organic layer was successively washed with brine and dried over magnesium sulfate.", "The solvent was evaporated in vacuo and the residue was purified by column chromatography on silica gel eluting with a mixture of dichloromethane and methanol (30:1).", "The eluted fractions containing the desired product were collected and evaporated in vacuo to give 4-(1-cyclohexyl-1,2,3,6-tetrahydro-4-pyridyl)benzonitrile (0.75 g).", "IR (Neat): 2931, 2854, 2220, 1649, 1541, 1504, 1456 cm−1 NMR (DMSO-d6, δ): 1.0-1.9 (10H, m), 2.2-2.5 (3H, m), 2.7-2.8 (2H, m), 3.2-3.3 (2H, m), 6.3-6.5 (1H, m), 7.61 (2H, d, J=8.5 Hz), 7.78 (2H, d, J=8.5 Hz) (+) APCI MASS: 267.20 (M++H) The following compounds [Preparation 113 to 125] were obtained according to a similar manner to that of Preparation 112.Preparation 113 Ethyl 4-[4-[4-(6-methoxyhexyloxy)cyclohexyl]-1-piperidyl]benzoate NMR (CDCl3, δ): 1.20-2.10 (20H, m), 2.80-3.00 (4H, m), 3.33 (3H, s), 3.35-3.50 (9H, m), 4.34 (2H, q, J=7.10 Hz), 6.86 (2H, t, J=8.96 Hz), 7.93 (2H, d, J=8.83 Hz) ESI MASS (Positive)(m/z): 447.5 (M++Na) Preparation 114 Ethyl 4-[4-(4-pyridylmethyl)-1-piperazinyl]benzoate NMR (CDCl3, δ): 1.37 (3H, t, J=7.1 Hz), 2.58-2.63 (0.4H, m), 3.32-3.37 (4H, m), 3.57 (2H, s), 4.32 (2H, q, J=7.1 Hz), 6.86 (2H, d, J=9 Hz), 7.31 (2H, d, J=5.9 Hz), 7.92 (2H, d, J=9 Hz), 8.56 (2H, d, J=5.9 Hz) APCI MASS (positive): 326.13 (MH+) Preparation 115 Ethyl 4-[4-(3,3-dimethyl-1,5-dioxaspiro[5.5]undec-9-yl)-1-piperazinyl]benzoate NMR (CDCl3, δ): 0.97 (6H, s), 1.33 (3H, t, J=7.1 Hz), 1.33-1.80 (5H, m), 2.0-2.5 (4H, m), 2.68-2.73 (4H, m), 3.29-3.34 (4H, m), 3.48-3.52 (4H, m), 4.32 (2H, q, J=7.1 Hz), 6.86 (2H, d, J=9 Hz), 7.92 (2H, d, J=9 Hz) APCI MASS: 417.13 (M++H) Preparation 116 Ethyl 4-(4-cyclopentyl-1-piperazinyl)benzoate NMR (CDCl3, δ): 1.36 (3H, t, J=7.1 Hz), 1.40-2.0 (8H, m), 2.49-2.62 (1H, m), 2.62-2.67 (4H, m), 3.32-3.37 (4H, m), 4.32 (2H, q, J=7.1 Hz), 6.86 (2H, d, J=9 Hz), 7.92 (2H, d, J=9 Hz) API-ES MASS: 303.3 (M++H) Preparation 117 Ethyl 4-(4-cycloheptyl-1-piperazinyl)benzoate NMR (CDCl3, δ): 1.36 (3H, t, J=7.1 Hz), 1.40-1.95 (12H, m), 2.50-2.70 (5H, m), 3.29-3.40 (4H, m), 4.32 (2H, q, J=7.1 Hz), 6.8.5 (2H, d, J=9 Hz), 7.91 (2H, d, J=9 Hz) APCI MASS: 331.27 (M++H) Preparation 118 Ethyl 4-[4-(1,4-dioxaspiro[4.5]dec-8-yl)-1-piperazinyl]benzoate NMR (CDCl3, δ): 1.36 (3H, t, J-7.1 Hz), 1.50-2.1 (8H, m), 2.47-2.55 (1H, m), 2.70-2.75 (4H, m), 3.30-3.35 (4H, m), 3.95 (4H, s), 4.32 (2H, q, J=7.1 Hz), 6.86 (2H, d, J=9 Hz), 7.92 (2H, d, J=9 Hz) APCI MASS: 375.2 (M++H) Preparation 119 Ethyl 4-(4-tetrahydro-2H-pyran-4-yl-1-piperazinyl)benzoate NMR (CDCl3, δ): 1.37 (3H, t, J=7.1 Hz), 1.5-2.0 (4H, m), 2.42-2.57 (1H, m), 2.68-2.73 (4H, m), 3.32-3.37 (4H, m), 3.60-4.20 (4H, m), 4.32 (2H, q, J=7.1 Hz), 6.86 (2H, d, J=9 Hz), 7.92 (2H, d, J=9 Hz) APCI MASS: 319.2 (M++H) Preparation 120 4-(1-Cyclohexyl-4-piperidyloxy)benzonitrile IR (Nujol): 2214, 1601, 1504, 1298, 1255, 1171, 1043 cm−1 NMR (DMSO-d6, δ): 0.9-1.4 (6H, m), 1.5-2.1 (9H, m), 2.2-2.6 (2H, m), 2.6-2.9 (2H, m), 4.4-4.6 (1H, m), 7.0-7.2 (2H, m), 7.6-7.8 (2H, m) ESI MASS (Positive): 285.3 (M++H) Preparation 121 4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyloxy]benzonitrile NMR (CDCl3, δ): 1.2-3.2 (24H, m), 3.33 (3H, s), 3.3-3.5 (6H, m), 4.3-4.5 (1H, m), 6.93 (2H, d, J=8.5 Hz), 7.5-7.7 (2H, m) (+) APCI MASS (Positive): 415.40 (M++H) Preparation 122 Ethyl 4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-4-piperidyloxy]benzoate NMR (CDCl3, δ): 1.2-3.0 (23H, m), 3.33 (3H, s), 3.3-3.5 (10H, m), 4.34 (2H, q, J=7.1 Hz), 4.3-4.5 (1H, m), 6.90 (2H, d, J=8.9 Hzl), 7.97 (2H, d, J=8.8 Hz) (+) APCI MASS (Positive): 462.53 (M++H) Preparation 123 Methyl 4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-4-piperidyl]benzoate NMR (CDCl3, δ): 1.2-2.7 (25H, m), 3.0-3.3 (2H, m), 3.33 (3H, s), 3.3-3.5 (4H, m), 3.90 (3H, s), 7.29 (2H, d, J=8.4 Hz), 7.96 (2H, d, J=8.3 Hz) (+) APCI MASS (Positive): 432.27 (M++H) Methyl 4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-4-piperidyl]benzoate NMR (CDCl3, δ): 1.2-2.1 (20H, m), 2.2-2.6 (5H, m), 3.0-3.2 (2H, m), 3.33 (3H, s), 3.3-3.5 (4H, m), 3.89 (3H, s), 7.29 (2H, d, J=8.3 Hz), 7.9-8.0 (2H, m) (+) APCI MASS (Positive): 432.47 (M++H), ESI MASS (Positive): 432.47 (M++H) Preparation 124 Ethyl 4-(1-cyclohexyl-4-piperidyloxy)benzoate IR (Nujol): 1701, 1601, 1504, 1311, 1248, 1163, 1105, 1039 cm−1 NMR (DMSO-d6, δ): 1.0-1.4 (9H, m), 1.5-2.1 (9H, m), 2.2-2.5 (2H, m), 2.7-2.9 (2H, m), 4.27 (2H, q, J=7.1 Hz), 4.4-4.6 (1H, m), 7.04 (2H, d, J=8.9 Hz), 7.88 (2H, d, J=8.9 Hz) ESI MASS (Positive): 332.4 (M++H) Preparation 125 Methyl 4′-[4-[cis-4-methoxy-4-(1-methoxycyclohexyl-1-yl)cyclohexyl-1-yl]-1-piperazinyl]-1,1′-biphenyl-4-carboxylate NMR (CDCl3, δ): 1.0-2.4 (19H, m), 2.7-2.85 (4H, m), 3.25-3.35 (4H, m), 3.43 (3H, s), 3.44 (3H, s), 3.93 (3H, s), 7.00 (2H, d, J=8.9 Hz), 7.5-7.7 (4H, m), 8.0-8.1 (2H, m) MASS (m/z): 521 (M++H) Methyl 4′-[4-[trans-4-methoxy-4-(1-methoxycyclohexyl-1-yl)cyclohexyl-1-yl]-1-piperazinyl]-1,1′-biphenyl-4-carboxylate NMR (CDCl3, δ): 0.8-2.3 (19H, m), 2.55-2.7 (4H, m), 3.2-3.35 (4H, m), 3.42 (3H, s), 3.43 (3H, s), 3.93 (3H, s), 7.00 (2H, d, J=8.9 Hz), 7.5-7.7 (4H, m), 8.0-8.15 (2H, m) MASS (m/z): 521 (M++H) Preparation 126 A mixture of 4-[1-(4-methoxyphenyl)-4-piperidyloxy]benzonitrile (0.59 g), thiosemicarbazide (0.44 g) and trifluoroacetic acid (3 ml) in toluene (6 ml) was stirred for 6 hours at 70° C. After being cooled to room temperature, the solvent was evaporated in vacuo.", "Then the residue was dissolved in tetrahydrofuran and poured into water.", "The solution was adjusted to pH 9 with stirring.", "The resulting precipitate was collected by filtration and washed with water and isopropyl ether to give 5-4-[1-(4-methoxyphenyl)-4-piperidyloxy]phenyl-1,3,4-thiadiazol-2-amine (0.71 g).", "IR (KBr): 3099, 1606, 1518, 1466, 1294, 1248, 1180, 1036 cm−1 NMR (DMSO-d6, δ): 1.6-1.9 (2H, m), 2.0-2.2 (2H, m), 2.8-3.0 (2H, m), 3.3-3.4 (2H, m), 3.68 (3H, s), 4.5-4.7 (1H, m), 6.6-7.0 (4H, m), 7.07 (2H, d, J=8.8 Hz), 7.29 (2H, s), 7.67 (2H, d, J=8.8 Hz) ESI MASS (Positive): 383.3 (M++H) The following compounds [Preparation 127 to 137] were obtained according to a similar manner to that of Preparation 126.Preparation 127 2-Amino-5-[4-[4-(4-methoxybutyloxymethyl)piperidin-1-yl]phenyl]-1,3,4-thiadiazole NMR (CDCl3+CD3OD, δ): 1.30-1.50 (2H, m), 1.50-1.80 (6H, m), 1.90-2.10 (2H, m), 2.90-3.10 (2H, m), 3.34 (3H, s), 3.35-3.70 (7H, m), 6.93 (2H, d, J=8.91 Hz), 7.63 (2H, d, J=8.83 Hz) APCI MASS (m/z): 377 (M+) Preparation 128 2-Amino-5-[4-(6-methoxyhexyl)piperazin-1-yl]phenyl]-1,3,4-thiadiazole IR (KBr): 3491, 3290, 3134, 2931, 1606, 1518 cm−1 NMR (DMSO-d6, δ): 1.20-1.40 (4H, m), 1.40-1.60 (4H, m), 2.50-2.70 (4H, m), 3.22 (3H, s), 3.20-3.50 (8H, m), 7.00 (2H, d, J=8.9 Hz), 7.22 (2H, br s), 7.57 (2H, d, J=8.7 Hz) MASS: 376 (M++H) Preparation 129 5-[4-[1-(6-Methoxyhexyl)-4-piperidyl]phenyl]-1,3,4-thiadiazol-2-amine trifluoroacetate IR (KBr): 3277, 3166, 2933, 2858, 1701, 1687, 1630, 1516, 1203, 1171, 1119 cm−1 NMR (DMSO-d6, δ): 1.2-2.0 (14H, m), 2.5-2.8 (5H, m), 3.22 (3H, s), 3.3-3.4 (2H, m), 7.3-7.4 (4H, m), 7.70 (2H, d, J=8.2 Hz) (+) APCI MASS: 375.13 (M++H) Preparation 130 5-[4-[4-(7-Methoxyheptyl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-amine trifluoroacetate IR (KBr): 3277, 3114, 2931, 1606, 1512, 1466, 1238, 1120 cm−1 NMR (DMSO-d6, δ): 1.2-1.6 (10H, m), 2.3-2.6 (6H, m), 3.21 (3H, s), 3.2-3.4 (6H, m), 6.98 (2H, d, J=8.8 Hz), 7.21 (2H, s), 7.57 (2H, d, J=8.8 Hz) (+) APCI MASS: 390.56 (M++H) Preparation 131 5-[4-[4-(5-Methoxypentyl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-amine trifluoroacetate IR (KBr): 3115, 2939, 1606, 1520, 1466, 1325, 1240, 1192, 1122, 1034, 824 cm−1 NMR (DMSO-d6, δ): 1.2-1.6 (6H, m), 2.2-2.6 (6H, m), 3.21 (3H, s), 3.2-3.4 (6H, m), 6.98 (2H, d, J=8.8 Hz), 7.20 (2H, s), 7.56 (2H, d, J=8.8 Hz), 8.05 (1H, s) (+) APCI MASS: 362.00 (M++H) Preparation 132 5-[4-(1-Phenyl-4-piperidyloxyphenyl]-1,3,4-thiadiazol-2-amine IR (KBr): 3269, 3097, 1603, 1518, 1468, 1246 cm−1 NMR (DMS-d6, δ): 1.6-1.9 (2H, m), 2.0-2.2 (2H, m), 3.0-3.2 (2H, m), 3.4-3.6 (2H, m), 4.6-4.7 (1H, m), 6.76 (1H, t, J=7.2 Hz), 6.9-7.4 (8H, m), 7.67 (2H, d, J=8.8 Hz) ESI MASS (Positive): 727.2 (2M++Na) Preparation 133 5-[4-(1-Cyclohexyl-1,2,3,6-tetrahydro-4-pyridyl)phenyl]-1,3,4-thiadiazol-2-amine trifluoroacetate IR (KBr): 3155, 2945, 1682, 1504, 1205, 1132 cm−1 NMR (DMSO-d6, δ): 1.0-2.2 (10H, m), 2.7-2.9 (2H, m), 3.1-3.5 (2H, m), 3.6-3.8 (1H, m), 3.8-4.0 (2H, m), 6.2-6.4 (1H, m), 7.46 (2H, s), 7.60 (2H, d, J=8.5 Hz), 7.77 (2H, d, J=8.5 Hz), 9.72 (1H, br s) (+) APCI MASS: 340.93 (M++H) Preparation 134 5-[4-(1-Cyclohexyl-4-piperidyloxy)phenyl]-1,3,4-thiadiazol-2-amine NMR (CDCl3, δ): 1.0-3.0 (19H, m), 4.3-4.5 (1H, m), 5.2-5.4 (2H, br s), 6.8-7.0 (2H, m), 7.4-7.6 (2H, m) (+) APCI MASS (Positive): 359.27 (M++H) Preparation 135 5-4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyloxy]phenyl-1,3,4-thiadiazol-2-amine NMR (CDCl31 6): 1.2-3.2 (24H, m), 3.33 (3H, s), 3.3-3.5 (6H, m), 4.3-4.5 (1H, m), 5.2-5.3 (2H, br s), 6.93 (2H, d, J=8.8 Hz), 7.71 (2H, d, J=8.6 Hz) (+) APCI MASS (Positive): 489.47 (M++H) Preparation 136 5-4-[4-(5-Methoxypentyloxymethyl)piperidin-1-yl]phenyl-1,3,4-thiadiazol-2-ylamine NMR (CDCl3, δ): 1.20-2.00 (11H, m), 2.65-2.90 (2H, m), 3.27 (2H, d, J=6.04 Hz), 3.33 (3H, s), 3.36 (2H, d, J=6.58 Hz), 3.44 (2H, d, J=6.43 Hz), 3.70-3.90 (2H, m), 5.52 (2H, br s), 6.90 (2H, d, J=8.93 Hz), 7.64 (2H, d, J=8.84 Hz) ESI MASS (Positive)(m/z): 413.3 (M++Na) Preparation 137 5-[4-(4-Cyclohexyl-4-methoxy-1-piperidyl)phenyl]-1,3,4-thiadiazol-2-amine NMR (DMSO-d6, δ): 0.8-2.0 (15H, m), 2.8-3.0 (2H, m), 3.08 (3H, s), 3.5-3.7 (2H, m), 6.9-7.6 (6H, m) (+) APCI MASS: 373.27 (M++H) Preparation 138 A mixture of phenyl 4-[4-(ethoxycarbonyl)phenyl]-1-piperazine carboxylate (13 g) and hydrazine monohydrate (49 ml) in a mixture of ethanol (130 ml) and tetrahydrofuran (65 ml) was refluxed for 24 hours.", "The reaction mixture was poured into water and the resulting precipitate was collected, washed with water, and dried to give ethyl 4-[4-(hydrazinocarbonyl)-1-piperazinyl]benzoate (6.28 g).", "IR (KBr): 3365.2, 2987.2, 1699.0, 1633.4, 1608.3, 1240.0 cm−1 NMR (CDCl3, δ): 1.37 (3H, t, J=7.1 Hz), 3.33-3.59 (8H, m), 3.83 (2H, br s), 4.34 (2H, q, J=7.1 Hz), 5.84 (1H, s), 6.81-6.87 (2H, m), 7.91-7.98 (2H, m) ESI MASS (Positive)(m/z): 315.23 (M++Na) The following compounds [Preparation 139 to 160]were obtained according to a similar manner to that of Preparation 138.Preparation 139 4-[4-[4-(6-Methoxyhexyloxy)cyclohexyl]-1-piperidyl]benzohydrazide (515 mg) NMR (CDCl3, δ): 1.15-1.70 (16H, m), 1.80-2.40 (4H, m), 2.65-2.75 (4H, m), 3.20-3.50 (12H, m), 4.05 (2H, md, J=3.75 Hz), 6.88 (2H, d, J=8.92 Hz), 7.20-7.30 (1H, br s), 7.65 (2H, d, J=8.86 Hz) ESI MASS (Positive)(m/z): 455.4 (M++Na), 433.5 (M++H) Preparation 140 4-[1-(8-Methoxyoctyl)-4-piperidyloxy]benzohydrazide IR (Nujol): 3290, 3275, 1626, 1500, 1325, 1255, 1119, 1036 cm−1 NMR (DMSO-d6, δ): 1.2-1.7 (14H, m), 1.8-2.0 (2H, m), 2.1-2.3 (4H, m), 2.6-2.8 (2H, m), 3.20 (3H, s), 3.29 (2H, t, J=6.4 Hz), 4.3-4.5 (3H, m), 6.97 (2H, d, J=8.8 Hz), 7.77 (2H, d, J=8.8 Hz), 9.58 (1H, s) ESI MASS (Positive): 378.3 (M++H) Preparation 141 4-[1-(7-Methoxyheptyl)-4-piperidyl]benzohydrazide IR (Nujol): 3325, 1624, 1524, 1122 cm−1 NMR (DMSO-d6, δ): 1.2-1.8 (14H, m), 1.8-2.0 (2H, m), 2.2-2.3 (2H, m), 2.4-2.7 (1H, m), 2.8-3.0 (2H, m), 3.21 (3H, s), 3.2-3.4 (2H, m), 4.44 (2H, br s), 7.30 (2H, d, J=8.2 Hz), 7.74 (2H, d, J=8.2 Hz), 9.67 (1H, s) ESI MASS (Positive): 348.5 (M++H) Preparation 142 4-[4-(4-Pyridylmethyl)-1-piperazinyl]benzohydrazide NMR (DMSO-d6, δ): 2.49-2.54 (4H, m), 3.24-3.28 (4H, m), 3.57 (2H, s), 4.35 (2H, s), 6.92 (2H, d, J=8.9 Hz), 7.35 (2H, d, J=5.9 Hz), 7.70 (2H, d, J=8.9 Hz), 8.52 (2H, d, J=5.9 Hz), 9.47 (1H, S) APCI MASS (Positive): 312 (M++H) Preparation 143 4-(1,4-Dioxa-8-azaspiro[4.5]dec-8-yl)benzohydrazide NMR (DMSO-d6, δ): 1.64-1.70 (4H, m), 3.35-3.41 (4H, m), 3.91 (4H, s), 4.35 (2H, s), 6.94 (2H, d, J=8.9 Hz), 7.68 (2H, d, J=8.9 Hz), 9.45 (1H, s) APCI MASS: 278.13 (M++H) Preparation 144 4-[4-(3,3-Dimethyl-1,5-dioxaspiro[5.5]undec-9-yl)-1-piperazinyl]benzohydrazide NMR (DMSO-d6, δ): 0.89 (6H, s), 1.20-1.70 (5H, m), 2.10-2.40 (4H, m), 2.58 (4H, br s), 3.20 (4H, br s), 3.41-3.43 (4H, m), 4.36 (2H, s), 6.91 (2H, d, J=8.9 Hz), 7.69 (2H, d, J=8.9 Hz), 9.46 (1H, s) API-EI MASS: 403.3 (M++H) Preparation 145 4-(4-Cyclopentyl-1-piperazinyl)benzohydrazide NMR (DMSO-d6, δ): 1.20-1.90 (8H, m), 2.4-2.6 (5H, m), 3.19-3.24 (4H, m), 4.36 (2H, s), 6.91 (2H, d, J=8.9 Hz), 7.69 (2H, d, J=8.9 Hz), 9.46 (1H, s) APCI MASS: 289.2 (M++H) Preparation 146 4-(4-Cycloheptyl-1-piperazinyl)benzohydrazide NMR (DMSO-d6, δ): 1.3-1.8 (12H, m), 2.5-2.65 (5H, m), 3.10-3.25 (4H, m), 4.35 (2H, s), 6.90 (2H, d, J=8.9 Hz), 7.69 (2H, d, J=8.9 Hz), 9.45 (1H, s) APCI MASS: 317.27 (M++H) Preparation 147 4-[4-(1,4-Dioxaspiro[4.5]dec-8-yl)-1-piperazinyl]benzohydrazide NMR (DMSO-d6, δ): 1.40-1.75 (8H, m), 2.22-2.45 (1H, m), 2.57-2.61 (4H, m), 3.18-3.23 (4H, m), 3.84 (4H, s), 4.35 (2H, s), 6.91 (2H, d, J=8.9 Hz), 7.69 (2H, d, J=8.9 Hz), 9.46 (1H, s) APCI MASS: 361.27 (M++H) Preparation 148 4-(4-Tetrahydro-2H-pyran-4-yl-1-piperazinyl)benzohydrazide NMR (DMSO-d6, δ): 1.30-1.50 (2H, m), 1.71-1.77 (2H, m), 2.30-2.50 (1H, m), 2.58-2.63 (4H, m), 3.20-3.40 (6H, m), 3.86-3.91 (2H, m), 4.38 (2H, s), 6.92 (2H, d, J=8.9 Hz), 7.69 (2H, d, J=8.9 Hz), 9.46 (1H, s) APCI MASS: 305.13 (M++H) Preparation 149 4-(1-Phenyl-4-piperidyloxy)benzohydrazide IR (KBr): 3261, 1601, 1498, 1250 cm−1 NMR (DMSO-d6, δ): 1.6-1.8 (2H, m), 2.0-2.2 (2H, m), 2.9-3.1 (2H, m), 3.4-3.6 (2H, m), 4.43 (2H, s), 4.6-4.7 (1H, m), 6.7-7.3 (7H, m), 7.79 (2H, d, J=8.7 Hz), 9.61 (1H, s) ESI MASS (Positive): 645.2 (2M++Na) Preparation 150 4-[1-(4-Methoxyphenyl)-4-piperidyloxy]benzohydrazide IR (KBr): 3319, 1606, 1510, 1254, 1190, 1119, 1036 cm−1 NMR (DMSO-d6, δ): 1.6-1.8 (2H, m), 2.0-2.2 (2H, m), 2.8-3.0 (2H, m), 3.2-3.4 (2H, m), 3.68 (3H, s), 4.41 (2H, s), 4.5-4.7 (1H, m), 6.7-7.0 (4H, m), 7.02 (2H, d, J=8.8 Hz), 7.78 (2H, d, J=8.8 Hz), 9.60 (1H, s) ESI MASS (Positive): 705.4 (2M++Na) Preparation 151 4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyloxy]benzohydrazide This compound was used in the next reaction without further purification.", "Preparation 152 4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyl]benzohydrazide NMR (CDCl3, δ): 1.2-1.7 (12H, m), 1.7-2.2 (8H, m), 2.3-2.7 (5H, m), 3.0-3.3 (2H, m), 3.33 (3H, s), 3.3-3.5 (4H, m), 4.0 (1H, br s), 7.29 (2H, d, J=8.8 Hz), 7.46 (2H, br s), 7.68 (2H, d, J=8.3 Hz) (+) APCI MASS (Positive): 432.27 (M++H) Preparation 153 4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyl]benzohydrazide NMR (CDCl3, δ): 1.2-2.1 (21H, m), 2.2-2.6 (3H, m), 3.0-3.2 (2H, m), 3.33 (3H, s), 3.3-3.5 (5H, m), 3.9-4.2 (1H, br s), 7.30 (2H, d, J=8.3 Hz), 7.41 (2H, br s), 7.67 (2H, d, J=8.3 Hz) (+) APCI MASS (Positive): 432.40 (M++H) Preparation 154 4-(1-Cyclohexyl-4-piperidyloxy)benzohydrazide NMR (CDCl3, δ): 1.0-1.4 (6H, m), 1.5-2.1 (8H, m), 2.2-2.6 (3H, m), 2.8-3.0 (2H, m), 3.8-4.2 (3H, br s), 4.2-4.4 (1H, m), 6.91 (2H, d, J=6.9 Hz), 7.6-7.8 (2H, m) (+) APCI MASS (Positive): 318.27 (M++H) Preparation 155 4-[4-(7-Methoxyheptyl)-1-piperazinyl]benzohydrazide NMR (CDCl3, δ): 1.2-1.7 (12H, m), 2.38 (2H, t, J=8.0 Hz), 2.58 (4H, t, J=5.2 Hz), 3.2-3.5 (4H, m), 3.33 (3H, s), 4.06 (2H, br s), 6.88 (2H, d, J=9.0 Hz), 7.68 (1H, br s), 7.92 (2H, d, J=9.0 Hz) (+) APCI MASS (Positive): 349.40 (M++H) Preparation 156 4-[4-(7-Methoxyheptyloxy)piperidin-1-yl]benzoylhydrazine NMR (CDCl3, δ): 1.22-1.46 (6H, m), 1.46-1.78 (6H, m), 1.88-2.04 (2H, m), 2.98-3.17 (2H, m), 3.33 (3H, s), 3.29-3.55 (5H, m), 3.55-3.72 (2H, m), 4.06 (2H, s), 6.89 (2H, d, J=9.0 Hz), 7.29 (1H, s), 7.64 (2H, d, J=9.0 Hz) MASS (m/z): 364 (M++H) Preparation 157 4-(4-Pentyloxypiperidin-1-yl)benzohydrazide IR (KBr): 3274.5, 2937.1, 1608.3, 1108.9 cm−1 NMR (CDCl3, δ): 0.90 (3H, t, J=6.8 Hz), 1.29-2.01 (12H, m), 3.01-4.05 (7H, m), 6.86-6.91 (2H, m), 7.32 (1H, s), 7.62-7.66 (2H, m) ESI MASS (Positive)(m/z): 306.20 (M++H) Preparation 158 4-(4-Butoxypiperidin-1-yl)benzohydrazide IR (KBr): 3270.7, 2952.5, 1606.4, 1103.1 cm−1 NMR (CDCl3, δ): 0.93 (3H, t, J=7.2 Hz), 1.33-2.03 (10H, m), 3.01-4.06 (7H, m), 6.86-6.92 (2H, m), 7.36 (1H, s), 7.61-7.68 (2H, m) ESI MASS (Positive)(m/z): 292.2 (M++H) Preparation 159 4-[4-(4-Methylpentyloxy)-1-piperidyl]benzohydrazide NMR (CDCl3, δ): 0.89 (6H, d, J=6.6 Hz), 1.1-1.3 (3H, m), 1.4-1.8 (4H, m), 1.8-2.1 (2H, m), 3.0-3.2 (2H, m), 3.4-3.5 (1H, m), 3.45 (2H, t, J-6.8 Hz), 3.5-3.7 (2H, m), 4.06 (2H, br s), 6.8-7.0 (2H, m), 7.33 (1H, br s), 7.5-7.7 (2H, m) (+) APCI MASS (Positive): 320.40 (M++H) Preparation 160 4-[4-(Cyclohexylmethoxy)-1-piperidyl]benzohydrazide NMR (CDCl3, δ): 0.8-2.1 (15H, m), 3.0-3.2 (2H, m), 3.26 (2H, d, J=6.4 Hz), 3.3-3.8 (3H, m), 4.07 (2H, br s), 6.88 (2H, d, J=8.9 Hz), 7.35 (1H, br s), 7.5-7.8 (2H, m) (+) APCI MASS (Positive): 332.40 (M++H) Preparation 161 To a solution of ethyl 4-(1-piperazinyl)benzoate (10 g) and pyridine (6.36 ml) in tetrahydrofuran (150 ml) was added dropwise phenylchloroformate (7.35 g) with stirring under ice-cooling, and the mixture was stirred at the ambient temperature for overnight.", "The reaction mixture was added water (750 ml) and the resulting precipitate collected, and dried to give phenyl 4-[4-(ethoxycarbonyl)phenyl]-1-piperazine carboxylate (13.49 g).", "IR (KBr): 2987.2, 1724.0, 1697.1, 1290.1 cm−1 NMR (CDCl3, δ): 1.38 (3H, t, J=7.1 Hz), 3.38-3.43 (4H, m), 3.78 (4H, br s), 4.34 (2H, q, J=7.1 Hz), 6.90 (2H, d, J=9.0 Hz), 7.09-7.42 (7H, m) ESI MASS (Positive)(m/z): 355.0 (M++H) The following compounds [Preparation 162 to 183] were obtained according to a similar manner to that of Preparation 161.Preparation 162 Methyl 4-[2-[4-4-[4-(6-methoxyhexyloxy)cyclohexyl-1-piperidyl]benzoyl]hydrazinocarbonyl]benzoate NMR (DMSO-d6, δ): 1.00-1.55 (14H, m), 1.70-2.35 (4H, m), 2.55-2.65 (4H, m), 3.21 (3H, s), 3.22-3.45 (8H, m), 3.89 (3H, s), 6.98 (2H, d, J=8.88 Hz), 7.80 (2H, d, J=8.72 Hz), 8.00-8.15 (4H, m), 10.26 (1H, s), 10.57 (1H, s) ESI MASS (Positive)(m/z): 617.4 (M++Na), 595.4 (M++H) Preparation 163 Methyl 4-[2-[4-[1-(8-methoxyoctyl)-4-piperidyloxy]benzoyl]hydrazinocarbonyl]benzoate IR (Nujol): 3213, 1720, 1684, 1653, 1281 cm−1 NMR (DMSO-d6, δ): 1.2-2.3 (20H, m), 2.9-3.4 (4H, m), 3.22 (3H, s), 3.90 (3H, s), 4.77 (1H, br s), 7.14 (2H, d, J=8.8 Hz), 7.92 (2H, d, J=8.8 Hz), 8.0-8.2 (4H, m) ESI MASS (Positive): 540.5 (M++H) Preparation 164 Methyl 4-[2-[4-[1-(7-methoxyheptyl)-4-piperidyl]benzoyl]hydrazinocarbonyl)benzoate NMR (CDCl3, δ): 1.2-1.4 (6H, m), 1.4-1.7 (4H, m), 1.7-1.9 (4H, m), 2.0-2.2 (2H, m), 2.3-2.7 (3H, m), 3.0-3.2 (2H, m), 3.33 (3H, s), 3.3-3.4 (2H, m), 3.94 (3H, s), 4.90 (2H, br s), 7.26 (2H, d, J=8.3 Hz), 7.77 (2H, d, J=8.2 Hz), 7.89 (2H, d, J=8.5 Hz), 8.05 (2H, d, J=8.5 Hz) (+) APCI MASS (Positive): 510.60 (M++H) Preparation 165 Methyl 4-[2-[4-[4-(4-pyridylmethyl)-1-piperazinyl]benzoyl]hydrazinocarbonyl]benzoate NMR (DMSO-d6, δ): 2.50-2.60 (4H, m), 3.2-3.4 (4H, m), 3.58 (2H, s), 3.89 (3H, s), 7.00 (2H, d, J=8.9 Hz), 7.37 (2H, d, J=5.9 Hz), 7.82 (2H, d, J=8.9 Hz), 8.00-8.11 (4H, m), 8.53 (2H, d, J=5.9 Hz), 10.28 (1H, s), 10.57 (1H, s) APCI MASS (Positive): 473.6 (MH+) Preparation 166 Methyl 4-[2-[4-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)benzoyl]hydrazinocarbonyl]benzoate NMR (DMSO-d6, δ): 1.66 (4H, m), 3.42-3.47 (4H, m), 3.90 (3H, s), 3.92 (4H, s), 7.02 (2H, d, J=9 Hz), 7.80 (2H, d, J=9 Hz), 8.00-8.12 (4H, m), 10.26 (1H, s), 10.58 (1H, s) APCI MASS: 440.2 (M++H) Preparation 167 Methyl 4-[2-[4-(2-phenyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl]benzoyl hydrazinocarbonyl]-benzoate NMR (DMSO-d6, δ): 2.86-3.00 (2H, m), 3.70-3.90 (2H, m), 3.90 (3H, s), 4.45-4.51 (2H, m), 7.09-8.31 (14H, m), 10.28 (1H, s), 10.59 (1H, s) APCI MASS: 495.93 (M+) Preparation 168 Methyl 4-[2-[4-[4-(3,3-dimethyl-1,5-dioxaspiro[5.5]-undec-9-yl)-1-piperazinyl]benzoyl]hydrazinocarbonyl]benzoate NMR (DMSO-d6, δ): 0.89 (6H, s), 1.2-1.8 (5H, m), 2.1-2.42 (4H, m), 2.60 (4H, br s), 3.26 (4H, br s), 3.42 (4H, s), 3.90 (3H, s), 6.98 (2H, d, J=8.9 Hz), 7.80 (2H, d, J=8.9 Hz), 8.02 (2H, d, J=8.7 Hz) 8.09 (2H, d, J=8.7 Hz), 10.26 (1H, s), 10.58 (1H, s) APCI MASS: 564.07 (M++H) Preparation 169 Methyl 4-[2-[4-(4-cyclopentyl-1-piperazinyl)benzoyl]hydrazinocarbonyl]benzoate NMR (DMSO-d6, δ): 1.2-1.9 (8H, m), 2.4-2.6 (5H, m), 3.1-3.4 (4H, m), 3.90 (3H, s), 6.99 (2H, d, J=8.9 Hz), 7.81 (2H, d, J=8.9 Hz), 8.00-8.12 (4H, m), 10.27 (1H, s), 10.58 (1H, s) APCI MASS: 451.27 (M++H) Preparation 170 Methyl 4-[2-[4-(4-cycloheptyl-1-piperazinyl)benzoyl]hydrazinocarbonyl]benzoate NMR (DMSO-d6, δ): 1.3-1.9 (12H, m), 2.5-2.7 (5H, m), 3.2-3.4 (4H, m), 3.90 (3H, s), 6.98 (2H, d, J=9 Hz), 7.80 (2H, d, J=9 Hz), 8.00-8.12 (4H, m), 10.26 (1H, s), 10.58 (1H, s) APCI MASS: 479.33 (M++H) Preparation 171 Methyl 4-[2-[4-[4-(1,4-dioxaspiro[4.5]dec-8-yl)-1-piperazinyl]benzoyl]hydrazinocarbonyl]benzoate NMR (DMSO-d6, δ): 1.40-1.80 (8H, m), 2.30-2.42 (1H, m), 2.50-2.70 (4H, m), 3.20-3.30 (4H, m), 3.85 (4H, s), 3.90 (3H, s), 6.98 (2H, d, J=8.9 Hz), 7.81 (2H, d, J=8.9 Hz), 8.02 (2H, d, J=8.7 Hz), 8.09 (2H, d, J=8.7 Hz), 10.26 (1H, s), 10.58 (1H, s) APCI MASS: 523.27 (M++H) Preparation 172 Methyl 4-[2-[4-(4-tetrahydro-2H-pyran-4-yl-1-piperazinyl)benzoyl]hydrazinocarbonyl]benzoate NMR (DMSO-d6, δ): 1.30-1.60 (2H, m), 1.72-1.78 (2H, m), 2.42-2.50 (1H, m), 2.62 (4H, br s), 3.20-3.40 (6H, m), 3.90 (5H, br s), 6.99 (2H, d, J=8.9 Hz), 7.81 (2H, d, J=8.9 Hz), 8.02 (2H, d, J=8.6 Hz), 8.09 (2H, d, J=8.6 Hz), 10.27 (1H, s), 10.58 (1H, s) APCI MASS: 467.2 (M++H) Preparation 173 Methyl 4-[2-[4-(1-phenyl-4-piperidyloxy)benzoyl]hydrazinocarbonyl]benzoate IR (Nujol): 1716, 1649, 1603, 1279, 1250 cm−1 NMR (DMSO-d6, δ): 1.6-1.9 (2H, m), 2.0-2.2 (2H, m), 3.0-3.2 (2H, m), 3.4-3.6 (2H, m), 3.90 (3H, s), 4.6-4.8 (1H, m), 6.7-7.3 (7H, m), 7.8-8.2 (6H, m), 10.43 (1H, s), 10.65 (1H, s) ESI MASS (Positive): 474.3 (M++H) Preparation 174 Methyl 4-[2-[4-[1-(4-methoxyphenyl)-4-piperidyloxy]benzoyl]hydrazinocarbonyl]benzoate IR (Nujol): 1720, 1649, 1601, 1512, 1286, 1254 cm−1 NMR (DMSO-d6, δ): 1.7-1.9 (2H, m), 2.0-2.2 (2H, m), 2.8-3.1 (2H, m), 3.3-3.5 (2H, m), 3.69 (3H, s), 3.90 (3H, s), 4.6-4.8 (1H, m), 6.8-7.0 (4H, m), 7.11 (2H, d, J-8.7 Hz), 7.91 (2H, d, J-8.7 Hz), 8.0-8.2 (4H, m), 10.43 (1H, s), 10.65 (1H, s) ESI MASS (Positive): 504.3 (M++H) Preparation 175 Methyl 4-[2-[4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-4-piperidyloxy]benzoyl]hydrazinocarbonyl]benzoate NMR (CDCl3, δ): 1.2-3.2 (26H, m), 3.33 (3H, s), 3.3-3.5 (6H, m), 3.95 (3H, s), 4.3-4.5 (1H, m), 6.90 (2H, d, J=8.6 Hz), 7.80 (2H, d, J=8.8 Hz), 7.91 (2H, d, J=8.5 Hz), 8.08 (2H, d, J=8.5 Hz) (+) APCI MASS (Positive): 610.47 (M++H) Preparation 176 Methyl 4-[2-[4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-4-piperidyl]benzoyl]hydrazinocarbonyl]benzoate NMR (CDCl3, δ): 1.2-2.2 (21H, m), 2.2-2.7 (3H, m), 3.0-3.2 (3H, m), 3.33 (3H, s), 3.3-3.5 (4H, m), 3.95 (3H, m), 7.29 (2H, d, J=8.1 Hz), 7.78 (2H, d, J=8.2 Hz), 7.91 (2H, d, J=8.4 Hz), 8.08 (2H, d, J=8.4 Hz) (+) APCI MASS (Positive): 594.33 (M++H) Preparation 177 Methyl 4-[2-[4-[1-(4-(6-methoxyhexyloxy)cyclohexyl]-4-piperidyl]benzoyl]hydrazinocarbonyl]benzoate NMR (CDCl3, δ): 1.2-2.1 (21H, m), 2.2-2.7 (3H, m), 3.0-3.2 (2H, m), 3.33 (3H, s), 3.3-3.5 (5H, m), 3.95 (3H, s), 4.80 (2H, br s), 7.28 (2H, d, J=7.5 Hz), 7.77 (2H, d, J=8.2 Hz), 7.90 (2H, d, J=8.5 Hz), 8.06 (2H, d, J=8.5 Hz) ESI MASS (Positive): 594.5 (M++H) Preparation 178 Methyl 4-[2-[4-(1-cyclohexyl-4-piperidyloxy)benzoyl]hydrazinocarbonyl]benzoate NMR (CDCl3, δ): 0.8-1.4 (6H, m), 1.5-2.2 (8H, m), 2.2-2.4 (3H, m), 2.8-3.0 (2H, m), 3.95 (3H, s), 4.3-4.5 (1H, m), 6.91 (2H, d, J=8.6 Hz), 7.7-8.2 (8H, m) (−) APCI MASS (Negative): 478.53 (M−−H) Preparation 179 Methyl 4-[2-[4-[4-(7-methoxyheptyl)-1-piperazinyl]benzoyl]hydrazinocarbonyl]benzoate NMR (CDCl3, δ): 1.2-1.7 (12H, m), 1.77 (2H, br s), 2.42 (2H, t, J=8.0 Hz), 2.62 (4H, t, J-4.9 Hz), 3.3-3.5 (4H, m), 3.33 (3H, s), 3.95 (3H, s), 6.88 (2H, d, J=9.0 Hz), 7.77 (2H, d, J=9.0 Hz), 7.92 (2H, d, J=8.4 Hz), 8.11 (2H, d, J=8.4 Hz) (+) APCI MASS (Positive): 511.47 (M++H) Preparation 180 Methyl 4-[2-[4-(4-pentyloxypiperidin-1-yl)benzoyl]hydrazinocarbonyl]benzoate IR (KBr): 3191.6, 1933.2, 1724.0, 1596.8, 1110.8 cm−1 NMR (CDCl3, δ): 0.91 (3H, t, J=6.8 Hz), 1.29-1.92 (10H, m), 3.03-3.69 (7H, m), 3.94 (3H, s), 6.81-6.85 (2H, m), 7.72-8.06 (6H, m), 9.51 (1H, d, J=5.2 Hz), 10.09 (1H, d, J=5.4 Hz) ESI MASS (Positive)(m/z): 468.33 (M++H) Preparation 181 N-[4-[4-(7-Methoxyheptyloxy)piperidin-1-yl]benzoyl-N′-(4-methoxycarbonylbenzoyl)hydrazine NMR (DMSO-d6, δ): 1.20-1.62 (12H, m), 1.81-2.01 (2H, m), 2.97-3.16 (2H, m), 3.20 (3H, s), 3.21-3.55 (5H, m), 3.55-3.72 (2H, m), 3.90 (3H, s), 6.99 (2H, d, J=9.0 Hz), 7.80 (2H, d, J=8.8 Hz), 8.03 (2H, d, J=8.7 Hz), 8.10 (2H, d, J=8.7 Hz), 10.24 (1H, s), 10.57 (1H, s) MASS (m/z): 526 (M++H) Preparation 182 Methyl 4-[2-[4-[4-(4-methylpentyloxy)-1-piperidyl]benzoyl]hydrazinocarbonyl]benzoate NMR (CDCl3, δ): 0.89 (6H, d, J=6.6 Hz), 1.1-1.3 (3H, m), 1.5-1.8 (4H, m), 1.8-2.1 (2H, m), 3.0-3.2 (2H, m), 3.4-3.8 (5H, m), 3.94 (3H, s), 6.84 (2H, d, J=9.0 Hz), 7.74 (2H, d, J-8.9 Hz), 7.90 (2H, d, J=8.5 Hz), 8.05 (2H, d, J=8.5 Hz), 9.3-9.5 (1H, m), 9.9-10.1 (1H, m) (+) APCI MASS (Positive): 482.47 (M++H) Preparation 183 Methyl 4-[2-[4-[4-(cyclohexylmethoxy)-1-piperidyl]benzoyl]hydrazinocarbonyl]benzoate NMR (CDCl3, δ): 0.8-2.0 (15H, m), 3.0-3.2 (2H, m), 3.23 (2H, d, J=7.7 Hz), 3.3-3.8 (3H, m), 3.95 (3H, s), 6.87 (2H, d, J=9.0 Hz), 7.75 (2H, d, J=8.9 Hz), 7.92 (2H, d, J=8.5 Hz), 8.09 (2H, d, J=8.4 Hz), 9.2-9.4 (1H, m), 9.7-9.8 (1H, m) (+) APCI MASS (Positive): 494.53 (M++H) Preparation 184 A mixture of 5-[4-[1-(4-methoxyphenyl)-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-amine (0.69 g) and ethyl 4-(bromoacetyl)benzoate (0.74 g) in ethanol (15 ml) was stirred for 6 hours at 90° C. After being cooled to room temperature, the reaction mixture was poured into isopropyl ether.", "The resulting precipitate was collected by filtration, washed with isopropyl ether and added to a solution of trifluoroacetic acid (2 ml) in xylene (20 ml).", "Then a mixture was stirred for 4 hours at 130° C. After being cooled to room temperature, the reaction mixture was poured into isopropyl ether.", "The resulting precipitate was collected by filtration and washed with isopropyl ether to give ethyl 4-[2-[4-[1-(4-methoxyphenyl)-4-piperidyloxy]phenyl]imidazo[2,1-b][,3,4]thiadiazol-6-yl]benzoate (1.06 g).", "IR (KBr): 1707, 1606, 1516, 1471, 1279, 1252, 1178, 1109, 1024, 833 cm−1 NMR (DMSO-d6, δ): 1.34 (3H, t, J=7.1 Hz), 2.0-2.4 (4H, m), 3.5-3.7 (4H, m), 3.80 (3H, s), 4.33 (2H, q, J=7.1 Hz), 4.8-5.0 (1H, m), 7.0-7.3 (4H, m), 7.5-7.7 (2H, m), 7.9-8.1 (6H, m), 8.90 (1H, s) ESI MASS (Positive): 555.3 (M++H) The following compounds [Preparation 185 to 196] were obtained according to a similar manner to that of Preparation 184.Preparation 185 Ethyl 4-[2-[4-[4-(4-methoxybutoxymethyl)-1-piperidyl]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate IR (KBr): 2935, 2862, 1703, 1608, 1471, 1282, 1176, 1115 cm−1 (+) APCI MASS: 549.47 (M++H) Preparation 186 Ethyl 4-[2-[4-[4-(6-methoxyhexyl)piperazin-1-yl]phenyl]imidazo[2,1-b][1,3,4′-thiadiazol-6-yl]benzoate IR (KBr): 3431, 2935, 2864, 1713, 1678, 1604, 1469 cm−1 NMR (DMSO-d6, δ): 1.20-1.40 (9H, m), 1.40-1.60 (2H, m), 1.60-1.80 (4H, m), 2.90-3.20 (6H, m), 3.26 (3H, s), 3.25-3.50 (2H, m), 4.32 (2H, q, J=7.1 Hz), 7.19 (2H, d, J=8.9 Hz), 7.84 (2H, d, J=8.8 Hz), 7.90-8.15 (4H, m), 8.86 (1H, s) MASS: 548 (M++H) Preparation 187 Ethyl 4-[2-[4-[1-(6-methoxyhexyl)-4-piperidyl]phenyl]-imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate trifluoroacetate IR (KBr): 2935, 1703, 1610, 1473, 1414, 1282, 1178, 1107 cm−1 NMR (DMSO-d6, δ): 1.2-2.2 (17H, m), 2.8-3.2 (5H, m), 3.23 (3H, s), 3.3-3.4 (2H, m), 4.33 (2H, q, J=7.0 Hz), 7.49 (2H, d, J=8.4 Hz), 7.9-8.1 (6H, m), 8.92 (1H, s), 9.32 (1H, br s) (+) APCI MASS: 547.60 (M++H) Preparation 188 Ethyl 4-[2-[4-[4-(1-methoxyheptyl)-1-piperazinyl]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate trifluoroacetate IR (KBr): 2933, 1701, 1606, 1471, 1404, 1281, 1178, 1109 cm−1 NMR (DMSO-d6, δ): 1.2-1.8 (13H, m), 3.0-3.2 (6H, m), 3.22 (3H, s), 3.31 (2H, t, J=6.3 Hz), 3.4-3.7 (2H, m), 4.0-4.2 (2H, m), 4.33 (2H, q, J=6.9 Hz), 7.1-7.3 (2H, m), 7.8-7.9 (2H, m), 7.9-8.1 (4H, m), 8.86 (1H, s), 9.64 (1H, br s) (+) APCI MASS: 562.47 (M++H) Preparation 189 Ethyl 4-[2-[4-[4-(5-methoxypentyl)-1-piperazinyl]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate trifluoroacetate IR (KBr): 2937, 1701, 1606, 1471, 1281, 1178, 1111 cm−1 NMR (DMSO-d6, δ): 1.2-1.8 (9H, m), 3.0-3.2 (6H, m), 3.24 (3H, s), 3.3-3.4 (2H, m), 3.5-4.2 (4H, m), 4.2-4.4 (2H, m), 7.1-7.2 (2H, m), 7.8-7.9 (2H, m), 7.9-8.1 (4H, m), 8.86 (1H, s), 9.54 (1H, br s) (+) APCI MASS: 534.53 (M++H) Preparation 190 Ethyl 4-[2-[4-(1-phenyl-4-piperidyloxy)phenyl]imidazo-[2,1-b][1,3,4]thiadiazol-6-yl]benzoate IR (KBr): 1707, 1606, 1471, 1279, 1250, 1178, 1109 cm−1 NMR (DMSO-d6, δ): 1.34 (3H, t, J=7.1 Hz), 2.0-2.4 (4H, m), 3.4-3.8 (4H, m), 4.33 (2H, q, J=7.1 Hz), 4.8-5.0 (1H, m), 7.2-8.2 (13H, m), 8.90 (1H, s) ESI MASS (Positive): 525.2 (M++H) Preparation 191 Ethyl 4-[2-[4-(1-cyclohexyl-1,2,3,6-tetrahydro-4-pyridyl)phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate trifluoroacetate IR (KBr): 2937, 1703, 1608, 1471, 1279, 1198, 1178, 1130, 1107 cm−1 NMR (DMSO-d6, δ): 1.0-2.3 (13H, m), 2.8-3.0 (2H, m), 3.1-3.9 (3H, m), 3.9-4.0 (2H, m), 4.33 (2H, q, J=6.8 Hz), 6.3-6.5 (1H, m), 7.4-8.1 (8H, m), 8.94 (1H, s), 9.59 (1H, br s) (+) APCI MASS: 513.07 (M++H) Preparation 192 Ethyl 4-[2-[4-(1-cyclohexyl-4-piperidyloxy)phenyl]-imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate IR (KBr): 2927, 1707, 1606, 1473, 1279, 1252, 1174, 1109 cm−1 NMR (CDCl3, δ): 1.1-3.3 (22H, m), 4.2-4.5 (2H, m), 4.7-4.9 (1H, m), 6.9-7.1 (2H, m), 7.5-8.3 (7H, m) (+) APCI MASS (Positive): 531.40 (M++H) Preparation 193 Ethyl 4-[2-[4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-4-piperidyloxy]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate IR (KBr): 2933, 1703, 1680, 1606, 1279, 1252, 1200, 1176, 1109 cm−1 NMR (CDCl3, δ): 1.2-3.6 (36H, m), 4.40 (2H, q, J=7.1 Hz), 4.8-4.9 (1H, m), 6.9-7.1 (2H, m), 7.8-8.0 (4H, m), 8.0-8.2 (3H, m) ESI MASS (Positive): 661.3 (M++H) Preparation 194 Ethyl 4-[2-[4-[4-(5-methoxypentyloxymethyl)-1-piperidyl]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate trifluoroacetic acid NMR (CDCl3, δ): 1.30-1.50 (5H, m), 1.50-1.70 (5H, m), 1.80-2.05 (5H, m), 2.90-3.10 (2H, m), 3.25-3.36 (5H, m), 3.36-3.47 (4H, m), 3.80-3.95 (2H, m), 4.38 (2H, q, J=7.14 Hz), 7.22 (2H, d, J=8.85 Hz), 7.77 (2H, d, J=8.75 Hz), 7.85 (2H, d, J=8.39 Hz), 8.08 (1H, s), 8.09 (2H, d, J=8.27 Hz) ESI MASS (Positive)(m/z): 563.3 (M++H) Preparation 195 Ethyl 4-[2-(5-methoxypentyloxyphenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate NMR (DMSO-d6, δ): 1.20-1.40 (3H, m), 1.40-1.80 (6H, m), 3.22 (3H, s), 3.20-3.60 (2H, m), 3.90-4.20 (2H, m), 4.20-4.40 (2H, m), 6.60-7.30 (3H, m), 7.50-8.40 (7H, m) API-ES MASS (Positive): 549, 511, 477, 465 (M++H) Preparation 196 Ethyl 4-[2-[4-(4-cyclohexyl-4-methoxy-1-piperidyl)phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate NMR (CDCl3, δ): 0.90-2.15 (15H, m), 3.00-3.30 (4H, m), 3.30-3.85 (3H, m), 6.80-7.05 (2H, m), 7.63 (1H, d, J=8.74 Hz), 7.72 (1H, d, J=8.80 Hz), 7.89 (2H, d, J=8.06 Hz), 7.95-8.15 (3H, m) Preparation 197 A mixture of methyl 4-[(2-(4-[1-(4-methoxyphenyl)-4-piperidyloxy]benzoyl]hydrazinocarbonyl]benzoate (1.71 g) and phosphorus pentasulfide (1.1 g) in ethylene glycol dimethyl ether (35 ml) was refluxed for 3 hours.", "After being added triethylamine, the reaction mixture was successively refluxed for 2.5 hours.", "After being cooled to room temperature, the reaction mixture was poured into ice-water.", "Then the solution was adjusted to pH 8 with 1N aqueous sodium hydroxide.", "The resulting precipitate was collected by filtration and washed with water to give methyl 4-[5-[4-[1-(4-methoxyphenyl)-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-yl]benzoate (1.5 g).", "NMR (DMSO-d6, δ): 1.7-1.9 (2H, m), 2.0-2.2 (2H, m), 2.8-3.4 (4H, m), 3.69 (3H, s), 3.91 (3H, s), 4.6-4.8 (1H, m), 6.8-7.0 (4H, m), 7.2-7.3 (2H, m), 7.9-8.3 (6H, m) The following compounds [Preparation 198 to 220] were obtained according to a similar manner to that of Preparation 197.Preparation 198 Ethyl 4-[4-[5-[4-(7-methoxyheptyloxy)phenyl]-1,3,4-thiadiazol-2-yl]-1-piperazinyl]benzoate IR (KBr): 2942.8, 1704.8, 1608.3, 1236.1, 1110.8 cm−1 ESI MASS (Positive) (m/z): 539.27 (M++H) Preparation 199 Methyl 4-[5-[4-[4-[4-(6-methoxyhexyloxy)cyclohexyl]-1-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoate ESI MASS (Positive) (m/z): 593.4 (M++H) Preparation 200 Methyl 4-[5-[4-[1-(8-methoxyoctyl)-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-yl]benzoate IR (Nujol): 1716, 1603, 1514, 1281, 1250, 1173, 1113 cm−1 NMR (DMOS-d6, δ): 1.0-2.3 (20H, m), 2.8-3.5 (4H, m), 3.21 (3H, s), 3.91 (3H, s), 4.75 (1H, br s), 7.20 (2H, d, J=8.7 Hz), 8.00 (2H, d, J=8.7 Hz), 8.1-8.2 (4H, m) ESI MASS (Positive): 538.3 (M++H) Preparation 201 Methyl 4-[5-[4-[1-(7-methoxyheptyl)-4-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 1.2-1.4 (6H, m), 1.4-1.7 (4H, m), 1.8-2.2 (6H, m), 2.3-2.5 (2H, m), 2.5-2.7 (1H, m), 3.0-3.2 (2H, m), 3.34 (3H, m), 3.3-3.4 (2H, m), 3.96 (3H, s), 7.34 (2H, d, J=8.3 Hz), 7.95 (2H, d, J=8.3 Hz), 8.0-8.2 (4H, m) (+) APCI MASS (Positive): 508.73 (M++H) Preparation 202 Methyl 4-[5-[4-[4-(4-pyridylmethyl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 2.61-2.64 (4H, m), 3.35-3.40 (4H, m), 3.59 (2H, s), 3.96 (3H, s), 6.96 (2H, d, J=9 Hz), 7.33 (2H, d, J=5.9 Hz), 7.90 (2H, d, J=9 Hz), 8.06 (2H, d, J=8.6 Hz), 8.15 (2H, d, J=8.6 Hz), 8.58 (2H, d, J=5.9 Hz) APCI MASS (Positive): 472 (M++H) Preparation 203 Methyl 4-[5-[4-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 1.81-1.87 (4H, m), 3.47-3.53 (4H, m), 3.96 (3H, s), 4.01 (4H, s), 6.97 (2H, d, J=9 Hz), 7.89 (2H, d, J=9 Hz), 8.06 (2H, d, J=8.6 Hz), 8.15 (2H, d, J=8.6 Hz) APCI MASS: 438.33 (M++H) Preparation 204 Methyl 4-[5-[4-(2-phenyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 3.0-3.10 (2H, m), 3.72-3.76 (2H, m), 3.96 (3H, s), 4.47 (2H, s), 7.02-8.28 (14H, m) APCI MASS: 494.4(M++H) Preparation 205 Methyl 4-[5-[4-[4-(3,3-dimethyl-1,5-dioxaspiro[5.5]undec-9-yl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 0.97 (6H, s), 1.2-1.9 (5H, m), 2.2-2.6 (4H, m), 2.78 (4H, br s), 3.37 (4H, br s), 3.48 (2H, s), 3.52 (2H, s), 3.96 (3H, s), 6.96 (2H, d, J=9 Hz), 7.90 (2H, d, J=9 Hz), 8.06 (2H, d, J=8.6 Hz), 8.15 (2H, d, J=8.6 Hz) APCI MASS: 563.27 (M++H) Preparation 206 Methyl 4-[5-[4-(4-cyclopentyl-1-piperazinyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 1.40-2.0 (8H, m), 2.50-2.60 (1H, m), 2.67-2.72 (4H, m), 3.35-3.40 (4H, m), 3.96 (3H, s), 6.96 (2H, d, J=9 Hz), 7.90 (2H, d, J=9 Hz), 8.06 (2H, d, J=8.6 Hz), 8.15 (2H, d, J=8.6 Hz) APCI MASS: 449.2 (M++H) Preparation 207 Methyl 4-[5-[4-(4-cycloheptyl-1-piperazinyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 1.2-2.4 (12H, m), 3.0-3.8 (9H, m), 3.96 (3H, s), 6.93 (2H, d, J=8.6H), 7.86 (2H, d, J=8.6H), 8.01-8.16 (4H, m) APCI MASS: 477.27 (M++H) Preparation 208 Methyl 4-[5-[4-[4-(1,4-dioxaspiro[4.5]dec-8-yl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]-benzoate NMR (CDCl3, δ): 1.4-2.0 (8H, m), 2.4-2.7 (1H, m), 2.7-2.9 (4H, m), 3.3-3.5 (4H, m), 3.95 (4H, s), 3.96 (3H, s), 6.96 (2H, d, J-9 Hz), 7.90 (2H, d, J=9 Hz), 8.06 (2H, d, J=8.6 Hz), 8.15 (2H, d, J=8.6 Hz) APCI MASS: 521.4 (M++H) Preparation 209 Methyl 4-[5-[4-(4-tetrahydro-2H-pyran-4-yl-1-piperazinyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 1.50-1.90 (4H, m), 2.4-2.6 (1H, m), 2.7-2.8 (4H, m), 3.36-3.46 (6H, m), 3.96 (3H, s), 4.0-4.1 (2H, m), 6.97 (2H, d, J=8.9 Hz), 7.91 (2H, d, J=8.9 Hz), 8.06 (2H, d, J=8.6 Hz), 8.15 (2H, d, J=8.6 Hz) APCI MASS: 465.27 (M++H) Preparation 210 Methyl 4-[5-[4-(1-phenyl-4-piperidyloxy)phenyl]-1,3,4-thiadiazol-2-yl)benzoate NMR (DMSO-d6, δ): 1.6-2.2 (4H, m), 3.0-4.0 (4H, m), 3.91 (3H, s), 4.6-4.9 (1H, m), 6.7-8.4 (13H, m) Preparation 211 Methyl 4-[5-[4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 1.2-3.2 (24H, m), 3.33 (3H, s), 3.3-3.5 (6H, m), 3.96 (3H, s), 4.3-4.5 (1H, m), 7.00 (2H, d, J=8.8 Hz), 7.94 (2H, d, J=8.7 Hz), 8.07 (2H, d, J=8.4 Hz), 8.16 (2H, d, J=8.4 Hz) (+) APCI MASS (Positive): 608.53 (M++H) Preparation 212 Methyl 4-[5-[4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-4-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 1.2-2.2 (24H, m), 2.2-2.6 (3H, m), 3.0-3.3 (3H, m), 3.33 (3H, s), 3.3-3.5 (4H, m), 7.37 (2H, d, J=8.3 Hz), 7.95 (2H, d, J=8.2 Hz), 8.0-8.3 (4H, m) (+) APCI MASS (Positive): 592.27 (M++H) Preparation 213 Methyl 4-[5-[4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-4-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 1.2-2.7 (24H, m), 3.0-3.2 (2H, m), 3.33 (3H, s), 3.3-3.5 (5H, m), 3.96 (3H, s), 7.38 (2H, d, J=8.3 Hz), 7.95 (2H, d, J=8.1 Hz), 8.0-8.2 (4H, m) (+) APCI MASS (Positive): 592.40 (M++H) Preparation 214 Methyl 4-[5-[4-(1-cyclohexyl-4-piperidyloxy)]phenyl]-1,3,4-thiadiazol-2-yl)benzoate NMR (CDCl3, δ): 1.0-1.4 (6H, m), 1.6-2.2 (8H, m), 2.2-2.6 (3H, m), 2.8-3.0 (2H, m), 3.96 (3H, s), 4.3-4.5 (1H, m), 7.00 (2H, d, J=8.8 Hz), 7.94 (2H, d, J=8.8 Hz), 8.07 (2H, d, J=8.6 Hz), 8.16 (2H, d, J=8.6 Hz) (+) APCI MASS (Positive): 478.47 (M++H) Preparation 215 Methyl 4-[5-[4-[4-(7-methoxyheptyl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 1.3-1.9 (12H, m), 2.3-2.5 (2H, m), 2.5-2.7 (4H, m), 3.34 (3H, s), 3.3-3.5 (4H, m), 3.96 (3H, s), 6.96 (2H, d, J=9.0 Hz), 7.90 (2H, d, J=8.8 Hz), 8.06 (2H, d, J=8.6 Hz), 8.15 (2H, d, J=8.6 Hz) (+) APCI MASS (Positive): 509.67 (M++H) Preparation 216 Methyl 4-(5-[4′-[4-(7-methoxyheptyloxy)piperidin-1-yl]phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 1.22-1.47 (6H, m), 1.47-1.82 (6H, m), 1.90-2.08 (2H, m), 3.03-3.22 (2H, m), 3.33 (3H, s), 3.28-3.56 (5H, m), 3.56-3.75 (2H, m), 3.97 (3H, m), 6.90-7.02 (2H, m), 7.82-7.94 (2H, m), 8.00-8.22 (4H, m) MASS (m/z): 524 (M++H) Preparation 217 Methyl 4-[5-[4-(4-pentyloxypiperidin-1-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoate IR (KBr): 2931.3, 1714.4, 1604.5, 1278.6, 1106.9 cm−1 NMR (CDCl3, δ): 0.91 (3H, t, J=6.8 Hz), 1.30-1.96 (10H, m), 3.06-3.73 (7H, m), 3.96 (3H, s), 6.89-6.99 (2H, m), 7.74-8.17 (6H, m) ESI MASS (Positive)(m/z): 466.53 (M++H) Preparation 218 Methyl 4-[5-[4-(4-butoxypiperidin-1-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoate IR (KBr): 2954.4, 1722.1, 1276.6, 1110.8 cm−1 NMR (CDCl3, δ): 0.93 (3H, t, J=7.2 Hz), 1.30-1.96 (8H, m), 3.07-3.73 (7H, m), 3.96 (3H, s), 6.94-6.99 (2H, m), 7.86-7.91 (2H, m), 8.04-8.17 (4H, m) ESI MASS (Positive)(m/z): 452.2 (M++H) Preparation 219 Methyl 4-[5-[4-[4-(4-methylpentyloxy)-1-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 0.90 (6H, d, J=6.6 Hz), 1.1-1.3 (2H, m), 1.4-1.9 (5H, m), 1.9-2.3 (2H, m), 3.1-3.3 (2H, m), 3.4-3.8 (5H, m), 3.96 (3H, s), 6.9-7.2 (2H, m), 7.8-8.2 (4H, m) ESI MASS (Positive): 480.2 (M++H) Preparation 220 Methyl 4-[5-[4-[4-(cyclohexylmethoxy)-1-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoate NMR (CDCl3, δ): 0.8-2.2 (15H, m), 3.1-3.3 (4H, m), 3.4-3.8 (3H, m), 3.96 (3H, s), 7.00 (2H, d, J-8.7 Hz), 7.89 (2H, d, J=8.8 Hz), 8.0-8.2 (4H, m) Preparation 221 A suspension of methyl 4-[5-[4-(4-butoxypiperidin-1yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoate (3.54 g) and 10% sodiumhydroxide in water (6.3 ml) in a mixture of ethanol (35 ml) and tetrahydrofuran (35 ml) was refluxed for 3.5 hours.", "The reaction mixture was poured into water, and the mixture was adjusted to pH 1-2 with 1N hydrochloric acid.", "The resulting precipitate was collected, washed with water, and dried to give 4-[5-[4-(4-butoxypiperidin-1-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid (2.76 g).", "IR (KBr): 2952.5, 1685.5, 1604.5, 1106.9 cm−1 NMR (DMSO-d6, δ): 0.89 (3H, t, J=7.1 Hz), 1.30-2.00 (8H, m), 3.00-3.80 (7H, m), 7.07-7.11 (2H, m), 7.82-7.87 (2H, m), 8.11 (4H, s) ESI MASS (Positive)(m/z): 438.47 (M++H) The following compounds [Preparation 222 to 234] were obtained according to a similar manner to that of Preparation 221.Preparation 222 4′-(4-Cyclohexylhexahydro-1H-1,4-diazepin-1-yl)-1,1′-biphenyl-4-carboxlic acid MASS (m/z): 379 (M++H) Preparation 223 4-[5-[4′-[4-(7-Methoxyheptyloxy)piperidin-1-yl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid hydrochloride NMR (DMSO-d6, δ): 1.10-1.60 (12H, m), 1.81-1.99 (2H, m), 3.00-3.79 (9H, m), 3.20 (3H, s), 7.08 (2H, d, J=9.0 Hz), 7.84 (2H, d, J=8.8 Hz), 8.10 (4H, s) MASS (m/z): 510 (M++H) Preparation 224 4′-(5-Cyclohexyl-2,5-diazabicyclo[2.2.1]hept-2-yl)-1,1′-biphenyl-4-carboxylic acid MASS (m/z): 377 (M++H) Preparation 225 4′-(4-Phenyl-1-piperidyl)-1,1′-biphenyl-4-carboxylic acid MASS (m/z): 356 (M++H) Preparation 226 4′-[4-(cis-4-Methylcyclohexyl)-1-piperazinyl]-1,1′-biphenyl-4-carboxylic acid MASS (m/z): 379 (M++H) Preparation 227 4′-[4-(trans-4-Methylcyclohexyl)-1-piperazinyl]-1,1′-biphenyl-4-carboxylic acid MASS (m/z): 379 (M++H) Preparation 228 4′-[4-[4-[4-(6-Methoxyhexyl)-1-piperazinyl]phenyl]-1-piperazinyl]-1,1′-biphenyl-4-carboxylic acid MASS (m/z): 557 (M++H) Preparation 229 4′-[4-[4-[1-(6-Methoxyhexyl)-4-piperidyloxy]phenyl]-1-piperazinyl]-1,1′-biphenyl-4-carboxylic acid MASS (m/z): 572 (M++H) Preparation 230 4′-[4-[trans-4-Methoxy-4-(1-methoxycyclohexyl-1-yl)cyclohexyl-1-yl]-1-piperazinyl)-1,1′-biphenyl-4-carboxylic acid MASS (m/z): 507 (M++H) Preparation 231 4′-[4-[cis-4-Methoxy-4-(1-methoxycyclohexyl-1-yl)cyclohexyl-1-yl]-1-piperazinyl]-1,1′-biphenyl-4-carboxylic acid MASS (m/z): 507 (M++H) Preparation 232 4′-[4-[4-[4-(6-Methoxyhexyl)-1-piperazinyl]phenyl]-1-piperazinyl]-1,1′-biphenyl-4-carboxylic acid MASS (m/z): 557 (M++H) Preparation 233 4-[5-[4-[4-(4-Methylpentyloxy)-1-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 0.86 (6H, d, J-6.5 Hz), 1.1-1.3 (2H, m), 1.3-1.6 (5H, m), 1.8-2.0 (2H, m), 2.9-3.3 (2H, m), 3.3-3.6 (3H, m), 3.6-3.8 (2H, m), 6.9-7.2 (2H, m), 7.7-8.2 (6H, m) (+) APCI MASS (Positive): 466.60 (M++H) Preparation 234 4-[5-[4-[4-(Cyclohexylmethoxy)-1-piperidyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d3, δ): 0.7-2.0 (15H, m), 3.0-4.9 (7H, m), 7.08 (2H, d, J=9.1 Hz), 7.84 (2H, d, J=8.7 Hz), 8.1-8.2 (4H, m) ESI MASS (Negative): 476.2 (M−−H) Preparation 235 A suspension of ethyl 4-[4-[5-[4-(7-methoxyheptyloxy)-phenyl]-1,3,4-thiadiazol-2-yl]-1-piperazinyl]benzoate (1.7 g) and 10% sodium hydroxide in water (19 ml) in a mixture of ethanol (34 ml) and tetrahydrofuran (51 ml) was refluxed for 13 hours.", "The reaction mixture was evaporated under reduced pressure.", "The residue was diluted with water and the mixture was adjusted to pH 1-2 with 1N hydrochloric acid.", "The resulting precipitate was collected, washed with water, and dried to give 4-[4-[5-[4-(7-methoxyheptyloxy)phenyl]-1,3,4thiadiazol-2-yl]-1-piperazinyl]benzoic acid (0.48 g).", "IR (KBr): 2935.1, 1675.8, 1602.6, 1234.2, 1116.6 cm−1 ESI MASS (Positive)(m/z): 533.3 (M++Na), 511.4 (M++H) The following compounds [Preparation 236 to 271] were obtained according to a similar manner to that of Preparation 235.Preparation 236 4-[4-(1,1′-Biphenyl)-4-yl-1H-pyrazol-1-yl]benzoic acid NMR (DMSO-d6, δ): 7.37-7.48 (3H, m), 7.71-7.76 (4H, m), 7.83-7.87 (2H, m), 7.98-8.10 (4H, m), 8.36 (1H, s), 9.19 (1H, s) MASS (m/z): 341 (MH+) Preparation 237 4-[4-(4-Hexyloxyphenyl)-1H-pyrazol-1-yl]benzoic acid IR (KBr): 1685.5, 1652.7, 1608.3 cm−1 NMR (DMSO-d6, δ): 0.80-0.95 (3H, m), 1.20-1.50 (6H, m), 1.6-1.8 (2H, m), 3.99 (2H, t, J=6.4 Hz), 6.98 (2H, d, J=8.6 Hz), 7.64 (2H, d, J=8.6 Hz), 7.97 (2H, d, J=8.6 Hz), 8.06 (2H, d, J=8.6 Hz), 8.21 (1H, s), 9.01.", "(1H, s) MASS (m/z): 365 (MH+) Preparation 238 4-[1-(4-Hexyloxyphenyl)-1H-pyrazol-4-yl]benzoic acid NMR (DMSO-d6, δ): 0.8-0.95 (3H, m), 1.2-1.5 (6H, m), 1.7-1.8 (2H, m), 4.01 (2H, t, J=6.42 Hz), 7.06 (2H, d, J=9 Hz), 7.63 (2H, d, J=8.1 Hz), 7.78 (2H, d, J=9 Hz), 7.87 (2H, d, J=8.1 Hz), 8.16 (1H, s), 8.89 (1H, s) EI-MS MASS (m/z): 365 (MH+) Preparation 239 4-[5-(4-[4-(4-Methylcyclohexyl)-1-piperazinyl]phenyl]-1,3,4-oxadiazol-2-yl]benzoic acid hydrochloride NMR (DMSO-d6, δ): 0.97 (3H, d, J=7.1 Hz), 1.5-2.0 (9H, m), 2.4-2.6 (1H, m), 3.2-3.4 (4H, m), 3.6-3.8 (2H, m), 4.0-4.2 (2H, m), 7.23 (2H, d, J=9 Hz), 8.03 (2H, d, J=9 Hz), 8.15 (2H, d, J=8.3 Hz), 8.24 (2H, d, J=8.3 Hz), 9.68 (1H, br s), 13.37 (1H, br s) API-ES MASS: 447.3 (MH+, free form) (+) Preparation 240 4-[5-[4-[4-[4-(6-Methoxyhexyloxy)cyclohexyl]-1-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid hydrochloride ESI MASS (Negative)(m/z): 577.3 (M+−H) Preparation 241 4-[2-[4-(6-Methoxyhexyl)piperazin-1-yl]phenyl]imidazo-[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid NMR (DMSO-d6, δ): 1.20-1.40 (6H, m), 1.40-1.60 (2H, m), 1.60-1.80 (4H, m), 3.10-3.30 (4H, m), 3.22 (3H, s), 3.70-3.90 (4H, m), 7.18 (2H, d, J=8.1 Hz), 7.84 (2H, d, J=7.9 Hz), 7.90-8.10 (4H, m), 8.83 (1H, s) MASS: 520 (M++H) Preparation 242 4-[5-[4-[1-(8-Methoxyoctyl)-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid hydrochloride IR (KBr): 2931, 2856, 1699, 1605, 1514, 1439, 1410, 1250, 1174, 1115, 1038 cm−1 NMR (DMSO-d6, δ): 1.0-2.3 (20H, m), 2.8-3.8 (4H, m), 3.21 (3H, s), 4.79 (1H, br s), 7.22 (2H, d, J=8.5 Hz), 8.00 (2H, d, J=8.5 Hz), 8.13 (4H, s) ESI MASS (Positive): 524.3 (M++H) Preparation 243 4-[5-[4-[1-(7-Methoxyheptyl)-4-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 1.2-2.1 (14H, m), 2.8-3.6 (12H, m), 7.49 (2H, d, J=8.3 Hz), 8.03 (2H, d, J=8.3 Hz), 8.1-8.2 (4H, m) (+) APCI MASS (Positive): 494.60 (M++H) Preparation 244 4-[5-[4-[4-(4-Pyridylmethyl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 2.6-4.1 (10H, m), 7.1-8.6 (12H, m) API-ES MASS (Negative): 456.3 (M+−H) Preparation 245 4-[5-[4-(1,4-Dioxa-8-azaspiro[4.5]dec-8-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid API-ES MASS: 422.2 (M+−H) Preparation 246 4-[5-[4-(2-Phenyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid API-ES MASS (Negative): 478.2 (M+−H) Preparation 247 4-[5-[4-[4-(3,3-Dimethyl-1,5-dioxaspiro[5.5]undec-9-yl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 0.90 (6H, s), 1.2-2.48 (9H, m), 2.8-3.8 (12H, m), 7.14-7.19 (2H, m), 7.9-7.95 (2H, m), 8.12 (4H, s) API-ES MASS: 549.3 (M++H) Preparation 248 4-[2-[4-[4-(4-Methoxybutoxymethyl)-1-piperidyl]phenyl]-imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid IR (KBr): 2937, 2854, 1684, 1608, 1470, 1421, 1284, 1250, 1200, 1109 cm−1 NMR (DMSO-d6, δ): 1.1-1.8 (9H, m), 2.7-2.9 (3H, m), 3.21 (3H, s), 3.1-3.6 (5H, m), 3.8-4.0 (2H, m), 7.0-7.1 (2H, m), 7.7-7.8 (2H, m), 7.9-8.0 (4H, m), 8.80 (1H, s) (+) APCI MASS: 521.20 (M++H) Preparation 249 4-[5-[4-(4-Cyclopentyl-1-piperazinyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 1.4-2.2 (8H, m), 3.0-3.75 (9H, m), 7.18 (2H, d, J=8.8 Hz), 7.93 (2H, d, J=8.8 Hz), 8.12 (4H, s) API-ES MASS: 435.3 (M++H) Preparation 250 4-[5-[4-(4-Cycloheptyl-1-piperazinyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 1.3-2.1 (12H, m), 2.6-4.0 (9H, m), 7.1-8.2 (9H, m) APCI MASS: 463.3 (M++H) Preparation 251 4-[2-[4-[1-(6-Methoxyhexyl)-4-piperidyl]phenyl]-imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid hydrochloride IR (KBr): 2935, 1709, 1610, 1473, 1414, 1371, 1255, 1221, 1176, 1099, 968 cm−1 NMR (DMSO-d6, δ): 1.2-2.1 (14H, m), 2.8-4.0 (7H, m), 3.23 (3H, s), 7.50 (2H, d, J=8.3 Hz), 7.9-8.1 (6H, m), 8.91 (1H, s) (+) APCI MASS: 519.47 (M++H) Preparation 252 4-[2-[4-[4-(7-Methoxyheptyl)-1-piperazinyl]phenyl]-imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid hydrochloride IR (KBr): 2933, 1699, 1606, 1471, 1402, 1373, 1246, 1174, 1101 cm−1 NMR (DMSO-d6, δ): 1.2-1.8 (10H, m), 3.0-3.8 (12H, m), 3.22 (3H, s), 7.1-7.2 (2H, m), 7.8-7.9 (2H, m), 7.9-8.1 (4H, m), 8.84 (1H, s) (+) APCI MASS: 534.47 (M++H) Preparation 253 4-[2-[4-[4-(5-Methoxypentyl)-1-piperazinyl]phenyl]-imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid hydrochloride IR (KBr): 1699, 1608, 1471, 1404, 1373, 1242, 1174, 1109 cm−1 NMR (DMSO-d6, δ): 1.2-1.8 (6H, m), 2.9-3.6 (12H, m), 3.23 (3H, s), 7.1-7.2 (2H, m), 7.7-7.9 (2H, m), 7.9-8.1 (4H, m), 8.83 (1H, s) (+) APCI MASS: 506.27 (M++H) Preparation 254 4-[5-[4-[4-(1,4-Dioxaspiro[4.5]dec-8-yl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 1.4-2.2 (4H, m), 2.8-3.8 (9H, m), 3.89 (4H, s), 7.17 (2H, d, J=8.9 Hz), 7.92 (2H, d, J=8.9 Hz), 8.12 (4H, s) APCI MASS: 507.3 (M++H) Preparation 255 4-[5-[4-(4-Tetrahydro-2H-pyran-4-yl-1-piperazinyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 1.50-2.10 (4H, m), 2.6-4.0 (14H, m), 7.18 (2H, d, J=8.9 Hz), 7.92 (2H, d, J=8.9 Hz), 8.12 (4H, s) APCI MASS: 451.2 (M++H) Preparation 256 4-[2-[4-(1-Phenyl-4-piperidyloxy)phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid IR (KBr): 1691, 1606, 1471, 1252, 1176 cm−1 NMR (DMSO-d6, δ): 2.0-2.4 (4H, m), 3.0-4.0 (4H, m), 4.8-5.0 (1H, m), 7.1-8.1 (13H, m), 8.87 (1H, s) ESI MASS (Positive): 497.2 (M++H) Preparation 257 4-[2-[4-(1-Cyclohexyl-1,2,3,6-tetrahydro-4-pyridyl)phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid hydrochloride NMR (DMSO-d6, δ): 1.00-2.20 (13H, m), 2.84 (2H, br s), 3.93 (2H, br s), 6.46 (1H, s), 7.75 (2H, d, J=8.2 Hz), 7.99 (2H, d, J=8.2 Hz), 8.01 (4H, s), 8.92 (1H, s) APCI-ES MASS (Positive): 485.2 (M++H) Preparation 258 4-[2-[4-(1-Cyclohexyl-4-piperidyloxy)phenyl]imidazo-[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid IR (KBr): 2937, 1687, 1606, 1471, 1416, 1309, 1252, 1174, 1113 cm−1 NMR (DMSO-d6, δ): 1.0-3.6 (21H, m), 7.22 (2H, d, J=8.7 Hz), 7.9-8.2 (6H, m), 8.87 (1H, s) (+) APCI MASS (Positive): 503.47 (M++H) Preparation 259 4-[2-[4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyloxy]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid NMR (DMSO-d6, δ): 1.0-3.6 (34H, m), 7.1-7.2 (2H, m), 7.8-8.0 (7H, m), 8.87 (1H, s) (+) APCI MASS (Positive): 633.47 (M++H) Preparation 260 4-[5-[4-(1-Phenyl-4-piperidyloxy)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid IR (KBr): 1680, 1603, 1514, 1296, 1252 cm−1 NMR (DMSO-d6, δ): 1.6-1.9 (2H, m), 2.0-2.2 (2H, m), 3.0-3.8 (4H, m), 4.6-4.8 (1H, m), 6.7-7.3 (7H, m), 7.9-8.3 (6H, m) ESI MASS (Negative): 456.1 (M−−H) Preparation 261 4-[5-[4-[1-(4-Methoxyphenyl)-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid IR (Nujol): 1680, 1512, 1294, 1252 cm−1 NMR (DMSO-d6, δ): 1.7-1.9 (2H, m), 2.0-2.2 (2H, m), 2.8-3.6 (4H, m), 3.69 (3H, s), 4.6-4.8 (1H, m), 6.7-7.0 (4H, m), 7.1-7.3 (2H, m), 7.8-8.3 (6H, m) ESI MASS (Negative): 486.1 (M−−H) Preparation 262 4-[2-[4-[1-(4-Methoxyphenyl)-4-piperidyloxy]phenyl]-imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid IR (KBr): 1680, 1605, 1516, 1471, 1423, 1302, 1248, 1176, 1122, 1030, 964, 831 cm−1 NMR (DMSO-d6, δ): 2.0-2.4 (4H, m), 3.0-3.7 (4H, m), 3.78 (3H, s), 4.8-5.0 (1H, m), 7.0-7.2 (2H, m), 7.2-7.4 (2H, m), 7.4-7.7 (2H, m), 7.8-8.2 (6H, m), 8.88 (1H, s) ESI MASS (Positive): 527.2 (M++H) Preparation 263 4-[5-(4-[1-[4-[(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 1.2-2.5 (20H, m), 3.0-3.6 (14H, m), 7.1-7.3 (2H, m), 8.01 (2H, d, J=8.6 Hz), 8.1-8.2 (5H, m) (+) APCI MASS (Positive): 594.40 (M++H) Preparation 264 4-[2-[4-[4-(5-Methoxypentyloxymethyl)-1-piperidyl]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid hydrochloride NMR (CDCl3, δ): 1.20-1.95 (11H, m), 2.70-3.00 (2H, m), 3.20-3.50 (6H, m), 3.55-3.80 (2H, m), 6.96 (2H, d, J=8.00 Hz), 7.73 (2H, d, J=8.28 Hz), 7.87 (2H, d, J=7.81 Hz), 8.00-8.15 (3H, m) APCI MASS (m/z): 535.2 (M+) Preparation 265 4-[5-[4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 1.1-1.7 (12H, m), 1.9-2.2 (14H, m), 2.8-3.6 (8H, m), 7.48 (2H, d, J=8.2 Hz), 8.04 (2H, d, J=8.3 Hz), 8.1-8.2 (4H, m) (+) APCI MASS (Positive): 578.33 (M++H) Preparation 266 4-[5-[4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl)-4-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 1.2-2.2 (20H, m), 2.2-2.6 (14H, m), 7.47 (2H, d, J=8.3 Hz), 8.04 (2H, d, J=8.2 Hz), 8.1-8.2 (4H, m) (+) APCI MASS (Positive): 578.40 (M++H) Preparation 267 4-[5-[4-(1-Cyclohexyl-4-piperidyloxy)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (DMSO-d6, δ): 1.0-3.6 (19H, m), 4.7-5.0 (1H, m), 7.1-7.3 (2H, m), 7.9-8.1 (2H, m), 8.1-8.3 (4H, m), 9.4-9.6 (1H, m) (+) APCI MASS (Positive): 464.33 (M++H) Preparation 268 4-[5-[4-[4-(7-Methoxyheptyl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid NMR (CDCl3, δ): 1.2-1.8 (10H, m), 3.0-3.8 (15H, m), 7.18 (2H, d, J=8.8 Hz), 7.93 (2H, d, J=8.8 Hz), 8.1-8.2 (4H, m) (+) APCI MASS (Positive): 495.60 (M++H) Preparation 269 4-[5-[4-(4-Pentyloxypiperidin-1-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid IR (KBr): 2931.3, 1685.5, 1604.5, 1108.9 cm−1 NMR (DMSO-d6, δ): 0.84-0.91 (3H, m), 1.29-2.00 (10H, m), 3.00-3.70 (7H, m), 7.07-7.11 (2H, m), 7.82-8.11 (6H, m) ESI MASS (Positive)(m/z): 452.40 (M++H) Preparation 270 4-[2-[4-(5-Methoxypentyloxy)phenyl]imidazo[1,2-b][1,3,4]thiadiazol-6-yl]benzoic acid NMR (DMSO-d6, δ): 1.35-1.80 (6H, m), 3.22 (3H, s), 3.10-3.40 (2H, m), 3.80-4.20 (2H, m), 6.60-7.70 (4H, m), 7.80-8.50 (6H, m) MASS: 517 (M++Br), 437 (M) Preparation 271 4-[2-[4-(4-Cyclohexyl-4-methoxy-1-piperidyl)phenyl]-imidazo[1,2-b][1,3,4]thiadiazol-6-yl]benzoic acid NMR (CDCl3+CD3OD, 6): 0.90-1.25 (6H, m), 1.42 (3H, t, J=70.10 Hz), 1.50-2.30 (9H, m), 3.20 (3H, s), 3.25-3.80 (4H, m), 4.39 (2H, q, J=7.12 Hz), 7.37 (2H, br d, J=8.66 Hz), 7.83 (2H, d, J=8.80 Hz), 7.90 (2H, d, J=8.44 Hz), 8.09 (2H, d, J=8.36 Hz), 8.11 (1H, s) Preparation 272 A mixture of 4-[5-[4-(2-phenyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid (1.39 g), O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (1.3 g) and N,N-diisopropylethylamine (1 ml) in 1-methyl-2-pyrrolidinone (30 ml) was stirred for 2 hours at 50° C. The reaction mixture was poured into water.", "Then the resulting precipitate was collected by filtration and washed with water to give 1-[4-[5-[4-(2-phenyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]-pyridin-5-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole (1.59 g).", "IR (KBr): 1778, 1601, 1504, 1414, 1230, 1188, 985 cm−1 NMR (CDCl3, δ): 2.9-3.1 (2H, m), 3.7-3.9 (2H, m), 4.4-4.6 (2H, m), 7.0-8.5 (18H, m) (+) APCI MASS: 596.73 (M++H) The following compounds [Preparation 273 to 279] were obtained according to a similar manner to that of Preparation 272.Preparation 273 1-[4′-(4-Cyclohexylhexahydro-1H-1,4-diazepin-1-yl)-1,1′-biphenyl-4-yl]carbonyloxy-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 0.8-4.0 (21H, m), 6.7-8.5 (12H, m) MASS (m/z): 496 (M++H) Preparation 274 1-[4′-(5-Cyclohexyl-2,5-diazabicyclo[2.2.1]hept-2-yl)-1,1′-biphenyl-4-yl]carbonyloxy-1H-1,2,3-benzotriazole MASS (m/z): 494 (M++H) Preparation 275 1-[4′-(4-Phenyl-1-piperidyl)-1,1′-biphenyl-4-yl]carbonyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1772, 1238, 1209, 976 cm−1 NMR (CDCl3, δ): 1.5-2.1 (4H, m), 2.6-3.05 (3H, m), 3.85-4.05 (2H, m), 7.09 (2H, d, J=8.9 Hz), 7.15-7.7 (10H, m), 7.80 (2H, d, J=8.6 Hz), 8.12 (1H, d, J=8.2 Hz), 8.30 (2H, d, J=8.6 Hz) MASS (m/z): 475 (M++H) Preparation 276 1-[4′-[4-(cis-4-Methylcyclohexyl)-1-piperazinyl]-1,1′-biphenyl-4-yl]carbonyloxy-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 0.96 (3H, d, J=6.9 Hz), 1.4-1.9 (9H, m), 2.3-2.5 (1H, m), 2.7-2.95 (4H, m), 3.3-3.5 (4H, m), 7.03 (2H, d, J=8.8 Hz), 7.4-7.7 (5H, m), 7.79 (2H, d, J=8.5 Hz), 8.15 (1H, d, J=6.0 Hz), 8.30 (2H, d, J=8.5 Hz) MASS (m/z): 496 (M++H) Preparation 277 1-[4′-[4-(trans-4-Methylcyclohexyl)-1-piperazinyl]-1,1′-biphenyl-4-yl]carbonyloxy-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 0.8-1.5 (8H, m), 1.6-2.1 (4H, m), 2.2-2.45 (1H, m), 2.7-2.9 (4H, m), 3.25-3.45 (4H, m), 7.03 (2H, d, J=8.9 Hz), 7.4-7.7 (5H, m), 7.79 (2H, d, J=8.6 Hz), 8.11 (1H, d, J=8.2 Hz), 8.30 (2H, d, J=8.6 Hz) MASS (m/z): 496 (M++H) Preparation 278 1-[4′-[4-[4-[4-(6-Methoxyhexyl)-1-piperazinyl)phenyl]-1-piperazinyl]-1,1′-biphenyl-4-yl]carbonyloxy-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 1.2-2.0 (8H, m), 2.41 (2H, t, J=7.4 Hz), 2.5-2.7 (4H, m), 3.0-3.5 (17H, m), 6.85-7.15 (6H, m), 7.35-7.7 (5H, m), 7.80 (2H, d, J=8.2 Hz), 8.12 (1H, d, J=8.3 Hz), 8.31 (2H, d, J=8.2 Hz) MASS (m/z): 674 (M++H) Preparation 279 1-[4′-[4-[4-[1-(6-Methoxyhexyl)-4-piperidyloxy]phenyl]-1-piperazinyl]-1,1′-biphenyl-4-yl]carbonyloxy-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 1.2-2.6 (16H, m), 2.7-2.9 (2H, m), 3.2-3.5 (13H, m), 4.2-4.35 (1H, m), 6.8-7.0 (4H, m), 7.09 (2H, d, J=8.9 Hz), 7.4-7.7 (5H, m), 7.80 (2H, d, J=8.5 Hz), 8.12 (1H, d, J=8.2 Hz), 8.31 (2H, d, J=8.5 Hz) MASS (m/z): 689 (M++H) Preparation 280 A suspension of 4-[5-[4-(4-butoxypiperidin-1-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid (2.75 g) in dichloromethane (55 ml) was treated with 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (959 mg) and 1-hydroxybenzotriazole (149 mg), and stirred for 16 hours at ambient temperature.", "The reaction mixture was extracted with dichloromethane.", "The extract was washed with brine and dried, and evaporated under reduced pressure to give 1-[4-[5-[4-(4-butoxypiperidin-1-yl)phenyl]-1,3,4-thiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole (3.35 g).", "IR (KBr): 2931.3, 1778.0, 1602.6, 1228.4, 1103.1 cm−1 NMR (CDCl3, δ): 0.94 (3H, t, J=7.2 Hz), 1.34-1.99 (8H, m), 3.09-3.83 (7H, m), 6.95-7.00 (2H, m), 7.47-8.42 (10H, m) ESI MASS (Positive)(m/z): 555.40 (M++H) The following compounds [Preparation 281 to 323] were obtained according to a similar manner to that of Preparation 280.Preparation 281 1-[4-[5-[4-(7-Methoxyheptyloxyphenyl]-1,3,4-thiadiazol-2-yl]-1-piperazinyl]benzoyl]-1H-1,2,3-benzotriazole IR (KBr): 2933.2, 1768.4, 1602.6, 1230.4, 1089.6 cm−1 NMR (CDCl3, δ): 1.22-1.81 (10H, m), 3.34 (3H, s), 3.35-4.03 (12H, m), 6.92-7.03 (4H, m), 7.39-8.20 (8H, m) ESI MASS (Positive)(m/z): 627.47 (M+) Preparation 282 1-[4-(7-Methoxyheptyloxy)benzoyl]-1H-1,2,3-benzotriazole IR (KBr): 2933.2, 1774.2, 1602.6, 1253.5 cm−1 NMR (CDCl3, δ): 1.41-1.92 (10H, m), 3.34 (3H, s), 3.36-3.42 (2H, m), 4.09 (2H, t, J=6.5 Hz), 7.03-7.08 (2H, m), 7.40-7.63 (4H, m), 8.20-8.26 (2H, m) ESI MASS (Positive)(m/z): 383.20 (M+) Preparation 283 1-[4-(4-[1,1′-Biphenyl]-4-yl-1H-pyrazol-1-yl)benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1774.2, 1602.6, 1571.7, 1513.8, 1403.9 cm−1 NMR (CDCl3, δ): 7.37-7.71 (12H, complex m), 8.03 (2H, d, J=8.8 Hz), 8.14 (1H, s), 8.1-8.14 (1H, m), 8.35 (1H, s), 8.42 (2H, d, J=8.8 Hz) MASS (m/z): 458 (MH+) Preparation 284 1-[4-[4-(4-Hexyloxyphenyl)-1H-pyrazol-1-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1776.1, 1602.6, 1504.2, 1402, 1247.7, 1234.2, 1176.4, 1087.7, 987.4, 944.9 cm−1 NMR (CDCl3, δ): 0.9-1.0 (3H, m), 1.4-1.6 (6H, m), 1.7-1.9 (2H, m), 4.00 (2H, t, J=6.5 Hz), 6.96 (2H, d, J=8.7 Hz), 7.46-7.60 (5H, m), 7.97-8.04 (3H, m), 8.12 (1H, d, J=8.2 Hz), 8.23 (1H, s), 8.39 (2H, d, J=8.8 Hz) MASS (m/z): 482 (MH+) Preparation 285 1-[4-[1-(4-Hexyloxyphenyl)-1H-pyrazol-4-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1770.3, 1608.3, 1567.8, 1519.6, 1240.0, 991.2 cm−1 NMR (CDCl3, δ): 0.9-1.0 (3H, m), 1.3-1.6 (6H, m), 1.70-1.90 (2H, m), 4.01 (2H, t, J=6.5 Hz), 7.01 (2H, d, J=9 Hz), 7.40-7.60 (3H, m), 7.64 (2H, d, J=9 Hz), 7.77 (2H, d, J=8.4 Hz), 8.09 (1H, s), 8.12 (1H, d, J=9 Hz), 8.23 (1H, s), 8.30 (2H, d, J=8.4 Hz) EI MASS (m/z): 482 (MH+) Preparation 286 1-[4-[5-[4-[4-(4-Methylcyclohexyl)-1-piperazinyl]-phenyl]-1,3,4-oxadiazol-2-yl]benzoyloxy)-1H-1,2,3-benzotriazole IR (KBr): 1780, 1610, 1496, 1242, 1230, 989 cm−1 NMR (CDCl3, δ): 0.93 (3H, d, J=6.9 Hz), 1.4-1.9 (9H, m), 2.35-2.6 (1H, m), 2.75-2.90 (4H, m), 3.4-3.55 (4H, m), 7.00 (2H, d, J=9 Hz), 7.45-7.65 (2H, m), 7.95-8.20 (4H, m), 8.35-8.48 (4H, m) Preparation 287 1-[4-[5-[4-[4-(6-Methoxyhexyloxy)-1-piperidyl]-phenyl]-1,3,4-oxadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 1.20-1.80 (14H, m), 1.85-2.50 (4H, m), 2.70-2.90 (4H, m), 3.33 (3H, s), 3.34-3.55 (8H, m), 6.97 (2H, d, J=8.90 Hz), 7.40-7.65 (3H, m), 7.92 (2H, d, J=8.68 Hz), 8.13 (2H, d, J=8.18 Hz), 8.23 (2H, d, J=8.44 Hz), 8.39 (2H, d, J=8.44 Hz) ESI MASS (Positive)(m/z): 696.4 (M++H) Preparation 288 4-[2-[4-(6-Methoxyhexyl)piperazin-1-yl]phenyl]imidazo-[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid benzotriazol-1-yl ester IR (KBr): 3458, 3425, 3404, 2931, 2854, 1776, 1603, 1471 cm−1 NMR (DMSO-d6, δ): 1.20-1.40 (6H, m), 1.40-1.60 (4H, m), 2.20-2.40 (4H, m), 3.15-3.30 (2H, m), 3.21 (3H, s), 3.70-4.00 (4H, m), 7.11 (2H, d, J=8.7 Hz), 7.20-7.50 (3H, m), 7.59 (2H, d, J=8.4 Hz), 7.70-8.20 (5H, m), 8.82 (1H, s) MASS: 637 (M++H), 534 (M−−103), Preparation 289 1-[4-[5-[4-[1-(8-Methoxyoctyl)-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2929, 2856, 1778, 1603, 1514, 1441, 1410, 1250, 1174, 1117, 1093 cm−1 NMR (CDCl3, δ): 1.1-1.8 (12H, m), 1.9-2.2 (2H, m), 2.2-2.4 (2H, m), 2.6-3.0 (6H, m), 3.3-3.4 (5H, m), 4.59 (1H, br s), 6.9-8.5 (12H, m) Preparation 290 1-[4-[5-[4-[1-(7-Methoxyheptyl)-4-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2929, 2854, 1776, 1433, 1230, 1119, 1090, 985, 739 cm−1 NMR (CDCl3, δ): 1.2-2.8 (21H, m), 3.33 (3H, s), 3.3-3.5 (2H, m), 7.3-7.8 (5H, m), 7.97 (2H, d, J=8.3 Hz), 8.1-8.2 (1H, m), 8.2-8.3 (2H, m), 8.3-8.5 (2H, m) (+) APCI MASS (Positive): 611.07 (M++H) Preparation 291 1-[4-[5-[4-[4-(4-Pyridylmethyl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl) benzoyloxy]-1H-1, 2, 3-benzotriazole IR (KBr): 1778, 1601, 1439, 1414, 1230 cm−1 NMR (CDCl3, δ): 2.6-2.7 (4H, m), 3.2-3.4 (4H, m), 3.60 (2H, s), 6.9-8.7 (16H, m) (+) APCI MASS: 574.93 (M++H) Preparation 292 8-[4-[5-[4-[(1H-1,2,3-Benzotriazol-1-yloxy)carbonyl]-phenyl]-1,3,4-thiadiazol-2-yl]phenyl]-1,4-dioxa-8-azaspiro[4.5]decane IR (KBr): 1778, 1599, 1524, 1441, 1414, 1228, 1180, 1099, 984 cm−1 NMR (CDCl3, δ): 1.8-1.9 (4H, m), 3.5-3.6 (4H, m), 4.02 (4H, s), 6.7-8.5 (12H, m) (+) APCI MASS: 541.00 (M++H) Preparation 293 1-[4-[5-[4-[4-(3,3-Dimethyl-1,5-dioxaspiro[5.5]undec-9-yl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2951, 1780, 1668, 1603, 1441, 1414, 1234, 1105, 982 cm−1 NMR (CDCl3, δ): 0.97 (6H, s), 1.2-2.5 (8H, m), 2.7-2.9 (5H, m), 3.3-3.6 (8H, m), 6.9-8.5 (12H, m) Preparation 294 1-[4-[2-[4-[4-(Methoxybutoxymethyl)-1-piperidyl]-phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2937, 2850, 1774, 1608, 1471, 1248, 1230, 1200, 1176, 1113, 1088, 984, 820 cm−1 NMR (CDCl3, δ): 1.1-2.1 (8H, m), 2.3-2.4 (1H, m), 2.7-3.0 (3H, m), 3.2-3.5 (5H, m), 3.28 (3H, s), 3.7-4.0 (2H, m), 6.8-8.3 (13H, m) (+) APCI MASS(Positive): 638.3 (M++H) Preparation 295 1-[4-[5-[4-(4-Cyclopentyl-1-piperazinyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2954, 1778, 1603, 1441, 1414, 1234, 985, 822 cm−1 NMR (CDCl3, δ): 1.4-2.3 (8H, m), 2.5-2.8 (5H, m), 3.3-3.5 (4H, m), 6.9-8.5 (12H, m) (+) APCI MASS: 551.93 (M++H) Preparation 296 1-[4-[5-[4-(4-Cycloheptyl-1-piperazinyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2924, 2852, 1780, 1603, 1441, 1414, 1232, 984 cm−1 NMR (CDCl3, δ): 1.2-2.1 (12H, m), 2.7-3.0 (5H, m), 3.3-3.6 (4H, m), 6.8-8.5 (12H, m) (+) APCI MASS: 580.00 (M++H) Preparation 297 1-[4-[2-[4-[1-(6-Methoxyhexyl)-4-piperidyl]phenyl]-imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2935, 1774, 1701, 1608, 1471, 1371, 1252, 1232, 1176, 1105, 972, 843 cm−1 NMR (CDCl3, δ): 1.2-2.3 (14H, m), 2.5-3.7 (7H, m), 3.31 (3H, s), 7.2-8.4 (13H, m) (+) APCI MASS: 636.13 (M++H) Preparation 298 1-[4-[2-[4-[4-(7-Methoxyheptyl)-1-piperazinyl]phenyl]-imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2929, 1774, 1606, 1471, 1387, 1232, 1200, 1173, 1117, 1088, 984, 820, 727 cm−1 NMR (DMSO-d6, δ): 1.2-1.7 (10H, m), 2.3-2.5 (2H, m), 2.5-2.7 (4H, m), 3.34 (3H, s), 3.3-3.5 (6H, m), 6.9-8.4 (13H, m) (+) APCI MASS: 651.13 (M++H) Preparation 299 1-[4-[2-[4-[4-(5-Methoxypentyl)-1-piperazinyl]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1774, 1701, 1608, 1471, 1390, 1232, 1198, 1174, 1115, 1090, 983 cm−1 NMR (CDCl3, δ): 1.2-2.0 (6H, m), 2.3-2.8 (6H, m), 3.2-3.5 (9H, m), 6.8-8.4 (13H, m) (+) APCI MASS (m/z): 623.20 (M++H) Preparation 300 1-[4-[5-[4-[4-(1,4-Dioxaspiro[4.5]dec-8-yl)-1-piperazinyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2951, 1778, 1603, 1441, 1416, 1232, 1101, 982 cm−1 NMR (CDCl3, δ): 1.5-2.6 (8H, m), 2.7-2.9 (5H, m), 3.3-3.5 (4H, m), 3.95 (4H, s), 6.9-8.5 (12H, m) (+) APCI MASS: 624.07 (M++H) Preparation 301 1-[4-[5-[4-(4-Tetrahydro-2H-pyran-4-yl-1-piperazinyl)phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2956, 2835, 1778, 1603, 1441, 1414, 1232 cm−1 NMR (CDCl3, δ): 1.5-2.2 (4H, m), 2.4-2.6 (1H, m), 2.7-2.8 (4H, m), 3.3-3.5 (6H, m), 4.0-4.2 (2H, m), 6.9-8.5 (12H, m) (+) APCI MASS: 567.93 (M++H) Preparation 302 1-[4-[2-[4-(1-Phenyl-4-piperidyloxy)phenyl]imidazo[2,1-b](1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1776, 1603, 1473, 1248, 1228, 1174, 982 cm−1 NMR (DMSO-d6, δ): 1.9-2.3 (4H, m), 3.1-3.3 (2H, m), 3.4-3.6 (2H, m), 4.5-4.7 (1H, m), 6.8-8.4 (18H, m) (+) APCI MASS: 614.13 (M++H) Preparation 303 1-[4-[2-[4-(1-Cyclohexyl-1,2,3,6-tetrahydro-4-pyridyl)phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2927, 1776, 1606, 1471, 1230, 1173, 982, 845 cm−1 NMR (CDCl3, δ): 1.0-2.2 (10H, m), 2.3-3.5 (7H, m), 6.2-6.3 (1H, m), 7.1-8.4 (13H, m) (+) APCI MASS: 601.93 (M++H) Preparation 304 1-[4-[2-[4-(1-Cyclohexyl-4-piperidyloxy)phenyl]-imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2931, 2515, 1680, 1606, 1471, 1427, 1252, 1174, 970 cm−1 NMR (DMSO-d6, δ): 0.8-3.3 (18H, m), 3.8-4.0 (1H, m), 4.6-4.8 (1H, m), 7.02 (2H, d, J=8.8 Hz), 7.4-7.7 (3H, m), 7.85 (2H, d, J=8.7 Hz), 8.0-8.2 (3H, m), 8.21 (1H, s), 8.33 (2H, d, J=8.4 Hz) (+) APCI MASS (Positive): 620.13 (M++H) This compound was used in the next reaction without further purification.", "Preparation 305 1-[4-[2-[4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4piperidyloxy]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole This compound was used in the next reaction without further purification.", "Preparation 306 1-[4-[5-[4-(1-Phenyl-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1780, 1601, 1500, 1439, 1410, 1304, 1250, 1178, 1030, 984 cm−1 NMR (CDCl3, δ): 1.9-2.3 (4H, m), 3.1-3.3 (2H, m), 3.4-3.6 (2H, m), 4.5-4.7 (1H, m), 6.8-8.5 (17H, m) Preparation 307 1-[4-[5-[4-[1-(4-Methoxyphenyl)-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1792, 1605, 1512, 1439, 1248, 1180, 1034, 987 cm−1 NMR (CDCl3, δ): 1.9-2.3 (4H, m), 2.9-3.1 (2H, m), 3.3-3.5 (2H, m), 3.78 (3H, s), 4.5-4.7 (1H, m), 6.8-8.5 (16H, m) Preparation 308 1-[4-[2-[4-[1-(4-Methoxyphenyl)-4-piperidyloxy]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1776, 1605, 1512, 1470, 1248, 1176, 1036, 980 cm−1 NMR (CDCl3, δ): 1.9-2.3 (4H, m), 2.9-3.1 (2H, m), 3.3-3.5 (2H, m), 3.78 (3H, s), 4.5-4.7 (1H, m), 6.8-8.4 (17H, m) Preparation 309 1-[4-[5-[4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyloxy]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1776, 1603, 1441, 1375, 1250, 1174, 1115, 1090, 984 cm−1 NMR (CDCl3, δ): 1.2-3.3 (20H, m), 3.32 (3H, s), 3.3-3.5 (10H, m), 4.3-4.5 (1H, m), 6.9-7.1 (2H, m), 7.4-7.7 (2H, m), 7.9-8.3 (7H, m), 8.41 (1H, d, J=8.4 Hz) ESI MASS (Positive): 711.3 (M++H) Preparation 310 1-[4-[2-[4-[4-(5-Methoxypentyloxymethyl)-1-piperidyl]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 1.20-1.95 (11H, m), 2.80-3.00 (2H, m), 3.25-3.50 (9H, m), 3.80-3.95 (2H, m), 6.95 (2H, d, J=8.97 Hz), 7.40-7.60 (3H, m), 7.74 (2H, d, J=8.80 Hz), 8.05 (2H, d, J=8.41 Hz), 8.11 (2H, d, J=8.29 Hz), 8.17 (1H, s), 8.31 (2H, d, J=8.43 Hz) APCI MASS (m/z): 674.3 (M++Na) Preparation 311 1-[4-[5-[4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2933, 2860, 1776, 1605, 1471, 1381, 1250, 1174, 1113, 1095 cm−1 NMR (CDCl3, δ): 1.2-2.8 (24H, m), 3.1-3.5 (7H, m), 3.33 (3H, s), 7.2-8.2 (8H, m), 8.26 (2H, d, J=8.5 Hz), 8.41 (2H, d, J=8.5 Hz) (+) APCI MASS (Positive): 695.33 (M++H) Preparation 312 1-[4-[5-[4-[1-[4-(6-Methoxyhexyloxy)cyclohexyl]-4-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2933, 2858, 1776, 1651, 1541, 1452, 1433, 1373, 1090, 987 cm−1 Preparation 313 1-[4-[5-[4-(1-Cyclohexyl-4-piperidyloxy)phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2929, 2507, 1776, 1603, 1514, 1412, 1377, 1250, 1173 cm−1 NMR (CDCl3, δ): 1.0-3.4 (20H, m), 6.9-8.2 (8H, m), 8.25 (2H, d, J=8.4 Hz), 8.42 (2H, d, J=8.4 Hz) (+) APCI MASS (Positive): 581.20 (M++H) Preparation 314 1-[4-[5-[4-[4-(7-Methoxyheptyl)-1-piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2929, 2854, 1776, 1603, 1441, 1414, 1232, 984 cm−1 NMR (CDCl3, δ): 1.2-2.4 (10H, m), 2.4-2.6 (2H, m), 2.6-2.8 (4H, m), 3.34 (3H, s), 3.3-3.5 (6H, m), 6.98 (2H, d, J=8.9 Hz), 7.4-7.7 (3H, m), 7.93 (2H, d, J=8.8 Hz), 8.13 (1H, d, J=8.1 Hz), 8.24 (2H, d, J=8.5 Hz), 8.40 (2H, d, J=8.5 Hz) (+) APCI MASS (Positive): 612.20 (M++H) Preparation 315 1-[4-[5-[4-[4-(7-Methoxyheptyloxy)-1-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 1.20-1.45 (6H, m), 1.45-1.80 (6H, m), 1.80-2.35 (4H, m), 3.00-3.20 (3H, m), 3.33 (3H, s), 3.37 (2H, t, J=6.42 Hz), 3.48 (2H, t, J=6.54 Hz), 3.55-3.75 (2H, m), 6.97 (2H, d, J=8.95 Hz), 7.40-7.65 (3H, m), 7.90 (2H, d, J=8.80 Hz), 8.12 (1H, d, J=8.17 Hz), 8.23 (2H, d, J=8.44 Hz), 8.40 (2H, d, J=8.43 Hz) Preparation 316 1-[4-[5-[4-(4-Pentyloxypiperidin-1-yl)phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2929.3, 1778.0, 1602.6, 1105.0 cm−1 NMR (CDCl3, δ): 0.91 (3H, t, J=6.9 Hz), 1.34-2.04 (10H, m), 3.11-3.83 (7H, m), 6.96-7.00 (2H, m), 7.36-8.42 (10H, m) ESI MASS (Positive)(m/z): 569.33 (M++H) Preparation 317 4-[2-(5-Methoxypentyloxy)phenyl]imidazo[1,2-b][1,3]-thiazol-6-yl]benzoic acid benzotriazol-1-yl ester IR (KBr): 2937, 2866, 1776, 1605, 1458 cm−1 NMR (DMSO-d6, δ): 1.30-1.80 (6H, m), 3.23 (3H, s), 3.50-3.70 (2H, m), 3.80-4.20 (2H, m), 6.90-7.70 (8H, m), 7.80-8.20 (4H, m), 8.30-8.60 (2H, m) MASS: 554 (M) Preparation 318 1-[1-[4′-[4-[trans-4-Methoxy-4-(1-methoxycyclohexyl-1-yl)cyclohexyl-1-yl]-1-piperazinyl]-1,1′-biphenyl-4-yl]carbonyloxy]-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 0.9-2.4 (19H, m), 2.6-2.8 (4H, m), 3.2-3.5 (10H, m), 7.03 (2H, d, J=8.9 Hz), 7.35-7.7 (5H, m), 7.79 (2H, d, J=8.6 Hz), 8.0-8.2 (1H, m), 8.30 (2H, d, J=8.6 Hz) MASS (m/z): 624 (M++H) Preparation 319 1-[1-[4′-[4-[cis-4-Methoxy-4-(1-methoxycyclohexyl-1-yl)cyclohexyl-1-yl]-1-piperazinyl]-1,1′-biphenyl-4-yl]carbonyloxy]-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 0.7-2.45 (19H, m), 2.7-2.9 (4H, m), 3.2-3.6 (10H, m), 7.03 (2H, d, J=8.9 Hz), 7.3-7.7 (5H, m), 7.79 (2H, d, J=8.5 Hz), 8.12 (1H, d, J=8.2 Hz), 8.30 (2H, d, J=8.5 Hz) MASS (m/z): 624 (M++H) Preparation 320 1-[1-[4′-[4-[4-[4-(6-Methoxyhexyl)-1-piperazinyl]-phenyl]-1-piperazinyl]-1,1′-biphenyl-4-yl]carbonyloxy]-1H-1,2,3-benzotriazole NMR (CDCl3, δ): 1.2-2.0 (8H, m), 2.41 (2H, t, J=7.4 Hz), 2.5-2.7 (4H, m), 3.0-3.5 (17H, m), 6.85-7.15 (6H, m), 7.35-7.7 (5H, m), 7.80 (2H, d, J=8.2 Hz), 8.12 (1H, d, J=8.3 Hz), 8.31 (2H, d, J=8.2 Hz) MASS (m/z): 674 (M++H) Preparation 321 1-[4-[5-[4-[4-(4-Methylpentyloxy)-1-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2951, 1780, 1605, 1439, 1228, 1107, 982 cm−1 NMR (CDCl3, δ): 0.90 (6H, d, J=6.6 Hz), 1.1-1.3 (2H, m), 1.5-1.9 (5H, m), 1.9-2.1 (2H, m), 3.0-3.3 (2H, m), 3.4-3.8 (5H, m), 6.97 (2H, d, J=9.0 Hz), 7.4-7.7 (3H, m), 7.90 (2H, d, J=7.9 Hz), 8.1-8.2 (1H, m), 8.2-8.3 (2H, m), 8.3-8.5 (2H, m) ESI MASS (Negative): 464.2 (M−−HOBT-H) Preparation 322 1-[4-[5-[4-[4-(Cyclohexylmethoxy)-1-piperidyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 2922, 2848, 1784, 1603, 1441, 1414, 1363, 1288, 1113, 1092 cm−1 NMR (CDCl3, δ): 0.8-2.2 (15H, m), 3.0-3.3 (4H, m), 3.4-3.8 (3H, m), 6.8-7.0 (2H, m), 7.3-7.6 (3H, m), 7.90 (2H, d, J-8.9 Hz), 8.0-8.2 (1H, m), 8.2-8.3 (2H, m), 8.3-8.5 (2H, m) ESI MASS (Negative): 476.2 (M−-HOBT-H) Preparation 323 1-[4-[2-[4-[(4-Cyclohexyl-4-methoxy)-1-piperidyl]-phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole IR (KBr): 1778, 1666, 1603, 1468, 1234 cm−1 NMR (CDCl3, δ): 0.90-2.10 (15H, m), 3.05-3.20 (2H, m), 3.20 (3H, s), 3.50-3.75 (2H, m), 6.95 (2H, d, J=90.10 Hz), 7.45-7.60 (3H, m), 7.73 (2H, d, J=8.90 Hz), 8.00-8.20 (4H, m), 8.31 (2H, d, J=8.60 Hz) Preparation 324 To a solution of tert-butyl 4-hydroxy-1-piperidinecarboxylate (15 g) in N,N-dimethylformamide (75 ml) was added sodium hydride (60% dispersion in mineral oil) (2.33 g).", "The solution was stirred for 2 hours at 60° C. After cooling to ambient temperature, to the solution was added 1-bromoethane (13.9 ml) and the mixture was stirred for 16 hours.", "The reaction mixture was added to a mixture of water and ethyl acetate.", "The organic layer was washed with brine and dried over magnesium sulfate.", "The magnesium sulfate was filtered off, and the filtrate was concentrated under reduced pressure.", "The residue was purified by silica gel chromatography (5:1 hexane-ethyl acetate elution) to give tert-butyl 4-ethoxy-1-piperidinecarboxylate (13.31 g).", "NMR (DMSO-d6, δ): 1.21 (3H, t, J=7.3 Hz), 1.45 (9H, s), 1.45-1.55 (2H, m), 1.75-1.9 (2H, m), 2.95-3.1 (2H, m), 3.35-3.5 (1H, m), 3.61 (2H, q, J=7.3 Hz), 3.75-3.85 (2H, m) MASS (m/z): 252.3 (M++Na) The following compounds [Preparation 325 and 326] were obtained according to a similar manner to that of Preparation 324.Preparation 325 Tert-Butyl 4-propoxy-1-piperidinecarboxylate NMR (DMSO-d6, δ): 0.92 (3H, t, J=7.4 Hz), 1.45 (9H, s), 1.45-1.65 (4H, m), 1.75-1.9 (2H, m), 3.0-3.15 (2H, m), 3.35-3.45 (3H, m), 3.7-3.85 (2H, m) MASS (m/z): 266.3 (M++Na) Preparation 326 Tert-Butyl 4-butoxy-1-piperidinecarboxylate NMR (DMSO-d6, δ): 0.92 (3H, t, J=7.3 Hz), 1.3-1.6 (6H, m), 1.45 (9H, s), 1.75-1.9 (2H, m), 3.0-3.15 (2H, m), 3.35-3.5 (3H, m), 3.7-3.85 (2H, m) MASS (m/z): 280.4 (M++Na) Preparation 327 To a solution of tert-butyl 4-ethoxy-1-piperidinecarboxylate (13.31 g) and anisole (44.2 ml) in dichloromethane (66.6 ml) was added dropwise with stirring trifluoroacetic acid (89.4 ml) at 0° C. The mixture was then stirred for 1.5 hours at room temperature.", "The solvent was evaporated to give 4-ethoxypiperidine trifluoroacetate (57.73 g).", "NMR (DMSO-d6, δ): 1.11 (3H, t, J=7.0 Hz), 1.55-1.7 (2H, m), 1.85-2.0 (2H, m), 2.9-3.05 (2H, m), 3.1-3.25 (2H, m), 3.45 (2H, q, J=7.0 Hz), 3.5-3.6 (1H, m), 8.2-8.5 (2H, m) MASS (m/z): 130.4 (M++H) The following compounds [Preparation 328 and 329] were obtained according to a similar manner to that of Preparation 327.Preparation 328 4-Propoxypiperidine trifluoroacetate NMR (DMSO-d6, δ): 0.87 (3H, t, J=7.4 Hz), 1.45-1.7 (4H, m), 1.85-2.0 (2H, m), 2.9-3.05 (2H, m), 3.1-3.2 (2H, m), 3.35 (2H, t, J=6.6 Hz), 3.5-3.6 (1H, m), 8.2-8.55 (2H, m) MASS (m/z): 144.3 (M++H) Preparation 329 4-Butoxypiperidine trifluoroacetate NMR (DMSO-d6, δ): 0.88 (3H, t, J=7.3 Hz), 1.25-1.55 (4H, m), 1.55-1.7 (2H, m), 1.85-2.0 (2H, m), 2.9-3.05 (2H, m), 3.1-3.2 (2H, m), 3.40 (2H, t, J=6.4 Hz), 3.45-3.55 (1H, m), 8.15-8.4 (2H, m) MASS (m/z): 158.4 (M++H) Preparation 330 To a suspension of 4-ethoxypiperidine trifluoroacetate (4 g) and potassium bicarbonate (6.14 g) in dimethylsulfoxide (16.5 ml) was added 4-fluorobenzonitrile (2.39 g) and stirred for 5 hours at 150° C. The reaction mixture was added to a mixture of water and ethyl acetate.", "The organic layer was washed with brine and dried over magnesium sulfate.", "The magnesium sulfate was filtered off, and the filtrate was concentrated under reduced pressure.", "The residue was purified by silica gel chromatography (5:1 hexane-ethyl acetate elution) to give 4-(4-ethoxy-1-piperidyl)benzonitrile (3.64 g).", "NMR (CDCl3, δ): 1.22 (3H, t, J=7.0 Hz), 1.6-1.75 (2H, m), 1.9-2.0 (2H, m), 3.05-3.2 (2H, m), 3.5-3.7 (5H, m), 6.8-6.9 (2H, m), 7.45-7.5 (2H, m) MASS (m/z): 253.4 (M++Na) The following compounds [Preparation 331 and 332] were obtained according to a similar manner to that of Preparation 330.Preparation 331 4-(4-Propoxy-1-piperidyl)benzonitrile NMR (CDCl3, δ): 0.93 (3H, t, J=7.4 Hz), 1.55-1.75 (4H, m), 1.9-2.0 (2H, m), 3.1-3.2 (2H, m), 3.43 (2H, t, J=6.7 Hz), 3.5-3.7 (3H, m), 6.8-6.9 (2H, m), 7.45-7.5 (2H, m) MASS (m/z): 267.3 (M++Na) Preparation 332 4-(4-Butoxy-1-piperidyl)benzonitrile NMR (CDCl3, δ): 0.93 (3H, t, J=7.3 Hz), 1.3-1.45 (2H, m), 1.5-1.75 (4H, m), 1.9-2.0 (2H, m), 3.1-3.2 (2H, m), 3.4-3.7 (5H, m), 6.8-6.9 (2H, m), 7.45-7.5 (2H, m) MASS (m/z): 281.2 (M++Na) Preparation 333 To a solution of 4-(4-ethoxy-1-piperidyl)benzonitrile (3.64 g) and hydrazinecarbothioamide (2.88 g) in toluene (36 ml) was added dropwise with stirring trifluoroacetic acid (18 ml) at room temperature.", "The mixture was then stirred for 10 hours at 65° C. After cooling to ambient temperature, the reaction mixture was poured into water and tetrahydrofuran and the mixture was adjusted to pH 9 with sodium hydroxide solution.", "The resulting precipitates were filtered, washed with water, diisopropyl ether, then dried to give 5-[4-(4-ethoxy-1-piperidyl)phenyl]-1,3,4-thiadiazol-2-amine (4.132 g).", "NMR (CDCl3, δ): 1.23 (3H, t, J=7.0 Hz), 1.6-1.8 (2H, m), 1.9-2.1 (2H, m), 2.9-3.1 (2H, m), 3.4-3.7 (5H, m), 5.08 (2H, br s), 6.92 (2H, d, J=8.9 Hz), 7.66 (2H, d, J=8.9 Hz) MASS (m/z): 327.3 (M++Na) The following compounds [Preparation 334 and 335] were obtained according to a similar manner to that of Preparation 333.Preparation 334 5-[4-(4-Propoxy-1-piperidyl)phenyl]-1,3,4-thiadiazol-2-amine NMR (CDCl3, δ): 0.94 (3H, t, J=7.4 Hz), 1.5-2.1 (6H, m), 2.95-3.15 (2H, m), 3.35-3.7 (5H, m), 5.22 (2H, br s), 6.92 (2H, d, J=8.9 Hz), 7.66 (2H, d, J=8.9 Hz) MASS (m/z): 341.2 (M++Na) Preparation 335 5-[4-(4-Butoxy-1-piperidyl)phenyl]-1,3,4-thiadiazol-2-amine NMR (CDCl3, δ): 0.93 (3H, t, J=7.4 Hz), 1.35-1.8 (6H, m), 1.95-2.05 (2H, m), 3.0-3.1 (2H, m), 3.4-3.7 (5H, m), 5.13 (2H, s), 6.9-6.95 (2H, m), 7.65-7.7 (2H, m) MASS (m/z): 355.2 (M++Na) Preparation 336 To a solution of 5-[4-(4-ethoxy-1-piperidyl)phenyl]-1,3,4-thiadiazol-2-amine (4.13 g) in ethanol (62 ml) was added ethyl 4-(bromoacetyl)benzoate (5.52 g).", "The mixture was stirred for 5 hours at 90° C. To the reaction mixture was added diisopropyl ether.", "The resulting precipitate was collected by filtration and washed by diisopropyl ether.", "To a solution of the crude in xylene (124 ml) was added trifluoroacetic acid (12 ml).", "The mixture was stirred for 6 hours at 130° C. To the reaction mixture was added diisopropyl ether.", "The resulting precipitate was collected by filtration and washed by diisopropyl ether to give ethyl 4-[2-[4-(4-ethoxy-1-piperidyl)phenyl]imidazo[2,1-b][1,3,4]-thiadiazol-6-yl]benzoate (6.864 g).", "NMR (CDCl3, δ): 1.27 (3H, t, J=7.0 Hz), 1.42 (3H, t, J=7.1 Hz), 1.95-2.15 (2H, m), 2.5-2.8 (2H, m), 3.35-3.5 (2H, m), 3.45 (2H, q, J=7.0 Hz), 3.65-3.8 (3H, m), 4.41 (2H, q, J=7.1 Hz), 7.7-8.0 (6H, m), 7.1-7.2 (3H, m) MASS (m/z): 477.2 (M++H) The following compounds [Preparation 337 and 338 were obtained according to a similar manner to that of Preparation 336.Preparation 337 Ethyl 4-[2-[4-(4-propoxy-1-piperidyl)phenyl]imidazo-[2,1-b][1,3,4]thiadiazol-6-yl]benzoate NMR (CDCl3, δ): 0.98 (3H, t, J=7.4 Hz), 1.42 (3H, t, J=7.1 Hz), 1.6-1.75 (2H, m), 1.95-2.15 (2H, m), 2.5-2.75 (2H, m), 3.35-3.55 (4H, m), 3.65-3.8 (3H, m), 4.41 (2H, q, J=7.1 Hz), 7.7-8.0 (6H, m), 8.1-8.2 (3H, m) Preparation 338 Ethyl 4-[2-[4-(4-butoxy-1-piperidyl)phenyl]imidazo-[2,1-b][1,3,4]thiadiazol-6-yl]benzoate NMR (CDCl3, δ): 0.96 (3H, t, J=7.2 Hz), 1.3-1.7 (7H, m), 1.9-2.1 (2H, m), 2.4-2.7 (2H, m), 3.3-3.9 (7H, m), 4.41 (2H, q, J=7.1 Hz), 7.6-8.0 (6H, m), 8.1-8.2 (3H, m) MASS (m/z): 505.4 (M++H) Preparation 339 A mixture of methyl ethyl 4-[2-[4-(4-ethoxy-1-piperidyl)phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoate (1 g) and 4 mol/l sodium hydroxide solution (10 ml) in a mixed solvent of methanol (20 ml) and tetrahydrofuran (10 ml) was refluxed for 5 hours.", "After cooling to ambient temperature, the reaction mixture was poured into cold water and the mixture was adjusted to pH 2 with 1.0 mol/l hydrochloric acid.", "The resulting precipitates were filtered, washed with water, isopropyl alcohol and diisopropyl ether, then dried to give 4-[2-[4-(4-ethoxy-1-piperidyl)phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoic acid (690.7 mg).", "MASS (m/z): 447.1 (M−−H) The following compounds [Preparation 340 and 341] were obtained according to a similar manner to that of Preparation 339.Preparation 340 4-[2-[4-(4-Propoxy-1-piperidyl)phenyl]imidazo[2,1-b]-[1,3,4]thiadiazol-6-yl]benzoic acid MASS (m/z): 461.2 (M−−H) Preparation 341 4-[2-[4-(4-Butoxy-1-piperidyl)phenyl]imidazo[2,1-b]-[1,3,4]thiadiazol-6-yl]benzoic acid MASS (m/z): 475.5 (M−−H) The Starting Compounds used and the Object Compounds obtained in the following Examples 1 to 79 are given in the table as below, in which the formulas of the starting compounds are in the upper column, and the formulas of the object compounds are in the lower column, respectively.", "Ex- ample No.", "Formula 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 EXAMPLE 1 A solution of Starting Compound (190 mg) in N,N-dimethylformamide (2 ml) was treated with 4-[4-[5-[4-(7-methoxyheptyloxy)phenyl]-1,3,4-thiadiazol-2-yl]-1-piperazinyl]benzoyl-1H-1,2,3-benzotriazole (100 mg) and stirred for 4 hours at ambient temperature.", "Piperidine (0.16 ml) was added the reaction mixture, and stirred for 2 hours at ambient temperature.", "Ethyl acetate (10 ml) was added, and resulting precipitate was collected, the precipitate was dissolved in a mixture of 10% acetonitrile in water and the solution was subjected to column chromatography on ODS (Daiso-gel, SP-120-40/60-ODS-B (Trademark: prepared by Daiso Co., Ltd.)) (60 ml) eluting with 40% acetonitrile in water.", "The fractions containing the object compound were collected and evaporated under reduced pressure to remove acetonitrile.", "The residue was lyophilized to give Object Compound (1).", "IR (KBr): 3361.3, 1666.2, 1631.5, 1610.3, 1511.9, 1234.2 cm−1 NMR (DMSO-d6+D2O, δ): 0.97 (3H, d, J=6.8 Hz), 1.08 (3H, d, J=6.0 Hz), 1.33-4.80 (59H, m), 6.70-7.87 (11H, m) ESI MASS (Negative)(m/z): 1455.6 (M+−H), 1456.6 (M+) Elemental Analysis Calcd.", "for C65H92N12O22S2.6H2O: C, 49.86; H, 6.69; N, 10.74 Found: C, 49.71; H, 6.70; N, 10.57 The following compounds [Example 2 to 62] were obtained according to a similar manner to that of Example 1.EXAMPLE 2 IR (KBr): 1664, 1628, 1444, 1431, 1408, 1269, 1192, 1043 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.11 (3H, d, J=5.8 Hz), 1.6-2.6 (7H, m), 2.8-4.6 (25H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.11 (1H, m), 7.3-7.6 (3H, m), 7.7-8.2 (10H, m), 8.36 (1H, s), 9.16 (1H, s) ESI MASS (Negative): 1286.3 (M−−H) Elemental Analysis Calcd.", "for C60H74N10O20S4.5H2O: C, 52.66; H, 6.11; N, 10.24 Found: C, 52.60; H, 6.09; N, 10.10 EXAMPLE 3 IR (KBr): 2933, 1659, 1628, 1547, 1462, 1444, 1246, 1196, 1041 cm−1 NMR (DMSO-d6+D2O, δ): 0.8-1.2 (9H, m), 1.2-1.6 (6H, m), 1.6-2.6 (9H, m), 2.8-4.5 (27H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.7-8.1 (6H, m), 8.26 (1H, s), 8.98 (1H, s) ESI MASS (Positive): 1333.2 (M++Na) Elemental Analysis Calcd.", "for C60H82N10O21S-4.5H2O: C, 51.75; H, 6.59; N, 10.06 Found: C, 51.75; H, 6.58; N, 10.04 EXAMPLE 4 IR (KBr): 3356, 2933, 1633, 1539, 1508, 1435, 1246, 1043 cm−1 NMR (DMSO-d6+D2O, δ): 0.89 (3H, t, J=6.6 Hz), 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=5.6 Hz), 1.2-1.5 (6H, m), 1.6-2.6 (9H, m), 2.8-4.5 (27H, m), 4.8-4.9 (2H, m), 6.7-6.9 (2H, m), 6.9-7.1 (3H, m), 7.65 (2H, d, J=8.6 Hz), 7.9-8.1 (4H, m), 8.21 (1H, s), 8.99 (1H, s) ESI MASS: 1333.3 (M++Na) (Positive), 1310.4 (M−−H) (Negative) Elemental Analysis Calcd.", "for C60H82N10O21S.6H2O: C, 50.77; H, 6.67; N, 9.87 Found: C, 50.72; H, 6.77; N, 9.87 EXAMPLE 5 IR (KBr): 2935, 1659, 1635, 1529, 1518, 1444, 1412, 1255, 1043 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.09 (3H, d, J=5.9 Hz), 1.2-2.6 (17H, m), 2.8-4.6 (26H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.14 (2H, d, J=8.9 Hz), 7.97 (2H, d, J=8.7 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1326.3 (M−−H) Elemental Analysis Calcd.", "for C59H78N10O21S2:.5H2O: C, 49.99; H, 6.26; N, 9.88 Found: C, 50.06; H, 6.14; N, 9.79 EXAMPLE 6 IR (KBr): 3356, 2931, 1630, 1529, 1516, 1439, 1271, 1217, 1043 cm−1 NMR (DMSO-d6+D2O, δ): 0.88 (3H, t, J=6.9 Hz), 0.97 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.8 Hz), 1.2-1.6 (8H, m), 1.6-2.6 (9H, m), 2.8-4.6 (27H, m), 4.7-4.9 (2H, m), 6.6-6.8 (2H, m), 7.0-7.1 (1H, m), 7.2-7.3 (1H, m), 7.3-7.4 (1H, m), 7.8-8.1 (3H, m), 8.43 (1H, s) ESI MASS: 1255.3 (M++Na) (Positive), 1232.4 (M−−H)(Negative) Elemental Analysis Calcd.", "for C56H80N8O21S.5H2O: C, 50.82; H, 6.85; N, 8.47 Found: C, 51.05; H, 6.48; N, 8.57 EXAMPLE 7 IR (KBr): 3352, 2933, 1630, 1531, 1516, 1441, 1271, 1236, 1217, 1043 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.09 (3H, d, J=5.7 Hz), 1.2-1.6 (8H, m), 1.6-2.5 (11H, m), 2.8-4.5 (32H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.2-7.3 (1H, m), 7.3-7.4 (1H, m), 7.8-8.0 (3H, m), 8.43 (1H, s) ESI MASS (Negative): 1276.4 (M−−H) Elemental Analysis Calcd.", "for C58H84N8O22S.4.5H2O: C, 51.28; H, 6.90; N, 8.25 Found: C, 51.37; H, 7.05; N, 8.28 EXAMPLE 8 IR (KBr): 3352, 1659, 1628, 1529, 1516, 1437, 1248, 1190, 1043 cm−1 NMR (DMSO-d6+D2O, δ): 0.8-1.2 (9H, m), 1.3-1.5 (4H, m), 1.6-2:6 (9H, m), 2.8-4.5 (27H, m), 4.8-4.9 (2H, m), 6.7-6.9 (3H, m), 7.0-7.1 (3H, m), 7.46 (1H, d, J=15.7 Hz), 7.6-7.8 (6H, m) ESI MASS: 1279.3 (M++Na) (Positive), 1256.3 (M−−H) (Negative) Elemental Analysis Calcd.", "for C58H80N8O21S.5H2O: C, 51.70; H, 6.73; N, 8.32 Found: C, 51.67; H, 6.81; N, 8.29 EXAMPLE 9 IR (KBr): 3356, 2929, 1633, 1539, 1514, 1495, 1437, 1257, 1043 cm−1 NMR (DMSO-d6+D2O, δ): 0.8-0.9 (3H, m), 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=6.2 Hz), 1.2-1.5 (10H, m), 1.6-2.5 (9H, m), 2.8-4.5 (27H, m), 4.8-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.18 (2H, d, J=8.9 Hz), 8.10 (4H, d, J=8.7 Hz), 8.22 (2H, d, J=8.5 Hz) ESI MASS (Negative): 1340.4 (M−−H) Elemental Analysis Calcd.", "for C61H84N10O22S.6H2O: C, 50.54; H, 6.68; N, 9.66 Found: C, 50.89; H, 6.71; N, 9.69 EXAMPLE 10 IR (KBr): 3356, 1633, 1543, 1516, 1489, 1452, 1439, 1271, 1248 cm−1 NMR (DMSO-d6+D2O, δ): 0.9-1.2 (9H, m), 1.6-2.6 (9H, m), 2.8-4.5 (27H, m), 4.8-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.75 (2H, d, J=8.7 Hz), 7.92 (2H, d, J=8.4 Hz), 8.12 (2H, d, J=8.5 Hz), 8.2-8.3 (4H, m) ESI MASS (Negative): 1246.4 (M−−H) Elemental Analysis Calcd.", "for C62H78N10O22S.6H2O: C, 51.16; H, 6.23; N, 9.62 Found: C, 51.06; H, 6.29; N, 9.58 EXAMPLE 11 IR (KBr): 3354, 2927, 1632, 1537, 1513, 1495, 1450, 1439, 1271, 1248 cm−1 NMR (DMSO-d6+D2O, δ): 0.8-0.9 (3H, m), 0.98 (3H, d, J=6.7 Hz), 1.09 (3H, d, J=5.8 Hz), 1.2-1.6 (12H, m), 1.6-2.6 (9H, m), 2.8-4.6 (27H, m), 4.8-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (3H, m), 7.6-7.8 (4H, m), 7.9-8.1 (2H, d, J=8.4 Hz) ESI MASS (Negative): 1286.4 (M−−H) Elemental Analysis Calcd.", "for C60H86N8O21S.5H2O: C, 52.31; H, 7.02; N, 8.13 Found: C, 52.27; H, 7.07; N, 8.14 EXAMPLE 12 IR (KBr): 3356, 2927, 1632, 1539, 1514, 1439, 1273, 1242, 1043 cm−1 NMR (DMSO-d6+D2O, δ): 0.8-0.9 (3H, m), 0.98 (3H, d, J=6.8 Hz), 1.09 (3H, d, J=6.1 Hz), 1.2-1.4 (8H, m), 1.5-2.6 (11H, m), 2.8-4.5 (25H, m), 4.8-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.31 (2H, d, J=8.2 Hz), 7.65 (2H, d, J=8.2 Hz), 7.75 (2H, d, J=8.3 Hz), 7.97 (2H, d, J=8.4 Hz) ESI MASS (Positive): 1265.3 (M++Na) Elemental Analysis Calcd.", "for C58H82N8O20S.5H2O: C, 52.24; H, 6.95; N, 8.40 Found: C, 52.35; H, 7.06; N, 8.43 EXAMPLE 13 IR (KBr): 1676, 1651, 1622, 1556, 1541, 1522, 1514, 1456 cm−1 NMR (DMSO-d6+D2O, δ): 0.8-1.2 (9H, m), 1.3-1.6 (2H, m), 1.6-2.6 (9H, m), 2.7-4.5 (27H, m), 4.8-4.9 (2H, m), 6.7-6.9 (3H, m), 7.0-7.1 (3H, m), 7.4-7.8 (7H, m) ESI MASS (Positive): 1265.3 (M++Na) Elemental Analysis Calcd.", "for C57H78N8O21S.6H2O: C 50.66, H 6.71, N 8.29 Found: C, 50.64; H, 6.67; N, 8.22 EXAMPLE 14 IR (KBr): 1676, 1649, 1632, 1554, 1539, 1514, 1456, 1439 cm−1 NMR (DMSO-d6+D2O, δ): 0.8-0.9 (3H, m), 0.97 (3H, d, J=6.8 Hz), 1.11 (3H, d, J=6.0 Hz), 1.2-1.4 (8H, m), 1.5-2.6 (9H, m), 2.4-4.6 (27H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.4-7.5 (1H, m), 7.75 (1H, s), 7.9-8.1 (3H, m), 8.46 (1H, s) ESI MASS (Positive): 1239.3 (M++Na) Elemental Analysis Calcd.", "for C56H80N8O20S.6H2O: C, 50.75; H, 7.00; N, 8.45 Found: C, 50.85; H, 6.79; N, 8.39 EXAMPLE 15 IR (KBr): 2935, 1666, 1649, 1632, 1541, 1504, 1454, 1437, 1273 cm−1 NMR (DMSO-d6+D2O, δ): 0.91 (3H, d, J=6.7 Hz), 0.98 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.5 Hz), 1.3-2.7 (20H, m), 2.8-4.5 (30H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.13 (2H, d, J=9.1 HZ), 7.97 (2H, d, J=8.8 Hz), 8.09 (2H, d, J=8.4 Hz), 8.20 (2H, d, J=8.6 Hz) ESI MASS (Negative): 1392.4 (M−−H) Elemental Analysis Calcd.", "for C64H88N12O21S.9H2O: C, 49.41; H, 6.87; N, 10.80 Found: C EXAMPLE 16 IR (KBr): 1649, 1632, 1603, 1541, 1514, 1450, 1514, 1450, 1439, 1275, 1228 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.8 Hz), 1.5-2.5 (12H, m), 2.7-4.7 (29H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (2H, m), 7.1-7.4 (5H, m), 7.9-8.2 (5H, m), 8.74 (1H, d, J=2.5 Hz) ESI MASS (Negative): 1388.4 (M−−H) Elemental Analysis Calcd.", "for C63H80N12O20S2.9.5H2O: C, 48.48; H, 6.39; N, 10.77 Found: C, 48.54; H, 6.20; N, 10.76 EXAMPLE 17 IR (KBr): 2931, 2856, 1676, 1651, 1608, 1556, 1541, 1514, 1452, 1441, 1419 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=5.6 Hz), 1.2-1.6 (10H, m), 1.6-2.6 (11H, m), 2.8-4.6 (37H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.85 (2H, d, J=8.8 Hz), 8.0-8.2 (4H, m) ESI MASS (Positive): 1478.4 (M++Na) Elemental Analysis Calcd.", "for C66H93N11O22S2.6H2O: C, 50.66; H, 6.76; N, 9.85 Found: C, 50.75; H, 6.77; N, 9.78 EXAMPLE 18 IR (KBr): 2927, 2856, 1678, 1651, 1556, 1541, 1514, 1456, 1439 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.0-1.2 (3H, m), 1.2-2.6 (23H, m), 2.6-4.6 (37H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.16 (2H, d, J=8.8 Hz), 7.97 (2H, d, J=8.7 Hz), 8.0-8.2 (4H, m) ESI MASS (Positive): 1471.4 (M++H) Elemental Analysis Calcd.", "for C67H95N11O22S2.5H2O: C, 51.56; H, 6.78; N, 9.87 Found: C, 51.53; H, 6.84; N, 9.69 EXAMPLE 19 IR (KBr): 3458, 3423, 3398, 3388, 3367, 2937, 1635, 1520, 1440, 1252 cm−1 NMR (DMSO-d6, δ): 0.80-1.00 (6H, m), 1.10 (3H, d, J=5.5 Hz), 1.20-1.50 (4H, m), 1.60-2.10 (7H, m), 2.20-2.50 (3H, m), 3.00-3.40 (4H, m), 3.40-4.60 (22H, m), 4.70-5.40 (10H, m), 6.60-6.80 (2H, m), 7.00 (1H, s), 7.08 (2H, d, J=8.8 Hz), 7.43 (1H, d, J=8.7 Hz), 7.55 (1H, d, J=8.9 Hz), 7.80 (2H, d, J=8.3 Hz), 7.80-8.05 (5H, m), 8.10-8.25 (2H, m), 8.25-8.40 (2H, m), 8.65-8.85 (2H, m) API-ES MASS (Negative): 1314 (M+), 1313 (M−−H), 1312 (M-2) Elemental Analysis Calcd.", "for C59H79N9O21S2.6H2O: C, 49.79; H, 6.40; N, 8.80 Found: C, 50.02; H, 6.41; N, 8.83 EXAMPLE 20 IR (KBr): 1676, 1651, 1632, 1556, 1541, 1524, 1514, 1452, 1441, 1419 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=6.1 Hz), 1.3-2.6 (15H, m), 2.7-4.5 (34H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.86 (2H, d, J=8.6 Hz), 8.0-8.2 (4H, m) ESI MASS (Positive): 1425.3 (M2++2 Na) Elemental Analysis Calcd.", "for C62H84N12O20S2.7H2O: C, 49.39; H, 6.55; N, 11.15 Found: C, 49.44; H, 6.43; N, 10.98 EXAMPLE 21 IR (KBr): 3444, 3421, 1699, 1678, 1651, 1558, 1541, 1524, 1514, 1456 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.7 Hz), 1.2-2.7 (20H, m), 2.7-4.5 (33H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.86 (2H, d, J=8.9 Hzl), 8.0-8.2 (4H, m) ESI MASS (Positive): 1454.5 (M2++2 Na) Elemental Analysis Calcd.", "for C64H88N12O20S2.7H2O: C, 50.05; H, 6.69; N, 10.94 Found: C, 50.29; H, 6.66; N, 10.81 EXAMPLE 22 IR (KBr): 3363, 1633, 1529, 1518, 1444, 1419, 1238, 1088, 1045 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.0-1.5 (11H, m), 1.6-2.7 (15H, m), 2.8-4.5 (42H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.02 (1H, br s), 7.08 (2H, d, J=9.0 Hz), 7.86 (2H, d, J=8.7 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1523.5 (M−−H) Elemental Analysis Calcd.", "for C70H100N12O22S2.7H2O: C, 50.90; H, 6.96; N, 10.18 Found: C, 50.70; H, 6.76; N, 10.04 EXAMPLE 23 IR (KBr): 3498, 3466, 3435, 1659, 1635, 1606, 1547, 1529, 1518, 1444, 1417 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz) 1.10 (3H, d, J=5.9 Hz), 1.2-2.6 (17H, m), 2.8-4.6 (40H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.87 (2H, d, J=8.7 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1440.5 (M−−H) Elemental Analysis Calcd.", "for C65H92N12O21S2.8H2: C, 49.23; H, 6.86; N, 10.60 Found: C, 49.54; H, 6.76; N, 10.41 EXAMPLE 24 IR (KBr): 3352, 1659, 1635, 1606, 1529, 1444, 1419, 1277, 1238 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=5.9 Hz), 1.2-2.8 (11H, m), 2.8-4.5 (38H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.87 (2H, d, J=8.8 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1396.4 (M−−H) Elemental Analysis Calcd.", "for C62H84N12O21S2.7H2O: C, 48.87; H, 6.48; N, 11.03 Found: C, 48.88; H, 6.50; N, 10.83 EXAMPLE 25 IR (KBr): 1659, 1628, 1606, 1529, 1444, 1417, 1281, 1240 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=5.7 Hz), 1.4-2.7 (15H, m), 2.7-4.5 (38H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.86 (2H, d, J=8.9 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1452.4 (M−−Na) Elemental Analysis Calcd.", "for C65H88N12O22S2.8H2O: C, 48.86; H, 6.56; N, 10.52 Found: C, 48.99; H, 6.47; N, 10.20 EXAMPLE 26 IR (KBr): 3464, 3429, 3373, 1659, 1628, 1606, 1529, 1444, 1419, 1281, 1238 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=5.8 Hz), 1.5-2.6 (7H, m), 2.8-4.5 (35H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.39 (2H, d, J=5.9 Hz), 7.87 (2H, d, J=8.8 Hz), 8.0-8.2 (4H, m), 8.53 (2H, d, j=5.8 Hz) ESI MASS (Negative): 1403.4 (M−−H) Elemental Analysis Calcd.", "for C63H81N13O20S2.8H2O: C, 48.86; H, 6.31; N, 11.76 Found: C, 49.02; H, 6.15; N, 11.42 EXAMPLE 27 IR (KBr): 3466, 3433, 3398, 2360, 2337, 1664, 1635, 1605, 1446, 1408, 1350 cm−1 ESI MASS (Negative): 1494.3 (M−−H) EXAMPLE 28 IR (KBr): 1664, 1628, 1605, 1529, 1444, 1417, 1279 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.9 Hz), 1.10 (3H, d, J=5.9 Hz), 1.6-2.5 (11H, m), 2.8-4.5 (32H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.15 (2H, d, J=8.8 Hz), 7.4-7.5 (4H, m), 7.87 (2H, d, J=8.8 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1452.3 (M−−H) Elemental Analysis Calcd.", "for C65H82N11O21S2.7H2O: C, 49.44; H, 6.13; N, 9.76 Found: C, 49.80; H, 6.06; N, 9.56 EXAMPLE 29 IR (KBr): 1645, 1632, 1608, 1539, 1514, 1443, 1419, 1273, 1232 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=5.9 Hz), 1.6-2.6 (11H, m), 2.7-4.5 (33H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.12 (2H, d, J=8.8 Hz), 7.86 (2H, d, J=8.9 Hz), 8.0-8.2 (4H, m) ESI MASS (Positive): 1392.3 (M++Na) Elemental Analysis Calcd.", "for C60H79N11O22S2.6H2O: C, 48.74; H, 6.20; N, 10.42 Found: C, 48.37; H, 6.25; N, 10.19 EXAMPLE 30 IR (KBr): 1649, 1633, 1608, 1539, 1512, 1450, 1443, 1419, 1238 cm−1 NMR (DMSO-d6+D2O, δ): 0.97 (3H, d, J=6.8 Hz), 1.11 (3H, d, J=5.2 Hz), 1.4-2.6 (7H, m), 2.7-4.6 (31H, m), 4.7-4.9 (2H, m), 6.6-6.9 (2H, m), 7.0-7.1 (1H, m), 7.21 (2H, d, J=9.2 Hz), 7.3-7.7 (6H, m), 7.90 (2H, d, J=8.7 Hz), 8.0-8.2 (4H, m) ESI MASS (Positive): 1470.2 (M2++2 Na) Elemental Analysis Calcd.", "for C65H79N13O20S2.7.5H2O: C, 49.99; H, 6.07; N, 11.66 Found: C, 49.90; H, 5.97; N, 11.34 EXAMPLE 31 ESI MASS (Negative): 1539.6 (M−−H) EXAMPLE 32 IR (KBr): 1666, 1649, 1632, 1554, 1541, 1514, 1450, 1441, 1254 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=6.0 Hz), 1.6-2.6 (11H, m), 2.8-4.5 (29H, m), 4.6-4.8 (3H, m), 6.7-6.9 (2H, m), 6.9-7.1 (3H, m), 7.1-7.3 (4H, m), 7.9-8.2 (7H, m) ESI MASS (Negative): 1403.4 (M−−H) Elemental Analysis Calcd.", "for C64H81N11O21S2.6.5H2O: C, 50.52; H, 6.23; N, 10.13 Found: C, 50.47; H, 6.19; N, 9.98 EXAMPLE 33 IR (KBr): 1676, 1649, 1632, 1556, 1541, 1514, 1452, 1441, 1250 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=5.9 Hz), 1.6-2.6 (11H, m), 2.8-4.8 (33H, m), 4.8-4.9 (2H, m), 6.6-7.1 (7H, m), 7.21 (2H, d, J=8.9 Hz), 7.99 (2H, d, J=8.7 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1433.4 (M−−Na) Elemental Analysis Calcd.", "for C65H83N11O22S2.6.5H2O: C, 50.31; H, 6.24; N, 9.93 Found: C, 50.22; H, 6.23; N, 9.81 EXAMPLE 34 IR (KBr): 3492, 3471, 3431, 3396, 1664, 1628, 1606, 1446, 1254 cm−1 NMR (DMSO-d6+D2O, δ): 0.9-1.3 (12H, m), 1.3-2.6 (15H, m), 2.8-4.6 (31H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.2-7.4 (2H, m), 7.9-8.2 (6H, m) ESI MASS: 1432.3 (M++Na)(Positive), 1409.6 (M−−H)(Negative) Elemental Analysis Calcd.", "for C64H87N11O21S2.7H2O: C, 50.02; H, 6.62; N, 10.03 Found: C, 50.00; H, 6.69; N, 9.85 EXAMPLE 35 IR (KBr): 2933, 2862, 1697, 1676, 1651, 1556, 1541, 1514, 1454, 1439 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.6 Hz), 1.0-1.2 (3H, m), 1.2-2.6 (22H, m), 2.6-4.5 (36H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.49 (2H, d, J=8.2 Hz), 7.98 (2H, d, J=8.4 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1439.6 (M−−H) Elemental Analysis Calcd.", "for C66H93N11O21S2.6H2O: C, 51.18; H, 6.83; N, 9.95 Found: C, 51.19; H, 6.89; N, 9.88 EXAMPLE 36 ESI MASS (Negative): 1523.6 (M−−H) EXAMPLE 37 IR (KBr): 3494, 3466, 3433, 3394, 2935, 1659, 1628, 1531, 1444, 1279 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.0-2.6 (26H, m), 2.7-4.5 (43H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.48 (2H, d, J=8.4 Hz), 7.9-8.2 (6H, m), 7.0-7.1 (1H, m), 7.48 (2H, d, J=8.4 Hz), 7.9-8.2 (6H, m) ESI MASS (Negative): 1523.6 (M−−H) Elemental Analysis Calcd.", "for C71H101N11O22S2.8H2O: C, 51.10; H, 7.07; N, 9.23 Found: C, 51.32; H, 7.04; N, 9.14 EXAMPLE 38 IR (KBr): 2935, 1632, 1608, 1535, 1516, 1464, 1439, 1248, 1084, 1045 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.09 (3H, d, J=5.8 Hz), 1.2-1.6 (11H, m), 1.6-2.6 (7H, m), 2.7-4.5 (38H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.75 (2H, d, J=8.8 Hz), 7.9-8.0 (4H, m), 8.78 (1H, s) ESI MASS (Negative): 1480.5 (M−−H) Elemental Analysis Calcd.", "for C67H92N12O22S2.6H2O: C, 50.62; H, 6.59; N, 10.57 Found: C, 50.51; H, 6.65; N, 10.46 EXAMPLE 39 IR (KBr): 1659, 1635, 1606, 1529, 1518, 1466, 1446, 1277, 1248 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.09 (3H, d, J=6.1 Hz), 1.1-2.6 (16H, m), 2.7-4.5 (38H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.75 (2H, d, J=8.6 Hz), 7.9-8.0 (4H, m), 8.77 (1H, s) ESI MASS (Negative): 1466.5 (M−−H) Elemental Analysis Calcd.", "for C66H90N12O22S2.6H2O: C, 50.31; H, 6.52; N, 10.67 Found: C, 54.61; H, 6.18; N, 11.45 EXAMPLE 40 IR (KBr): 3464, 3435, 3394, 1659, 1628, 1529, 1514, 1468, 1444, 1250 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.8 Hz), 1.5-2.6 (11H, m), 2.8-4.7 (33H, m), 4.7-4.9 (2H, m), 6.6-7.1 (7H, m), 7.22 (2H, d, J=8.9 Hz), 7.8-8.1 (6H, m), 8.83 (1H, s) ESI MASS (Negative): 1472.4 (M−−H) Elemental Analysis Calcd.", "for C67H84N12O22S2.7H2O: C, 50.30; H, 6.17; N, 10.51 Found: C, 50.12; H, 6.19; N, 10.33 EXAMPLE 41 IR (KBr): 3494, 3465, 3433, 3367, 1659, 1628, 1529, 1518, 1468, 1444, 1254 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.11 (3H, d, J=5.5 Hz), 1.6-2.6 (11H, m), 2.8-4.6 (30H, m), 4.7-4.9 (2H, m), 6.7-6.9 (3H, m), 6.9-7.1 (3H, m), 7.2-7.4 (4H, m), 7.8-8.1 (6H, m), 8.83 (1H, s) ESI MASS: 1465.3 (M++Na) (Positive), 1442.4 (M−−H) (Negative) Elemental Analysis Calcd.", "for C66H82N12O21S2.7.5H2O: C, 50.21; H, 6.19; N, 10.65 Found: C, 50.37; H, 6.23; N, 10.54 EXAMPLE 42 IR (KBr): 1666, 1649, 1632, 1554, 1539, 1516, 1466, 1458, 1250 cm−1 NMR (DMSO-d6+D2O, δ): 0.97 (3H, d, J=6.8 Hz), 1.0-1.3 (9H, m), 1.5-2.6 (15H, m), 2.7-4.6 (31H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.16 (2H, d, J=8.9 Hz), 7.89 (2H, d, J=9.0 Hz), 7.9-8.1 (4H, m), 8.83 (1H, s) ESI MASS (Negative): 1448.5 (M−−H) Elemental Analysis Calcd.", "for C66H88N12O21S2.8H2O: C, 49.74; H, 6.58; N, 10.55 Found: C, 54.68; H, 6.12; N, 11.59 EXAMPLE 43 IR (KBr): 1676, 1649, 1633, 1556, 1541, 1514, 1471, 1458, 1435 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.0-2.6 (22H, m), 2.7-4.5 (30H, m), 4.7-4.9 (2H, m), 6.3-6.5 (1H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.6-9.2 (9H, m) ESI MASS (Positive): 1475.7 (M2++2 Na) Elemental Analysis Calcd.", "for C66H86N12O20S2.8H2O: C, 50.31; H, 6.52; N, 10.67 Found: C, 50.55; H, 6.30; N, 10.58 EXAMPLE 44 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.9 Hz), 1.2-2.5 (19H, m), 2.7-4.5 (37H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.50 (2H, d, J=8.6 Hz), 7.8-8.1 (6H, m), 8.86 (1H, s) ESI MASS (Negative): 1464.4 (M−−H) EXAMPLE 45 IR (KBr): 3493, 3469, 3435, 1664, 1635, 1606, 1446 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.6 Hz), 1.09 (3H, d, J=6.0 Hz), 1.2-2.6 (13H, m), 2.8-4.5 (40H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.78 (2H, d, J=8.7 Hz), 7.9-8.0 (4H, m), 8.78 (1H, s) ESI MASS (Negative): 1451.4 (M−−H) Elemental Analysis Calcd.", "for C65H89N13O2lS2.7H2O: C, 49.45; H, 6.58; N, 11.53 Found: C, 49.34; H, 6.64; N, 11.18 EXAMPLE 46 IR (KBr): 3464, 3425, 3386, 3365, 2935, 1635, 1614, 1523 cm−1 NMR (DMSO-d6, δ): 0.97 (3H, d, J-6.8 Hz), 1.10 (3H, d, J-5.7 Hz), 1.20-1.40 (4H, m), 1.40-1.60 (5H, m), 1.70-2.10 (4H, m), 2.20-2.40 (6H, m), 2.80-3.00 (1H, m), 3.22 (3H, s), 3.40-4.50 (18H, m), 4.70-5.10 (4H, m), 5.10-5.40 (6H, m), 6.71 (1H, d, J=8.1 Hz), 6.70-6.90 (1H, m), 7.00 (1H, br s), 7.08 (2H, d, J=9.0 Hz), 7.40-7.60 (2H, m), 7.77 (2H, d, J=8.8 Hz), 7.80-8.00 (6H, m), 8.20-8.40 (1H, m), 8.60-8.80 (2H, m), 8.80 (1H, s) API-ES MASS (Negative): 1466(M), 1465(M+), 1464(M−−2) Elemental Analysis Calcd.", "for C66H91N13O21S2.6H2O: C, 50.32; H, 6.54; N, 11.56 Found: C, 50.38; H, 6.66; N, 11.43 EXAMPLE 47 IR (KBr): 1658, 1635, 1606, 1529, 1518, 1468, 1446, 1431, 1238 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.9 Hz), 1.09 (3H, d, J=5.5 Hz), 1.2-2.6 (17H, m), 2.8-4.5 (40H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.78 (2H, d, J=8.7 Hz), 7.9-8.1 (4H, m), 8.78 (1H, s) ESI MASS (Negative): 1479.4 (M−−H) Elemental Analysis Calcd.", "for C67H93N13O21S2.6H2O: C, 50.65; H, 6.66; N, 11.46 Found: C, 50.82; H, 6.90; N, 11.17 EXAMPLE 48 IR (KBr): 3464, 3460, 3425, 3400, 3367, 2939, 1633, 1522, 1454, 1248 cm−1 NMR (DMSO-d6, δ): 0.98 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.6 Hz), 1.30-2.00 (10H, m), 3.23 (3H, s), 2.80-4.45 (20H, m), 4.60-5.40 (10H, m), 6.60-6.80 (1H, m), 6.71 (1H, d, J=8.1 Hz), 7.00 (1H, s), 7.04 (2H, d, J=8.9 Hz), 7.46 (1H, m), 7.60 (2H, d, J=8.7 Hz), 7.70-8.00 (3H, m), 8.37 (1H, br s), 8.71 (1H, s) API-ES MASS (Negative): 1383(M), 1382(M−−H), 1381(M−−2) Elemental Analysis Calcd.", "for C62H82N10O22S2.10H2O: C, 47.60; H, 6.52; N, 8.96 Found: C, 47.36; H, 6.12; N, 8.78 EXAMPLE 49 IR (KBr): 1632, 1539, 1514, 1452, 1236 cm−1 NMR (DMSO-d6, δ): 0.98 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.6 Hz), 1.6-2.6 (9H, m), 2.8-4.6 (35H, m), 4.7-5.4 (9H, m), 6.65-7.05 (7H, m), 7.11 (2H, d, J=8.8 Hz), 7.3-8.0 (9H, m), 8.0-8.45 (3H, m), 8.6-8.8 (2H, m) MASS (m/z): 1333 (M+−H) Elemental Analysis Calcd.", "for C62H82N10O21S-9H2O: C, 49.73; H, 6.73; N, 9.35 Found: C, 49.78; H, 6.54; N, 9.60 EXAMPLE 50 IR (KBr): 1666, 1649, 1632, 1539, 1512, 1452, 1232 cm−1 NMR (DMSO-d6, δ): 0.97 (2H, d, J=6.6 Hz), 1.0-2.6 (29H, m), 2.6-4.6 (40H, m), 4.7-5.4 (9H, m), 6.65-7.2 (9H, m), 7.4-8.05 (9H, m), 8.1-8.8 (5H, m) MASS (m/z): 1516 (M+−H) Elemental Analysis Calcd.", "for C73H103N11O22S-7H2O: C, 53.31; H, 7.17; N, 9.37 Found: C, 53.18; H, 7.14; N, 9.56 EXAMPLE 51 NMR (DMSO-d6, δ): 0.97 (3H, d, J=6.9 Hz), 0.9-1.4 (8H, m), 1.4-2.7 (18H, m), 2.8-4.6 (29H, m), 4.7-5.5 (9H, m), 6.6-7.1 (5H, m), 7.4-8.4 (12H, m), 8.5-8.8 (2H, m) MASS (m/z): 1321 (M+−H) EXAMPLE 52 IR (KBr): 1668, 1649, 1632, 1539, 1514, 1456, 1238 cm−1 NMR (DMSO-d6, δ): 0.90 (3H, d, J=6.8 Hz), 0.97 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=5.9 Hz), 1.3-2.75 (23H, m), 2.8-4.6 (28H, m), 4.7-5.4 (9H, m), 6.6-7.2 (5H, m), 7.3-8.5 (12H, m), 8.6-8.8 (2H, m) MASS (m/z): 1325 (M++H) Elemental Analysis Calcd.", "for C62H88N10O20S-9H2O: C, 50.06; H, 7.18; N, 9.42 Found: C, 50.14; H, 7.11; N, 9.36 EXAMPLE 53 IR (KBr): 1645, 1632, 1539, 1514, 1454, 1236 cm−1 NMR (DMSO-d6, δ): 0.86 (3H, d, J=6.4 Hz), 0.97 (3H, d, J=6.8 Hz), 1.0-1.4 (8H, m), 1.6-3.0 (19H, m), 3.1-3.6 (27H, m), 3.7-5.4 (9H, m), 6.7-7.1 (5H, m), 7.3-8.8 (14H, m) MASS (m/z): 1325 (M++H) Elemental Analysis Calcd.", "for C62H88N10O20S-9H2O: C, 50.06; H, 7.18; N, 9.42 Found: C, 50.33; H, 7.13; N, 9.37 EXAMPLE 54 IR (KBr): 1664, 1628, 1605, 1547, 1531, 1497, 1446, 1277 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.09 (3H, d, J=5.7 Hz), 1.6-4.5 (41H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.2-7.4 (5H, m), 7.63 (2H, d, J=8.8 Hz), 7.71 (2H, d, J=8.3 Hz), 7.94 (2H, d, J=8.4 Hz) ESI MASS (Negative): 1303.3 (M−−H) Elemental Analysis Calcd.", "for C62H81N9O20S.6H2O: C, 52.72; H, 6.64; N, 8.92 Found: C, 52.90; H, 6.71; N, 8.82 EXAMPLE 55 IR (KBr): 1659, 1628, 1605, 1529, 1444, 1417, 1277, 1228 cm−1 NMR (DMSO-d6+D2O, δ): 0.89 (3H, t, J=7.2 Hz), 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=6.0 Hz), 1.2-1.6 (4H, m), 1.7-2.6 (11H, m), 2.8-4.5 (32H, m), 4.7-4.9 (2H, m), 6.6-6.8 (2H, m), 7.0-7.1 (1H, m), 7.09 (2H, d, J=9.0 Hz), 7.85 (2H, d, J=8.7 Hz), 7.9-8.2 (4H, m) ESI MASS (Negative): 1383.4 (M−−H) Elemental Analysis Calcd.", "for C62H85N11O21S2.7H2O: C, 49.29; H, 6.61; N, 10.20 Found: C, 49.69; H, 6.37; N, 10.29 EXAMPLE 56 IR (KBr): 1664, 1628, 1605, 1529, 1444, 1417, 1277, 1228 cm−1 NMR (DMSO-d6+D2O, δ): 0.87 (3H, t, J=6.7 Hz), 0.98 (3H, d, J=6.6 Hz), 1.10 (3H, d, J=5.8 Hz), 1.2-1.6 (6H, m), 1.7-2.6 (11H, m), 2.8-4.5 (32H, m), 4.7-4.9 (2H, m), 6.6-6.8 (2H, m), 7.0-7.1 (1H, m), 7.09 (2H, d, J=8.9 Hz), 7.85 (2H, d, J=8.7 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1396.4 (M2—2H) Elemental Analysis Calcd.", "for C63H87N11O21S2.7H2O: C, 49.63; H, 6.68; N, 10.11 Found: C, 49.82; H, 6.55; N, 10.10 EXAMPLE 57 IR (KBr): 1664, 1635, 1605, 1446, 1412, 1350, 1281, 1043 cm−1 NMR (DMSO-d6+D2O, δ): 0.86 (6H, d, J=6.5 Hz), 0.98 (3H, d, J=6.8 Hz), 1.1-1.3 (5H, m), 1.4-2.6 (14H, m), 2.8-4.5 (32H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.85 (2H, d, J=8.6 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1410.3 (M2—2H) Elemental Analysis Calcd.", "for C64H89N11O21S2.7H2O: C, 49.96; H, 6.75; N, 10.01 Found: C, 49.89; H, 6.53; N, 9.92 EXAMPLE 58 IR (KBr): 1662, 1628, 1605, 1529, 1444, 1417, 1277, 1227, 1043 cm−1 NMR (DMSO-d6+D2O, δ): 0.8-1.4 (12H, m), 1.4-2.6 (16H, m), 2.8-4.5 (32H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.1 (1H, m), 7.09 (2H, d, J=9.1 Hz), 7.84 (2H, d, J=8.8 Hz), 8.0-8.2 (4H, m) ESI MASS (Negative): 1423.5 (M−−H) Elemental Analysis Calcd.", "for C65H89N11O21S2.7H2O: C, 50.34; H, 6.69; N, 9.94 Found: C, 50.75; H, 6.56; N, 9.96 EXAMPLE 59 IR (KBr): 2360, 1662, 1635, 1606, 1529, 1466, 1446, 1240 cm−1 NMR (DMSO-d6+D2O, δ): 0.8-1.3 (12H, m), 1.5-2.6 (16H, m), 2.8-4.5 (32H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 7.0-7.2 (3H, m), 7.75 (2H, d, J=8.8 Hz), 7.9-8.0 (4H, m), 8.78 (1H, s) ESI MASS (Negative): 1462.5 (M−−H) Elemental Analysis Calcd.", "for C67H90N12O21S2.8H2O: C, 50.05; H, 6.65; N, 10.45 Found: C, 50.15; H, 6.38; N, 10.45 EXAMPLE 60 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.5 Hz), 1.2-2.6 (15H, m), 2.8-4.5 (48H, m), 4.7-4.9 (2H, m), 6.7-7.0 (6H, m), 7.0-7.2 (3H, m), 7.6-7.8 (4H, m), 7.94 (2H, d, J=8.1 Hz) ESI MASS (Negative): 1502.6 (M−−H) EXAMPLE 61 IR (KBr): 1664, 1635, 1605, 1589, 1446, 1408, 1350 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.09 (3H, d, J=5.7 Hz), 1.2-2.8 (25H, m), 2.8-4.5 (40H, m), 4.7 4.9 (2H, m), 6.7-6.9 (2H, m), 6.9-7.1 (3H, m), 7.63 (2H, d, J=8.8 Hz), 7.71 (2H, d, J=8.8 Hz), 7.92 (2H, d, J=8.0 Hz) ESI MASS (Negative): 1452.6 (M−−H) EXAMPLE 62 IR (KBr): 2972, 1664, 1628, 1606, 1446, 1279, 1240, 1082, 1047 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.7 Hz), 1.09 (3H, d, J=5.9 Hz), 1.2-2.6 (25H, m), 2.8-4.5 (40H, m), 4.7-4.9 (2H, m), 6.7-6.9 (2H, m), 6.9-7.1 (3H, m), 7.63 (2H, d, J=8.8 Hz), 7.71 (2H, d, J=8.3 Hz), 7.93 (2H, d, J=8.3 Hz) ESI MASS (Negative): 1452.6 (M−−H) Elemental Analysis Calcd.", "for C69H100N10O22S.7H2O: C, 52.46; H, 7.27; N, 8.87 Found: C, 57.01; H, 6.93; N, 9.64 EXAMPLE 63 To a solution of Starting Compound (190 mg) was added 1-[4-[2-[4-[4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-piperidyloxy]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole (120 mg) and hunings base (0.042 ml) and the mixture was stirred for 16 hours.", "1-[4-[2-[4-[4-[1-[4-(6-methoxyhexyloxy)cyclohexyl]-piperidyloxy]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy]-1H-1,2,3-benzotriazole (240 mg) was added to the above mixture to complete the reaction.", "After stirring for 5 hours, EtOAc (100 ml) was added dropwise to the solution to give crude precipitation.", "Usual workup followed by preparative liquid chromatography (ODS, CH3CN: H2O=40:60) gave, in order of elution, Minor Compound (63) (11 mg) and Major Compound (63) (23 mg), which were used in the next reaction without further purification.", "The following compound was obtained according to a similar manner to that of Example 63.EXAMPLE 64 MASS (m/z): 1545 (M+−H) EXAMPLE 65 To a solution of Minor Compound (63) (11 mg) was added piperidine (0.006 ml) and the solution was stirred for 1 hour.", "EtOAc (100 ml) was added dropwise to the above solution to give crude precipitation, which was collected and usual workup followed by chromatography (ODS, CH3CN:H2O=50:50) gave Object Compound (65) (1 mg).", "ESI MASS (Negative): 1578.6 (M−−H) The following compounds [Example 66: and 67] were obtained according to a similar manner to that of Example 65.EXAMPLE 66 ESI MASS (Negative): 1578.8 (M−−H) EXAMPLE 67 MASS (m/z): 1323 (M+−H) EXAMPLE 68 To a solution of 4-[5-[4-[4-(cis-4-methylcyclohexyl)piperazinyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoic acid (8.29 kg) in N-methyl-2-pyrrolidone (140 l) were added N,N-diisopropylethylamine (DIPEA) (4.0 kg) and 0-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) (7.05 kg) at room temperature, and the mixture was stirred at 40-50° C. for 2.5 hours.", "After cooling to 20° C., DIPEA (2.0 kg) and Starting Compound (68) (14.0 kg) were added and stirring was continued at 25-30° C. for 2 hours.", "The resulting mixture was poured into water (980 l) at 30-35° C. for 1 hour and stirred for 0.5 hour.", "The resulting crystals were filtered and washed with water (140 l).", "The crystals were dried overnight in vacuo to give Object Compound (68) (20.6 kg).", "The product was used in the next step without further purification.", "IR (KBr): 1676, 1645, 1635, 1630, 1533, 1515, 1446, 1439, 1425 cm−1 NMR (DMSO-d6, δ): 0.90-1.26 (12H, m), 0.96 (6H, d, J=7.0 Hz), 1.10 (3H, d, J=5.8 Hz), 1.23-5.37 (61H, m), 6.41-9.15 (17H, m) ESI MASS (m/z)(Negative): 1348.4 (M-DIPEA+) EXAMPLE 69 To a solution of Starting Compound (69) (17.0 kg) in N-methyl-2-pyrrolidone (85 l) were added pyridine (3.87 kg) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (8.82 kg) with stirring at 0-10° C. After stirring for 20 hours at 50-60° C., the mixture was added to water (850 l).", "The pH value of the mixture were maintained at 4-5 during the addition with 1N—HCl (about 50 l) at 20-30° C. and the stirring was kept for 1 hour.", "The pH of the suspension was raised to 10.4-10.6 with 1N-NaOH (55 l) and then the suspension was heated to 40-45° C. and stirred for 7 hours.", "The resulting solution was cooled to 20-30° C. and 1N—HCl was added to the pH range 6.5-7.0 to precipitate the product.", "After stirring for 1 hour, the precipitate was filtered and washed with water (170 l).", "The precipitate without drying was dissolved in alkaline water (pH 9.5-9.7: about 700 l) and acetonitrile (170 l) mixture and the solution was chromatographed on HP20SS (Mitsubishi Chemical Corporation) (340 l) using 20-40% aqueous acetonitrile as an eluting solvent to be fractionated.", "The fractions that contained desired product were combined, adjusted to pH 6.8-7.0 with 1N—HCl and concentrated under reduced pressure at 30-45° C. until 1500 l. The condensed solution was adjusted to pH 5.5-5.7 with 1N—HCl and the resulting yellow suspension was stirred at 30-35° C. for 30 minutes and at 15-20° C. for 1 hour.", "Yellow precipitates were filtered, washed with water (85 l) and dried at 35-45° C. for 18 hours.", "Object Compound (69) was obtained as yellow powder (8.42 kg).", "IR (KBr): 1645, 1635, 1533, 1516, 1446, 1269, 1200 cm−1 NMR (DMSO-d6, δ): 0.96 (6H, d, J=6.9 Hz), 1.11 (3H, d, J=5.5 Hz), 1.20-5.85 (52H, m), 6.41-9.15 (17H, m) ESI MASS (m/z)(Negative): 1330.4 (M+) EXAMPLE 70 To a solution of tetrahydrofuran (160 l), water (40 l), 25% aqueous NH3 solution (26.4 l) and Starting Compound (70) (6.4 kg) was added a slurry of tetrahydrofuran (20 l), water (51) and Rh/Al2O3 (5% Rh, 6.4 kg).", "The resulting black slurry was treated with hydrogen (4.0 kg/cm2) at 30° C. for 35.5 hours.", "After completion of the reaction, the reaction mixture was filtered through a pad of KC-floc (powder of cellulose 2 kg), and washed with a mixture of tetrahydrofuran (51 l) and water (13 l).", "The filtrate was concentrated at 30-40° C. under reduced pressure to 80 l and yellow crystals precipitated in the residual solution.", "The precipitates were re-dissolved at pH 9.5-10.0 with 4N-NaOH (about 25 l) at 35-40° C. The pH of the solution was adjusted to 6.4-6.6 by the slow addition of 1N-HCl (2bout 24 l) at 35-40° C. to precipitate the product.", "It took more than 30 minutes to adjust the pH.", "After stirring for more than 30 minutes at 15-20° C., the precipitates were filtered with centrifuge, washed and dried at 35-45° C. under reduced pressure for 15 hours.", "Object Compound (70) was obtained as yellow powder (7.22 kg).", "IR (KBr): 1645, 1635, 1630, 1533, 1516, 1446, 1269, 1240 cm−1 NMR (DMSO-d6, δ): 0.90 (3H, d, J=6.8 Hz), 0.98 (3H, d, J=6.8 Hz), 1.11 (3H, d, J=5.6 Hz), 1.43-5.22 (56H, m), 6.69-8.92 (17H, m) ESI MASS (m/z)(Negative): 1334.5 (M+) EXAMPLE 71 To a solution of dimethylformamide (47 l), dihydroxyacetone (1.06 kg) and Starting Compound (71) (5.2 kg) was added a slurry of Pt/C (5% Pt, 1.04 kg) in dimethylformamide (5 l).", "The resulting black slurry was treated with hydrogen (4.0 kg/cm2) at 30° C. for 34 hours.", "After completion of the reaction, methanol (52 l) was added under N2 atmosphere, and the reaction mixture was filtered through a pad of KC-floc (powder of cellulose, 2 kg) and washed with dimethylformamide (10 l).", "The filtrate was added slowly to acetonitrile (745 l) in 1000-liter reactor.", "The mixture was stirred for 0.5 hour, and resulting precipitate was filtered and washed with acetonitrile (26 l).", "The precipitate was dried overnight in vacuo to give crude Object Compound (71) (5.07 kg).", "The crude-Object Compound (71) (4.90 kg) and water (260 l) were stirred for 0.5 hour, and the precipitate was filtered and washed with water (52 l).", "The precipitate was dissolved in a mixture of water (104 l) and 1N sodium hydroxide and the solution was subjected to column chromatography on adsorption resin (HP20SS (Trademark: prepared by Mitsubishi Chemical Co., Ltd.)) (260 l) eluting with 75% methanol in water.", "The fractions containing the object compound were collected and evaporated under reduced pressure to remove methanol.", "The suspension was stirred at 5° C. for 1 hour, and resulting precipitate was filtered and washed with water (140 l).", "The precipitate was dried overnight in vacuo to give pure-Object Compound (71) (1.36 kg).", "IR (KBr): 1645, 1635, 1630, 1533, 1516, 1446, 1425, 1271, 1238 cm−1 NMR (DMSO-d6, δ): 0.90 (3H, d, J=6.8 Hz), 0.98 (3H, d, J=6.8 Hz), 1.11 (3H, d, J=5.6 Hz), 1.43-5.23 (62H, m), 6.69-8.88 (17H, m) ESI MASS (m/z) (Negative): 1408.5 (M+) EXAMPLE 72 To a suspension of Starting Compound (72) (100 mg) in N,N-dimethylformamide (1 ml) was added 4-[2-[4-[4-(6-methoxyhexyloxy)piperadin-1-yl]phenyl]imidazo[2,1-b][1,3,4]thiadiazol-6-yl]benzoyloxy-1H-1,2,3-benzotriazole (53.6 mg) and N,N-diisopropylethylamine (22 μl), and stirred for overnight at ambient temperature.", "To the reaction mixture was added piperidine (83.3 μl), and stirred for 3.5 hours at ambient temperature, then added ethyl acetate (100 ml).", "The resulting precipitate was collected by filtration, washed with diisopropylethylether (10 ml) to give a crude yellow powder (124.3 mg).", "The crude powder was purified by column chromatography on ODS (Daisogel SP-120 (40/60 μm)-ODS-B (Trademark: prepared by Daiso Co., Ltd.)) (40% acetonitrile aqueous solution).", "The fractions containing the object compound were combined, and evaporated under reduced pressure to remove acetonitrile.", "The residue was lypophilized to give Object Compound (72) (42.6 mg).", "IR (KBr): 3353.6, 1631.1, 1606.4, 1517.7, 1463.7, 1436.7, 1268.9, 1228.4, 1195.6, 1087.7, 1045.2 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=6.1 Hz), 1.2-1.45 (4H, m), 1.4-1.6 (6H, m), 1.6-4.9 (48H, m), 6.73 (1H, d, J=8.0 Hz), 6.75-6.85 (1H, m), 7.03 (1H, d, J=1.7 Hz), 7.09 (2H, d, J=9.1 Hz), 7.76 (2H, d, J=8.8 Hz), 7.94 (2H, d, J=8.8 Hz), 7.97 (2H, d, J=8.6 Hz), 8.76 (1H, s) MASS (m/z): 1479.4 (M−−H) EXAMPLE 73 A solution of Starting Compound (73) (500 mg) 1-[4-[5-[4-(7-methoxyheptyloxy)phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole (240 mg) and N,N-diisopropylethylamine (0.11 ml) in DMF (5 ml) was stirred at room temperature for 5.5 hours.", "To the reaction mixture was added piperizine (0.42 ml) and stirred at room temperature for 1 hour.", "To the reaction mixture was added ethyl acetate (50 ml).", "The resulting precipitate was collected by filtration.", "The precipitate was dissolved in 20% acetonitrile in water (10 ml), and the solution was subjected to column chromatography on ODS (Daiso-gel, SP-120-40/60-ODS-B (Trademark: prepared by Daiso Co., Ltd.)) (25 ml) eluting with 35% acetonitrile in water.", "The fractions containing the object compound were collected and evaporated under reduced pressure to remove acetonitrile.", "The residue was lyophilized to give Object Compound (73) (500 mg).", "IR (KBr): 3342.0, 1631.5, 1515.8, 1442.5, 1257.4 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=5.9 Hz), 1.33-4.83 (51H, m), 6.71-8.14 (11H, m) ESI MASS (m/z)(Negative): 1372.4, 1371.4 (M−−H) Elemental Analysis Calcd.", "for C61H84N10O22S2.5H2O: C, 50.06; H, 6.47; N, 9.57 Found: C 49.83, H 6.71, N 9.49 EXAMPLE 74 A solution of Starting Compound (74) (100 mg), 1-[4-[5-[4-[4-(2-ethoxyethoxy)phenyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole (49.8 mg) and N,N-diisopropylethylamine (16.3 mg) in DMF (1 ml) was stirred at room temperature overnight.", "To the reaction mixture was added piperizine (0.08 ml) and stirred at room temperature for 4 hours.", "To the reaction mixture was added ethyl acetate (10 ml).", "The resulting precipitate was collected by filtration.", "The precipitate was dissolved in 20% acetonitrile in water (10 ml), and the solution was subjected to column chromatography on ODS (Daiso-gel, SP-120-40/60-ODS-B (Trademark: prepared by Daiso Co., Ltd.)) (25 ml) eluting with 40% acetonitrile in water.", "The fractions containing the object compound were collected and evaporated under reduced pressure to remove acetonitrile.", "The residue was lyophilized to give Object Compound (74) (110 mg).", "IR (KBr): 3371.0, 1633.4, 1535.1, 1442.5, 1249.6 cm−1 NMR (DMSO-d6+D2O, δ): 0.99 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.5 Hz), 1.15-4.83 (47H, m), 6.72-8.18 (11H, m) ESI MASS (m/z)(Negative): 1392.4, 1391.4 (M−−H) Elemental Analysis Calcd.", "for C63H80N10O22S2.5.5H2O: C, 50.70; H, 6.14; N, 9.38 Found: C, 50.71; H, 6.48; N, 9.35 EXAMPLE 75 A solution of Starting Compound (75) (100 mg), 1-[4-[5-[4-[4-(2-methoxyethoxy)phenyl]phenyl]-1,3,4-thiadiazol-2-yl]benzoyloxy]-1H-1,2,3-benzotriazole (69.4 mg) and N,N-diisopropylethylamine (21.8 mg) in DMF (1 ml) was stirred at room temperature overnight.", "To the reaction mixture was added piperizine (0.08 ml) and stirred at room temperature for 4 hours.", "To the reaction mixture was added ethyl acetate (10 ml).", "The resulting precipitate was collected by filtration.", "The precipitate was dissolved in 20% acetonitrile in water (10 ml), and the solution was subjected to column chromatography on ODS (Daiso-gel, SP-120-40/60-ODS-B (Trademark: prepared by Daiso Co., Ltd.)) (25 ml) eluting with 35% acetonitrile in water.", "The fractions containing the object compound were collected and evaporated under reduced pressure to remove acetonitrile.", "The residue was lyophilized to give Object Compound (75) (100 mg).", "IR (KBr): 3351.7, 1633.4, 1537.0, 1511.9, 1442.5, 1249.6 cm−1 NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.11 (3H, d, J=5.9 Hz), 1.21-4.81 (45H, m), 6.70-8.18 (11H, m) ESI MASS (m/z)(Negative): 1378.5, 1377.4 (M−−H) Elemental Analysis Calcd.", "for C62H78N10O22S2.6H2O: C, 50.06; H, 6.10; N, 9.42 Found: C, 49.99; H, 6.29; N, 9.24 EXAMPLE 76 To a solution of Starting Compound (76) (100 mg) and 4-[2-[4-(4-ethoxy-1-piperidyl)phenyl]imidazo[2,1-b][1,3,4]-thiadiazol-6-yl]benzoic acid (37.8 mg) and 1-hydroxybenzotriazole (17.1 mg) and 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (32.3 mg) in N,N-dimethylformamide (1 ml) was added diisopropylethylamine (44 μl) at room temperature.", "The solution was stirred for 24 hours at the same temperature.", "Then to the reaction mixture was added piperidine and the mixture was stirred for 4 hours.", "Ethyl acetate was added to the reaction mixture.", "The resulting precipitates were collected by filtration and dried in vacuo.", "The precipitates were purified by column chromatography on ODS to give Object Compound (76) (51.1 mg).", "NMR (DMSO-d6+D2O, δ): 0.98 (3H, d, J=6.8 Hz), 1.0-1.2 (6H, m), 1.35-4.55 (43H, m), 4.75-4.9 (2H, m), 6.7-6.85 (2H, m), 7.0-7.2 (3H, m), 7.77 (2H, d, J=8.9 Hz), 7.85-8.05 (4H, m), 8.76 (1H, s) MASS (m/z): 1393.4 (M−−H) Elemental Analysis Calcd.", "for C62H82N12O21S2.8H2O: C, 48.37; H, 6.42; N, 10.92 Found: C, 48.64; H, 6.39; N, 10.89 The following compounds [Example 77 and 78] were obtained according to a similar manner to that of Example 76.EXAMPLE 77 NMR (DMSO-d6+D2O, δ): 0.88 (3H, t, J=7.4 Hz), 0.98 (3H, d, J=6.7 Hz), 1.10 (3H, d, J=5.9 Hz), 1.4-4.55 (45H, m), 4.7-4.9 (2H, m), 6.65-6.85 (2H, m), 6.95-7.15 (3H, m), 7.76 (2H, d, J=8.8 Hz), 7.85-8.05 (4H, m), 8.77 (1H, s) MASS (m/z): 1407.4 (M−−H) Elemental Analysis Calcd.", "for C63H84N12O21S2.7H2O: C, 49.28; H, 6.43; N, 10.95 Found: C, 49.37; H, 6.54; N, 11.03 EXAMPLE 78 NMR (DMSO-d6+D2O, δ): 0.89 (3H, t, J=7.2 Hz), 0.98 (3H, d, J=6.8 Hz), 1.10 (3H, d, J=5.8 Hz), 1.2-4.5 (47H, m), 4.75-4.9 (2H, m), 6.65-6.85 (2H, m), 7.0-7.2 (3H, m), 7.76 (2H, d, J=8.8 Hz), 7.85-8.05 (4H, m), 8.78 (1H, s) MASS (m/z): 1421.5 (M−−H) Elemental Analysis Calcd.", "for C64H86N12O21S2.7H2O: C, 49.60; H, 6.50; N, 10.85 Found: C, 49.52; H, 6.49; N, 10.75 EXAMPLE 79 A solution of Starting Compound (79) (100 mg) 1-(5-methyl-2-oxo-1,3-dioxol-4-yl)methoxycarbonyloxy-2,5-pyrrolidinedione (28.6 mg) and N,N-diisopropylethylamine (13.6 mg) in DMF (1 ml) was stirred at room temperature overnight.", "To the reaction mixture was added ethyl acetate (10 ml).", "The resulting precipitate was collected by filtration.", "The precipitate was dissolved in phosphoric acid buffer solution (pH 6.86) (10 ml), and the solution was subjected to column chromatography on ODS (Daiso-gel, SP-120-40/60-ODS-B (Trademark: prepared by Daiso Co., Ltd.)) (25 ml) eluting with 30% acetonitrile in water.", "The fractions containing the object compound were collected and evaporated under reduced pressure to remove acetonitrile.", "To the residue was added diluted HCl, and lyophilized to give Object Compound (79) (40 mg).", "IR (KBr): 3417.2, 1814.7, 1639.2, 1515.8, 1442.5, 1238.1 cm−1 NMR (DMSO-d6+D2O, δ): 0.96 (3H, d, J=6.7 Hz), 1.08 (3H, d, J=5.8 Hz), 1.33-4.79 (54H, m), 6.64-8.14 (11H, m) ESI MASS (m/z)(Negative): 1578.6 (M−−H) Elemental Analysis Calcd.", "for C71H93N11O26S2.8H2O: C, 49.44; H, 6.37; N, 8.93 Found: C, 49.29; H, 6.41; N, 8.89" ] ]	Patent_10469233
[ [ "Variable light wave function circuit and variable light wave function device", "Characteristics are rendered variable and high-functional by using the side-pressure inductive polarization mode coupling of a PMF to thereby change the position and magnitude of a side pressure.", "An input light is incident via a polarizer (2), and an outgoing light is output via the PMF (1) and another polarizer (3).", "Light may enter and go out in an opposite way.", "The PMF (1) has two polarization axes orthogonal to each other, and the polarization axis of the polarizer (2) is coupled so as to agree with one end of the polarization axis of the PMF (1).", "The polarization axis of the polarizer (3) is coupled so as to agree with one end of the polarization axis of the PMF (1).", "The PMF (1) induces polarization mode coupling when a polarization light tilted a specified angle with respect to the polarization axis is incident to apply a side pressure to the PMF (1).", "Characterstics/functions can be changed by changing the position and the magnitude of a side pressure by an application unit (5) so that the length of the PMF (1) of a basic structure can be easily set precisely." ], [ "1.A variable lightwave functional circuit comprising: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; a first polarizer arranged at one end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said first polarizer is inclined at a predetermined angle with respect to the polarization axis of said polarization-maintaining optical fiber; and a second polarizer arranged at the other end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said second polarizer is made coincident with the polarization axis of said polarization-maintaining optical fiber; wherein: laser light is entered via any one of said first polarizer and said second polarizer to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed through any one of said first polarizer and said second polarizer, which is not arranged on the side of laser entering port thereof.", "2.A variable lightwave functional circuit comprising: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; and a branching/coupling device having a first port to a fourth port, for branching laser light inputted from the first port to both said second port and said third port, and also for projecting laser light inputted from said second port to the fourth port; wherein: one end of said polarization-maintaining optical fiber is connected to one of said second port and said third port in such a manner that the polarization axis is inclined at a predetermined angle to an axial direction parallel to a branching plane of said branching/coupling device; the other end of said polarization-maintaining optical fiber is connected to the other of said second port and said third port in such a manner that the polarization axis is made coincident with said axial direction parallel to said branching plane of said branching/coupling device; laser light is entered from the first port of said branching/coupling device via the third port to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed from said fourth port of said branching/coupling device.", "3.A variable lightwave functional circuit as claimed in claim 2 wherein: said branching/coupling device is a polarization beam splitter and said variable lightwave functional circuit further comprising: a polarizer arranged between either said second port or said third port of said branching/coupling device and said polarization-maintaining optical fiber, the polarization axis of which is adjusted in such a manner that the laser light whose characteristic has been changed passes through said branching/coupling device.", "4.A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, further comprising: a rotator for inclining a polarization axis of entered laser light at a predetermined angle with respect to the polarization axis of said polarization-maintaining optical fiber.", "5.A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, wherein: said applying unit applies the lateral pressure to said polarization-maintaining optical fiber at either one point or plural points in equal intervals.", "6.A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, wherein: said applying unit applies the lateral pressure to said polarization-maintaining optical fiber at positions defined from a zero-th position to an N-th (symbol “N” being integer) position, where an interval from one end of said polarization-maintaining optical fiber up to the zero-th position is defined as 2NL (symbol “L” being predetermined length), an interval between the zero-th position and a first position is defined as 2N-1L, - - - , an interval between a(k-1)-th position and a k-th position is defined as 2N-kL, - - - , and an interval between an (N-1)-th position and the N-th position is defined as 20L; and said applying unit applies the lateral pressure in such a manner that equal rotations are applied at the respective positions, so that a repetition frequency of a pulse stream of entered laser light is multiplexed.", "7.A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, wherein: either the characteristic to be changed or a function to be changed includes one of a time period, a center wavelength, a bandwidth; a wavelength to be derived, a repetition frequency, a gain, a group velocity, and a dispersion.", "8.A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, wherein: the entered laser light corresponds to wavelength division multiplexed laser light.", "9.A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, wherein: a predetermined angle of mutual polarization axes defined between said polarization-maintaining optical fiber and said first polarizer, said second polarizer, or said branching/coupling device is either 45 degrees or approximately 45 degrees.", "10.A variable lightwave functional apparatus comprising: an optical amplifier for outputting wavelength multiplexed light containing a plurality of pulses having a first wavelength interval; a polarization branching/coupling device having a first port to a third port, for branching the output from said optical amplifier, which is entered from said first port into the polarization branching/coupling device, to both said second port and said third port; a coupler arranged at the second port of said polarization branching/coupling device; and the variable lightwave functional circuit recited in any one of claim 1 to claim 3, and arranged between said third port of said polarization branching/coupling device and said coupler; wherein: the wavelength multiplexed light containing said plural pulses having said first wavelength interval is converted into wavelength multiplexed light containing a plurality of pulses having a second wavelength interval in response to lateral pressure applied to said polarization-maintaining optical fiber, and then said converted wavelength multiplexed light is outputted from said coupler 11.A variable lightwave functional apparatus comprising: an acousto-optical modulator for outputting wavelength multiplexed light containing a plurality of pulses having a first wavelength interval; a coupler arranged at an output of said acousto-optical modulator; a polarization branching/coupling device having a first port to a third port, for branching the output form said coupler, which is entered from said first port into the polarization branching/coupling device, to both said second port and said third port; an optical amplifier connected to said second port of said polarization branching/coupling device; and the variable lightwave functional circuit recited in any one of claim 1 to claim 3, and arranged between said second port of said polarization branching/coupling device and said optical amplifier; wherein: multi-wavelength oscillation light is outputted from said coupler by laser light from said acousto-optical modulator.", "12.A variable lightwave functional apparatus as claimed in claim 10, further comprising: a polarizer arranged between either said second port or said third port of said polarization branching/coupling device and said polarization-maintaining optical fiber, for adjusting a polarization axis thereof in such a manner that laser light whose characteristic has been changed is transmitted by said polarization branching/coupling device.", "13.A variable lightwave functional apparatus as claimed in claim 11, further comprising: a polarizer arranged between either said second port or said third port of said polarization branching/coupling device and said polarization-maintaining optical fiber, for adjusting a polarization axis thereof in such a manner that laser light whose characteristic has been changed is transmitted by said polarization branching/coupling device.", "14.A method of making a variable lightwave functional circuit comprising: providing an applying unit for applying lateral pressure; providing a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; providing a first polarizer arranged at one end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said first polarizer is inclined at a predetermined angle with respect to the polarization axis of said polarization-maintaining optical fiber; and providing a second polarizer arranged at the other end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said second polarizer is made coincident with the polarization axis of said polarization-maintaining optical fiber; wherein laser light is entered via any one of said first polarizer and said second polarizer to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed through any one of said first polarizer and said second polarizer, which is not arranged on the side of laser entering port thereof 15.A method for making a variable lightwave functional circuit, said method comprising: providing an applying unit for applying lateral pressure; providing a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; providing a branching/coupling device having a first port to a fourth port, for branching laser light inputted from the first port to both said second port and said third port, and also for projecting laser light inputted from said second port to the fourth port; wherein one end of said polarization-maintaining optical fiber is connected to one of said second port and said third port in such a manner that the polarization axis is inclined at a predetermined angle to an axial direction parallel to a branching plane of said branching/coupling device; the other end of said polarization-maintaining optical fiber is connected to the other of said second port and said third port in such a manner that the polarization axis is made coincident with said axial direction parallel to said branching plane of said branching/coupling device; and laser light is entered from the first port of said branching/coupling device via the third port to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed from said fourth port of said branching/coupling device.", "16.A method of using a variable lightwave functional circuit, said variable lightwave functional circuit comprising: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; a first polarizer arranged at one end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said first polarizer is inclined at a predetermined angle with respect to the polarization axis of said polarization-maintaining optical fiber; and a second polarizer arranged at the other end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said second polarizer is made coincident with the polarization axis of said polarization-maintaining optical fiber; wherein laser light is entered via any one of said first polarizer and said second polarizer to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed through any one of said first polarizer and said second polarizer, which is not arranged on the side of laser entering port thereof; said method comprising transmitting laser light into said polarization-matching optical fiber; and applying lateral pressure to said polarization-matching optical fiber.", "17.A method of using a variable lightwave functional circuit, said variable lightwave functional circuit comprising: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; a branching/coupling device having a first port to a fourth port, for branching laser light inputted from the first port to both said second port and said third port, and also for projecting laser light inputted from said second port to the fourth port; wherein one end of said polarization-maintaining optical fiber is connected to one of said second port and said third port in such a manner that the polarization axis is inclined at a predetermined angle to an axial direction parallel to a branching plane of said branching/coupling device; the other end of said polarization-maintaining optical fiber is connected to the other of said second port and said third port in such a manner that the polarization axis is made coincident with said axial direction parallel to said branching plane of said branching/coupling device; laser light is entered from the first port of said branching/coupling device via the third port to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed from said fourth port of said branching/coupling device; said method comprising transmitting laser light into said polarization-matching optical fiber; and applying lateral pressure to said polarization-matching optical fiber." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention is related to a variable lightwave functional circuit and a variable lightwave functional apparatus.", "More specifically, the present invention is directed to a variable lightwave functional circuit and a variable lightwave functional apparatus, using a mode coupling within a polarization-maintaining optical fiber.", "Network technology of optical fiber communication systems has been conducted in practical fields in connection with appearances of optical fiber amplifiers in a front half decade in 1990, and is being rapidly developed.", "In the present stage, there are certain possibilities that ultra-long distance (e.g., up to 10,000 Km) and very high-speed (e.g., up to 40 Gb/s) wavelength division multiplexed transmission systems (WDM system operable up to, e.g., 64 wavelengths) could be realized.", "Then, optical fiber information communication networks may be conceivable as the most important infrastructure in the beginning stage of 21 century.", "However, up to now, while optical fiber communication systems are mainly employed only in point-to-point correspondence trunk line systems, conventional coaxial cables and semiconductor integrated circuits/electronic devices may constitute major basic elements in network portions.", "Since the Internet has been currently developed in explosive manners, extensions of transfer capacities which are required for future's networks could be predicted by that the transfer capacities are extended twice per 12 months.", "This extension rate of the transfer capacities exceeds a so-called “Moore's law (twice per 18 months)” related to semiconductor integrated circuits.", "Soon or later, there is no doubt about such a fact that WDM optical networks using light are necessarily required also in these network portions.", "However, under the present situation, optical devices employed in such WDM optical networks have not yet been well-developed, so that very rapid development of these optical devices is necessarily needed." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>That is to say, more specifically, the present invention is featured by realizing a “variable optical transversal filter using a lateral-pressure induced polarization mode of a polarization-maintaining optical fiber (PMF)”.", "In general, a transversal filter is equipped with a repeated structure and a summation circuit, in which a delay line and a branching/weighting circuit are employed as a basic unit.", "Different from an electric summation circuit, since a summation circuit can be hardly realized in an optical field, a two-port-pair cascade connecting mode optical lattice type filter may be employed [1] (note that symbol “[ ]” indicates below-mentioned reference publication number).", "Also, the optical lattice filter constructed of the planar lightwave circuit (PLC) with employment of the repeated Mach-Zehnder optical circuit structure has been proposed by JINGUJI et al.", "of NTT [2], and various sorts of the above-explained functions have be realized.", "Also, such a fact has been disclosed by KOSEKI of Sophia University.", "That is, the optical lattice filter could be manufactured even by the PMF rotational connection structure which utilizes the delays occurred between the polarization modes of the PMF as the delay, and also the rotational connection of the PMF as the branching/weighting operations [1] and [3].", "However, as to these structures which have been conventionally realized, the functions and the characteristics of these structures have already been determined when the structures are manufactured, so that these functions and characteristics cannot be changed.", "Also, in the PMF rotational connection structures, it is practically difficult that the PMF lengths of the basic structures cannot be made coincident in higher precision.", "The present invention has been made to solve the above-described difficulties, and has an object to provide both an optical fiber type variable lightwave functional circuit and a variable lightwave functional apparatus, capable of properly accepting structural changes in a WDM optical network, and furthermore, capable of having flexibilities as to various functions.", "As a consequence, the present invention owns such an object to provide both a variable lightwave functional circuit and a variable lightwave functional apparatus, which are capable of realizing a large number of functions, and also can be applied to optical communication apparatus such as WDM optical networks and optical transmitting/receiving devices.", "These various functions involve optical filtering, wavelength add/drop operation, pulse multiplexing operation, optical amplifier gain equalizing operation, optical fiber wavelength dispersion compensating operation, and the like.", "Also, an object of the present invention is to provide both a variable lightwave functional circuit and a variable lightwave functional apparatus, such as an optical transversal filter whose characteristic is variable and which owns higher functions, while a lateral-pressure induced polarization mode coupling of a PMF is used instead of a PMF rotational connection, and thus, both a position and a magnitude of lateral pressure are changed.", "According to a first solving means of the present invention, such a variable lighwave functional circuit is provided which is comprised of: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by the applying unit; a first polarizer arranged at one end of the polarization-maintaining optical fiber in such a manner that a polarization axis of the first polarizer is inclined at a predetermined angle with respect to the polarization axis of the polarization-maintaining optical fiber; and a second polarizer arranged at the other end of the polarization-maintaining optical fiber in such a manner that a polarization axis of the second polarizer is made coincident with the polarization axis of the polarization-maintaining optical fiber; in which: laser light is entered via any one of the first polarizer and the second polarizer to the polarization-maintaining optical fiber, the polarization-maintaining optical fiber changes a characteristic of the variable lighwave functional circuit by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by the applying unit, or plural conditions thereof; and the polarization-maintaining optical fiber projects the laser light whose characteristic has been changed through any one of the first polarizer and the second polarizer, which is not arranged on the side of laser entering port thereof.", "According to a second solving means of the present invention, such a variable lightwave functional apparatus is provided which is comprised of: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by the applying unit; and a branching/coupling device having a first port to a fourth port, in which laser light entered from the first port is branched to both the second port and the third port; one end of the polarization-maintaining optical fiber is arranged between the second port and the third port in such a manner that polarization axes thereof are inclined at a predetermined angle to the branching/coupling device; the other end of the polarization-maintaining optical fiber is arranged in such a manner that polarization axes thereof are made coincident with the branching/coupling device; and laser light entered from either the first port or the second port is projected to the fourth port; in which: laser light is entered from the third port of the branching/coupling device into the polarization-maintaining optical fiber; the polarization-maintaining optical fiber changes a characteristic of the variable lightwave functional circuit by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by the applying unit, or plural conditions thereof; and the polarization-maintaining optical fiber projects the laser light whose characteristic has been changed from the fourth port of the branching/coupling device.", "According to a third solving means of the present invention, such a variable lightwave functional apparatus is provided which is comprised of: an optical amplifier for outputting wavelength multiplexed light containing a plurality of pulses having a first wavelength interval; a polarization branching/coupling device having a first port to a third port, for branching the output from the optical amplifier, which is entered from the first port into the polarization branching/coupling device, to both the second port and the third port; a coupler arranged at the second port of the polarization branching/coupling device; and the above-described variable lightwave functional circuit arranged between the third port of the polarization branching/coupling device and the coupler; in which: the wavelength multiplexed light containing the plural pulses having the first wavelength interval is converted into wavelength multiplexed light containing a plurality of pulses having a second wavelength interval in response to lateral pressure applied to the polarization-maintaining optical fiber, and then the converted wavelength multiplexed light is outputted from the coupler.", "According to a fourth solving means of the present invention, such a variable lightwave functional apparatus is provided which is comprised of: an acousto-optical modulator for outputting wavelength multiplexed light containing a plurality of pulses having a first wavelength interval; a coupler arranged at an output of the acousto-optical modulator; a polarization branching/coupling device having a first port to a third port, for branching the output form the coupler, which is entered from the first port into the polarization branching/coupling device, to both the second port and the third port; an optical amplifier connected to the second port of the polarization branching/coupling device; and the above-described variable lightwave functional circuit arranged between the second port of the polarization branching/coupling device and the optical amplifier; in which: multi-wavelength oscillation light is outputted from the coupler by laser light from the acousto-optical modulator." ], [ "BACKGROUND OF THE INVENTION The present invention is related to a variable lightwave functional circuit and a variable lightwave functional apparatus.", "More specifically, the present invention is directed to a variable lightwave functional circuit and a variable lightwave functional apparatus, using a mode coupling within a polarization-maintaining optical fiber.", "Network technology of optical fiber communication systems has been conducted in practical fields in connection with appearances of optical fiber amplifiers in a front half decade in 1990, and is being rapidly developed.", "In the present stage, there are certain possibilities that ultra-long distance (e.g., up to 10,000 Km) and very high-speed (e.g., up to 40 Gb/s) wavelength division multiplexed transmission systems (WDM system operable up to, e.g., 64 wavelengths) could be realized.", "Then, optical fiber information communication networks may be conceivable as the most important infrastructure in the beginning stage of 21 century.", "However, up to now, while optical fiber communication systems are mainly employed only in point-to-point correspondence trunk line systems, conventional coaxial cables and semiconductor integrated circuits/electronic devices may constitute major basic elements in network portions.", "Since the Internet has been currently developed in explosive manners, extensions of transfer capacities which are required for future's networks could be predicted by that the transfer capacities are extended twice per 12 months.", "This extension rate of the transfer capacities exceeds a so-called “Moore's law (twice per 18 months)” related to semiconductor integrated circuits.", "Soon or later, there is no doubt about such a fact that WDM optical networks using light are necessarily required also in these network portions.", "However, under the present situation, optical devices employed in such WDM optical networks have not yet been well-developed, so that very rapid development of these optical devices is necessarily needed.", "SUMMARY OF THE INVENTION That is to say, more specifically, the present invention is featured by realizing a “variable optical transversal filter using a lateral-pressure induced polarization mode of a polarization-maintaining optical fiber (PMF)”.", "In general, a transversal filter is equipped with a repeated structure and a summation circuit, in which a delay line and a branching/weighting circuit are employed as a basic unit.", "Different from an electric summation circuit, since a summation circuit can be hardly realized in an optical field, a two-port-pair cascade connecting mode optical lattice type filter may be employed [1] (note that symbol “[ ]” indicates below-mentioned reference publication number).", "Also, the optical lattice filter constructed of the planar lightwave circuit (PLC) with employment of the repeated Mach-Zehnder optical circuit structure has been proposed by JINGUJI et al.", "of NTT [2], and various sorts of the above-explained functions have be realized.", "Also, such a fact has been disclosed by KOSEKI of Sophia University.", "That is, the optical lattice filter could be manufactured even by the PMF rotational connection structure which utilizes the delays occurred between the polarization modes of the PMF as the delay, and also the rotational connection of the PMF as the branching/weighting operations [1] and [3].", "However, as to these structures which have been conventionally realized, the functions and the characteristics of these structures have already been determined when the structures are manufactured, so that these functions and characteristics cannot be changed.", "Also, in the PMF rotational connection structures, it is practically difficult that the PMF lengths of the basic structures cannot be made coincident in higher precision.", "The present invention has been made to solve the above-described difficulties, and has an object to provide both an optical fiber type variable lightwave functional circuit and a variable lightwave functional apparatus, capable of properly accepting structural changes in a WDM optical network, and furthermore, capable of having flexibilities as to various functions.", "As a consequence, the present invention owns such an object to provide both a variable lightwave functional circuit and a variable lightwave functional apparatus, which are capable of realizing a large number of functions, and also can be applied to optical communication apparatus such as WDM optical networks and optical transmitting/receiving devices.", "These various functions involve optical filtering, wavelength add/drop operation, pulse multiplexing operation, optical amplifier gain equalizing operation, optical fiber wavelength dispersion compensating operation, and the like.", "Also, an object of the present invention is to provide both a variable lightwave functional circuit and a variable lightwave functional apparatus, such as an optical transversal filter whose characteristic is variable and which owns higher functions, while a lateral-pressure induced polarization mode coupling of a PMF is used instead of a PMF rotational connection, and thus, both a position and a magnitude of lateral pressure are changed.", "According to a first solving means of the present invention, such a variable lighwave functional circuit is provided which is comprised of: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by the applying unit; a first polarizer arranged at one end of the polarization-maintaining optical fiber in such a manner that a polarization axis of the first polarizer is inclined at a predetermined angle with respect to the polarization axis of the polarization-maintaining optical fiber; and a second polarizer arranged at the other end of the polarization-maintaining optical fiber in such a manner that a polarization axis of the second polarizer is made coincident with the polarization axis of the polarization-maintaining optical fiber; in which: laser light is entered via any one of the first polarizer and the second polarizer to the polarization-maintaining optical fiber, the polarization-maintaining optical fiber changes a characteristic of the variable lighwave functional circuit by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by the applying unit, or plural conditions thereof; and the polarization-maintaining optical fiber projects the laser light whose characteristic has been changed through any one of the first polarizer and the second polarizer, which is not arranged on the side of laser entering port thereof.", "According to a second solving means of the present invention, such a variable lightwave functional apparatus is provided which is comprised of: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by the applying unit; and a branching/coupling device having a first port to a fourth port, in which laser light entered from the first port is branched to both the second port and the third port; one end of the polarization-maintaining optical fiber is arranged between the second port and the third port in such a manner that polarization axes thereof are inclined at a predetermined angle to the branching/coupling device; the other end of the polarization-maintaining optical fiber is arranged in such a manner that polarization axes thereof are made coincident with the branching/coupling device; and laser light entered from either the first port or the second port is projected to the fourth port; in which: laser light is entered from the third port of the branching/coupling device into the polarization-maintaining optical fiber; the polarization-maintaining optical fiber changes a characteristic of the variable lightwave functional circuit by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by the applying unit, or plural conditions thereof; and the polarization-maintaining optical fiber projects the laser light whose characteristic has been changed from the fourth port of the branching/coupling device.", "According to a third solving means of the present invention, such a variable lightwave functional apparatus is provided which is comprised of: an optical amplifier for outputting wavelength multiplexed light containing a plurality of pulses having a first wavelength interval; a polarization branching/coupling device having a first port to a third port, for branching the output from the optical amplifier, which is entered from the first port into the polarization branching/coupling device, to both the second port and the third port; a coupler arranged at the second port of the polarization branching/coupling device; and the above-described variable lightwave functional circuit arranged between the third port of the polarization branching/coupling device and the coupler; in which: the wavelength multiplexed light containing the plural pulses having the first wavelength interval is converted into wavelength multiplexed light containing a plurality of pulses having a second wavelength interval in response to lateral pressure applied to the polarization-maintaining optical fiber, and then the converted wavelength multiplexed light is outputted from the coupler.", "According to a fourth solving means of the present invention, such a variable lightwave functional apparatus is provided which is comprised of: an acousto-optical modulator for outputting wavelength multiplexed light containing a plurality of pulses having a first wavelength interval; a coupler arranged at an output of the acousto-optical modulator; a polarization branching/coupling device having a first port to a third port, for branching the output form the coupler, which is entered from the first port into the polarization branching/coupling device, to both the second port and the third port; an optical amplifier connected to the second port of the polarization branching/coupling device; and the above-described variable lightwave functional circuit arranged between the second port of the polarization branching/coupling device and the optical amplifier; in which: multi-wavelength oscillation light is outputted from the coupler by laser light from the acousto-optical modulator.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a structural diagram of an optical fiber type variable lightwave functional circuit of a first embodiment mode.", "FIG.", "2 is a structural diagram of a variable lightwave functional circuit of a second embodiment mode.", "FIG.", "3 is a structural diagram of a variable lightwave functional circuit of a third embodiment mode.", "FIG.", "4 shows a characteristic diagram of a periodic optical filter.", "FIG.", "5 is a structural diagram of a variable lightwave functional circuit of a fourth embodiment mode.", "FIG.", "6 is a diagram for showing a wavelength-interval variable multi-wavelength oscillation spectrum (1).", "FIG.", "7 is a structural diagram of a variable lightwave functional circuit of a fifth embodiment mode.", "FIG.", "8 is a diagram for showing a wavelength-interval variable multi-wavelength oscillation spectrum (2).", "FIG.", "9 is a structural diagram of a variable lightwave functional circuit of a sixth embodiment mode.", "FIG.", "10 is an explanatory diagram for explaining an optical pulse stream to be inputted and an optical pulse stream to be outputted.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A.", "First Embodiment Mode FIG.", "1 shows a structural diagram for showing an optical fiber type variable lightwave functional circuit according to a first embodiment mode of the present invention.", "This variable lightwave functional circuit is equipped with a polarization-maintaining optical fiber (PMF) 1 having a certain length, polarizers 2 and 3, a rotator 4, and an applying unit 5 for applying lateral pressure.", "In this functional circuit, incident light is entered via the polarizer 2, and output light is projected via the PMF 1 and the other polarizer 3.Also, this optical fiber type variable lightwave functional circuit may be employed in such a reversible manner that incident light is entered into the polarizer 3, and output light is projected via the PFM 1 and the other polarizer 2.The input light is, for instance, signal light employed in an optical communication.", "Normally, this incident light corresponds to laser light emitted from a semiconductor laser is modulated by a signal.", "Also, while the PMF 1 owns two polarization axes perpendicular to each other, a polarization axis of the polarizer 2 is coupled in such a manner that this polarization axis is made coincident with one of the polarization axes of the PMF 1.In the PMF 1, when polarized light which is inclined at a predetermined angle with respect to the polarization axis is entered, polarization mode coupling is induced by applying lateral pressure to the PMF 1.As this predetermined angle, when an angle of, for example, 45 degrees is set, a polarization mode coupling may be effectively induced.", "To this end, the rotator 4 of 45 degrees is provided on the side of one polarizer 2 in this drawing.", "Alternatively, instead of the rotator 4, any one of the polarizers 2 and 3 may be connected, or arranged in such a manner that this polarizer is inclined at an angle of 45 degrees with respect to the polarization axis of the PMF 1.Also, since the lateral pressure is applied to the PMF 1, the polarization axis of the polarizer 2 may be inclined by an angle of 45 degrees to be coupled to the polarization axis of the PMF 1.Furthermore, the predetermined angle is not limited to 45 degrees, but may be selected to be a proper inclination.", "Also, the polarization axis of the polarizer 3 may be coupled to one of the polarization axes of the PMF 1 in such a manner that this polarization axis of the polarizer 3 is made coincident with the second-mentioned polarization axis.", "As a means for applying the lateral pressure to the PMF 1 by the applying unit 5, for example, there is such a method that force is applied from a side surface of the PMF 1 by using a member such as a wood, plastics, and a metal.", "It should be noted that the PMF 1 may be nipped by way of a clip such as a binder clip.", "In the applying unit 5 or in the lateral pressure, a magnitude of force may be adjusted according to a clipping direction.", "Next, the polarization mode coupling will now be described in detail.", "When lateral pressure is applied to the PMF 1 by the applying unit 5, the polarization mode coupling is induced, which is equivalent to such a fact that the PMF 1 is rotary- connected so as to induce the polarization mode coupling.", "As a result, a transfer matrix (Jones matrix) of a photoelectric field vector at a point to which the lateral pressure is applied may be expressed by a rotational matrix.", "A Jones matrix “Mf” of a PMF portion when the points to which lateral pressure is applied are provided in an equi-interval “1” is expressed by the following formulae: M f = S ⁡ ( m ) ⁢ ∏ i = N - 1 0 ⁢ ⁢ ( R ⁡ ( θ i ) ⁢ C ⁡ ( ϕ i ) ⁢ S ⁡ ( m ) ) ( 1 ) S ⁡ ( m ) = ⁢ ( m 0 0 m * ) , C ⁡ ( ϕ i ) = ( ⅇ jϕ i 0 0 ⅇ - ⅈϕ i ) , R ⁡ ( θ i ) = ⁢ ( cos ⁢ ⁢ θ i - sin ⁢ ⁢ θ i sin ⁢ ⁢ θ i cos ⁢ ⁢ θ i ) , m = ⅇ ⅈ ⁢ B ⁢ ⁢ ϖ 2 ⁢ c ⁢ l ( 2 ) In this case, symbol “B=nx−ny indicates a modal birefringence index, symbol “ω” shows an angular frequency, and symbol “c” denotes a light velocity.", "A rotation angle “θi” at this time may be changed based upon the magnitude of the lateral pressure.", "Also, symbol “S(m)” represents a transfer matrix of a PMF having a length of “l”, and symbol “m” indicates a parameter indicative of a delay between an x-polarized wave and a y-polarized wave.", "Symbol “C(φi)” shows a matrix for applying a shift by a phase “φ”, and a phase shift may be realized by not providing the pressure-applied positions in the completely equi-interval, but by fine-adjusting the pressure-applied positions.", "Symbol “R(θ1)” represents a rotational matrix for rotating a polarization by an angle of “θ”, and a polarization rotation is induced by applying pressure.", "In this case, since the Jones matrix “Mf” may be expressed by the following formula (3) as a unitary matrix, the below-mentioned formula (4) may be obtained as a Jones matrix “T” of the entire optical fiber type variable lightwave functional circuit: M f = ( a - b * b a * ) ,  a  2 +  b  2 = 1 ( 3 ) T = ( 1 0 0 0 ) ⁢ M f ⁢ R ⁡ ( 45 ⁢ ° ) ⁢ ( 1 0 0 0 ) = a - b * 2 ⁢ ( 1 0 0 0 ) ( 4 ) As a consequence, in accordance with the present invention, such a function may be realized which is similar to the optical lattice circuits [1] and [3] based upon the PMF rotary connection structure which has been proposed by KOSEKI et al.", "of Sophia University.", "In other words, in both an optical fiber type variable lightwave functional circuit and a variable lightwave function (optical lattice circuit) according to the present invention, characteristics and functions thereof can be varied by changing the positions and the magnitutes of the lateral pressure, as compared with those manufactured based upon the PMF rotary connection structure, and the PMF length 1 of the basic structure can be easily coincident in higher precision.", "The functions which can be realized by the optical fiber type variable lightwave functional circuit of the present invention will now be exemplified as follows: (1) A periodic optical filter: An optical filter in which transmission wavelengths are arranged in an equi-interval period.", "The period may be varied and this periodic optical filter may be employed in a multi-wavelength optical source.", "(2) An optical bandpass filter: A so-called “Solc type optical filter.” Both a central wavelength and a bandwidth of this optical bandpass filter may be varied.", "(3) A wavelength add/drop circuit: A circuit for deriving either one wavelength or several wavelengths from a WDM signal.", "A wavelength to be derived may be varied.", "(4) A pulse multiplexing circuit: A circuit for multiplying a repetition frequency of a pulse stream derived from a pulse light source.", "The multiplication number may be varied.", "(5) An optical amplifier gain equalizing circuit: A circuit for equalizing a wavelength characteristic of a gain of an optical fiber amplifier and for flattening the equalized wavelength characteristic.", "The wavelength characteristic of the circuit may be varied in response to characteristics of the respective amplifiers.", "(6) An optical fiber wavelength dispersion compensating circuit: The wavelength dispersion of the optical fiber can be compensated by flattening a wavelength characteristic of an amplitude of a circuit, and by setting a wavelength characteristic of a group speed to a reverse characteristic of an optical fiber.", "The wavelength characteristic of the group speed may be varied in response to a sort/length of an optical fiber.", "It should be understood that the optical lattice circuit, the characteristic/function of which cannot be varied, has already been realized by JINGUJI et al.", "of NTT by employing the planar lightwave circuit (PLC) with employment of the repeated Mach-Zehnder optical circuit structure.", "Also, the designing method capable of realizing various functions has been proposed [1] and [2].", "When a variable lightwave functional circuit of the present invention used to realize these functions is designed, for example, the designing method may be employed which have been proposed by JINGUJI et al.", "of NTT.", "B.", "Second Embodiment Mode Since the optical fiber type variable lightwave functional circuit of the first embodiment mode employs the polarizers 2 and 3, this functional circuit owns the polarization dependent characteristic.", "To solve this problem, such a structure of a variable lightwave functional circuit capable of realizing a polarization independent characteristic will now be explained.", "FIG.", "2 shows a structural diagram of a variable lightwave functional circuit according to a second embodiment mode of the present invention.", "This second embodiment mode corresponds to a polarization independent optical fiber type variable lightwave functional circuit.", "First, FIG.", "2(a) indicates a structural diagram in which the optical fiber type variable lightwave functional circuit of FIG.", "1 is rearranged so as to realize the polarization independent characteristic.", "This variable lightwave functional circuit is equipped with a PMF 21, a polarization beam splitter (PBS) 22, a rotator 23, and an applying unit 25 of lateral pressure.", "Since the PMF 21 is jointed to each other by the applying unit 25, or is obliquely jointed to the PBS 22, a polarization axis of light derived from the PBS 22 is inclined at an angle of, for example, 45 degrees, and then is entered into one end of the PMF 21 to be outputted to the other end thereof.", "The light outputted from the PMF 21 is adjusted in such a manner that the polarization axis thereof is further inclined by the rotator 23 at an angle of 45 degrees, and then this light with the inclined polarization axis passes through the PBS 22.Thus, output light is outputted from the PBS 22 in this manner.", "It should be noted that the rotator 23 may be omitted.", "It is alternative case, as to the light outputted from the PMF 1, a light component along the polarization axial direction may be outputted.", "Furthermore, the polarization axis may be adjusted by way of the applying unit 25.Now, a description will be made of the polarization independent characteristic.", "Assuming now that a right-turned Jones matrix of this PMF 21 portion is given as “Mf” of the above-described formula (3), a left-turned Jones matrix may be given by the following formula (5).", "As a result, a Jones matrix “T” of the entire variable lightwave functional circuit shown in FIG.", "2(a) is given by the following formula (6), and thus, becomes the polarization independent characteristic: M b = ( a - b b * a * ) ( 5 ) T = ⁢ ( 1 0 0 0 ) ⁢ M f ⁢ R ⁡ ( 45 ⁢ ° ) ⁢ ( 1 0 0 0 ) + ( 0 0 0 1 ) ⁢ R ⁡ ( 45 ⁢ ° ) ⁢ M b ⁡ ( 0 0 0 1 ) = ⁢ 1 2 ⁢ ( a - b * 0 0 a * - b ) ( 6 ) Also, FIG.", "2(b) shows a structural diagram of a Sagnac interferometer mode optical fiber type variable light-wave functional circuit [5] and [6].", "Although operations of this structure of the optical fiber type variable lightwave functional circuit is slightly different from the operation of each of the above-described variable lightwave functional circuits, this variable lightwave functional circuit of FIG.", "2(b) has such a merit that this entire functional circuit can be constituted by the optical fiber.", "In other words, this Sagnac interferometer mode optical fiber type variable lightwave functional circuit is provided with a 50%-fiber coupler 32 which is formed by using a PMF 31, and also an applying unit 35 of lateral pressure.", "A Jones matrix “T” of the entire functional circuit shown in FIG.", "2(b) may have such a polarization independent characteristic in the form of the following formula (7): T = 1 2 × 1 2 ⁢ M f + j 2 × j 2 ⁢ M b = b - b * 2 ⁢ ( 0 1 1 0 ) ( 7 ) As previously explained, since the structure of the second embodiment mode is employed, it is possible to realize the polarization independent optical fiber type variable lightwave functional circuit.", "C. Third Embodiment Mode Next, FIG.", "3 is a structural diagram for representing a variable lightwave functional circuit according to a third embodiment mode of the present invention.", "This third embodiment mode shows a periodic optical filter made by an optical fiber type variable lightwave functional circuit.", "In this embodiment mode, a periodic optical filter having the above-described function (1) is formed by employing the optical fiber type variable lightwave function of FIG.", "1, so that a multi-wavelength light source whose wavelength interval is variable could be realized.", "At this time, as shown in this drawing, as to a position of lateral pressure applied by the applying unit 5, a single lateral-pressure applying point may be selected.", "Assuming now that the position of the lateral pressure applied by the applying unit 5 is selected to be such a position apart from the light entering port of the PMF 1 by a distance of “L”, this transmission characteristic may become a sine waveform as expressed by the following formula (8), while the rotation angle is “θ”:  T  2 = ⁢ 1 2 ⁢ ( 1 - sin ⁢ ⁢ 2 ⁢ θcosΔβ ⁢ ⁢ L ) , Δβ = ⁢ β x - β y = B ⁢ ⁢ ω c , ( 8 ) A wavelength interval “Δλ” obtained at this time is inversely proportional to the distance “L” as defined in the below-mentioned (9): Δλ=λ2/(BL) (9) FIG.", "4 represents a characteristic diagram of the periodic optical filter.", "FIG.", "4(a) shows a periodic transmission characteristic diagram of a periodic optical filter which is actually manufactured.", "When the distance “L” is selected to be 2.0 m, the resulting periodic transmission characteristic diagram is indicated by a solid line.", "When the distance “L” is selected to be 8.0 m, the resulting periodic transmission characteristic diagram is indicated by a broken line.", "From this drawing, such a fact can be understood that the periodic transmission characteristic having the sine wave shape could be obtained by the variable lightwave functional circuit of the present invention.", "Also, FIG.", "4(b) represents a lateral pressure/position dependent characteristic diagram of a wavelength interval.", "As shown in this drawing, such a fact may be understood that the wavelength interval “Δλ” is inversely proportional to the lateral pressure position “L”.", "In this example, a relationship between the wavelength interval “Δλ” and the lateral pressure position “L” is given as the following formula (10) from FIG.", "4(b): Δλ = 5.81 × 10 - 9 × 1 L .", "( 10 ) D. Fourth Embodiment Mode FIG.", "5 shows a structural diagram of a variable lightwave functional circuit according to a fourth embodiment mode of the present invention.", "This fourth embodiment mode is a structural diagram of a wavelength-interval variable multi-wavelength optical fiber laser.", "In this fourth embodiment mode, such a periodic optical filter as shown in the above-explained embodiment mode was inserted into an erbium doped optical fiber laser resonator shown in this drawing, and thus, such a multi-wavelength optical fiber laser could be realized, the wavelength interval of which could be varied by lateral pressure applied to a PMF.", "This corresponds to, as one example, an application example in which the structure of either FIG.", "1 or FIG.", "2 is employed as an optical fiber filter.", "In other words, in this embodiment mode, while this optical fiber filter is inserted into the resonator of the optical fiber laser, a multi-wavelength oscillation in a time period corresponding to the transmission characteristic may be realized.", "In addition to the above-explained periodic optical filter, this optical fiber laser is equipped with a Faraday rotation mirror (FRM) 51, an erbium doped optical fiber (EDF) 52, a wavelength division multiplexing (WDM) coupler 53, a PBS 54, a pumping semiconductor laser 55, isolators 56/58, a polarization controller 57, a 10%-coupler 59, and a spectrum analyzer 60.While as the periodic optical fiber, a portion of a thick line, namely the PMF 1, the polarizers 2 and 3, and the applying unit 5 are equipped, since the applying unit 5 applies pressure to, as one example, one point within the PMF 1, such an optical fiber filter having a periodic transmission characteristic is realized.", "The FRM 51 corresponds to a mirror capable of applying a polarization rotation due to the Faraday effect.", "The EDF 52 oscillates such a laser light having a wavelength of, for example, 1.55 μm.", "The pumping semiconductor laser 55 oscillates such a pumping light having a wavelength of, for example, 1.48 μm.", "The WDM coupler 53 corresponds to such an optical fiber coupler used to combine, for example, the pumping light having the wavelength of 1.48 μm oscillated from the pumping semiconductor laser 55 with the laser light having the wavelength of 1.55 μm oscillated by the EDF 52.It should be understood that a spectrum analyzer 60 corresponds to such a measuring device which monitors not a portion of the optical fiber laser according to this embodiment mode, but a spectrum of such a laser light which is outputted via both the coupler 59 and the isolator 58, and thus, confirms that the laser light is actually oscillated in the multi-wavelength mode.", "Next, operations of such an optical fiber laser will now be explained.", "The erbium doped optical fiber laser (EDFA) is constituted by the FRM 51, the EDF 52, the WDM coupler 53, the pumping semiconductor laser 55, and the like.", "This EDFA is pumped by multiplexing the pumping semiconductor laser 55 having the wavelength of 1.48 μm by way of the WDM coupler 53 so as to oscillate laser.", "The output laser light of the EDFA is branched by the PBS 54.One branched light is entered via both the isolator 56 and the polarization controller 57 into the periodic optical filter equipped with the PMF 1 and the polarizers 2 and 3.Output light of this periodic optical filter is outputted via the 10%-coupler 59 as a laser output.", "It should be noted that in this case, this laser output is entered into the spectrum analyzer 60 so as to be measured.", "On the other hand, the output light is also inputted from the 10%-coupler 54 to the PBS 54.In this case, since the polarization axis is controlled by the PC 57, this controlled polarization axis is made coincident with the polarization axis passing through the PBS 54, and the entered light is propagated to the side of the EDFA so as to be resonated.", "It should also be noted that the propagation of the other branched light which is branched by the PBS 54 is blocked off by the isolator 56.FIG.", "6 shows a diagram of a wavelength-interval variable multi-wavelength oscillation spectrum (1).", "This is such a spectrum which is obtained when a multi-wavelength oscillation is realized, while the erbium doped optical fiber is cooled by using liquid nitrogen so as to be employed as a gain medium having inhomogeneous broadening.", "FIG.", "6(a) indicates a wavelength-interval variable multi-wavelength oscillation spectrum obtained in such a case that the length of the PMF 1 is selected to be 10 m, and the distance “L” over which the lateral pressure is applied is equal to 4 m. FIG.", "6(b) indicates a wavelength-interval variable multi-wavelength oscillation spectrum obtained in such a case that the length of the PMF 1 is selected to be 10 m, and the distance “L” over which the lateral pressure is applied is equal to 8 m. As shown in this drawing, it maybe understood that the laser light is oscillated in different wavelength intervals corresponding to the respective applied positions of the lateral pressure by the applying unit 5.E.", "Fifth Embodiment Mode FIG.", "7 shows a structural diagram of a variable lightwave functional circuit according to a fifth embodiment mode of the present invention.", "This fifth embodiment mode represents a wavelength-interval variable multi-wavelength optical fiber laser.", "In this fifth embodiment mode, since an acousto-optic modulator (AOM) is inserted into a resonator instead of the liquid nitrogen cooling, a multi-wavelength oscillation may be realized at a room temperature.", "This optical fiber laser is equipped with the above-explained periodic optical filter, an FRM 71, an AOM 72, a 10%-coupler 73, a PBS 74, an erbium doped optical fiber laser (EDFA) 75, and PCs 76 and 77.As previously explained, a portion of a thick line, namely both the PMF 1 and the applying unit 5 may realize such an optical fiber filter having a periodic transmission characteristic.", "Also, in this case, the optical fiber laser is oscillated in a proper wavelength interval in correspondence with a position of lateral pressure to the PMF 1 and/or the lateral pressure applied by the applying unit 5.The AOM 72 may shift a frequency of light by such a frequency of an acoustic wave.", "Due to the characteristic aspect of the EDFA 75, this EDFA 75 does not oscillate in the multi-wavelength mode in the room temperature, but must be cooled up a temperature of −196° C. by using, for example, liquid nitrogen (LN2).", "However, since the AOM 72 is inserted, an oscillation threshold value becomes infinite and thus the EDFA 75 is not oscillated as a matter of fact.", "As a result, the multi-wavelength laser output may be obtained even in the room temperature.", "FIG.", "8 shows a diagram of a wavelength-interval variable multi-wavelength oscillation spectrum (2).", "This is such a spectrum obtained when the multi-wavelength oscillation at the room temperature is realized by the AOM 72.In this example, FIG.", "8(a) indicates a wavelength-interval variable multi-wavelength oscillation spectrum obtained in such a case that the length of the PMF 1 is selected to be 10 m, and the distance “L” over which the lateral pressure is applied is equal to 2.3 m. FIG.", "8(b) indicates a wavelength-interval variable multi-wavelength oscillation spectrum obtained in such a case that the length of the PMF 1 is selected to be 10 m, and the distance “L” over which the lateral pressure is applied is equal to 8.25 m. As shown in this drawing, it may be understood that the laser light is oscillated in such wavelength intervals corresponding to the respective applied positions 6f the lateral pressure and the lateral pressure applied by the applying unit 5.F.", "Sixth Embodiment Mode FIG.", "9 shows a structural diagram of a variable lightwave functional circuit according to a sixth embodiment mode of the present invention.", "This sixth embodiment mode represents a pulse multiplexing circuit.", "A pulse multiplexing circuit having the above-described function (4) is realized as shown in this drawing by way of this optical fiber type variable lightwave functional circuit.", "While as to the respective positions from a zero-th position to an N-th (“N” being an integer) position where lateral pressure is applied to a PMF 11, an interval from one end of the PMF 11 up to the zero-th position is defined as “2NL” (“L” being a predetermined length); an interval from the zero-th position up to a first position is defined as “2N−1L”; - - - ; an interval from a (k-1)-th position up to a k-th position is defined as 2N-kL”; - - - ; and an interval from an (N-1)-th position up to an N-th position is defined as 20L, the applying unit 5 applies lateral pressure in such a way that equal rotations are applied to the respective positions.", "As a result, the pulse multiplexing circuit multiplexes a repetition frequency of a pulse stream of inputted light to output the multiplexed pulse stream.", "In this example, the polarization axis is rotated by an angle of 45 degrees at the respective positions by applying the lateral pressure by the applying unit 5.However, the present invention is not limited to this example, but a proper rotation may be applied.", "As described above, instead of the rotator 4, the polarization axis may be rotated by a predetermined angle by changing a method of applying lateral pressure, or a jointing method.", "FIG.", "10 shows an explanatory diagram for explaining an optical pulse stream to be inputted, and an optical pulse stream to be outputted.", "Such an optical pulse stream as shown in FIG.", "10(a) is inputted from a left side as viewed in this drawing.", "In the case of FIG.", "10(a), as an example, a repetition frequency of the optical pulse is 10 GHz, and this optical pulse is employed in a communication of 10 Gbit/s.", "When this optical pulse is inputted to the variable lightwave functional circuit of the embodiment mode shown in FIG.", "6, and proper pressure is applied at N places, the repetition frequency may become “2N” due to a delay characteristic of xy polarization axes of the PMF 11, and thus, the optical pulse is multiplexed.", "As a consequence, this optical pulse may be employed in a communication at a higher speed than the input laser light.", "FIG.", "10(b) shows an output optical pulse stream in the case of N=1, and represents that an optical pulse stream having a repetition frequency of 20 GHz.", "As a consequence, such an optical pulse stream may be maintained in which the optical pulse stream (FIG.", "10(a)) having the repetition frequency of 10 GHz is multiplexed to obtain a repetition frequency of 20 GHz.", "G. Others It should be understood that as to the various sorts of setting conditions such as the branching/coupling ratios of the respective couplers, the wavelengths/power of the laser light, the lengths of the PMFs, the magnitudes/positions/temporal changes of the lateral pressure applied by the applying unit, the joint angles/joint means between either the polarizers or the rotators and the PMFs, one example is merely indicated in the above-explained respective embodiment modes, and thus, these setting conditions may be properly changed.", "REFERENCE PUBLICATIONS [1] “OPTICAL TRANSMISSION CIRCUIT” written by T. OZEKI, The Institute of Electronics, Information, Communication Engineers, 2000.INDUSTRIAL APPLICABILITY As previously described, according to the present invention, both the optical fiber type variable lightwave functional circuit and the variable lightwave functional apparatus can be provided which are capable of properly accepting the structural changes in the WDM optical network, and furthermore, capable of having the flexibilities as to various functions.", "As a consequence, in accordance with the present invention, a large number of functions can be realized, and moreover, the present invention can be applied to optical communication apparatus such as WDM optical networks and optical transmitting/receiving devices.", "These various functions involve the optical filtering, the wavelength add/drop operation, the pulse multiplexing operation, the optical amplifier gain equalizing operation, the optical fiber wavelength dispersion compensating operation, and the like.", "Also, in accordance with the present invention, it is possible to realize both the variable lightwave functional circuit and the variable lightwave functional apparatus, such as the optical transversal filter whose characteristic is variable and which owns the higher functions, while the lateral-pressure induced polarization mode coupling of the PMF is used instead of the PMF rotational connection, and thus, both the position and the magnitude of the lateral pressure are changed." ] ]	Patent_10469243
[ [ "Use of n-acetyl-d-glucosamine in the manufacture of pharmaceutical useful for adjuvant treatment of perianal disease", "The present invention has disclosed a use of N-acetyl-D-glucosamine in the manufacture of a medicine for auxiliary treatment of the peri-anal disease.", "Through stabilizing celluar lysosome membrance, the degree and scope of the injure extended by the release of various enzymes in the cellular lysosome are decreased; improving the healing of the injured tissues; resist the field planting of the microorganism on the traumatic surface so as to prevent the occurrence of infection.", "The preparation with N-acetyl-D-glucosamine as a main active component can be used in the auxiliary treatment of the peri-anal diseases, with a remarkable curative effect." ], [ "1-4.", "(canceled) 5.A method for treating perianal tissue injury, comprising: administering to the site of the perianal tissue injury a therapeutically effective amount of a medicament that contains N-acetyl-D-glucosamine and/or a pharmaceutically acceptable salt thereof.", "6.A method as recited in claim 5, wherein the medicament is in the form of a liquid.", "7.A method as recited in claim 5, wherein the medicament is sprayed on the injured tissue.", "8.A method as recited in claim 5, wherein the medicament is applied to a subject's perineum.", "9.A method as recited in claim 5, wherein the medicament is applied to a subject's perianal mucousal membrane." ], [ "<SOH> BACKGROUND ART <EOH>The peri-anal disease includes anal fissure, peri-anal abscess, fistula cannulas, haemorrhoids, polyp of rectum, carcinoma of the rectum and so on, the common feature of which is that there is an tissue injure in-situ, so, normally, the symptom is light, but when it acutely break out, there will be pain, red and swollen, pruritus, exudation increasing and so on, which would bring many worries and pains to the people's life and work.", "The treatment of these diseases needs eliminating acute oedema, lightening pain, stabilizing membrane structure, so as to prevent the inflammation to be more intensive, at the same time, it is needed to control the infection.", "Said disease is known as a little disease but a big problem clinically for many years.", "Therefore, the medicament for auxiliary treatment of peri-anal disease is needed all along in the field.", "In the research of “bio-waves” theory, the present inventor has set up a bacterial wave growth model.", "Through researching, it is known that this wave is of its intrinsic regulation mechanism: some chemical substances are able to participate the regulation in the bio-wave process, so as to transform an abnormal periodic slow wave into a normal physiological chaotic quick wave, and this kind of substances are known as promoting wave factors.", "Through separating, purifying and identifying, it is determined that one of the factors is N-acetyl-D-glucosamine, the promoting wave function of which is shown in lubricating and protecting the cell.", "Many biochemical and physiological process of human body need the participation of the promoting wave factors, and it would lead to an abnormal state, if this kind of promoting wave factors is lacked in the living body.", "N-acetyl-D-glucosamine is a chemical reagent.", "From the 1990's, it is continually used to treat pericementitis (WO9102530A1), microbiological infection (WO9718790A3), intestinal inflammation (WO9953929A1), cornea disease (JP10287570A2), hypertrophy of the prostate (U.S. Pat.", "No.", "5,116,615) and so on.", "It is also applied in cosmetology (JP59013708A2), shampoo preparation (JP2011505A2), tissue growth regulation agent (WO/A 8 702244), and etc., but it has not been used in the manufacture of a medicament for auxiliary treatment of peri-anal disease up to now." ], [ "TECHNICAL FIELD The present invention relates to the use of N-acetyl-D-glucosane and pharmaceutical acceptable salts thereof in the manufacture of a medicament for treating the peri-anal disease.", "BACKGROUND ART The peri-anal disease includes anal fissure, peri-anal abscess, fistula cannulas, haemorrhoids, polyp of rectum, carcinoma of the rectum and so on, the common feature of which is that there is an tissue injure in-situ, so, normally, the symptom is light, but when it acutely break out, there will be pain, red and swollen, pruritus, exudation increasing and so on, which would bring many worries and pains to the people's life and work.", "The treatment of these diseases needs eliminating acute oedema, lightening pain, stabilizing membrane structure, so as to prevent the inflammation to be more intensive, at the same time, it is needed to control the infection.", "Said disease is known as a little disease but a big problem clinically for many years.", "Therefore, the medicament for auxiliary treatment of peri-anal disease is needed all along in the field.", "In the research of “bio-waves” theory, the present inventor has set up a bacterial wave growth model.", "Through researching, it is known that this wave is of its intrinsic regulation mechanism: some chemical substances are able to participate the regulation in the bio-wave process, so as to transform an abnormal periodic slow wave into a normal physiological chaotic quick wave, and this kind of substances are known as promoting wave factors.", "Through separating, purifying and identifying, it is determined that one of the factors is N-acetyl-D-glucosamine, the promoting wave function of which is shown in lubricating and protecting the cell.", "Many biochemical and physiological process of human body need the participation of the promoting wave factors, and it would lead to an abnormal state, if this kind of promoting wave factors is lacked in the living body.", "N-acetyl-D-glucosamine is a chemical reagent.", "From the 1990's, it is continually used to treat pericementitis (WO9102530A1), microbiological infection (WO9718790A3), intestinal inflammation (WO9953929A1), cornea disease (JP10287570A2), hypertrophy of the prostate (U.S. Pat.", "No.", "5,116,615) and so on.", "It is also applied in cosmetology (JP59013708A2), shampoo preparation (JP2011505A2), tissue growth regulation agent (WO/A 8 702244), and etc., but it has not been used in the manufacture of a medicament for auxiliary treatment of peri-anal disease up to now.", "CONTENTS OF THE INVENTION The applicant of the present invention finds that N-acetyl-D-glucosamine is able to resist in situ bacterial field planting of microorganism and stabilize the membrane of cellular lysosome, so it has an auxiliary effect in the treatment of peri-anal disease.", "Therefore, the present invention is related to the use of N-acetyl-D-glucosamine and pharmaceutical acceptable salt thereof in the manufacture of a medicament for auxiliary treatment of peri-anal disease.", "On the other hand, the present invention is related to a method for treating peri-anal disease, including administrating to a patient who is in need thereof an effective amount of N-acetyl-D-glucosamine or pharmaceutical acceptable salts thereof.", "The molecular formula of N-acetyl-D-glucosamine is C8H15NO6, its structure is as follows: N-acetyl-D-glucosamine can be purchased in the market or prepared according to known methods.", "For instance, patent application WO97/31121 has disclosed a method for preparing N-acetyl-D-glucosamine from chitin by enzyme method, Japanese patent application JP63273493 has disclosed a method in which chitin is partially hydrolyzed into N-acetyl-chitose, and then it is treated with enzyme to obtain N-acetyl-D-glucosamine.", "The pharmaceutical acceptable salts of N-acetyl-D-glucosamine that can be mentioned are the salts formed with pharmaceutical acceptable acids, for instance, the salts formed with inorganic acids, such as hydrochloride, hydrobromide, borate, phosphate, sulfate sulfite and hydrophosphate, and the salts formed with organic acids, such as citrate, benzoate, ascorbate, methyl sulfate, naphthalene-2-sulfonate, picrate, fumarate, maleate, malonate, oxalate, succinate, acetate, tartrate, mesylate, tosylate, isethionate, α-ketoglutarate, α-glyceryl phosphate and glucose-1-phosphate.", "N-acetyl-D-glucosamine and the pharmaceutical acceptable salts thereof can be formulated in the form of liquid preparation and sprayed on a local part for auxiliary treatment of peri-anal disease, it can also be formulated into a preparation combined with other active components capable of treating peri-anal disease, so as to treat the peri-anal disease.", "The medicament of the present invention can be used in the sanitation of skin mucous membrane at perineum part of peri-anal for men or women, so as to relieve odor and itching, anti-inflammation, relieve pain, anti-infection, and quickly relieve and diminish the red-swollen, pruritus, pus secretion exuding condition etc.", "caused by anal fissure, peri-anal abscess, anal fistula, inner and outer haemorrhoids, polyp of rectum and carcinoma of the rectum.", "It can quickly and effectively relieve the uncomfortableness of perineum caused by seating for a long time of work.", "OPTIMAL MODE FOR CARRYING OUT THE INVENTION The following experimental examples are used to illustrate that the compound of the present invention (the compound of formula (I)) have promoting wave function, with low toxicity, and resist in situ bacterial field planting of microorganism and stabilize the membrane of cellular lysosome.", "I.", "Promoting Wave Test of the Compound of Formula (I) 1.1 Experimental Materials and Method: 1.1 Samples: Pure Compound of Formula (I) 1.2 Experimental Materials: Strain: Proteus Mirabilis (which should comply with the following biological reaction characteristics: dynamics (+), urease (+), lactose (−), glucose (+), H2S (−), phenylalanine deaminase (+).", "Culture medium: modified LB culture medium (the component of the composition are: trytones of 1%, yeast extract of 0.5%, sodium chloride 1%, glucose of 0.1%, TTC of 0.002% and pH=7.2˜7.4).", "1.3 Experimental Method: The Proteus Mirabilis were inoculated at the center of LB plate, incubating at 37° C. for 9 hours, then there were concentric rings emerged, which were extended outward continually with an interval of 3 hours, and this was taken as a control; adding the compound of formula (I) with final concentration of 0.5% onto the LB plate, the Proteus Mirabilis were innoculated by the same method, cultured at 37° C., and the result showed that not only the concentric rings formed with an interval of 3 hours were emerged, comparing with the control, it can be seen that there were also many fine waves on each ring emerged.", "2.Experimental Results and Evaluation: The experiment adopts a bio-wave model which is used to research the promoting wave function of the compound of formula (I).", "It can be seen from the result that the compound of formula (I) was not only able to cause bacterial cell to reveal a normal bio-wave characteristic, but also cause the wave reveal finer wave mode, and these indicated that the compound of formula (I) have promoting function to bio-waves, and the promoting wave function is able to modulate the wave of the smooth muscle of intestines and the wave of the bacterial colony in intestines.", "II.", "Toxicological Test of the Compound of Formula (I), Including: 1.acute toxicity test: including tests of administrating medicine by oral, Intravenous injection and maximum limit amount for administration; 2.Ames test; 3.micronucleus test of bone marrow cell of mouse; 4.abnormal sexual test for the sperm of mouse; 5.abnormal aberrance test for the chromosin of mouse's testis; 6.chronic lethal test; 7.subchronic toxicity (feed for 90 days) test; 8.traditional aberrance-inducing test; The results from these tests show that, in the acute toxicity test of the compound of formula (I), the dosage more than 2 g/kg is taken, which is 300 times than the injection dosage for human being, but acute toxicosis reaction had not appeared; in the long-period toxicity test, the maximum dosage has reached up to 1 g/kg, and after the treatment and observation for four weeks, there is no toxicosis reaction yet; and in the reproduction test, the mouse was fed with routine dosages from 7 mg/kg for 3 generations, it has been proved that the compound of formula (I) has no influence on the pregnancy, birth, nurse and the growth of baby mice, so it is proved that the compound of formula (I) is a substance without toxicity.", "III.", "Test of Resistance to Field Planting 1.Animal: Mice of Kunming Species, Provided by the 3rd Military Medical University P.R.", "China.", "2.Method: Firstly, the animals used for experiment were infused abdominally with a suspension of Pseudomonas aeruginosa so as to make a Pseudomonas aeruginosa planting model, the mice that have Pseudomonas aeruginosa in the feces specimen cultured continuously for three days are deemed as positive.", "32 mices which were positive in the experiment for making model were selected and divided into four groups, that is, three experimental groups with different dosages and one control group, eight mice in each group.", "The experimental groups are infused abdominally with N-acetyl-D glucosamine each day according to a schedule, while the control group is infused with saline solution.", "Cultivate the excrement every day, observe the saturation of Pseudomonas aeruginosa planting growth.", "3.Results: All of three dosages with high, middle and low concentrations of N-acetyl-Dglucosamine were able to effectively resist Pseudomonas aeruginosa in intestines.", "IV.", "Test of Stabilizing Lysosome Membrane: 1.Animals: wistar rat, provided by the 3rd Military Medical University P.R.", "China.", "2.Method: setting up a rat model of endotoxin shock, at the same time, for the liver lysosome of rat, carry out a test of stabilizing membranes in vivo and in vitro.", "The model animals were divided into two groups, in the experimental group, N-acetyl-D-glucosamine was used to carry out the treatment, in the control group, saline solution was used to carry out the same treatment.", "The condition of liver lysosome mambrane was reflected by the activity of acidic phosphatase (ACP).", "3.Result: the activity of free ACP in the control group and that in the experimental group are obviously different.", "4.Conclusion: N-acetyl-D-glucosamine is able to effectively stabilize liver lysosome membrane of rat with endotoxin shock, and decrease the release of acidic phosphatase.", "V. Human on Trial The experiment is based on the clinic test for the human who have suffered from peri-anal diseases, it is showed that the pharmaceutical preparations made from the compound of formula (I) are able to eliminate or relieve quickly the peri-anal uncomfortableness, pain, exudation, pruritus and the like caused by various reasons." ] ]	Patent_10469284
[ [ "Apparatus for heating a food product and heating devices and a feed assembly therefor", "A device (22) for heating a food product (P).", "The device (22) including an oven enclosure (52) at lest one magnetron for emitting microwave energy and a microwave energy focusing device (68, 70, 72) associated with at least one magnetron (56, 58, 60, 62, 64 and 66) and adapted to focus microwave energy towards the food product.", "The magnetron(s) is/are disposed external the enclosure (52) and the microwave energy focusing device(s) is/are disposed internal the enclosure (52).", "Also disclosed is a food product support device (116) of a substantially truncated conical external shape with a first larger base surface (118) adapted to be positioned adjacent the base of the oven enclosure and a second smaller supporting surface (120) adapted to support a food product (P) in a position vertically displaced from the base of the oven enclosure for heating and a sloping side surface (122) extending between the base and support surfaces.", "The food product (P) is able to exit the oven enclosure by sliding down the side surface (122) when pushed from the support surface towards (120) an opening in the oven enclosure.", "Also disclosed is a food product positioning and ejection device (138) adapted for reciprocal movement towards and away from an opening in the oven enclosure." ], [ "1.An device for heating a food product, the device including: an oven enclosure; at least one magnetron for emitting microwave energy; and a microwave energy focusing device associated with each magnetron and adapted to focus microwave energy towards the food product, wherein the magnetron(s) is/are disposed external the enclosure and the microwave energy focusing device(s) is/are disposed internal the enclosure.", "2.The device as claimed in claim 1, wherein the microwave energy focusing device(s) are a horn with a first end adjacent the magnetron(s) and a second end directed towards the food product.", "3.The device as claimed in claim 2, wherein the horn(s) has/have a rectangular cross-section or is/are substantially conical.", "4.The device as claimed in claim 2, wherein the horn(s) taper from a smaller first end to a larger second end.", "5.The device as claimed in claim 1, wherein the device includes a first (upper) magnetron.", "6.The device as claimed in claim 5, wherein the device includes second and third (side) magnetrons.", "7.The device as claimed in claim 1, wherein the oven enclosure includes a door, the door being associated with an interlock such that the magnetron(s) can not be energised whilst the door is open.", "8.An device for heating a food product, the apparatus including: a microwave oven enclosure; a food product support device within the oven enclosure, the device being of a substantially truncated conical external shape with a first larger base surface adapted to be positioned adjacent the base of the oven enclosure and a second smaller supporting surface adapted to support a food product in a position vertically displaced from the base of the oven enclosure for heating and a sloping side surface extending between the base and support surfaces, whereby the food product is able to exit the oven enclosure by sliding down the side surface when pushed from the support surface towards an opening in the oven enclosure.", "9.The device as claimed in claim 8, wherein the device includes: at least one magnetron for emitting microwave energy disposed external the enclosure; and a microwave energy focusing device disposed internal the enclosure and associated with each magnetron and adapted to focus microwave energy towards the support surface.", "10.The device as claimed in claim 8, wherein the support device includes a hollow polyethylene base portion that includes the base and side surfaces and a Teflon support portion that includes the support surface.", "11.The device as claimed in claim 10, wherein the Teflon support surface includes a dipole antenna adapted above which the food product is, in use, positioned, the antenna being adapted to focus the microwave energy towards the food product.", "12.The device as claimed in claim 11, wherein the antenna is a metal screw which is screwed into the surface of the Teflon support plate that is, in use, remote the food product.", "13.An device for heating a food product, the device including: an oven enclosure with an opening; a food product positioning and ejection device adapted for reciprocal movement towards and away from the opening between retracted and an extended positions; and a chute sloping downwardly into the enclosure through the opening, wherein when the device is adapted, when in the retracted position, to limit the movement of the food product into the enclosure by abutment with same further adapted to push the food product from the enclosure during movement towards the extended position.", "14.The device as claimed in claim 13, wherein the positioning and ejection device includes a food product head having a leading edge substantially complimentary to the food product and a sliding mechanism adapted to extend and retract the head.", "15.The device as claimed in claim 14, wherein the sliding mechanism includes a pair of guide rods and an expandable/retractable drive rod.", "16.The device as claimed in claim 15, wherein the drive rod is attached to a pneumatic cylinder or electrical solenoid.", "17.The device as claimed in claim 13, wherein the enclosure includes a door over the opening and the chute is adapted to pivot downwardly to present its lower edge towards the opening when the door is open and to pivot upwardly and away from the opening to allow the door to be opened and closed.", "18.The device as claimed in claim 13, wherein the enclosure includes a slide therein that is adapted to convey the food product from the chute to the retracted food product positioning and ejection device.", "19.The device as claimed in claim 13, wherein the device includes at least one internal magazine adapted to receive a substantially vertical stack of food products therein, the bottom of the magazine having an open end alignable with the chute.", "20.The device as claimed in claim 19, wherein the device includes a carousel with a multiplicity of said magazines therein.", "21.The device as claimed in claim 19, wherein the carousel is rotatable about a substantially vertical axis and adapted for indexed stopping in positions aligning the open end of each of said magazines with the chute.", "22.An apparatus for heating a food product, the apparatus including: a device as claimed in claim 1." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Food product vending machines are common at many public venues such as public transport stations, sporting fields, shopping centres and the like.", "The vending machines can be broadly categorised into three man types, namely: Refrigerating for products such as cool drinks and ice creams; Ambient for products such as sweets and crisps; and Heating for food products such as hot chips and prepared meals.", "A disadvantage of existing heating food product vending machines is that they are slow in operation and produce a cooked product that is inferior to that cooked by conventional methods.", "For example, most heating food product vending machines rely on a conventional microwave type oven which results in soggy food and require the customer to purchase the food product from a vending machine and then place it in, and subsequently remove it from, a separate microwave oven." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with a first aspect of the invention there is provided a device for heating a food product, the device including: an oven enclosure; at least one magnetron for emitting microwave energy; and a microwave energy focusing device associated with each magnetron and adapted to focus microwave energy towards the food product, wherein the magnetron(s) is/are disposed external the enclosure and the microwave energy focusing-device(s) is/are disposed internal the enclosure.", "The microwave energy focusing device(s) are preferably a horn with a first end adjacent the magnetron(s) and a second end directed towards the food product.", "In a preferred embodiment, the horn(s) has/have a substantially rectangular cross-section.", "In another embodiment, the horn(s) is/are substantially conical.", "In one form, the horns taper from a smaller first end to a larger second end.", "In another form, the first and second ends are the same size, which results in the horn(s) having the form of a parallel rectangular tube.", "In one embodiment, the device includes a first (upper) magnetron and a second (lower) magnetron which are desirably substantially (vertically) aligned to face one another.", "The long axis of the horn of the first magnetron is preferably oriented with the short axis of the second magnetron.", "The device preferably includes a third (lower) magnetron which is desirably substantially (horizontally) aligned side-by-side with the second magnetron.", "The device preferably includes an infra red heater which is desirably substantially (vertically) aligned facing the third magnetron.", "The device preferably includes a fourth (upper) magnetron and a fifth (upper) magnetron either side of the infra red heater &hat are desirably disposed at an angle to facing the third magnetron.", "The device optionally includes a sixth (lower) magnetron which is desirably substantially (horizontally) aligned side-by-side with the third magnetron.", "The infra red heater is desirably a single or a pair of halogen lamps.", "The oven enclosure desirably includes an inlet door and, an outlet door, the doors being associated with interlocks such that the magnetrons/heater can not be energised whilst either door is open.", "In another embodiment, the device includes a first (upper) magnetron and, desirably, second and third (side) magnetrons.", "The oven enclosure desirably includes a door, the door being associated with an interlock such that the magnetron(s) can not be energised whilst the door is open.", "The device desirably includes a conveyor in the oven enclosure between the inlet door and the outlet door.", "The conveyor is preferably controllable to convey a food product from the inlet door to a first heating position between the first and second magnetrons and then to a second heating position between the third, fourth and fifth magnetrons, and the heater and the fifth magnetron and then to the outlet door.", "The conveyor is optionally controllable to convey a food product from the second heating position to a third heating position above the sixth magnetron and then to the outlet door.", "The conveyor is preferably also controllable to hold the food product stationary in the first heating position for a first predetermined period of time.", "The conveyor is preferably also controllable to oscillate the food product in the second heating position a predetermined distance for a second predetermined period of time.", "The conveyor is preferably optionally controllable to hold the food product stationary in the third heating position for a third predetermined period of time.", "In accordance with a second aspect of the invention there is provided a food product feed assembly for a device for heating the food product, the assembly including: an apparatus for storage of a plurality of food products, the apparatus having an outlet opening with a movable sealing device; and a transfer means with a first receptacle adapted to receive the sealing device therein and a second receptacle adapted to receive one of the food products therein, the second receptacle having an outlet door adapted, upon opening, to release any food product in the second receptacle towards the device for heating the food product, the first receptacle being adjacent the outlet open when the transfer means is in a first position for receiving the sealing device and the second outlet being adjacent the outlet opening when the transfer means is in a second position for receiving a food product, wherein the second receptacle outlet door is only openable when the transfer means is in the second position such that outlet opening is maintained substantially sealed by the first receptacle's sealing device when the transfer means is in the first position and is maintained substantially sealed by the second receptacle's outlet door when the transfer means is in the second position.", "The transfer means is preferably adapted to receive the sealing device into the first receptacle in the first position, move to the second position and receive a food product in the second receptacle, then return to the second position for replacing the sealing device in the outlet and opening the outlet door to release the food product from the second receptacle.", "The transfer means is preferably adapted to reciprocally slide between the first and second positions.", "In an embodiment, the storage apparatus preferably includes at least one internal magazine adapted to receive a substantially vertical stack of food products therein, the bottom of the magazine having an open end alignable with the apparatus' outlet opening.", "The storage apparatus preferably includes a carousel with a multiplicity of said magazines therein.", "The carousel is desirably rotatable about a substantially vertical axis and adapted for indexed stopping in positions aligning the open end of each of said magazines with the apparatus' outlet opening.", "In accordance with a third aspect of the invention, there is provided a device for heating a food product, the apparatus including: a microwave oven enclosure; a food product support device within the oven enclosure, the device being of a substantially truncated conical external shape with a first larger base surface adapted to be positioned adjacent the base of the oven enclosure and a second smaller supporting surface adapted to support a food product in a position vertically displaced from the base of the oven enclosure for heating and a sloping side surface extending between the base and support surfaces, whereby the food product is able to exit the oven enclosure by sliding down the side surface when pushed from the support surface towards an opening in the oven enclosure.", "The device preferably includes: at least one magnetron for emitting microwave energy disposed external the enclosure; and a microwave energy focusing device disposed the enclosure and associated with each magnetron and adapted to focus microwave energy towards the food product on the support surface.", "The device preferably includes a hollow polyethylene base portion that includes the base and side surfaces and a teflon support portion that includes the support surface.", "The teflon support surface desirably includes a dipole antenna above which the food product is, in use, positioned, the antenna being adapted to focus the microwave energy towards the food product.", "The antenna is preferably in the form of a metal screw which is screwed into the side of the teflon support plate that is, in use, remote the food product.", "In accordance with a fourth aspect of the invention, there is provided a device for heating a food product, the apparatus including: an oven enclosure with an opening; a food product positioning and ejection device adapted for reciprocal movement towards and away from the opening between retracted and an extended positions; and a chute sloping downwardly into the enclosure through the opening, wherein when the device is adapted, when in the retracted position, to limit the movement of the food product into the enclosure by abutment with same further adapted to push the food product from the enclosure during movement towards the extended position.", "The position and ejection device preferably also includes a food product head having a leading edge substantially complimentary to the food product and a sliding mechanism adapted to extend and retract the head.", "The sliding mechanism desirably includes a pair of guide rods and an expandable/retractable drive rod.", "The drive rod is preferably attached to a pneumatic cylinder or electrical solenoid.", "The enclosure preferably includes a door over the opening and the chute is adapted to pivot downwardly to present its lower edge towards the opening when the door is open and to pivot upwardly to and away from the opening to allow the door to be opened and closed.", "Further, the enclosure desirably also includes a slide therein that is adapted to convey the food product from the chute to the retracted food product positioning and ejection device.", "In an embodiment, the apparatus includes at least one internal magazine adapted to receive a substantially vertical stack of food products therein, the bottom of the magazine having an open end alignable with the chute.", "The apparatus preferably includes a carousel with a multiplicity of said magazines therein.", "The carousel is desirably rotatable about a substantially vertical axis and adapted for indexed shipping in positions aligning the open end of each of said magazines with the chute.", "In accordance with a fifth aspect of the invention there is provided an apparatus for heating a food product, the apparatus including a device for heating a food product in accordance with the first aspect of the invention; a device for heating a food product in accordance with the third aspect of the invention and a device for heating a food product in accordance with the fourth aspect of the invention." ], [ "FIELD OF THE INVENTION The present invention relates to an apparatus for heating a food product and heating devices and a feed assembly therefor.", "The apparatus, device and assembly have been primarily developed for use in a hot pie vending machine and will be described hereinafter with reference to that application.", "However, it will be appreciated that the apparatus, device and assembly are not limited to that particular application.", "BACKGROUND OF THE INVENTION Food product vending machines are common at many public venues such as public transport stations, sporting fields, shopping centres and the like.", "The vending machines can be broadly categorised into three man types, namely: Refrigerating for products such as cool drinks and ice creams; Ambient for products such as sweets and crisps; and Heating for food products such as hot chips and prepared meals.", "A disadvantage of existing heating food product vending machines is that they are slow in operation and produce a cooked product that is inferior to that cooked by conventional methods.", "For example, most heating food product vending machines rely on a conventional microwave type oven which results in soggy food and require the customer to purchase the food product from a vending machine and then place it in, and subsequently remove it from, a separate microwave oven.", "OBJECT OF THE INVENTION It is an object of the present invention to substantially overcome or at least ameliorate the prior art deficiencies.", "SUMMARY OF THE INVENTION In accordance with a first aspect of the invention there is provided a device for heating a food product, the device including: an oven enclosure; at least one magnetron for emitting microwave energy; and a microwave energy focusing device associated with each magnetron and adapted to focus microwave energy towards the food product, wherein the magnetron(s) is/are disposed external the enclosure and the microwave energy focusing-device(s) is/are disposed internal the enclosure.", "The microwave energy focusing device(s) are preferably a horn with a first end adjacent the magnetron(s) and a second end directed towards the food product.", "In a preferred embodiment, the horn(s) has/have a substantially rectangular cross-section.", "In another embodiment, the horn(s) is/are substantially conical.", "In one form, the horns taper from a smaller first end to a larger second end.", "In another form, the first and second ends are the same size, which results in the horn(s) having the form of a parallel rectangular tube.", "In one embodiment, the device includes a first (upper) magnetron and a second (lower) magnetron which are desirably substantially (vertically) aligned to face one another.", "The long axis of the horn of the first magnetron is preferably oriented with the short axis of the second magnetron.", "The device preferably includes a third (lower) magnetron which is desirably substantially (horizontally) aligned side-by-side with the second magnetron.", "The device preferably includes an infra red heater which is desirably substantially (vertically) aligned facing the third magnetron.", "The device preferably includes a fourth (upper) magnetron and a fifth (upper) magnetron either side of the infra red heater &hat are desirably disposed at an angle to facing the third magnetron.", "The device optionally includes a sixth (lower) magnetron which is desirably substantially (horizontally) aligned side-by-side with the third magnetron.", "The infra red heater is desirably a single or a pair of halogen lamps.", "The oven enclosure desirably includes an inlet door and, an outlet door, the doors being associated with interlocks such that the magnetrons/heater can not be energised whilst either door is open.", "In another embodiment, the device includes a first (upper) magnetron and, desirably, second and third (side) magnetrons.", "The oven enclosure desirably includes a door, the door being associated with an interlock such that the magnetron(s) can not be energised whilst the door is open.", "The device desirably includes a conveyor in the oven enclosure between the inlet door and the outlet door.", "The conveyor is preferably controllable to convey a food product from the inlet door to a first heating position between the first and second magnetrons and then to a second heating position between the third, fourth and fifth magnetrons, and the heater and the fifth magnetron and then to the outlet door.", "The conveyor is optionally controllable to convey a food product from the second heating position to a third heating position above the sixth magnetron and then to the outlet door.", "The conveyor is preferably also controllable to hold the food product stationary in the first heating position for a first predetermined period of time.", "The conveyor is preferably also controllable to oscillate the food product in the second heating position a predetermined distance for a second predetermined period of time.", "The conveyor is preferably optionally controllable to hold the food product stationary in the third heating position for a third predetermined period of time.", "In accordance with a second aspect of the invention there is provided a food product feed assembly for a device for heating the food product, the assembly including: an apparatus for storage of a plurality of food products, the apparatus having an outlet opening with a movable sealing device; and a transfer means with a first receptacle adapted to receive the sealing device therein and a second receptacle adapted to receive one of the food products therein, the second receptacle having an outlet door adapted, upon opening, to release any food product in the second receptacle towards the device for heating the food product, the first receptacle being adjacent the outlet open when the transfer means is in a first position for receiving the sealing device and the second outlet being adjacent the outlet opening when the transfer means is in a second position for receiving a food product, wherein the second receptacle outlet door is only openable when the transfer means is in the second position such that outlet opening is maintained substantially sealed by the first receptacle's sealing device when the transfer means is in the first position and is maintained substantially sealed by the second receptacle's outlet door when the transfer means is in the second position.", "The transfer means is preferably adapted to receive the sealing device into the first receptacle in the first position, move to the second position and receive a food product in the second receptacle, then return to the second position for replacing the sealing device in the outlet and opening the outlet door to release the food product from the second receptacle.", "The transfer means is preferably adapted to reciprocally slide between the first and second positions.", "In an embodiment, the storage apparatus preferably includes at least one internal magazine adapted to receive a substantially vertical stack of food products therein, the bottom of the magazine having an open end alignable with the apparatus' outlet opening.", "The storage apparatus preferably includes a carousel with a multiplicity of said magazines therein.", "The carousel is desirably rotatable about a substantially vertical axis and adapted for indexed stopping in positions aligning the open end of each of said magazines with the apparatus' outlet opening.", "In accordance with a third aspect of the invention, there is provided a device for heating a food product, the apparatus including: a microwave oven enclosure; a food product support device within the oven enclosure, the device being of a substantially truncated conical external shape with a first larger base surface adapted to be positioned adjacent the base of the oven enclosure and a second smaller supporting surface adapted to support a food product in a position vertically displaced from the base of the oven enclosure for heating and a sloping side surface extending between the base and support surfaces, whereby the food product is able to exit the oven enclosure by sliding down the side surface when pushed from the support surface towards an opening in the oven enclosure.", "The device preferably includes: at least one magnetron for emitting microwave energy disposed external the enclosure; and a microwave energy focusing device disposed the enclosure and associated with each magnetron and adapted to focus microwave energy towards the food product on the support surface.", "The device preferably includes a hollow polyethylene base portion that includes the base and side surfaces and a teflon support portion that includes the support surface.", "The teflon support surface desirably includes a dipole antenna above which the food product is, in use, positioned, the antenna being adapted to focus the microwave energy towards the food product.", "The antenna is preferably in the form of a metal screw which is screwed into the side of the teflon support plate that is, in use, remote the food product.", "In accordance with a fourth aspect of the invention, there is provided a device for heating a food product, the apparatus including: an oven enclosure with an opening; a food product positioning and ejection device adapted for reciprocal movement towards and away from the opening between retracted and an extended positions; and a chute sloping downwardly into the enclosure through the opening, wherein when the device is adapted, when in the retracted position, to limit the movement of the food product into the enclosure by abutment with same further adapted to push the food product from the enclosure during movement towards the extended position.", "The position and ejection device preferably also includes a food product head having a leading edge substantially complimentary to the food product and a sliding mechanism adapted to extend and retract the head.", "The sliding mechanism desirably includes a pair of guide rods and an expandable/retractable drive rod.", "The drive rod is preferably attached to a pneumatic cylinder or electrical solenoid.", "The enclosure preferably includes a door over the opening and the chute is adapted to pivot downwardly to present its lower edge towards the opening when the door is open and to pivot upwardly to and away from the opening to allow the door to be opened and closed.", "Further, the enclosure desirably also includes a slide therein that is adapted to convey the food product from the chute to the retracted food product positioning and ejection device.", "In an embodiment, the apparatus includes at least one internal magazine adapted to receive a substantially vertical stack of food products therein, the bottom of the magazine having an open end alignable with the chute.", "The apparatus preferably includes a carousel with a multiplicity of said magazines therein.", "The carousel is desirably rotatable about a substantially vertical axis and adapted for indexed shipping in positions aligning the open end of each of said magazines with the chute.", "In accordance with a fifth aspect of the invention there is provided an apparatus for heating a food product, the apparatus including a device for heating a food product in accordance with the first aspect of the invention; a device for heating a food product in accordance with the third aspect of the invention and a device for heating a food product in accordance with the fourth aspect of the invention.", "BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the invention will now be described, by way of example only; with reference to the accompanying drawings, in which; FIG.", "1 is a schematic front view of an apparatus for heating a food product in accordance with a first embodiment of the invention; FIG.", "2 is a schematic top view of the apparatus shown in FIG.", "1; FIG.", "3 is a schematic side view of the apparatus shown in FIG.", "1; FIG.", "4 is a schematic side view of an embodiment of a transfer means used in a food product feed assembly used in the apparatus shown in FIG.", "1; FIG.", "5 is a schematic top view of the transfer means shown in FIG.", "4; FIG.", "6 is a schematic side view of a sealing device used with the transfer means shown in FIG.", "4; FIG.", "7 is a schematic top view of the sealing device shown in FIG.", "6; FIGS.", "8 to 11 are schematic cross sectional side views of an embodiment of a food product feed assembly utilising the transfer means shown in FIG.", "4 and the sealing device shown in FIG.", "6 in various progressive stages of operation; FIG.", "12 is a schematic side view of an embodiment of a device for heating a food product used in the apparatus shown in FIG.", "1; FIG.", "13 is a schematic left hand end view of the device shown in FIG.", "12; FIG.", "14 is a schematic right hand end view of the device shown in FIG.", "12; FIG.", "15 is a schematic top view of the device shown in FIG.", "12; FIG.", "16 is a schematic side view of a first embodiment of a microwave energy focusing device used in the food product heating device shown in FIG.", "12; FIG.", "17 is a schematic top view of the focusing device shown in FIG.", "16; FIG.", "18 is a schematic side view of a second embodiment of a microwave energy focusing device; FIG.", "19 is a schematic top view of the focusing device shown in FIG.", "18.FIG.", "20 is a schematic front view of a second embodiment of a device for heating a food product used in a second embodiment of an apparatus for heating a food product according to the invention; FIG.", "21 is a schematic top view of the device shown in FIG.", "20.FIG.", "22 is a schematic font view of the device shown in FIG.", "20 with the oven door open during delivery of a pie; FIG.", "23 is a schematic front view of the device shown in FIG.", "20 with the oven door closed during cooking of the pie; FIG.", "24 is a schematic front view of the device shown in FIG.", "20 with the oven door open showing ejection of the pie; and FIG.", "25 is an enlarged cross sectional view of a top plate used in the device shown in FIG.", "20.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring firstly to FIGS.", "1 to 3, there is shown an apparatus 20 for heating a food product, which in the preferred embodiment shown is a frozen pie P (see FIGS.", "8 to 11 and FIGS.", "13 to 14), according to a first embodiment of the invention.", "The apparatus 20 includes a device for heating the pie P, designated generally by the reference numeral 22, and a frozen pie feed assembly, designated generally by the reference numeral 24.The device 22 and assembly 24 will be described individually in more detail below.", "Referring to FIGS.", "1 to 11, the feed assembly 24 basically comprises a sealable storage apparatus 26 which, in the preferred embodiment shown, is both refrigerated and insulated.", "The storage apparatus 26 includes a rotatable carousel 28 with an outer ring of twelve open-ended cylindrical magazines 30 therein that are each able to hold a stack of up to twenty-five frozen pies P. The magazines 30 have removable sliding closures (not shown) on each end to retain the pies P therein during transport, storage and installation and to prevent theft.", "The closures are removed during installation of the magazines 30 into the carousel 28.The storage apparatus 26 has an outlet opening 32 at its lower end which is adapted to receive a moveable sealing device 34 (see FIG.", "8) therein, as will be described in more detail below.", "The carousel 28 is rotatable about a vertical axis 36 and adapted for indexed stopping in twelve positions in which the lower open end of each of the magazines 30 is respectively aligned with the outlet opening 32 of the storage apparatus 26.As best seen in FIGS.", "4 and 5, the assembly 24 also includes a transfer means, in the form of block 38.The block 38 includes a first circular receptacle 40 adapted to receive the sealing device 34 therein and a second circular receptacle 42 adapted to receive one of the frozen pies P therein.", "As best seen in FIGS.", "8 to 11, the underside of the second receptacle 42 has an outlet door 44 which is pivotable in the direction of arrow 46 (see FIG.", "11) to open or close the second receptacle 42.The block 38 reciprocally slides between a first position (see FIGS.", "8 and 9) in which the first receptacle 40 is fully vertically aligned with the outlet opening 32 of the storage apparatus and a second position (see FIG.", "10) in which the second receptacle 42 is substantially vertically aligned with the outlet opening 32.When the block is in the fist position the second receptacle 42 is substantially vertically aligned with a transfer chute 48 which leads to a transfer conveyor 50 (see FIG.", "3) which itself leads to the food product heating device 22, which will now be described with reference to FIGS.", "12 to 17.The food product heating device 22 is basically comprised of a metal oven enclosure 52 which has first, second, third, fourth, fifth and optionally sixth microwave energy omitting magnetrons 56, 58, 60, 62, 64 and 66 respectively mounted thereon.", "The magnetrons, in the preferred embodiment shown, are of 1 to 2 KW capacity.", "The first, second and third magnetrons 56, 58 and 60 have their microwave energy directed by first embodiments of focusing horns 68, 70 and 72 respectively.", "As best seen in FIGS.", "16 and 17, the horns 68, 70 and 72 taper from a smaller end 74 adjacent the magnetron to a larger end 76 and have a rectangular cross section.", "The longer axis L of the first horn 68 is oriented with the shorter axis of the second horn 70.FIGS.", "18 and 19 show second embodiments of focusing horns 68a, 70a and 72a of parallel rectangular tube form that can alternatively be used with the food product heating device 22 of the apparatus 20.Returning to FIGS.", "12 to 15, the oven enclosure 52 also includes an infra red heater 77 in the form of a pair of halogen lamps 78 covered by a mesh screen 80, and an inlet and an outlet door 82 and 84 respectively.", "A controllable conveyor 86 driven by an electric drive motor 88 is provided between the inlet and outlet doors 82 and 84.As best shown in FIGS.", "1 and 3, the transfer conveyor 50 leads to the oven inlet door 82.In the preferred form shown, the first and second magnetrons 56 and 58 are substantially vertically aligned, the second, third and sixth magnetrons 58, 60 and 66 are substantially horizontally aligned and the fourth and fifth magnetrons 62 and 64 are angled with respect to the third magnetron 60 and are positioned either side of the infra red heater 77.A control system (not shown) is also included in the apparatus 20, preferably a Programmable Logic Controller (PLC) which are well known in the art and will not be described in any further detail.", "The operation of the food product heating apparatus 20 will now be described.", "Firstly, the carousel 28 is loaded with magazines 30 of frozen pies P, which are preferably at a temperature of −18° C. to −20° C. This reduces the required cooling capacity of the refrigeration device associated with the storage chamber 26 as it does not have to freeze/chill the food products, only maintain them at their pre-chilled delivery temperature.", "Different styles or flavours of pie P can be loaded into different magazines 30.Secondly, a customer requests a particular style or flavour of pie P and places an appropriate payment into the apparatus 20.Various payment accepting mechanisms are also well known in the art and will not be described in any further detail.", "The control system then energises a stepper motor (not shown) to rotate the carousel 28 until the magazine 30 with the desired pie P therein is above the outlet 32, as is best seen in FIGS.", "8 to 11.The control system then causes the sealing device 34 to be driven, preferably by a pneumatic cylinder (not shown), from within the outlet opening 32 to within the first receptacle 40.The sealing device 34 carries an adjacent pie P with it under the influence of gravity from the bottom of the magazine 30 into the outlet opening 32, as indicated by arrow 89.The control system then causes the block 38 to be driven, again preferably by a pneumatic cylinder (not shown), in the direction of arrow 92 from the first position (see FIGS.", "8 and 9) to the second position (see FIG.", "10) which results in the pie falling under the influence of gravity into the second receptacle 42.The outlet door 44 associated with the second opening 42 remains closed whilst the block 38 is in the second position.", "The control system then causes the block 38 to be driven in the direction of arrow 94 back to the first position (see FIG.", "11) and energises a further pneumatic cylinder (not shown) to cause the door 44 to pivot open, in the direction of arrow 46.This results in the pie P, falling onto the transfer chute 48 for travel, in the direction indicated by arrow 98, towards the transfer conveyor 50 (see FIG.", "1).", "Simultaneously, the sealing device 34 is driven back into the outlet opening 32 to close same, as indicated by arrow 100.One reciprocal cycle of movement of the block 38 occurs within about 2 to 3 seconds.", "Turning now to FIGS.", "12 to 15, the inlet door 82 is opened and the conveyor 50 (see FIG.", "1) is activated by the control system to drive the pie P into the oven enclosure 52 and onto the conveyor 86.The control system then actuates the drive motor 88 to move the conveyor 86 and bring the pie P to a first heat position (see FIG.", "12) between the first and second magnetrons 56 and 58 the magnetrons 56 and 58 are then activated for between 10 to 30 seconds.", "The position of the pie P is preferably engaged by an electronic beam (not shown) communicating with the control system.", "Whilst in this position, the horns 68 and 70 (which are oriented at 90 degrees to one another) direct the microwave energy (which are thus also oriented at 90 degree to one another) emitted from the first and second magnetrons 56 and 58 into the frozen centre of the pie P to where maximum heating is required.", "The 90° orientation mentioned above advantageously avoids cross coupling of the energy of one magnetron to the other, as well as increasing the uniformity of heating of the central core of the pie P. The pie P is then advanced to a second heating position between the third, fourth and fifth magnetrons 60, 62 and 64 and the infra red heater 77 where it oscillates fore and aft over an amplitude of 40 millimetres for a further 10 to 30 seconds.", "Whilst in this position, the third, fourth and fifth magnetrons 60, 62 and 64 continue to heat the remainder of the pie P, including the previously thawed centre, and the infra red heater 77 heats, browns and crisps the top layer of pastry.", "The magnetrons/infra red heater are then de-energised and the pie P is advanced to and through the outlet door 84 for pick up by the customer.", "It should be noted that all the magnetrons and the infra red heater are interlocked with the inlet and outlet doors 82 and 84 and thus cannot be energised whilst either of the doors 82, 84 are open, either intentionally or inadvertently.", "If further heating is required the control system can also be configured to place the pie above the sixth optional magnetron 66.The embodiment of the invention described above has the following advantages over prior art devices.", "Firstly, the product feed assembly 24 is able to quickly deliver a selected pie from within the substantially sealed storage and refrigeration apparatus to the oven enclosure in 2 to 3 seconds.", "In this connection, it should be noted that the integrity of the seal of the refrigerated storage apparatus 26 is always maintained by either the sealing device when the block is in the first position or by the second receptacle outlet door when the block is in the second position, thereby ensuring minimal temperature losses.", "Also, if the invention is used to deliver a food product that is not refrigerated, then the same sealing arrangement advantageously maintains a substantially hermetic seal of the food storage chamber.", "Secondly, the two stage heating process (ie.", "high intensity focused microwave energy and infra red heat) of the preferred embodiment is able to quickly thaw and heat a frozen food product whilst minimising degradation of the food product.", "Thirdly, the total process from ordering a pie to receiving the heated pie takes only about 30 to 45 seconds and does not require any assistance from the customer.", "FIGS.", "20 to 24 show a second embodiment of a device, designated generally by the reference numeral 100, for heating a pie P. The device 100 includes an oven enclosure 102 that has one (1000 Watt) upper magnetron 104 and two (800 Watt) side magnetrons 106 and 108 respectively, which are located externally of the enclosure 102.Each of the magnetrons 104, 106 and 108 have an associated focusing horn.", "110, 112 and 114 respectively, which are located within the oven enclosure 102.The horns 110, 112 and 114 taper from a smaller end adjacent to their respective magnetron to a larger end inside the enclosure 102 focus the microwave energy from the magnetrons 104, 106 and 108 towards the pie P which is, during heating, positioned atop a food product support device, designated generally by the reference numeral 116, which will be described in more detail below.", "The oven enclosure 102 is similar to known household microwave ovens, except for the addition of the magnetrons 104 and 106 and the horns 110, 112 and 114 previously described.", "The support device 116 has a substantially truncated conical external shape with a large annular base surface 118, a smaller circular support 120 and all outwardly downwardly sloping side surface 122 therebetween.", "The support device 116 is formed from two components, namely a polyethylene hollow base portion 124 and a teflon top plate 126.The base portion 124 sits on, and in use in rotated by, the oven's internal electric motor driven base carousel 127, which are well known.", "The top plate 126 has a short dipole antenna, in the form of a steel screw 128, screwed into it.", "The screw 128 serves to assist in focusing the microwave energy emitted from the focusing horns 110, 112 and 114, which are themselves directed towards the (elevated) heating position of the pie P when atop the support device 116.As best shown in FIG.", "25, the top plate 126 has a central rebate 127 to assist locating the pie P thereon as the pie P slides into the oven enclosure 102.The diameter of the rebate 127 matches the size of the pie base.", "The front of the enclosure 102 has a door 130 adapted to be opened and closed under control from the previously described PLC.", "External the enclosure 102 is a pivotally mounted chute 132 having a distal end that rests on the top edge of the door 130.The chute 132 pivots to a raised position (see FIG.", "23) by being pushed upwards by the door 130 as the door 130 is closed.", "When the door 130 is opened, the chute 132 pivots, under the influence of gravity, to a lower position (see FIGS.", "22 and 24), where the lower most edge of the chute 132 is directed into the oven enclosure 102.In the lower position the lower edge of the chute 132 is substantially adjacent to the upper edge of a slide 134 suspended within the enclosure 102.As is shown by arrow 136, the lowered chute 132 and slide 134 co-operate to provide a path for a pie P down the chute 132, across the slide 134 and onto the teflon top plate 126.The chute 132 is fed from the previously described carousel 28.A food product positioning and ejection device, designated generally by the reference numeral 138, also extends into the oven enclosure 102.The device 138 includes a polyethylene positioning head 140 whose leading edge 142 is complimentary to that of the pie P. The head 140 is able to be extended and retracted in the direction of double headed arrow 144 by expandable/retractable pneumatic cylinder 146.The device 138 also includes guide rods 148 to maintain the head 140 in the orientation shown.", "The operation of the device 100 will now be described.", "Firstly, a modified form (not shown) of the previously described carousel 28 is loaded with frozen pies P. The modified carousel uses fixed guide rods in place of the removable magazines 30.Secondly, a customer requests a particular style or flavour of pie P and places an appropriate payment into the apparatus, also as previously described.", "The control system then energises a ratchet type index arm (not shown), operated by a pneumatic cylinder (not shown), to rotate the carousel 28 until the desired pie P is above the chute 132, again as previously described.", "The control system then causes a device (not shown) similar to the moveable sealing device 34 to be driven away from the cylinder outlet opening 32 so that an adjacent pie P falls, under the influence of gravity, from the bottom of the cylinder 30 and on to the chute 132.In preparation for the pie P entering the chute 132, the door 130 is opened, which allows the chute 132 to pivot downwardly into the lowered position shown in FIG.", "22.The head 140 is then extended to the position shown in FIG.", "22.The pie P then slides down the chute 132, across the slide 134 and onto the top plate 126, all in the direction of arrow 136.The movement of the pie P is stopped by its abutment with the surface 142 of the head 140, thereby positioning the pie P generally in the centre of the top plate 126 adjacent the rebate 127 and, importantly, above the screw 128 and between the horns 110, 112 and 114.The door 130 is then closed which allows the chute 132 to pivot upwardly to the raised position shown in FIG.", "23.During this time the head 140 is retracted to the position shown in FIG.", "23 so that all available microwave energy can be received by the pie P. The magnetrons 104, 106 and 108 are then energised and the pie P is then defrosted and heated.", "The antenna (screw 128) helps to further focus and concentrate the microwave energy in the base of the pie P. When the heating is complete, the door 130 is open and the head 140 is extended towards the pie P and across the top plate 126 so as to push the cooked pie P from the plate onto the sloping side wall 122.The pie P slides down the wall 122 into a customer delivery chute, in the direction of arrow 146.The head is then retracted to the position shown in FIG.", "22, ready for receipt of another pie from the chute 132 and slide 134.The second embodiment of the invention described above possesses the advantages of the first described embodiment.", "The second embodiment also has the advantage of lower construction costs due to the use of a modified form of a readily available domestic microwave oven enclosure 102 and the simplified positioning and ejection device 138.Another advantage is the focusing and concentrating of the microwave energy in the base of the pie P caused by the antenna (screw 128).", "Although the invention has been described with reference to preferred embodiments, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.", "As examples, the invention is also able to be used with other frozen and non-frozen food products tat are best served with a crisped or browned upper surface such as pizzas, pastries, sausage rolls, calzones and turnovers.", "Also, more than one type of food product may be provided in a single apparatus.", "For example, with reference to FIG.", "2, pies can be provided in the outer row of small cylindrical chambers and pizzas may be provided in the inner row of larger cylindrical chambers.", "The inner row of magazines being associated with a second feed assembly (not shown) similar to the feed assembly described above." ] ]	Patent_10469312
[ [ "Methods and primers for evaluating hiv-1 mutations", "Primer sequences and a method of using such sequences for the genotyping of HIV-1-containing samples, particularly those which have failed genotyping analysis are provided using primer sequences designed for analysis of Group B subtype of the Group M type virus.", "For example, a combination of primers, including at least one species of forward primer and at least one species of reverse primer where the forward primer(s) can be represented by the degenerate sequence: RARRARGGGCTGYTGGARATGTS (Seq.", "ID No.", "9) and the reverse primer(s) can be represented by the degenerate sequence: BCHTYACYTTRATCCCSGVRTARATYTGACT (Seq.", "ID No.", ": 10) or BCHTYACYTTRATCCCSGVRTARATYTGAC (Seq.", "ID No.", "12) are suitably employed.", "The selected primers, one or more from each group, can be used as reverse transcription, amplification and sequencing primers and are suitably packaged in a genotyping kit.", "Such a kit may include reagents in addition to the primers, such as an RNase inhibitor, a reverse transcriptase, a polymerase, and/or dNTP and ddNTP feedstocks." ], [ "1.A primer combination comprising, in a single solution, at least one forward HIV-1 primer selected from among primers comprising a sequence represented by the degenerate sequence RARRARGGGCTGYTGGARATGTS (Seq ID No.", "9) and at least one reverse HIV-1 primer selected from among primers comprising a sequence represented by the degenerate sequence AGTCARATYTAYBCWGGGATYAARGTRADGV (Seq.", "ID No.", ": 10) or GTCARATYTAYBCWGGGATYAARGTRADGV.", "(Seq.", "ID No.", "12) 2.The primer combination of claim 1, wherein at least one forward primer comprises a sequence selected from the group consisting of: GGAAAAAGGGCTGTTGGAAATGTGG (Seq.", "ID No.", ": 17) GGAAAAAGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 18) GGAAAAAGGGCTGTTGGAAATGTCG (Seq.", "ID No.", ": 19) GGAAAAAGGGCTGTTGGAAATGTC (Seq.", "ID No.", ": 11) GGAAAAAGGGCTGTTGGAAATGYG (Seq.", "ID No.", ": 1) GRARRARGGGCTGTTGGAAATGTGG (Seq.", "ID No.", ": 2) GRARRARGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 3) GAAAAAGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 15) GAAAAAGGGCTGTTGGAAATGCG.", "(Seq.", "ID No.", ": 16) 3.The primer combination of claim 1, wherein at least one reverse primer comprises a sequence selected from the group consisting of: AGTCAGATTTACCCAGGGATTAAAGTAAGGV (Seq.", "ID No.", "4) AGTCAGATTTACCCAGGGATTAAGGTAAGGV (Seq.", "ID No.", "5) AGTCAGATTTACCCAGGGATCAAAGTAAGGV (Seq.", "ID No.", "6) GYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", "7) AGYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", "8) GTCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", ": 13) GCCAGATTTACCCAGGGATTAAAGTAAGGC.", "(Seq.", "ID No.", ": 14) 4.The primer combination of claim 3, wherein at least one forward primer comprises a sequence selected from the group consisting of: GGAAAAAGGGCTGTTGGAAATGTGG (Seq.", "ID No.", ": 17) GGAAAAAGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 18) GGAAAAAGGGCTGTTGGAAATGTCG (Seq.", "ID No.", ": 19) GGAAAAAGGGCTGTTGGAAATGTC (Seq.", "ID No.", ": 11) GGAAAAAGGGCTGTTGGAAATGYG (Seq.", "ID No.", ": 1) GRARRARGGGCTGTTGGAAATGTGG (Seq.", "ID No.", ": 2) GRARRARGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 3) GAAAAAGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 15) GAAAAAGGGCTGTTGGAAATGCG.", "(Seq.", "ID No.", ": 16) 5.The primer combination of claim 4, wherein the forward and reverse primers are members of a set of degenerate forward and reverse primers, and the primer combination includes at least two species of degenerate forward and at least two species of degenerate reverse primers.", "6.The primer combination of claim 5, wherein the forward and reverse primers comprise sequences as set forth in Seq.", "ID Nos.", "1 and 7, respectively.", "7.The primer combination of claim 5, wherein the forward primers are members of the set of degenerate primers comprising the sequence: GGAAAAAGGGCTGTTGGAAATGYG.", "(Seq.", "ID No.", ": 1) 8.The primer combination of claim 7, wherein the forward primers have sequences as set forth in Seq.", "ID Nos.", "15 and 16.9.The primer combination of claim 7, wherein the reverse primers are members of the set of degenerate primers comprising the sequence: GYCAGATTTACCCAGGGATTAAAGTAAGGC.", "(Seq.", "ID No.", "7) 10.The primer combination of claim 9, wherein the reverse primers have the sequence as set forth in Seq.", "ID Nos.", "13 and 14.11.The primer combination of claim 5, wherein the reverse primers are members of the set of degenerate primers comprising the sequence: GYCAGATTTACCCAGGGATTAAAGTAAGGC.", "(Seq.", "ID No.", "7) 12.The primer combination of claim 11, wherein the reverse primers have the sequence as set forth in Seq.", "ID Nos.", "13 and 14.13.The primer combination of claim 1, wherein the forward primers or the reverse primers are labeled with a detectable label.", "14.The primer combination of claim 13, wherein the detectable label is a fluorescent label.", "15.A genotyping kit comprising at least one forward HIV-1 primer selected from among primers comprising a sequence represented by the degenerate sequence RARRARGGGCTGYTGGARATGTS (Seq ID No.", "9) and at least one reverse HIV-1 primer selected from among primers comprising a sequence represented by the degenerate sequence AGTCARATYTAYBCWGGGATYAARGTRADGV (Seq.", "ID No.", ": 10) or GTCARATYTAYBCWGGGATYAARGTRADGV.", "(Seq.", "ID No.", "12) 16.The kit of claim 15, wherein at least one forward primer is selected from the group consisting of: GGAAAAAGGGCTGTTGGAAATGTGG (Seq.", "ID No.", ": 17) GGAAAAAGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 18) GGAAAAAGGGCTGTTGGAAATGTCG (Seq.", "ID No.", ": 19) GGAAAAAGGGCTGTTGGAAATGTC (Seq.", "ID No.", ": 11) GGAAAAAGGGCTGTTGGAAATGYG (Seq.", "ID No.", ": 1) GRARRARGGGCTGTTGGAAATGTGG (Seq.", "ID No.", ": 2) GRARRARGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 3) GAAAAAGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 15) GAAAAAGGGCTGTTGGAAATGCG.", "(Seq.", "ID No.", ": 16) 17.The kit of claim 15, wherein at least one reverse primer is selected from the group consisting of: AGTCAGATTTACCCAGGGATTAAAGTAAGGV (Seq.", "ID No.", "4) AGTCAGATTTACCCAGGGATTAAGGTAAGGV (Seq.", "ID No.", "5) AGTCAGATTTACCCAGGGATCAAAGTAAGGV (Seq.", "ID No.", "6) GYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", "7) AGYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", "8) GTCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", ": 13) GCCAGATTTACCCAGGGATTAAAGTAAGGC.", "(Seq.", "ID No.", ": 14) 18.The kit of claim 17, wherein at least one forward primer is selected from the group consisting of: GGAAAAAGGGCTGTTGGAAATGTGG (Seq.", "ID No.", ": 17) GGAAAAAGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 18) GGAAAAAGGGCTGTTGGAAATGTCG (Seq.", "ID No.", ": 19) GGAAAAAGGGCTGTTGGAAATGTC (Seq.", "ID No.", ": 11) GGAAAAAGGGCTGTTGGAAATGYG (Seq.", "ID No.", ": 1) GRARRARGGGCTGTTGGAAATGTGG (Seq.", "ID No.", ": 2) GRARRARGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 3) GAAAAAGGGCTGTTGGAAATGTG (Seq.", "ID No.", ": 15) GAAAAAGGGCTGTTGGAAATGCG.", "(Seq.", "ID No.", ": 16) 19.The kit of claim 15, wherein the forward and reverse primers are members of a set of degenerate forward and reverse primers, and the primer combination includes at least two species of degenerate forward and at least two species of degenerate reverse primers.", "20.The kit of claim 19, wherein the forward primers are members of the set of degenerate primers comprising the sequence: GGAAAAAGGGCTGTTGGAAATGYG.", "(Seq.", "ID No.", ": 1) 21.The kit of claim 20, wherein the forward primers have the sequence as set forth in Seq.", "ID Nos.", "15 and 16.22.The kit of claim 20, wherein the reverse primers are members of the set of degenerate primers comprising the sequence: GYCAGATTTACCCAGGGATTAAAGTAAGGC.", "(Seq.", "ID No.", "7) 23.The kit of claim 22, wherein the forward primers have the sequence as set forth in Seq.", "ID Nos.", "15 and 16.24.The kit of claim 23, wherein the reverse primers have the sequence as set forth in Seq.", "ID Nos.", "13 and 14.25.The kit of claim 19, wherein the reverse primers are members of the set of degenerate primers comprising the sequence: GYCAGATTTACCCAGGGATTAAAGTAAGGC.", "(Seq.", "ID No.", "7) 26.The kit of claim 25, wherein the reverse primers have the sequence as set forth in Seq.", "ID Nos.", "13 and 14.27.The kit of claim 15, wherein the forward primers or the reverse primers are labeled with a detectable label.", "28.The kit of claim 27, wherein the detectable label is a fluorescent label.", "29.The kit of claim 15, wherein the kit further comprises one or more reagents selected from the group consisting of an RNase inhibitor, a reverse transcriptase, a polymerase, and dNTP and ddNTP feedstocks.", "30.The kit of claim 29, wherein the forward primers or the reverse primers are labeled with a detectable label.", "31.The kit of claim 30, wherein the detectable label is a fluorescent label.", "32.A method for evaluating a sample suspected of containing a non-B Group M HIV-1 virus or a Group O HIV-1 virus to assess the type of the virus, comprising the steps of: treating the sample to recover viral RNA; reverse transcribing the recovered viral RNA; sequencing the reverse transcription product; and using the results of the sequencing step to establish the genotype of the tested virus, wherein at least one of the reverse transcription step and the sequencing step is performed using a primer combination in accordance with claim 1.33.The method of claim 32, further comprising the step of performing a parallel genotyping procedures that is designed to evaluate B-subtype virus.", "34.The method of claim 32, wherein the sample is one that has previously been the subject of a failed genotyping attempt using genotyping procedures that are designed to evaluate B-subtype virus." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The present application relates to methods and primers for evaluating mutations in human immunodeficiency virus (HIV-1).", "Human immunodeficiency virus is the primary causative agent of Acquired Immune Deficiency Syndrome (AIDS), or AIDS-related complex (ARC).", "AIDS is an infectious disease characterized by generalized immune suppression, multiple opportunistic infections, and neurological disease.", "Although HIV is regarded to be the primary causative agent of AIDS, multiple co-infecting clinical viral and bacterial pathogens are responsible for the cluster of clinical syndromes seen in AIDS patients.", "The clinical course of HIV infection is remarkable for its great variability.", "The clinical effects include increased susceptibility to opportunistic infections and rare cancers, such as Kaposi's sarcoma, neurological dysfunctions, leading to AIDS related dementias, and generalized immune dysfunctions.", "The HIV-1 virus is a member of the lentivirus group of the retroviruses.", "Like all other retroviruses, it has an RNA genome which is replicated via the viral reverse transcriptase, into a DNA provirus which becomes integrated into the host cell genome.", "Various drugs are presently available to treat HIV.", "They fall into three different classes—nucleoside reverse transcriptase inhibitors, or NRTI's such as zidovudine, didanosine, zalcitabine, lamivudine, stavudine, abacavir, tenofovir, foscamet; non-nucleoside reverse transcriptase inhibitors or NNRTI's such as nevirapine, delavirdine, efavirenz; and protease inhibitors or PI's such as saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, and lopinavir with ritonavir.", "Although some of these drugs may have similar modes of actions, resistance to one does not necessarily confer resistance to another.", "Each of the presently available anti-retroviral compounds used to treat AIDS suffers from some disadvantages, including transient CD4 cell count effects, incomplete inhibition of viral replication, toxicity at prescribing doses, and emergence of resistant forms of the virus.", "As a result, combination therapies are being used to treat patients.", "Several in vitro studies have suggested that the combination of two or more anti-HIV compounds will more effectively inhibit HIV replication than each drug alone.", "Over the last several years, the standard of patient care has evolved such that HIV patients are routinely treated with triple drug combination therapy.", "Combination therapy has significantly decreased HIV associated morbidity and mortality.", "However, a large number of patients are not able to achieve or maintain complete viral suppression even with combination therapy.", "Drug resistance is the consequence of this incomplete viral suppression.", "The very high mutagenicity rate of HIV virus (due to the error-prone nature of the viral reverse transcriptase) and the genetic variability of the virus have led to many HIV variants with decreased drug susceptibility.", "HIV-1 replication depends on a virally encoded enzyme, reverse transcriptase (RT) that copies the single-stranded viral RNA genome into a double-stranded DNA/RNA hybrid.", "The HIV-1 RT enzyme lacks a 3′ exonuclease activity which normally helps the “proof-reading” function of a polymerase enzyme to repair errors.", "HIV-1 has a 9200-base genome and, on average, RT makes at least one error during every transcription of 10,000 bases copied.", "Therefore, each progeny virus produced may be slightly different from its predecessor.", "The inaccuracy of RT results in an estimated in vivo forward mutation rate of 3×10 −5 per base incorporated.", "Mansky LM.", "Virology.", "1996; 222:391-400.Many mutations introduced into the HIV-1 genome will compromise the infectivity of the virus; while some are compatible with virus infectivity.", "The frequency with which genetic variants of HIV-1 are detected in patients is a function of each variant's replicative vigor (fitness) and the nature of the selective pressures that may be acting on the population within the infected patient, Volberding PA, et al., Antiretroviral therapy for HIV infection: promises and problems.", "JAMA.", "1998;279:1343-4.Selective pressures existing in HIV-1 infected persons include anti-HIV-1 immune responses, the availability of host cells that are susceptible to virus infection in different tissues, and the use of antiretroviral drug treatments.", "The mutagenicity of the virus represents a significant barrier to treatment of the disease.", "Moreover, the mutagenicity of the virus makes testing for genetic changes in the virus very difficult.", "Testing for changes in DNA sequence can proceed via complete sequencing of a target nucleic acid molecule, although many persons in the art believe that such testing is too expensive to ever be routine.", "Attention has been increasingly focused on failure to achieve or maintain viral suppression.", "Several factors may contribute to drug failure, including poor patient adherence to treatment regimen, drug potency, pharmacokinetic issues (related to antiretroviral drug absorption, metabolism, excretion, and drug-drug interactions) and drug resistance Vella S, et al.Aids.", "1998; 12:S147-8.b.", "Although multiple combinations of antiretroviral drugs may suppress HIV-1 below the level of HIV-1 RNA detection, this does not mean that the virus is not replicating in “sanctuary” compartments.", "A therapy regimen may decrease HIV-1 RNA to below detectable levels, but within months the HIV-1 viral load may increase again.", "If HIV-1 is replicating, resistance to therapy can develop.", "Because HIV-1 replication occurs rapidly, large numbers of virus variants, including those that display diminished sensitivity to antiretroviral drugs, are generated.", "Mutations that confer resistance to antiretroviral drugs can be present in HIV-1 infected persons before antiretroviral therapy is initiated due to transmission from an individual having had prior therapy or due to spontaneously arising mutations.", "Once drug therapy is initiated, the pre-existing population of drug-resistant viruses can rapidly predominate because of a selective advantage.", "For drugs such as lamivudine or nevirapine (and other NNRTIs), a single nucleotide change in the HIV-1 RT gene can confer 100- to 1,000-fold reductions in drug susceptibility (Schinazi RF, et al Int Antiviral News.", "1997;5:129-42).", "In vivo antiretroviral activity of these drugs, when used alone, is largely lost within 4 weeks of starting therapy due to the rapid outgrowth of drug-resistant variants, Richman DD, et al.", "Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy.", "J Virol.", "1994; 68:1660-6.Some mutations selected by antiretroviral drugs directly affect viral enzymes and cause resistance via decreased drug binding, whereas others have indirect effects., Condra JH, et al.", "J Virol.", "1996; 70:8270-6, and Harrigan PR, et al.", "J Virol.", "1996; 70:5930-4.Treatment with different antiretroviral drugs may select for HIV-1 variants that harbor the same, or related, mutations.", "Treatments may even select for the outgrowth of HIV-1 variants that are resistant to drugs to which the patient has not yet been exposed (cross-resistance).", "Mutations can be detected by a technique called “single stranded conformational polymorphism” (SSCP) described by Orita et al., Genomics 5: 874-879 (1989), or by a modification thereof referred to as dideoxy-fingerprinting (“ddF”) described by Sarkar et al, Genomics 13: 441-443 (1992).", "SSCP and ddF both evaluate the pattern of bands created when DNA fragments are electrophoretically separated on a non-denaturing electrophoresis gel.", "This pattern depends on a combination of the size of the fragments and the three-dimensional conformation of the undenatured fragments.", "Thus, the pattern can not be used for sequencing, because the theoretical spacing of the fragment bands is not equal.", "Others have attempted to determine the genetic status of the virus by probe-based analyses, in which the presence or absence of a specific viral mutation is determined by whether or not an inquiry probe hybridizes to the viral nucleic acid under specific hybridization conditions.", "For example, Stuyver et al.", "(PCT International Publication No.", "WO 99/67428) describe the use of nucleic acid probe panels in a reverse hybridization assay, and Gingeras et al.", "describe the use of probes to detect pairs of mutations (PCT International Publication No.", "WO 92/16180).", "Such assays may suffer from several deficiencies, including being unable to detect new viral mutants, and may not be sensitive enough to cope with the complexity of many mutations within a region.", "Other methods include the use of resistance test vectors to culture host cells with virus derived from a patient.", "The vector may include an indicator gene, such that when a test amount of an anti-HIV drug is added to the cell culture, in an attempt to measure the resistance of the cloned virus to the drug in the cell culture system.", "(Parkin et al, U.S. Pat.", "No.", "5,837,464).", "By far, the most direct information about the genetic composition of the virus in a patient is to directly determine the sequence of the virus (genotyping).", "The positive clinical benefit of genotyping has been demonstrated in controlled retrospective and prospective intervention based studies such as the Genotypic Antiretroviral Resistance Testing (GART)(Baxter JD, et al.", "A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy.", "AIDS; 2000;14;F83-F93 and VIRADAPT studies, Durant J, et al.", "Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial, Lancet.", "1999; 353:2195-9 and Lancet 1999 September 25;354(9184): 1128.The greater reduction in viral load when the identification of mutations associated with resistance to specific antiretroviral drugs is used as an adjunct to standard of care in treated patients has demonstrated the clinical benefit of the adjunctive use of genotyping to guide therapeutic decisions.", "One of the difficulties of genotyping is the inherent variability and heterogeneity of the virus.", "Viruses have been found to be serologically different on the basis of reactivity of the host immune system to the virus, and on the basis of ELISAs, antibody dependent cellular cytotoxicity assays (ADCC's), and CD4 inactivation procedures.", "The extensive serologic heterogeneity of the virus is also mirrored in the genetic sequences of the virus.", "As a result, the HIV-1 virus has been categorized into two genetic groups, based on phylogenetic reconstruction using the viral DNA sequences.", "Group O (outlier) represents a minority of the HIV-1, and is thought to originate in West Africa, perhaps in Cameroon.", "The vast majority of HIV-1 sequences that are associated with clinical AIDS are of the Group M (major) type.", "Within the M group, there are various subtypes (also referred to as clades), having different geographic distributions, as shown below.", "HIV-1 Group M Subtype Predominant geographical location A (including A1 and A2) Central Africa B Europe, North and South America, Australia, and Asia C East and South Africa, India D Central Africa E Southeast Asia (Thailand) F (including F1 and F2) South America (Brazil) and Eastern Europe (Romania) G Central Africa, Russia, and Portugal H Central Africa and Taiwan I Cyprus J Central Africa and Europe K N O Each subtype differs from the others in amino acid composition by at least 20% in the viral envelope region, and at least 15% in the viral gag region.", "Within each subtype, the differences in env can be up to 10%, while the differences in gag can be Up to 8%.", "The viral reverse transcriptase and protease genes, the sites known to be associated with drug resistance, are found on the viral pol transcript.", "It is estimated that there is only a 75% similarity in amino acids between subtypes for HIV-1 pol.", "The variability at the nucleic acid sequence level is even greater.", "Retroviruses have propensity to recombine with related retroviruses.", "If one cell is infected with multiple viruses, recombination events may occur, leading to recombinant subtypes that may then infect other individuals.", "In addition to the various subtypes known, circulating recombinant subtypes have been observed, such as A/E (Central Africa), A/G (West and Central Africa), A/B (Kalingrad), A/G/H/K (Cyprus/Greece) as well as D/F, and B/D recombinants.", "To date, the majority of clinical research in North America and Western Europe has been directed to the Group M subtype B, due to its relative prevalence over the other Group M subtypes.", "However, as the AIDS epidemic has spread, non-B subtypes are appearing with increasing frequency in North America and Europe.", "In some instances, for example, an initial infected person with a non-B infection may serve as the infection focal point for a local group, such that in some North American centers (which remain predominantly B subtype), there can be entire localized population groups infected with non-B subtypes.", "For example, Group O and Subtype G of Group M have recently been found in AIDS patients arriving in the United States from Africa." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides primer sequences, and a method of using such sequences for the genotyping of HIV-1-containing samples, particularly those which have failed genotyping analysis using primer sequences designed for analysis of Group B subtype of the Group M type virus.", "Thus, a first aspect of the present invention is a combination of primers, including at least one species of forward primer and at least one species of reverse primer.", "The forward primer(s) can be represented by the degenerate sequence: RARRARGGGCTGYTGGARATGTS (Seq ID No.", "9) optionally with an additional G at either or both ends, where the non-standard letters (those others than A, C, G and T) reflect choices of bases in accordance with conventional nomenclature as outlined below.", "There are a total of 128 possible sequences represented by this sequence.", "Variations of these sequences may also be employed.", "For example, Seq.", "ID Nos.", "11, 15 and6 show primers where one G is added.", "Similarly, the reverse primer(s) can be represented by the degenerate sequence: AGTCARATYTAYBCWGGGATYAARGTRADGV (Seq.", "ID No.", ": 10) or GTCARATYTAYBCWGGGATYAARGTRADGV (Seq.", "ID No.", "12) In the former case, there are a total of 3456 possible primer species within this definition.", "In the latter case, the degenerate sequence also represents 3456 possible sequences, differing only in the initial A.", "The selected primers, one or more from each group, can be used as reverse transcription, amplification and sequencing primers.", "The primers are suitably packaged in a genotyping kit.", "Such a kit may include reagents in addition to the primers, such as an RNase inhibitor, a reverse transcriptase, a polymerase, and/or dNTP and ddNTP feedstocks.", "The primers are suitably employed in the method of the invention.", "In accordance with this method, a sample suspected of containing a non-B Group M HIV-1 virus or a Group O HIV-1 virus is treated to recover viral RNA.", "The recovered viral RNA is reverse transcribed to DNA, which is sequenced using the primers of the invention.", "The resulting sequence information is used to establish the genotype of the tested virus, i.e., to determine to which subtype the virus in the sample belongs.", "The method of the invention may be practiced in parallel with genotyping procedures that are designed to evaluate B-subtype virus.", "Alternatively, the method of the invention is practiced on samples that have previously been the subject of a failed genotyping attempt using genotyping procedures that are designed to evaluate B-subtype virus.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "BACKGROUND OF THE INVENTION The present application relates to methods and primers for evaluating mutations in human immunodeficiency virus (HIV-1).", "Human immunodeficiency virus is the primary causative agent of Acquired Immune Deficiency Syndrome (AIDS), or AIDS-related complex (ARC).", "AIDS is an infectious disease characterized by generalized immune suppression, multiple opportunistic infections, and neurological disease.", "Although HIV is regarded to be the primary causative agent of AIDS, multiple co-infecting clinical viral and bacterial pathogens are responsible for the cluster of clinical syndromes seen in AIDS patients.", "The clinical course of HIV infection is remarkable for its great variability.", "The clinical effects include increased susceptibility to opportunistic infections and rare cancers, such as Kaposi's sarcoma, neurological dysfunctions, leading to AIDS related dementias, and generalized immune dysfunctions.", "The HIV-1 virus is a member of the lentivirus group of the retroviruses.", "Like all other retroviruses, it has an RNA genome which is replicated via the viral reverse transcriptase, into a DNA provirus which becomes integrated into the host cell genome.", "Various drugs are presently available to treat HIV.", "They fall into three different classes—nucleoside reverse transcriptase inhibitors, or NRTI's such as zidovudine, didanosine, zalcitabine, lamivudine, stavudine, abacavir, tenofovir, foscamet; non-nucleoside reverse transcriptase inhibitors or NNRTI's such as nevirapine, delavirdine, efavirenz; and protease inhibitors or PI's such as saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, and lopinavir with ritonavir.", "Although some of these drugs may have similar modes of actions, resistance to one does not necessarily confer resistance to another.", "Each of the presently available anti-retroviral compounds used to treat AIDS suffers from some disadvantages, including transient CD4 cell count effects, incomplete inhibition of viral replication, toxicity at prescribing doses, and emergence of resistant forms of the virus.", "As a result, combination therapies are being used to treat patients.", "Several in vitro studies have suggested that the combination of two or more anti-HIV compounds will more effectively inhibit HIV replication than each drug alone.", "Over the last several years, the standard of patient care has evolved such that HIV patients are routinely treated with triple drug combination therapy.", "Combination therapy has significantly decreased HIV associated morbidity and mortality.", "However, a large number of patients are not able to achieve or maintain complete viral suppression even with combination therapy.", "Drug resistance is the consequence of this incomplete viral suppression.", "The very high mutagenicity rate of HIV virus (due to the error-prone nature of the viral reverse transcriptase) and the genetic variability of the virus have led to many HIV variants with decreased drug susceptibility.", "HIV-1 replication depends on a virally encoded enzyme, reverse transcriptase (RT) that copies the single-stranded viral RNA genome into a double-stranded DNA/RNA hybrid.", "The HIV-1 RT enzyme lacks a 3′ exonuclease activity which normally helps the “proof-reading” function of a polymerase enzyme to repair errors.", "HIV-1 has a 9200-base genome and, on average, RT makes at least one error during every transcription of 10,000 bases copied.", "Therefore, each progeny virus produced may be slightly different from its predecessor.", "The inaccuracy of RT results in an estimated in vivo forward mutation rate of 3×10−5 per base incorporated.", "Mansky LM.", "Virology.", "1996; 222:391-400.Many mutations introduced into the HIV-1 genome will compromise the infectivity of the virus; while some are compatible with virus infectivity.", "The frequency with which genetic variants of HIV-1 are detected in patients is a function of each variant's replicative vigor (fitness) and the nature of the selective pressures that may be acting on the population within the infected patient, Volberding PA, et al., Antiretroviral therapy for HIV infection: promises and problems.", "JAMA.", "1998;279:1343-4.Selective pressures existing in HIV-1 infected persons include anti-HIV-1 immune responses, the availability of host cells that are susceptible to virus infection in different tissues, and the use of antiretroviral drug treatments.", "The mutagenicity of the virus represents a significant barrier to treatment of the disease.", "Moreover, the mutagenicity of the virus makes testing for genetic changes in the virus very difficult.", "Testing for changes in DNA sequence can proceed via complete sequencing of a target nucleic acid molecule, although many persons in the art believe that such testing is too expensive to ever be routine.", "Attention has been increasingly focused on failure to achieve or maintain viral suppression.", "Several factors may contribute to drug failure, including poor patient adherence to treatment regimen, drug potency, pharmacokinetic issues (related to antiretroviral drug absorption, metabolism, excretion, and drug-drug interactions) and drug resistance Vella S, et al.Aids.", "1998; 12:S147-8.b.", "Although multiple combinations of antiretroviral drugs may suppress HIV-1 below the level of HIV-1 RNA detection, this does not mean that the virus is not replicating in “sanctuary” compartments.", "A therapy regimen may decrease HIV-1 RNA to below detectable levels, but within months the HIV-1 viral load may increase again.", "If HIV-1 is replicating, resistance to therapy can develop.", "Because HIV-1 replication occurs rapidly, large numbers of virus variants, including those that display diminished sensitivity to antiretroviral drugs, are generated.", "Mutations that confer resistance to antiretroviral drugs can be present in HIV-1 infected persons before antiretroviral therapy is initiated due to transmission from an individual having had prior therapy or due to spontaneously arising mutations.", "Once drug therapy is initiated, the pre-existing population of drug-resistant viruses can rapidly predominate because of a selective advantage.", "For drugs such as lamivudine or nevirapine (and other NNRTIs), a single nucleotide change in the HIV-1 RT gene can confer 100- to 1,000-fold reductions in drug susceptibility (Schinazi RF, et al Int Antiviral News.", "1997;5:129-42).", "In vivo antiretroviral activity of these drugs, when used alone, is largely lost within 4 weeks of starting therapy due to the rapid outgrowth of drug-resistant variants, Richman DD, et al.", "Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy.", "J Virol.", "1994; 68:1660-6.Some mutations selected by antiretroviral drugs directly affect viral enzymes and cause resistance via decreased drug binding, whereas others have indirect effects., Condra JH, et al.", "J Virol.", "1996; 70:8270-6, and Harrigan PR, et al.", "J Virol.", "1996; 70:5930-4.Treatment with different antiretroviral drugs may select for HIV-1 variants that harbor the same, or related, mutations.", "Treatments may even select for the outgrowth of HIV-1 variants that are resistant to drugs to which the patient has not yet been exposed (cross-resistance).", "Mutations can be detected by a technique called “single stranded conformational polymorphism” (SSCP) described by Orita et al., Genomics 5: 874-879 (1989), or by a modification thereof referred to as dideoxy-fingerprinting (“ddF”) described by Sarkar et al, Genomics 13: 441-443 (1992).", "SSCP and ddF both evaluate the pattern of bands created when DNA fragments are electrophoretically separated on a non-denaturing electrophoresis gel.", "This pattern depends on a combination of the size of the fragments and the three-dimensional conformation of the undenatured fragments.", "Thus, the pattern can not be used for sequencing, because the theoretical spacing of the fragment bands is not equal.", "Others have attempted to determine the genetic status of the virus by probe-based analyses, in which the presence or absence of a specific viral mutation is determined by whether or not an inquiry probe hybridizes to the viral nucleic acid under specific hybridization conditions.", "For example, Stuyver et al.", "(PCT International Publication No.", "WO 99/67428) describe the use of nucleic acid probe panels in a reverse hybridization assay, and Gingeras et al.", "describe the use of probes to detect pairs of mutations (PCT International Publication No.", "WO 92/16180).", "Such assays may suffer from several deficiencies, including being unable to detect new viral mutants, and may not be sensitive enough to cope with the complexity of many mutations within a region.", "Other methods include the use of resistance test vectors to culture host cells with virus derived from a patient.", "The vector may include an indicator gene, such that when a test amount of an anti-HIV drug is added to the cell culture, in an attempt to measure the resistance of the cloned virus to the drug in the cell culture system.", "(Parkin et al, U.S. Pat.", "No.", "5,837,464).", "By far, the most direct information about the genetic composition of the virus in a patient is to directly determine the sequence of the virus (genotyping).", "The positive clinical benefit of genotyping has been demonstrated in controlled retrospective and prospective intervention based studies such as the Genotypic Antiretroviral Resistance Testing (GART)(Baxter JD, et al.", "A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy.", "AIDS; 2000;14;F83-F93 and VIRADAPT studies, Durant J, et al.", "Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial, Lancet.", "1999; 353:2195-9 and Lancet 1999 September 25;354(9184): 1128.The greater reduction in viral load when the identification of mutations associated with resistance to specific antiretroviral drugs is used as an adjunct to standard of care in treated patients has demonstrated the clinical benefit of the adjunctive use of genotyping to guide therapeutic decisions.", "One of the difficulties of genotyping is the inherent variability and heterogeneity of the virus.", "Viruses have been found to be serologically different on the basis of reactivity of the host immune system to the virus, and on the basis of ELISAs, antibody dependent cellular cytotoxicity assays (ADCC's), and CD4 inactivation procedures.", "The extensive serologic heterogeneity of the virus is also mirrored in the genetic sequences of the virus.", "As a result, the HIV-1 virus has been categorized into two genetic groups, based on phylogenetic reconstruction using the viral DNA sequences.", "Group O (outlier) represents a minority of the HIV-1, and is thought to originate in West Africa, perhaps in Cameroon.", "The vast majority of HIV-1 sequences that are associated with clinical AIDS are of the Group M (major) type.", "Within the M group, there are various subtypes (also referred to as clades), having different geographic distributions, as shown below.", "HIV-1 Group M Subtype Predominant geographical location A (including A1 and A2) Central Africa B Europe, North and South America, Australia, and Asia C East and South Africa, India D Central Africa E Southeast Asia (Thailand) F (including F1 and F2) South America (Brazil) and Eastern Europe (Romania) G Central Africa, Russia, and Portugal H Central Africa and Taiwan I Cyprus J Central Africa and Europe K N O Each subtype differs from the others in amino acid composition by at least 20% in the viral envelope region, and at least 15% in the viral gag region.", "Within each subtype, the differences in env can be up to 10%, while the differences in gag can be Up to 8%.", "The viral reverse transcriptase and protease genes, the sites known to be associated with drug resistance, are found on the viral pol transcript.", "It is estimated that there is only a 75% similarity in amino acids between subtypes for HIV-1 pol.", "The variability at the nucleic acid sequence level is even greater.", "Retroviruses have propensity to recombine with related retroviruses.", "If one cell is infected with multiple viruses, recombination events may occur, leading to recombinant subtypes that may then infect other individuals.", "In addition to the various subtypes known, circulating recombinant subtypes have been observed, such as A/E (Central Africa), A/G (West and Central Africa), A/B (Kalingrad), A/G/H/K (Cyprus/Greece) as well as D/F, and B/D recombinants.", "To date, the majority of clinical research in North America and Western Europe has been directed to the Group M subtype B, due to its relative prevalence over the other Group M subtypes.", "However, as the AIDS epidemic has spread, non-B subtypes are appearing with increasing frequency in North America and Europe.", "In some instances, for example, an initial infected person with a non-B infection may serve as the infection focal point for a local group, such that in some North American centers (which remain predominantly B subtype), there can be entire localized population groups infected with non-B subtypes.", "For example, Group O and Subtype G of Group M have recently been found in AIDS patients arriving in the United States from Africa.", "SUMMARY OF THE INVENTION The present invention provides primer sequences, and a method of using such sequences for the genotyping of HIV-1-containing samples, particularly those which have failed genotyping analysis using primer sequences designed for analysis of Group B subtype of the Group M type virus.", "Thus, a first aspect of the present invention is a combination of primers, including at least one species of forward primer and at least one species of reverse primer.", "The forward primer(s) can be represented by the degenerate sequence: RARRARGGGCTGYTGGARATGTS (Seq ID No.", "9) optionally with an additional G at either or both ends, where the non-standard letters (those others than A, C, G and T) reflect choices of bases in accordance with conventional nomenclature as outlined below.", "There are a total of 128 possible sequences represented by this sequence.", "Variations of these sequences may also be employed.", "For example, Seq.", "ID Nos.", "11, 15 and6 show primers where one G is added.", "Similarly, the reverse primer(s) can be represented by the degenerate sequence: AGTCARATYTAYBCWGGGATYAARGTRADGV (Seq.", "ID No.", ": 10) or GTCARATYTAYBCWGGGATYAARGTRADGV (Seq.", "ID No.", "12) In the former case, there are a total of 3456 possible primer species within this definition.", "In the latter case, the degenerate sequence also represents 3456 possible sequences, differing only in the initial A.", "The selected primers, one or more from each group, can be used as reverse transcription, amplification and sequencing primers.", "The primers are suitably packaged in a genotyping kit.", "Such a kit may include reagents in addition to the primers, such as an RNase inhibitor, a reverse transcriptase, a polymerase, and/or dNTP and ddNTP feedstocks.", "The primers are suitably employed in the method of the invention.", "In accordance with this method, a sample suspected of containing a non-B Group M HIV-1 virus or a Group O HIV-1 virus is treated to recover viral RNA.", "The recovered viral RNA is reverse transcribed to DNA, which is sequenced using the primers of the invention.", "The resulting sequence information is used to establish the genotype of the tested virus, i.e., to determine to which subtype the virus in the sample belongs.", "The method of the invention may be practiced in parallel with genotyping procedures that are designed to evaluate B-subtype virus.", "Alternatively, the method of the invention is practiced on samples that have previously been the subject of a failed genotyping attempt using genotyping procedures that are designed to evaluate B-subtype virus.", "DETAILED DESCRIPTION OF THE INVENTION While the terminology used in this application is standard within the art, the following definitions of certain terms are provided to assure clarity.", "The term “allele” as used herein means a specific version of a nucleotide sequence at a polymorphic genetic locus.", "The term “polymorphic site” as used herein means a given nucleotide location in a genetic locus which is variable within a population.", "The term “gene” or “genetic locus” as used herein means a specific nucleotide sequence within a given genome.", "The term “location” or “position” of a nucleotide in a genetic locus means the number assigned to the nucleotide in the gene, generally taken from the cDNA sequence of the genomic sequence of a gene.", "The nucleotides adenosine, cytosine, guanine and thymine are represented by their one-letter codes A, C, G, and T respectively.", "In representations of degenerate primers, the symbol R refers to either G or A, the symbol Y refers to either T/U or C, the symbol M refers to either A or C, the symbol K refers to either G or T/U, the symbol S refers to G or C, the symbol W refers to either A or T/U, the symbol B refers to “not A”, the symbol D refers to “not C”, the symbol H refers to “not G”, the symbol V refers to “not T/U” and the symbol N refers to any nucleotide.", "In the specification and claims of this application, a degenerate primer refers to any or all of the combinations of base choices and to either DNA or the corresponding RNA sequence (i.e., with T replaced by U).", "Thus, a degenerate primer may represent a single species, or a mixture of two species which fall within the choices, or a mixture of three choices which fall with the choices, and so on up to a mixture containing all the possible combinations.", "The tenn “oligonucleotide primer” as used herein defines a molecule comprised of more than three deoxyribonucleotides or ribonucleotides.", "Its exact length will depend on many factors relating to the ultimate function and use of the oligonucleotide primer, including temperature, source of the primer and use of the method.", "The oligonucleotide primer is capable of acting as an initiation point for synthesis when placed under conditions which induce synthesis of a primer extension product complementary to a nucleic acid strand.", "The conditions can include the presence of nucleotides and an inducing agent such as a DNA polymerase at a suitable temperature and pH.", "In the preferred embodiment, the primer is a SS oligodeoxyribonucleotide of sufficient length to prime the synthesis of an extension product from a specific sequence in the presence of an inducing agent.", "In the preferred embodiment, the oligonucleotide primers are at least 18 nucleotides long.", "Sensitivity and specificity of the oligonucleotide primers are determined by the primer length and uniqueness of sequence within a given sample of template nucleic acid.", "Primers which are too short, for example, may show non-specific binding to a wide variety of sequences.", "A first aspect of the present invention is a primer combination comprising, in a single solution, at least one forward HIV-1 primer selected from among primers comprising a sequence as represented by the degenerate sequence of Seq ID Nos.", "9, for example Seq.", "ID.", "Nos.", "11, 15 or 16, and preferably from among primers with exactly these sequences, at least one reverse HIV-1 primer selected from among primers comprising a sequence as represented by the degenerate sequences of Seq.", "ID Nos.", "10 or 12, for example Seq.", "ID.", "Nos.", "13 or 14, and preferably from among primers with exactly these sequences.", "Where the primers are to be used as sequencing primers, the forward primers or the reverse primers are labeled with a detectable label.", "For most common sequencing instruments, a fluorescent label is desirable, although other labels types including colored, chromogenic, fluorogenic (including chemiluminescent) and radiolabels could also be employed.", "The primer combination may include other reagents appropriate for reverse transcription, amplification or sequencing, and may, of course, include HIV-1 genetic material for analysis.", "Specific forward primers for use in the primer combinations of the invention are: GGAAAAAGGGCTGTTGGAAATGYG (Seq.", "ID No.", "1) GRARRARGGGCTGTTGGAAATGTGG (Seq.", "ID No.", "2) GRARRARGGGCTGTTGGAAATGTG (Seq.", "ID No.", "3) including without limitation the following non-degenerate primer sequences: GGAAAAAGGGCTGTTGGAAATGTGG (Seq.", "ID No.", "17) GGAAAAAGGGCTGTTGGAAATGTG (Seq.", "ID No.", "18) GGAAAAAGGGCTGTTGGAAATGTCG (Seq.", "ID No.", "19) GGAAAAAGGGCTGTTGGAAATGTC (Seq.", "ID No.", "11) GAAAAAGGGCTGTTGGAAATGTG (Seq.", "ID No.", "15) GAAAAAGGGCTGTTGGAAATGCG.", "(Seq.", "ID No.", "16) Specific reverse primers for use in the primer combinations of the invention are: AGTCAGATTTACCCAGGGATTAAAGTAAGGV (Seq.", "ID No.", "4) AGTCAGATTTACCCAGGGATTAAGGTAAGGV (Seq.", "ID No.", "5) AGTCAGATTTACCCAGGGATCAAAGTAAGGV (Seq.", "ID No.", "6) GYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", "7) AGYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", "8) including without limitation the following non-degenerate primer sequences: GTCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", ": 13) GCCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", ": 14) The primer combinations described above can be used in a method in accordance with the invention for a sample suspected of containing a non-B Group M HIV-1 virus or a Group O HIV-1 virus to assess the subtype and genotype of the virus.", "The method comprises the steps of treating the sample to recover viral RNA; reverse transcribing the recovered viral RNA; sequencing the reverse transcription product; and using the results of the sequencing step to establish the genotype of the tested virus.", "In this method, either or both of the reverse transcription step and the sequencing step are performed using a primer combination as described above.", "The method of the invention can include the step of performing a parallel genotyping procedure that is designed to evaluate B-subtype virus.", "Alternatively, the method can be utilized with a sample that has previously been the subject of a failed genotyping attempt using genotyping procedures that are designed to evaluate B-subtype virus.", "This alternative method provides the advantage of not performing needless testing on a sample that proves to be the presently more common B-subtype.", "EXAMPLE 1 Degenerate mixtures of forward primers of the sequence GGAAAAAGGGCTGTTGGAAATGYG (Seq.", "ID No.", "1) and reverse primers of the sequence GYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.", "ID No.", "7) were used to salvage non-B subtypes in samples that could not be genotyped using other primers.", "The TRUGENE HIV-1 genotyping kit (Visible Genetics Inc., Toronto, Canada) was used to determine the genotype of certain non-B subtypes of HIV-1 virus.", "RNA was extracted from patient plasma samples according to the package instructions in a TRUPREP Extraction Kit forviral RNA.", "17 ul of RNA is added to the amplification master mix.", "The master mix contains (per reaction) 0.2 ul of SEQ ID No.", "1 (30 pmole/ul), 0.4 ul of SEQ ID No.", "7 (30 pmole/ul), 6.4 ul of water, 1.75 ul of dNTP, 1.165 ul of DTT, and 0.58 ul of Rnase inhibitor.", "The reactions are thermocycled using a Perkin-Elmer 9700 thermocycler using the following temperature/time cycle program: 20 cycles 15 cycles 90 C. 50 C. 94 C. 94 C. 57 C. 72 C. 94 C. 60 C. 70 C. 70 C. 4 C. 2 60 2 30 30 1.5 30 30 2 7 hold After incubating the amplification master mix and the RNA together for five minutes at 50 C, 4 ul of a second master mix is added and the RT-PCR cycle program is continued.", "The second master mix contains 10 ul of RT-PCR buffer, 5 ul of Rnase inhibitor, 1 ul of reverse transcriptase enzyme (Superscript, Invitrogen Corporation), and 17.5 ul of DNA polymerase.", "After the PCR cycle program was completed, samples were sequenced according to the protocol in the TRUGENE HIV-1 package insert (Visible Genetics Inc.).", "The following samples were successfully amplified and sequenced.", "Viral load numbers are provided as copies of viral RNA per milliliter of patient plasma.", "“Origin” refers to the residence of the patient at the time the plasma was collected.", "Sample ID Subtype ORIGIN TYPE Viral Load 1 I-001 B USA co-culture unknown 2 I-002 A/G USA co-culture unknown 3 I-003 F USA co-culture 7.5 × 107 4 I-004 B USA co-culture Unknown 5 I-005 Group O USA co-culture Unknown 6 I-006 E USA co-culture 6.4 × 106 7 I-007 E USA co-culture 2.6 × 109 8 I-008 B USA co-culture Unknown 9 I-009 E USA co-culture 4.4 × 105 10 I-010 E USA co-culture unknown 11 I-011 E USA co-culture 1.1 × 107 12 I-012 C USA co-culture 6.9 × 106 13 I-013 B USA co-culture Unknown 14 I-014 F USA co-culture Unknown 15 I-015 F/B USA co-culture 2.1 × 106 16 I-016 B USA co-culture Unknown 17 I-017 C USA co-culture 5.6 × 105 18 1747 A NIH Repos.", "co-culture 2.5 × 104 19 2386 D NIH Repos.", "co-culture 4 × 104 20 W1-1 A Europe patient plasma 2.1 × 104 21 W1-2 A Europe patient plasma 1.4 × 105 22 W1-3 C Europe patient plasma 2.1 × 104 23 W1-4 D Europe patient plasma 6.2 × 104 24 W1-5 C Europe patient plasma 9.4 × 104 25 W1-6 A Europe patient plasma 8.5 × 104 26 W1-7 D Europe patient plasma 5.6 × 105 27 W1-8 F Europe patient plasma 5.7 × 103 28 W1-9 C Europe patient plasma Unknown 29 W1-10 G Europe patient plasma Unknown 30 W1-11 F Europe patient plasma 1.6 × 105 31 W1-12 G Europe patient plasma 5.6 × 105 32 W1-13 G Europe patient plasma 2.3 × 103 33 W2-1 A Europe patient plasma 2.2 × 104 34 W2-2 A Europe patient plasma 1.4 × 105 35 W2-3 C Europe patient plasma 2.1 × 104 36 13866 B Israel patient plasma 1.4 × 104 37 15089 B Israel patient plasma 1.0 × 103 38 15214 A Israel patient plasma 2.0 × 103 39 15422 C Israel patient plasma 1.1 × 106 40 16100 C Israel patient plasma 7.2 × 105 41 16242 C Israel patient plasma 7.2 × 105 42 16360 C Israel patient plasma 2.6 × 105 43 16361 C Israel patient plasma 2.2 × 105 44 NA A/G Russia patient plasma 2.0 × 105 45 NA C Africa patient plasma 6.3 × 103 46 NA B Israel patient plasma 7.6 × 104 47 NA C Ethiopia patient plasma 9.2 × 104 48 NA C Ethiopia patient plasma 4.1 × 105 49 NA C Ethiopia patient plasma 2.5 × 104 50 NA unknown Argentina patient plasma 7.3 × 103 51 NA unknown Argentina patient plasma 3.6 × 104 52 NA C Ethiopia patient plasma 7.3 × 104 53 NA unknown Argentina patient plasma 4.8 × 105 54 NA C Ethiopia patient plasma 1.2 × 105 55 NA C Ethiopia patient plasma 2.7 × 105 56 NA C Ethiopia patient plasma 1.9 × 105 57 NA C Ethiopia patient plasma 5.3 × 104 58 NA C Ethiopia patient plasma 1.6 × 104 59 NA C Ethiopia patient plasma 1.1 × 105 60 NA C Ethiopia patient plasma 9.3 × 104 61 NA C Ethiopia patient plasma 3.8 × 104 62 NA C Ethiopia patient plasma 1.0 × 103 63 NA C Ethiopia patient plasma 2.8 × 104 64 NA C Ethiopia patient plasma 1.4 × 104 65 NA C Ethiopia patient plasma 7.5 × 105 66 NA B Israel patient plasma 5.5 × 105 67 NA unknown Israel patient plasma 3.2 × 104 68 NA C Ethiopia patient plasma unknown 69 NA B Europe patient plasma unknown 70 NA K Ivory Coast patient plasma unknown EXAMPLE 2 Oligonucleotide primers in accordance with the present invention were tested to determine what percentage of non-B subtypes could be genotyped using samples that could not be genotyped using the TRUGENE HIV-1 genotyping kit.", "74% of those samples were successfully genotyped.", "Similarly, greater than 95% of all samples known to be non-B samples were successfully genotyped using the oligonucleotide primers of the present invention.", "Panels of non-B subtype viruses were generated, using pLAI as a positive control.", "After a viral load for each sample was obtained (Roche Amplicor or Organon Teknika NASBA), samples were diluted, using a serial dilution procedure, down to 25 copies per reverse transcriptase reaction.", "In each case, successful base calling and mutation detection was achieved.", "EXAMPLE 3 Using the primers of the present invention, successful amplification and sequencing of HIV-1 viruses from both Group M (various subtypes and recombinants) and Group O was achieved.", "Mean % HIV-1 Type Number non-B's Sucessfully or Subtype tested Amplified A (Group M) n = >7 >95% B (Group M) n = >200 >95% C (Group M) n = >29 >95% D (Group M) n = >3 >95% E (Group M) n = >5 >95% F (Group M) n = >25 >95% G (Group M) n = 3 >95% K (Group M) n = 1 100% Group O n = 2 100% Group M n = 6 100% Recombinants" ] ]	Patent_10469313
[ [ "Process for the preparation of citalopram", "The present invention relates to an improved and industrially advantageous process for the preparation of citalopram represented by the following Formula I, and pharmaceutically acceptable acid addition salt thereof." ], [ "1.A process for the preparation of citalopram of Formula I, comprising reacting 5-halophthalane compound of Formula III, wherein X is bromo or iodo with a cyanide source in a suitable solvent in the presence of an organic base and isolating citalopram of Formula I, as the free base or in the form of a pharmaceutically acceptable acid addition salt thereof.", "2.The process according to claim 1 wherein the cyanide source is any cyanide ion donor.", "3.The process according to claim 2 wherein the cyanide ion donor is selected from the group consisting of potassium cyanide, sodium cyanide, ammonium cyanide, cuprous cyanide, zinc cyanide, ammonium cynide, tetra alkylammonium cyanide, and mixtures thereof.", "4.The process according to claim 1 wherein the suitable solvent is a polar aprotic solvent.", "5.The process according to claim 4 wherein the polar aprotic solvent is selected from the group consisting of dimethylformamide, dimethylacetamide, N-methylpyrrolidone, N-methylpiperidinone, 1,3-dimethyl-3,4,5,6-tetrahydro (2H) pyrimidinone (DMPU), and mixtures thereof.", "6.The process according to claim 5 wherein the polar aprotic solvent is dimethylformamide.", "7.The process according to claim 1 wherein the organic base is selected from the group consisting of trimethylamine, triethylamine, dilsopropylamine, picolines, pyridine, pyridine derivatives (wherein pyridine derivatives are 2,6-lutidine or 4-methyl pyridine), quinoline, 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), piperidine, aryl substituted amines (e.g.", "aniline), dicyclohexylamine, and mixtures thereof.", "8.The process according to claim 7 wherein the organic base is pyridine or quinoline.", "9.The process according to claim 7 wherein the organic base is used in stoichiometric amount or in excess ranging from about 1-5 molar equivalents per equivalent of the compound of Formula III.", "10.The process according to claim 1 wherein the reaction is carried out at a temperature ranging from about 120° C. to 170° C. 11.The process according to claim 10 wherein the reaction is carried out at a temperature ranging from about 135 to 145° C. 12.The process according to claim 1 wherein the citalopram is isolated as the hydrobromide salt." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Citalopram is a well known anti-depressant drug and is chemically known as 1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro-5-isobenzofurancarbonitrile.", "It is a selective centrally acting serotonin (5-hydroxy-tryptamine; 5-HT) re-uptake inhibitor and was described for the first time in U.S. Pat.", "No.", "4,136,193.Citalopram is further used in the treatment of dementia and cerebrovascular disorders as disclosed in European Patent No.", "474,580.A method for preparing citalopram is described in U.S. Pat.", "No.", "4,136,193.According to the invention, 4-halo-2-(hydroxymethyl)phenyl-(4′-fluorophenyl)-(3-dimethylaminopropyl)methanol represented by the following Formula II, wherein X represents halogen, is reacted with a dehydrating agent to effect ring closure for obtaining 5-halophthalane compound represented by the following Formula III, wherein X represents halogen.", "The compound of Formula III is reacted with cuprous cyanide in an inert organic solvent to give citalopram of Formula I.", "However, the process is unsuitable for accomplishment on an industrial scale since exchange reaction of the 5-halophthalane compound and cuprous cyanide does not go to completion even after refluxing them overnight in dimethylformamide thereby making it very difficult to separate the resulting citalopram from the corresponding 5-halo compound.", "WO 00/13648 discloses the preparation of citalopram by reacting the 5-halophthalane compound of Formula III wherein X is bromo or iodo or the corresponding triflate compound with a cyanide source in the presence of a palladium catalyst and a catalytic amount of Cu + or Zn 2+ or with zinc cyanide in the presence of a palladium catalyst, and isolation of the corresponding 5-cyano phthalane compound i.e.", "citalopram.", "The cyanide source is chosen from potassium cyanide, sodium cyanide, ammonium cyanide and tetra alkyl ammonium cyanide.", "A variant of this process is described in another PCT application, WO 00/11926, wherein the cyanide exchange is achieved with a cyanide source in the presence of a nickel catalyst.", "The processes described in the above PCT applications for the manufacture of citalopram suffer from the following limitations and for various reasons stated below are not suitable for commercial purposes.", "The reaction is carried out in the presence of palladium or nickel complexes which are very expensive, inconvenient to handle at commercial scale as they are air sensitive and light sensitive, highly flammable, cancer suspect agents and have limited commercial availability.", "The reaction conditions are unsafe and are burdened with the risk of explosion and fire as the processes make use of solvents like tetrahydrofuran and diethyl ether.", "Another process described in PCT application WO 01/02383 comprises the conversion of 5-halophthalane of Formula III to the corresponding Grignard reagent which is then converted to citalopram via reaction with compounds containing a cyano group bound to a leaving group.", "An alternative process involves obtaining an aldehyde from the Grignard reagent and its transformation to cyano group via an oxime or hydrazone intermediate.", "The process described in WO 01/02383 involves many steps and make use of raw materials which are not available commercially.", "Accordingly, none of the processes described heretofore are completely satisfactory at a commercial scale." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to solve the problems associated with the prior art and to provide an efficient and commercially viable process for producing citalopram via an improved cyano exchange process.", "The process is simple and provides obvious benefits with respect to economics and convenience to operate at a commercial scale.", "More particularly, the present invention relates to a process for the preparation of citalopram of Formula I comprising reacting 5-halophthalane compound of Formula III, wherein X is bromo or iodo with a cyanide source in a suitable solvent, in the presence of an organic base and isolating corresponding 5-cyano compound i.e.", "citalopram of Formula I as the free base or in the form of a pharmaceutically acceptable acid addition salt thereof.", "In a further aspect the invention relates to the above process which produces S-enantiomer of Formula I.", "The cyanide source may be any source which is a cyanide ion donor.", "Preferred sources are potassium cyanide, sodium cyanide, ammonium cyanide, cuprous cyanide, zinc cyanide, tetra-alkylammonium cyanide or mixtures thereof.", "More preferred sources are cuprous cyanide and zinc cyanide.", "The cyanide source may be used in stoichiometric amount or in excess.", "Preferably, 1 to 2 molar equivalents per equivalent of compound of Formula III is used.", "The term “suitable solvent” means any polar aprotic solvent.", "Preferably, the solvent may be selected from the group consisting of dimethylformamide, dimethylacetamide, N-methylpyrrolidone, N-methylpiperidinone, 1,3-dimethyl-3,4,5,6-tetrahydro(2H) pyrimidinone (DMPU), or mixtures thereof.", "Suitable organic base includes trimethylamine, triethylamine, diisopropylamine, picolines, pyridine, pyridine derivatives such as 2,6-lutidine, 4-methylpyridine morpholine, morpholine derivatives, quinoline, 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU), piperidine, aryl substituted amines such as aniline and dicyclohexylamine, or mixtures thereof.", "Preferably, pyridine or quinoline is used.", "The organic base may be used in stoichiometric amount or in excess.", "Preferably, about 1 to 5 molar equivalents per equivalent of starting material of Formula III is used.", "We believe that nitrogen containing organic base plays a crucial role and facilitates the completion of reaction.", "The base is believed to form a complex of Formula IV in case of cuprous cyanide, with the cyanide source which facilitates the exchange of halogen with nitrile via a transient state which involves a coordination complex of formula V, The reaction is generally carried out at a temperature ranging from about 120° C. to 170° C., preferably, at 135° C. to 145° C. The reaction completion may take from about 3 hours to several hours.", "The intermediate of Formula III wherein X is bromo or iodo may be prepared from bromo or iodophthalide respectively, as described in U.S. Pat.", "No.", "4,136,193, which is hereby incorporated herein by reference.", "Citalopram of Formula I may be obtained as the free base or converted into its pharmaceutically acceptable acid addition salts.", "Examples of such salts include those formed with organic acids such as maleic, fumaric, benzoic, ascorbic, succinic, oxalic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, acetic, propionic, tartaric, salicylic, citric, gluconic, lactic, malic, mandelic, cinnamic, aspartic, stearic, palmitic, itaconic, glycolic, glutamic and benzene sulfonic acids or with inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric and nitric acid.", "The acid addition salts of the compounds may be prepared by methods known in the art.", "The base is reacted with either the calculated amount of acid in a water miscible solvent such as ethanol or acetone and the salt is isolated after concentration and cooling or with an excess of the acid in a water immiscible solvent such as ether, dichloromethane or toluene with the salt separating out spontaneously.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates to an improved and industrially advantageous process for the preparation of citalopram represented by the following Formula I, and pharmaceutically acceptable acid addition salts thereof.", "BACKGROUND OF THE INVENTION Citalopram is a well known anti-depressant drug and is chemically known as 1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro-5-isobenzofurancarbonitrile.", "It is a selective centrally acting serotonin (5-hydroxy-tryptamine; 5-HT) re-uptake inhibitor and was described for the first time in U.S. Pat.", "No.", "4,136,193.Citalopram is further used in the treatment of dementia and cerebrovascular disorders as disclosed in European Patent No.", "474,580.A method for preparing citalopram is described in U.S. Pat.", "No.", "4,136,193.According to the invention, 4-halo-2-(hydroxymethyl)phenyl-(4′-fluorophenyl)-(3-dimethylaminopropyl)methanol represented by the following Formula II, wherein X represents halogen, is reacted with a dehydrating agent to effect ring closure for obtaining 5-halophthalane compound represented by the following Formula III, wherein X represents halogen.", "The compound of Formula III is reacted with cuprous cyanide in an inert organic solvent to give citalopram of Formula I.", "However, the process is unsuitable for accomplishment on an industrial scale since exchange reaction of the 5-halophthalane compound and cuprous cyanide does not go to completion even after refluxing them overnight in dimethylformamide thereby making it very difficult to separate the resulting citalopram from the corresponding 5-halo compound.", "WO 00/13648 discloses the preparation of citalopram by reacting the 5-halophthalane compound of Formula III wherein X is bromo or iodo or the corresponding triflate compound with a cyanide source in the presence of a palladium catalyst and a catalytic amount of Cu+ or Zn2+ or with zinc cyanide in the presence of a palladium catalyst, and isolation of the corresponding 5-cyano phthalane compound i.e.", "citalopram.", "The cyanide source is chosen from potassium cyanide, sodium cyanide, ammonium cyanide and tetra alkyl ammonium cyanide.", "A variant of this process is described in another PCT application, WO 00/11926, wherein the cyanide exchange is achieved with a cyanide source in the presence of a nickel catalyst.", "The processes described in the above PCT applications for the manufacture of citalopram suffer from the following limitations and for various reasons stated below are not suitable for commercial purposes.", "The reaction is carried out in the presence of palladium or nickel complexes which are very expensive, inconvenient to handle at commercial scale as they are air sensitive and light sensitive, highly flammable, cancer suspect agents and have limited commercial availability.", "The reaction conditions are unsafe and are burdened with the risk of explosion and fire as the processes make use of solvents like tetrahydrofuran and diethyl ether.", "Another process described in PCT application WO 01/02383 comprises the conversion of 5-halophthalane of Formula III to the corresponding Grignard reagent which is then converted to citalopram via reaction with compounds containing a cyano group bound to a leaving group.", "An alternative process involves obtaining an aldehyde from the Grignard reagent and its transformation to cyano group via an oxime or hydrazone intermediate.", "The process described in WO 01/02383 involves many steps and make use of raw materials which are not available commercially.", "Accordingly, none of the processes described heretofore are completely satisfactory at a commercial scale.", "SUMMARY OF THE INVENTION It is an object of the present invention to solve the problems associated with the prior art and to provide an efficient and commercially viable process for producing citalopram via an improved cyano exchange process.", "The process is simple and provides obvious benefits with respect to economics and convenience to operate at a commercial scale.", "More particularly, the present invention relates to a process for the preparation of citalopram of Formula I comprising reacting 5-halophthalane compound of Formula III, wherein X is bromo or iodo with a cyanide source in a suitable solvent, in the presence of an organic base and isolating corresponding 5-cyano compound i.e.", "citalopram of Formula I as the free base or in the form of a pharmaceutically acceptable acid addition salt thereof.", "In a further aspect the invention relates to the above process which produces S-enantiomer of Formula I.", "The cyanide source may be any source which is a cyanide ion donor.", "Preferred sources are potassium cyanide, sodium cyanide, ammonium cyanide, cuprous cyanide, zinc cyanide, tetra-alkylammonium cyanide or mixtures thereof.", "More preferred sources are cuprous cyanide and zinc cyanide.", "The cyanide source may be used in stoichiometric amount or in excess.", "Preferably, 1 to 2 molar equivalents per equivalent of compound of Formula III is used.", "The term “suitable solvent” means any polar aprotic solvent.", "Preferably, the solvent may be selected from the group consisting of dimethylformamide, dimethylacetamide, N-methylpyrrolidone, N-methylpiperidinone, 1,3-dimethyl-3,4,5,6-tetrahydro(2H) pyrimidinone (DMPU), or mixtures thereof.", "Suitable organic base includes trimethylamine, triethylamine, diisopropylamine, picolines, pyridine, pyridine derivatives such as 2,6-lutidine, 4-methylpyridine morpholine, morpholine derivatives, quinoline, 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU), piperidine, aryl substituted amines such as aniline and dicyclohexylamine, or mixtures thereof.", "Preferably, pyridine or quinoline is used.", "The organic base may be used in stoichiometric amount or in excess.", "Preferably, about 1 to 5 molar equivalents per equivalent of starting material of Formula III is used.", "We believe that nitrogen containing organic base plays a crucial role and facilitates the completion of reaction.", "The base is believed to form a complex of Formula IV in case of cuprous cyanide, with the cyanide source which facilitates the exchange of halogen with nitrile via a transient state which involves a coordination complex of formula V, The reaction is generally carried out at a temperature ranging from about 120° C. to 170° C., preferably, at 135° C. to 145° C. The reaction completion may take from about 3 hours to several hours.", "The intermediate of Formula III wherein X is bromo or iodo may be prepared from bromo or iodophthalide respectively, as described in U.S. Pat.", "No.", "4,136,193, which is hereby incorporated herein by reference.", "Citalopram of Formula I may be obtained as the free base or converted into its pharmaceutically acceptable acid addition salts.", "Examples of such salts include those formed with organic acids such as maleic, fumaric, benzoic, ascorbic, succinic, oxalic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, acetic, propionic, tartaric, salicylic, citric, gluconic, lactic, malic, mandelic, cinnamic, aspartic, stearic, palmitic, itaconic, glycolic, glutamic and benzene sulfonic acids or with inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric and nitric acid.", "The acid addition salts of the compounds may be prepared by methods known in the art.", "The base is reacted with either the calculated amount of acid in a water miscible solvent such as ethanol or acetone and the salt is isolated after concentration and cooling or with an excess of the acid in a water immiscible solvent such as ether, dichloromethane or toluene with the salt separating out spontaneously.", "DETAILED DESCRIPTION OF THE INVENTION The invention is further illustrated by the following example which should not be construed to be limiting the scope of the present invention.", "EXAMPLE 1 Preparation of Citalopram Base 1-(4′-Fluorophenyl)-1-(3-dimethylaminopropyl)-5-iodophthalane (7.5 g, 18 mmol), cuprous cyanide powder (2.4 g, 27 mmol) and pyridine (5.6 g, 71 mmol) were added to dimethylformamide (40ml) and the mixture so obtained was heated to 140-141° C. The reaction mixture was further stirred at 140-145° C. for about 3 hours.", "The reaction mixture was then cooled to 35° C., and diluted with a cooled mixture of toluene and water.", "The organic layer was separated, washed with ammonia solution and water.", "The toluene was recovered completely under vacuum to get the product as a free base in the form of an oil (6.0 g) EXAMPLE 2 Preparation of Citalopram Hydrobromide Toluene (40 ml) was added to the above obtained free base of citalopram (6.0 g) and stirred to obtain a homogeneous solution.", "To this solution, was added aqueous HBr solution (48%, 3.6 g).", "The reaction mixture so obtained was then stirred for about 4 hours at 5-10° C. and toluene layer was decanted off.", "Fresh toluene (40 ml) was added to it and further stirred at 5-10° C. The separated solid was filtered, washed with toluene and dried to obtain citalopram hydrobromide (6.7 g, yield 93.7%, purity >98,5% by HPLC) as a crystalline powder.", "While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention." ] ]	Patent_10469329
[ [ "Nucleic acids coding for a protein interacting with a porin channel", "The invention relates to an isolated nucleic acid coding for at least a partial sequence of a protein kinase of the mitogenic signaling cascade, in particular c-raf, wherein the partial sequence interacts with VDAC or BAX channels in mitochondrial membranes, or with a nucleic acid hybridizing said nucleic acid or homologues or derivatives of said nucleic acid, to proteins or peptides coded for by such a nucleic acid and to a screening method." ], [ "1.An isolated nucleic acid that encodes at least one partial sequence of a protein kinase of the mitogenic signaling cascade, wherein the partial sequence interacts with a porin channel or a nucleic acids capable of hybridizing with said nucleic acid or homologues or derivatives of said nucleic acid.", "2.An isolated nucleic acid according to claim 1, wherein the interaction is a reduction in the permeability of porin channels comprising, VDAC, BAX or bacterial porin channels.", "3.An isolated nucleic acid according to claim 1, wherein the nucleic acid codes for a protein or peptide containing the sequence c-raf-1 (338 to 627) or an active fragment thereof or a nucleic acid that hybridizes such a nucleic acid.", "4.A cDNA prepared from a nucleic acid according to one of claims 1 to 3.5.An isolated recombinant vector containing a nucleic acid according to one of claims 1 to 3.6.A protein or peptide encoded by a nucleic acid according to one of claims 1 to 3.7.A method for treating AIDS, neurodegenerative diseases, for protecting non-transformed cells in a tissue containing cancer cells, or treating bacterial infections comprising administering a pharmaceutical composition comprising a nucleic acid or a protein or peptide encoded by a nucleic acid according to one of claims 1 to 3.8.A screening method for determining a substance causing the closure of VDAC or BAX or bacterial channels comprising administering a pharmaceutical composition comprising a nucleic acid according to one of claims 1 to 3 or of a protein or peptide coded for hereby.", "9.A method for screening for substances capable of modulating porin channels comprising, VDAC, of BAX, or bacterial porin channels, comprising the following steps: a) providing a solution comprising a porin further comprising, VDAC, BAX, or a bacterial porin that is incubated with a partial sequence of raf, b) contacting the solution of step a) with a membrane positioned in an electrolyte, c) applying an electrical potential difference between different sides of the membrane, at time t=0 and beginning with t=0 or at a defined time after t=0, measuring an electrical current caused by the potential difference or a charge transport through the membrane as a function of time, d) comparing a value measured in step c) to a value which has been measured with an inactive form or an active form of raf.", "10.A method for identifying raf activating substances for preparing a pharmaceutical composition for treating a disease according to claim 7, comprising the following steps: a) reacting target cells with a prospective active ingredient or a mixture of prospective active ingredients, and cultivating the target cells, b) measuring the expression and/or concentration of raf protein after a pre-defined time, and the obtained measured value is compared to a measured value which has been obtained without addition of a prospective active ingredient, and c) selecting a prospective active ingredient or a mixture of prospective active ingredients for which an increase of in the expression and/or concentration of raf protein was found in step b).", "11.A method for identifying raf activating substances for preparing a pharmaceutical composition for treating a disease according claim 7, comprising the following steps: a) identifying raf inhibitors naturally occurring in target cells comprising substances that bind to the kinase domain of raf or substances that down-regulate the expression of raf, b) reacting a substance identified in step a) in a binding assay with a prospective active ingredient or a mixture of prospective active ingredients, and examining the binding capability of the substance with the prospective active ingredient, and c) selecting a prospective active ingredient or a mixture of prospective active ingredients for which a binding event has been detected in step b).", "12.The method of claim 9, wherein the porin comprises a defined partial sequence of VDAC, BAX, or a bacterial porin.", "13.The method of claim 10, wherein the raf comprises c-raf-1.14.The method of claim 10, wherein in step c), a mixture of prospective active ingredients is selected and a deconvolution is performed comprising individual measurement of the active ingredients of the mixture.", "15.The method of claim 11, wherein the substance identified in step a) is isolated or purified.", "16.The method of claim 11, wherein in step c), a mixture of prospective active ingredients is selected and a deconvolution is performed comprising individual measurement of the active ingredients of the mixture.", "17.The method of claim 11, wherein the method further comprises the steps according to claim 10.18.An isolated nucleic acid according to claim 3, wherein the interaction is a reduction in the permeability of the porin channels comprising VDAC, BAX or bacterial porin channels." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The raf protein in its various isoforms c-raf, B-raf and A-raf plays an important role in the regulation of the proliferation and differentiation of cells.", "raf is an effector kinase of ras and an important member in the mitogenic cytoplasmic protein kinase (MAPK) signaling cascade (see e.g.", "Daum, O., et al., Trends Biochem.", "Sci.", "19:474-479 (1994)).", "raf proto-oncogenes are highly conserved genes coding for serine/threonine specific kinases of the cytoplasm.", "These kinases have functions in the mitogenic signal transduction.", "This cascade transfers signals from receptor tyrosine kinases via ras, raf, MEK and ERK to targets in the cytoplasm and basically for the regulation of the proliferation and differentiation of the cells.", "The role of c-raf-1 in the classic mitogenic MAP kinase cascade is rather well researched.", "For details, reference is made here as an example only to U. R. Rapp in The Oncogene Handbook, T. Curran et al., Eds, Elsevier Science Publishers, The Netherlands, 1988, pages 115-154.c-raf-1 is practically ubiquitous in the human organism.", "With the transduction of an apoptosis signal into a cell, there are changes of the permeability of membranes of the mitochondria.", "An increase of the permeability of these membranes will lead to a translocation of the apoptotic protein cytochrome C into the cytoplasm, thereby finally proteolytic proteins called caspase being activated.", "These proteins lead to the apoptosis.", "The permeability of mitochondrial membranes is determined, among other factors, by the permeability of the mitochondrial porin channels VDAC (voltage-dependent anion channel).", "The permeability is in addition influenced by a protein family to which Bcl-2, Bcl-X L , BAX and BAK belong.", "Bcl-2 acts protectively and binds probably to VDAC.", "BAX is an integral membrane protein and promotes apoptosis.", "BAX and Bcl-2 can heterodimerize, and an overexpression of BAX overcompensates the protective effect of Bcl-2.For a deeper insight, reference is made as an example only to Zhou, M., et al.", "J. Biol.", "Chem.", "273:11930-11936 (1998) and Shimizu, M., Nature (Asia) 399:483 (1999).", "It is common to the members of the Bcl-2 family that there are four regions with amino acid homology, BH1, BH2, BH3 and BH4.These domains are essential for the dimerization and/or the function of the different members.", "In particular the presence or absence of BH4 seem to play an important role for the antiapoptotic or proapoptotic effect (see e.g.", "Wang, H.-G. et al., Cell 87:629-638 (1996)).", "For instance Bcl-2 comprises BH4, whereas BAX does not comprise BH4.From the document Wang, H.-G. et al., Cell 87:629-638 (1996), it is known in the art that raf-1 interacts with Bcl-2.According to this document, BH4 is essential for this interaction.", "An association raf-1/BAX has not been found, rather has been negated.", "The results are subject to discussion.", "Various diseases are accompanied by a disturbance of the natural cell death programming.", "A lacking function of the natural cell death is for instance associated with cancer or autoimmune diseases.", "In contrast thereto, an excessive cell death comes with diseases such as AIDS and neurodegenerative diseases.", "But infections with pathogenic bacteria may also be accompanied by a disturbance of the natural cell death, bacterial porins being translocated into a cellular membrane and triggering apoptosis of the target cells there.", "For instance from the document The EMBO Journal 18:339-352 (1999) it is known in the art that the porin PorB in its different variants translocates from Neisseria gonorrhoeae into artificial membranes as well as into cellular target membranes, in particular the outer mitochondrial membrane.", "These considerations generally apply to Gram-negative bacteria.", "The relationship of natural mitochondrial porins to bacterial porins has by the way been described already for instance for Escherichia coli , Salmonella typhimurium and Pseudomonas aeruginosa in the document Biochimica et Biophysica Acta 686:204-214 (1982).", "Technical Object.", "The invention is based on the technical object to inhibit an undesired cell death and to provide suitable active ingredients for treating diseases accompanied by an undesired cell death, in particular AIDS, neurodegenerative diseases, or infections accompanied by an undesired cell death.", "Finding the Invention is Based On.", "Various experiments with regard to membrane permeability of VDAC or BAX channels were performed in synthetic membranes.", "Herein, it was found that with an incubation of VDAC or BAX with c-raf, the channels remain closed.", "In contrast thereto, the channels are open with an incubation of VDAC or BAX with an inactive form of the c-raf.", "The found interaction of raf with VDAC or BAX may be performed directly or indirectly by mediating substances, possibly Bcl-2.It can further be assumed that due to the similarity between on the one hand natural mitochondrial porins, VDAC and/or BAX, and on the other hand bacterial porins, the latter can also be inhibited or closed by interaction with raf.", "Basics of the Invention.", "The invention relates to a nucleic acid coding for at least a partial sequence of a protein kinase of the mitogenic signaling cascade, the partial sequence interacting with porins, in particular VDAC, BAX channels in mitochondrial membranes or with bacterial porin channels or a nucleic acid hybridizing such a nucleic acid or homologues or derivatives of said nucleic acids.", "The term nucleic acid in particular includes DNA, RNA and PNA.", "Further subsumed to the term are double-stranded nucleic acids as well as single-stranded nucleic acids and thus also nucleic acids being complementary to each other.", "Silent mutations are also nucleic acids according to the invention.", "Silent mutations are variants in the sequence which do not lead to a functional difference, referred to the interaction with VDAC or BAX, of the variant compared to the natural non-mutated sequence.", "Silent mutations may be alleles or artificial mutations.", "Derivatives are also covered by the invention.", "Derivatives are non-natural chemical modifications.", "Nucleic acids hybridizing nucleic acids according to the invention are such ones which hybridize under stringent conditions.", "Stringency typically occurs in a temperature range of 5° C. to 25° C. below the melting temperature at the hybridization.", "By stringent conditions is meant a hybridization at at least 95% sequence identity, preferably 98%.", "With regard to the hybridization method on which these definitions are based, reference is made to the document Sambrook, J. M. et al., A laboratory manual, Cold Spring Harbor Laboratory Press (1989) and Southern, E. M., J. Mol.", "Biol.", "98:503 (1975).", "As protein kinases of the mitogenic signaling cascade, in particular A-raf, B-raf or c-raf may be used.", "The nucleic acid according to the invention may also be a raf partial sequence only, namely the kinase domain CR3 or active partial sequences therefrom as well as silent mutations and/or derivatives herefrom.", "CR3 is above the amino acid 302 in the range up to 648.Insofar the nucleic acid may for instance be a nucleic acid coding for Δraf(26-302).", "It seems to be important that the nucleic acid codes for an active form of the kinase or kinase-active part hereof or a homologous protein or peptide.", "By means of nucleic acids according to the invention, a search can be performed for substances interacting with VDAC, BAX or bacterial porin channels.", "On the one hand, in case of an indirect interaction, substances can be searched which bind to that part of raf having before been found as essential for the interaction.", "On the other hand, such nucleic acids may also serve as a model for substances of the same function having a higher efficiency.", "Therefore, the invention also relates to a screening method for determining substances modulating VDAC, BAX or bacterial porin channels, comprising the following steps: a) a VDAC, BAX or bacterial porin channel, as an option a defined partial sequence thereof only, is incubated with a sample substance, in particular a partial sequence of raf, b) the solution of step a) is brought into contact with a membrane positioned in an electrolyte, c) the membrane of step b) is subjected at a time t=0 to an electrical potential difference between different sides of the membrane, and beginning with t=0 or at a defined time after t=0, the current caused by the potential difference or the charge transport through the membrane is measured preferably in dependence of the time, d) the value measured in step c) is compared to a value which has been measured under identical conditions, however with an inactive or active form of raf.", "Hereby, it can be determined, for instance with regard to the inactive form, whether there is any inhibition at all, and for instance with regard to the active form, whether the inhibition is stronger than for active raf.", "Preferred is a nucleic acid according to the invention wherein the interaction is a reduction of the permeability of VDAC, BAX or bacterial porin channels.", "Preferably, the nucleic acid codes for an active protein or peptide comprising the sequence c-raf-1 (338 to 627) or an active fragment herefrom or is a nucleic acid hybridizing such a nucleic acid or codes for a protein or peptide consisting of the sequence c-raf-1 (338 to 627) or is a nucleic acid hybridizing such a nucleic acid.", "It may also be a cDNA.", "Preferably, for the purpose of this invention, it is human raf.", "For research purposes, it may however also be raf of non-human mammals.", "The invention further relates to an isolated recombinant vector comprising a nucleic acid according to the invention or an expression plasmid comprising this nucleic acid.", "For the purpose of a stable expression, a DNA fragment, for instance gag, coding for a suitable viral protein may also be used herein (fusion gag with for instance c-raf or c-raf fragments).", "By means of the expression plasmid, a transformant may be formed which in turn can be used for preparing the protein or peptide coded for by the nucleic acid.", "For this purpose, the transformant is cultivated in a suitable way following conventional methods.", "The invention further relates to a protein or peptide coded for by a nucleic acid according to the invention.", "Subject matter of the invention is finally in particular the use of a nucleic acid according to the invention or of a protein or peptide coded for thereby for preparing a pharmaceutical composition for treating AIDS or neurodegenerative diseases or for protecting non-transformed cells in a tissue containing cancer cells or bacterial infections.", "It is made use herein of that the nucleic acid according to the invention or the protein or peptide coded for thereby acts in an antiapoptotic manner and prevents a (premature) cell death with the consequence of a corresponding disease.", "In the case of AIDS, the T-lymphocytes are protected.", "In the case of neurodegenerative diseases, nerve cells are protected from a (irreparable) cell death.", "For a cancer therapy using cell poisons directed to cells proliferating in an uncontrolled manner, normal healthy cells can be protected from the undesired cell death by the action of the cell poison.", "The invention finally relates to a screening method for determining substances activating raf and thus being suitable for preparing pharmaceutical compositions for treating the above diseases, said substances preferably being low-molecular (<10,000 da, preferably <5,000 da).", "As a screening method is insofar also understood a method for verifying the raf activation by a prospective active ingredient.", "In principle, such a screening method can be directed to substances which directly or indirectly cause in a cell model an increase of the raf concentration.", "This may for instance be substances regulating the raf expression up.", "They may however also be substances which in turn inhibit the raf expression or raf itself.", "In detail, such screening methods may be configured as follows.", "A screening method for identifying raf activating substances may comprise the following steps: a) target cells are reacted with a prospective active ingredient or a mixture of prospective active ingredients, and the target cells are cultivated, b) after a defined time, the expression and/or concentration of raf protein is measured, and the obtained measured value is compared to a measured value which has been obtained under identical conditions, however without addition of a prospective active ingredient, c) a prospective active ingredient or a mixture of prospective active ingredients are selected such that an increase of the expression and/or concentration of raf protein was found in step b), d) as an option, in the case of the use of a mixture of prospective active ingredients, a deconvolution is performed, for instance by individual measurement of the active ingredients of the mixture.", "A screening method for identifying raf activating substances may however also comprise the following steps: a) raf inhibitors naturally occurring in target cells, in particular substances binding to the kinase domain of raf or substances regulating the raf expression down, are identified, b) a substance identified in step a) is as an option isolated and/or purified, reacted in a binding assay with a prospective active ingredient or a mixture of prospective active ingredients, and the binding capability of the substance with the prospective active ingredient is examined, c) a prospective active ingredient or a mixture of prospective active ingredients, for which a binding event has been detected in step b), is selected, d) as an option, in the case of the use of a mixture of prospective active ingredients, a deconvolution is performed, for instance by individual measurement of the active ingredients of the mixture, e) as an option, a selected prospective active ingredient or a selected mixture of prospective active ingredients is subjected to a method according to claim 10 for verifying the raf activity.", "In principle, all methods for determining a raf concentration and/or a binding assay known to the man skilled in the art can be used.", "In conjunction with the raf determination, it may be suitable if the used target cells are genetically altered such that expressed raf carries a reporter group, which can easily be detected by measurement and does naturally not exist in the target cells.", "Additionally, it is remarked that by an inhibition of raf, to which reference is made in the literature, on the other hand an inhibition of the natural cell death can be prevented for cancer cells, i.e.", "for lack of raf.", "VDAC and/or BAX channels are not closed, and apoptosis occurs.", "Insofar the invention also covers a pharmaceutical composition containing at least one inhibitor of raf for treating diseases, for which the natural apoptosis in cells does not take place, as for instance in cancer cells.", "The explanations for one claim category of the invention apply in a corresponding manner for other claim categories.", "The invention finally also relates to healing methods, for instance classically by administration of pharmaceutical preparations, but also gene therapeutically, by means of which one or several substances according to the invention are brought into a target cell or are produced in the target cell and the expression of which is excited or increased.", "In the following, the invention is explained in more detail, based on figures and experiments representing embodiments only.", "There are: FIG.", "1 a, b : the permeability of VDAC (a) and BAXΔTM (b) channels after incubation with GST-c-rafL375W (a), FIG.", "2 a, b : the permeability of VDAC (a) and BAXΔTM (b) channels after incubation with GST-c-rafYY340/341DD, and FIG.", "3 : the permeability of BCL2ΔTM channels after incubation with GST-c-rafYY340/341DD.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The invention relates to nucleic acids coding for a protein interacting with porins, in particular VDAC, BAX channels or bacterial porins, and to the uses of such nucleic acids or proteins or peptides coded for by such a nucleic acid in screening methods or to the preparation of pharmaceutical compositions.", "BACKGROUND OF THE INVENTION The raf protein in its various isoforms c-raf, B-raf and A-raf plays an important role in the regulation of the proliferation and differentiation of cells.", "raf is an effector kinase of ras and an important member in the mitogenic cytoplasmic protein kinase (MAPK) signaling cascade (see e.g.", "Daum, O., et al., Trends Biochem.", "Sci.", "19:474-479 (1994)).", "raf proto-oncogenes are highly conserved genes coding for serine/threonine specific kinases of the cytoplasm.", "These kinases have functions in the mitogenic signal transduction.", "This cascade transfers signals from receptor tyrosine kinases via ras, raf, MEK and ERK to targets in the cytoplasm and basically for the regulation of the proliferation and differentiation of the cells.", "The role of c-raf-1 in the classic mitogenic MAP kinase cascade is rather well researched.", "For details, reference is made here as an example only to U. R. Rapp in The Oncogene Handbook, T. Curran et al., Eds, Elsevier Science Publishers, The Netherlands, 1988, pages 115-154.c-raf-1 is practically ubiquitous in the human organism.", "With the transduction of an apoptosis signal into a cell, there are changes of the permeability of membranes of the mitochondria.", "An increase of the permeability of these membranes will lead to a translocation of the apoptotic protein cytochrome C into the cytoplasm, thereby finally proteolytic proteins called caspase being activated.", "These proteins lead to the apoptosis.", "The permeability of mitochondrial membranes is determined, among other factors, by the permeability of the mitochondrial porin channels VDAC (voltage-dependent anion channel).", "The permeability is in addition influenced by a protein family to which Bcl-2, Bcl-XL, BAX and BAK belong.", "Bcl-2 acts protectively and binds probably to VDAC.", "BAX is an integral membrane protein and promotes apoptosis.", "BAX and Bcl-2 can heterodimerize, and an overexpression of BAX overcompensates the protective effect of Bcl-2.For a deeper insight, reference is made as an example only to Zhou, M., et al.", "J. Biol.", "Chem.", "273:11930-11936 (1998) and Shimizu, M., Nature (Asia) 399:483 (1999).", "It is common to the members of the Bcl-2 family that there are four regions with amino acid homology, BH1, BH2, BH3 and BH4.These domains are essential for the dimerization and/or the function of the different members.", "In particular the presence or absence of BH4 seem to play an important role for the antiapoptotic or proapoptotic effect (see e.g.", "Wang, H.-G. et al., Cell 87:629-638 (1996)).", "For instance Bcl-2 comprises BH4, whereas BAX does not comprise BH4.From the document Wang, H.-G. et al., Cell 87:629-638 (1996), it is known in the art that raf-1 interacts with Bcl-2.According to this document, BH4 is essential for this interaction.", "An association raf-1/BAX has not been found, rather has been negated.", "The results are subject to discussion.", "Various diseases are accompanied by a disturbance of the natural cell death programming.", "A lacking function of the natural cell death is for instance associated with cancer or autoimmune diseases.", "In contrast thereto, an excessive cell death comes with diseases such as AIDS and neurodegenerative diseases.", "But infections with pathogenic bacteria may also be accompanied by a disturbance of the natural cell death, bacterial porins being translocated into a cellular membrane and triggering apoptosis of the target cells there.", "For instance from the document The EMBO Journal 18:339-352 (1999) it is known in the art that the porin PorB in its different variants translocates from Neisseria gonorrhoeae into artificial membranes as well as into cellular target membranes, in particular the outer mitochondrial membrane.", "These considerations generally apply to Gram-negative bacteria.", "The relationship of natural mitochondrial porins to bacterial porins has by the way been described already for instance for Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa in the document Biochimica et Biophysica Acta 686:204-214 (1982).", "Technical Object.", "The invention is based on the technical object to inhibit an undesired cell death and to provide suitable active ingredients for treating diseases accompanied by an undesired cell death, in particular AIDS, neurodegenerative diseases, or infections accompanied by an undesired cell death.", "Finding the Invention is Based On.", "Various experiments with regard to membrane permeability of VDAC or BAX channels were performed in synthetic membranes.", "Herein, it was found that with an incubation of VDAC or BAX with c-raf, the channels remain closed.", "In contrast thereto, the channels are open with an incubation of VDAC or BAX with an inactive form of the c-raf.", "The found interaction of raf with VDAC or BAX may be performed directly or indirectly by mediating substances, possibly Bcl-2.It can further be assumed that due to the similarity between on the one hand natural mitochondrial porins, VDAC and/or BAX, and on the other hand bacterial porins, the latter can also be inhibited or closed by interaction with raf.", "Basics of the Invention.", "The invention relates to a nucleic acid coding for at least a partial sequence of a protein kinase of the mitogenic signaling cascade, the partial sequence interacting with porins, in particular VDAC, BAX channels in mitochondrial membranes or with bacterial porin channels or a nucleic acid hybridizing such a nucleic acid or homologues or derivatives of said nucleic acids.", "The term nucleic acid in particular includes DNA, RNA and PNA.", "Further subsumed to the term are double-stranded nucleic acids as well as single-stranded nucleic acids and thus also nucleic acids being complementary to each other.", "Silent mutations are also nucleic acids according to the invention.", "Silent mutations are variants in the sequence which do not lead to a functional difference, referred to the interaction with VDAC or BAX, of the variant compared to the natural non-mutated sequence.", "Silent mutations may be alleles or artificial mutations.", "Derivatives are also covered by the invention.", "Derivatives are non-natural chemical modifications.", "Nucleic acids hybridizing nucleic acids according to the invention are such ones which hybridize under stringent conditions.", "Stringency typically occurs in a temperature range of 5° C. to 25° C. below the melting temperature at the hybridization.", "By stringent conditions is meant a hybridization at at least 95% sequence identity, preferably 98%.", "With regard to the hybridization method on which these definitions are based, reference is made to the document Sambrook, J. M. et al., A laboratory manual, Cold Spring Harbor Laboratory Press (1989) and Southern, E. M., J. Mol.", "Biol.", "98:503 (1975).", "As protein kinases of the mitogenic signaling cascade, in particular A-raf, B-raf or c-raf may be used.", "The nucleic acid according to the invention may also be a raf partial sequence only, namely the kinase domain CR3 or active partial sequences therefrom as well as silent mutations and/or derivatives herefrom.", "CR3 is above the amino acid 302 in the range up to 648.Insofar the nucleic acid may for instance be a nucleic acid coding for Δraf(26-302).", "It seems to be important that the nucleic acid codes for an active form of the kinase or kinase-active part hereof or a homologous protein or peptide.", "By means of nucleic acids according to the invention, a search can be performed for substances interacting with VDAC, BAX or bacterial porin channels.", "On the one hand, in case of an indirect interaction, substances can be searched which bind to that part of raf having before been found as essential for the interaction.", "On the other hand, such nucleic acids may also serve as a model for substances of the same function having a higher efficiency.", "Therefore, the invention also relates to a screening method for determining substances modulating VDAC, BAX or bacterial porin channels, comprising the following steps: a) a VDAC, BAX or bacterial porin channel, as an option a defined partial sequence thereof only, is incubated with a sample substance, in particular a partial sequence of raf, b) the solution of step a) is brought into contact with a membrane positioned in an electrolyte, c) the membrane of step b) is subjected at a time t=0 to an electrical potential difference between different sides of the membrane, and beginning with t=0 or at a defined time after t=0, the current caused by the potential difference or the charge transport through the membrane is measured preferably in dependence of the time, d) the value measured in step c) is compared to a value which has been measured under identical conditions, however with an inactive or active form of raf.", "Hereby, it can be determined, for instance with regard to the inactive form, whether there is any inhibition at all, and for instance with regard to the active form, whether the inhibition is stronger than for active raf.", "Preferred is a nucleic acid according to the invention wherein the interaction is a reduction of the permeability of VDAC, BAX or bacterial porin channels.", "Preferably, the nucleic acid codes for an active protein or peptide comprising the sequence c-raf-1 (338 to 627) or an active fragment herefrom or is a nucleic acid hybridizing such a nucleic acid or codes for a protein or peptide consisting of the sequence c-raf-1 (338 to 627) or is a nucleic acid hybridizing such a nucleic acid.", "It may also be a cDNA.", "Preferably, for the purpose of this invention, it is human raf.", "For research purposes, it may however also be raf of non-human mammals.", "The invention further relates to an isolated recombinant vector comprising a nucleic acid according to the invention or an expression plasmid comprising this nucleic acid.", "For the purpose of a stable expression, a DNA fragment, for instance gag, coding for a suitable viral protein may also be used herein (fusion gag with for instance c-raf or c-raf fragments).", "By means of the expression plasmid, a transformant may be formed which in turn can be used for preparing the protein or peptide coded for by the nucleic acid.", "For this purpose, the transformant is cultivated in a suitable way following conventional methods.", "The invention further relates to a protein or peptide coded for by a nucleic acid according to the invention.", "Subject matter of the invention is finally in particular the use of a nucleic acid according to the invention or of a protein or peptide coded for thereby for preparing a pharmaceutical composition for treating AIDS or neurodegenerative diseases or for protecting non-transformed cells in a tissue containing cancer cells or bacterial infections.", "It is made use herein of that the nucleic acid according to the invention or the protein or peptide coded for thereby acts in an antiapoptotic manner and prevents a (premature) cell death with the consequence of a corresponding disease.", "In the case of AIDS, the T-lymphocytes are protected.", "In the case of neurodegenerative diseases, nerve cells are protected from a (irreparable) cell death.", "For a cancer therapy using cell poisons directed to cells proliferating in an uncontrolled manner, normal healthy cells can be protected from the undesired cell death by the action of the cell poison.", "The invention finally relates to a screening method for determining substances activating raf and thus being suitable for preparing pharmaceutical compositions for treating the above diseases, said substances preferably being low-molecular (<10,000 da, preferably <5,000 da).", "As a screening method is insofar also understood a method for verifying the raf activation by a prospective active ingredient.", "In principle, such a screening method can be directed to substances which directly or indirectly cause in a cell model an increase of the raf concentration.", "This may for instance be substances regulating the raf expression up.", "They may however also be substances which in turn inhibit the raf expression or raf itself.", "In detail, such screening methods may be configured as follows.", "A screening method for identifying raf activating substances may comprise the following steps: a) target cells are reacted with a prospective active ingredient or a mixture of prospective active ingredients, and the target cells are cultivated, b) after a defined time, the expression and/or concentration of raf protein is measured, and the obtained measured value is compared to a measured value which has been obtained under identical conditions, however without addition of a prospective active ingredient, c) a prospective active ingredient or a mixture of prospective active ingredients are selected such that an increase of the expression and/or concentration of raf protein was found in step b), d) as an option, in the case of the use of a mixture of prospective active ingredients, a deconvolution is performed, for instance by individual measurement of the active ingredients of the mixture.", "A screening method for identifying raf activating substances may however also comprise the following steps: a) raf inhibitors naturally occurring in target cells, in particular substances binding to the kinase domain of raf or substances regulating the raf expression down, are identified, b) a substance identified in step a) is as an option isolated and/or purified, reacted in a binding assay with a prospective active ingredient or a mixture of prospective active ingredients, and the binding capability of the substance with the prospective active ingredient is examined, c) a prospective active ingredient or a mixture of prospective active ingredients, for which a binding event has been detected in step b), is selected, d) as an option, in the case of the use of a mixture of prospective active ingredients, a deconvolution is performed, for instance by individual measurement of the active ingredients of the mixture, e) as an option, a selected prospective active ingredient or a selected mixture of prospective active ingredients is subjected to a method according to claim 10 for verifying the raf activity.", "In principle, all methods for determining a raf concentration and/or a binding assay known to the man skilled in the art can be used.", "In conjunction with the raf determination, it may be suitable if the used target cells are genetically altered such that expressed raf carries a reporter group, which can easily be detected by measurement and does naturally not exist in the target cells.", "Additionally, it is remarked that by an inhibition of raf, to which reference is made in the literature, on the other hand an inhibition of the natural cell death can be prevented for cancer cells, i.e.", "for lack of raf.", "VDAC and/or BAX channels are not closed, and apoptosis occurs.", "Insofar the invention also covers a pharmaceutical composition containing at least one inhibitor of raf for treating diseases, for which the natural apoptosis in cells does not take place, as for instance in cancer cells.", "The explanations for one claim category of the invention apply in a corresponding manner for other claim categories.", "The invention finally also relates to healing methods, for instance classically by administration of pharmaceutical preparations, but also gene therapeutically, by means of which one or several substances according to the invention are brought into a target cell or are produced in the target cell and the expression of which is excited or increased.", "In the following, the invention is explained in more detail, based on figures and experiments representing embodiments only.", "There are: FIG.", "1a, b: the permeability of VDAC (a) and BAXΔTM (b) channels after incubation with GST-c-rafL375W (a), FIG.", "2a, b: the permeability of VDAC (a) and BAXΔTM (b) channels after incubation with GST-c-rafYY340/341DD, and FIG.", "3: the permeability of BCL2ΔTM channels after incubation with GST-c-rafYY340/341DD.", "SEQUENCE INFORMATION The sequence of BAX is known and ready to be called for under the accession number L22473 from www.ncbi.nlm.nih.gov.", "The sequence of BCL-2 is known and ready to be called for under the accession number M13995 from www.ncbi.nlm.nih.", "gov.", "The sequence of VDAC is known and ready to be called for under the accession number NM003374 from www.ncbi.nlm.nih.gov.", "The sequence of c-raf is known and ready to be called for under the accession number X03484 from www.ncbi.", "nlm.nih.gov.", "The sequence of A-raf is known and ready to be called for under the accession number X04790 from www.ncbi.nlm.nih.gov.", "The sequence of B-raf is known and ready to be called for under the accession number M95712 from www.ncbi.nlm.nih.gov.", "Example 1 Preparation of Plasmids BAX and BCL-2 cDNAs are used which code for proteins where the COOH-terminal transmembrane domain is lacking (ATM).", "In the case of BAX this is BAXAC19, i.e.", "the amino acids 172-191 are lacking.", "In the case of BCL-2 this is BCL-2ΔC21, i.e.", "the amino acids 219-239 are lacking.", "cDNA coding for BAXΔTM was cut out by digestion with EcoRI/XhoI from the vector pJG4-5 and subcloned into the EcoRI/XhoI sites of pGEX-4T1.cDNA coding for BCL-2ΔTM was expressed in the pGEX-4T1 plasmid (obtained from J. Reed).", "GST-c-rafYY340/341DD and GST-c-rafL375W were cloned in pFastBac baculoviruses for the expression in Sf9 insect cells.", "Example 2 Expression and Purification of the Recombinant Proteins Escherichia coli BL21 DE3 (pLysS) were transformed with BAXΔTM or BCL-2ΔTM constructs and bred on over night at 37° C. in presence of 0.1 mg/ml ampicilin.", "A single colony of every vector was cultivated in 2×Y-TG medium containing 0.1 mg/ml ampicilin at 37° C. to give an OD (600 nm) value of 0.6.Protein expression was induced by means of 0.1 mM isopropyl-β-D-thiogalactopyranoside for 16 h at 25° C. The cells were obtained by means of centrifugation at 4,000 rpm for 30 min at 4° C. and resuspended in lysis buffer (Tris 50 mM pH 8, NaCl 100 mM, EDTA 1 mM, benzamidine 1 mM, phenylmethylsulfonyl fluoride 1 mM, leupeptin 10 μg/ml, aprotinin 10 μg/ml, β-mercaptoethanol 10 mM).", "Homogenates of the cells were frozen in liquid nitrogen in presence of 1 mg lysozyme and thawed again prior to ultrasonic treatment.", "After centrifugation at 28,000 g at 4° C. and for 30 min, the supernatant was incubated with glutathione sepharose beads (Pharmacia Amersham) for 2 h at 4° C. The beads were washed with lysis buffer containing Triton-X100 (0.1%) and NaCl (20 mM) and incubated over night in a buffer (Tris 50 mM pH 8, β-mercaptoethanol 20 mM) containing thrombin (Sigma).", "Cleaved BAXΔTM and BCL-2ΔTM was further purified on an anion exchange column Resource Q (Pharmacia Amersham), and the proteins were eluted with a NaCl gradient (0-0.5 M).", "For obtaining the GST-C-raf proteins, the Sf9 insect cells were infected with the baculoviruses, and cell pellets were frozen and kept at −70° C. The pellets were resuspended in lysis buffer (Tris 25 mM pH 7.6, NaCl 150 mM, Na4P2O7 10 mM, β-glycerophosphate 25 mM, glycerol 10%, NP-40 0.75%, leupeptin 10 μg/ml, aprotinin 10 μg/ml, benzamidine 1 mM, phenylmethylsulfonyl fluoride 1 mM), homogenized and incubated for 40 min at 4° C. Cell debris was removed by centrifugation at 15,000 g at 4° C. and for 30 min.", "Supernatants were incubated with glutathione sepharose beads (Pharmacia Amersham) for 2 h at 4° C. The beads were washed the first time with lysis buffer and a second time with lysis buffer containing NaCl (500 mM).", "GST fusion proteins were eluted with buffer (Tris 50 mM pH 8, aprotinin, leupeptin, benzamidine) containing 20 mM glutathione.", "VDAC protein was purified from rat liver under denaturating/renaturating conditions.", "Example 3 Execution of the Experiments on Lipid Bilayers The channel-forming proteins were reconstituted in an artificial lipid bilayer.", "The experimental structure contained a Teflon chamber with two compartments containing aqueous KCl buffers (0.3 M) having a volume of 5 ml, the compartments being connected by a circular hole with a cross section of 0.2 mm2.The membrane was generated from a solution of 1% (w/v) diphytanoylphosphateidylcholine (DiphPC, Avanti Polar Lipids, Alabaster, Ala.) in n-decane.", "The bilayer formation was detectable by that the membrane became optically black with regard to reflected light.", "BAXΔTM, BCL-2ΔTM or VDAC protein was added to the KCl buffer in both compartments.", "The single-channel conductance of the generated pores or channels was measured after application of a constant membrane potential (20 mV) by introduction of one Ag/AgCl electrode each with salt bridges into the buffer of the two compartments.", "The detected current values were amplified with a current amplifier and recorded as a function of the time.", "In order to test the effect of C-Raf kinase on BAXΔTM, BCL-2ΔTM or VDAC channels, the channel-forming proteins (BAXΔTM: 2.4 μg, BCL-2ΔTM: 1.8 μg) were incubated in kinase buffer (Hepes 25 mM pH 7,4, NaCl 150 mM, β-glycerophosphate 25 mM, DTT 1 mM, MgCl2 10 mM) in presence of ATP (0.1 mM) with GST-c-rafYY340/341DD (active form of c-raf, GST-c-rafK375W (inactive form of c-raf) or GST alone for 30 min at 30° C. Samples were applied on both sides of the DiphPC membrane in KCl buffer of the compartments, and the single-channel formation was measured.", "By control measurements it was found that GST-c-RafYY340/341DD, GST-c-rafK375W or GST, each alone, did not form channels in the artificial bilayer membrane.", "Example 4 Results In FIG.", "1 a, b are shown the results of the permeability measurements with an experimental set as in example 3, VDAC/GST-c-rafL375W (a) and BAXΔTM/GST-c-raf375W (b).", "It can be seen that channels are formed, i.e.", "there is no inhibition.", "In contrast thereto, it can be taken from FIGS.", "2 a, b that the use of the active forms of c-raf, GST-c-rafXX340/341DD in lieu of the inactive form according to FIG.", "1 a, b leads to a considerable inhibition of the channel form ation.", "This is based on the interaction according to the invention of the active c-raf protein with VDAC or BAXΔTM.", "Using the active form of c-raf, further a potential influence on BCL-2ΔTM was tested.", "These results are shown in FIG.", "3.It can be seen that obviously there is a channel formation, i.e.", "c-raf interacts with BCL-2 at any case in a manner not inhibiting the channel formation." ] ]	Patent_10469375
[ [ "Method for the preparation of reagents for amplification and/or detection of nucleic acids that exhibit no significant contamination by nucleic acids", "The present invention decribes reagents free of detectable contaminating nucleic acids for performing highly sensitive and specific nucleic acids amplification and/or detection.", "It relates to an improvement in the technology of nucleic acid inactivation prior to nucleic acid testing (NAT) in order to prevent false-positive results.", "Specifically, this invention describes optimized and standardized reagents and ultra-violet treatment to achieve an effective and highly reproducible nucleic acid inactivation prior to NAT without substantially affecting the performance of the assay.", "More specifically, this nucleic acid inactivation process resulted in a reduction of up to four logs of the background signal associated with the PCR (polymerase chain reaction) amplification of DNA contaminating PCR reagents.", "This optimized and standardized method is also adaptable for use with NAT technologies other than PCR." ], [ "1.A reagent to be put in contact with nucleic acids of interest, said reagent having a treatable surface, wherein the concentration of amplifiable contaminating nucleic acids is below a level that interferes with an amplification and/or detection reaction conducted with said nucleic acids of interest, said reagent comprising a furocoumarin compound and having been submitted to a UV light treatment capable of reducing contaminating nucleic acids below said level, with the standardization of the wavelength spectrum of the UV source and the total energy of the treatment per unit of surface, the combination of furocoumarin and UV light treatment inactivating the contaminating nucleic acids by rendering them unamplifiable; said treatment having no substantial detrimental effect on the performance of said amplification and/or detection reaction.", "2.A reagent as defined in claim 1, which is obtainable by a UV light treatment equivalent to a treatment conducted in the presence of 8-MOP as the furocoumarin, with a Spectrolinker™XL-1000 apparatus, equipped with a UV sensor and a UV source of a wavelength spectrum of about 300 to 400 nm, and providing a total energy of about 750 to 4500 mJoules per square centimeter as measured by the UV sensor located at about 17.6 cm of the UV source while a reagent is disposed in 0.6 ml MaxyClear flip cap conical plastic tubes purchased from Axygen, located at about 10.8 cm from the UV source.", "3.A reagent as defined in claim 1, which is obtainable by a UV light treatment equivalent to a treatment conducted in the presence of Trioxsalen as the furocoumarin, with a Spectrolinker™ XL-1000 apparatus, equipped with a UV sensor and a UV source of a wavelength spectrum of about 300 to 400 nm, and providing a total energy of about 500 to 1500 mJoules per square centimeter as measured by the UV sensor located at about 17.6 cm of the UV source while a reagent is disposed in 0.6 ml MaxyClear flip cap conical plastic tubes purchased from Axygen, located at about 10.8 cm from the UV source.", "4.A reagent as defined in claim 1, which further comprises a level of contaminating nucleic acids (either spiked or naturally present in the reagent(s)), the presence of which can be detected if its concentration is not below said level; said contaminating nucleic acids being used as a standard to monitor and optimize the conditions for nucleic acids inactivation.", "5.A reagent as defined in claim 1, which comprises a protein, the function of which is not substantially affected by said treatment.", "6.The reagent of claim 1, wherein said reagent comprises a component selected from the group consisting of: a nucleotide and/or nucleotide analog; an oligonucleotide primer and/or probe; a buffer solution; a monovalent and/or divalent ion; an enzyme selected from the group consisting of DNA polymerase, RNA polymerase, reverse transcriptase, DNA ligase, restriction enzyme DNAase, RNAase, protease and an enzyme used for NAT or in test sample preparation for NAT; an amplification facilitator; a cryoprotector; a stabilizer; a solvent; and any suitable combination thereof.", "7.The reagent of claim 6, wherein at least two components are mixed together in a common vial.", "8.The reagent of claim 1, which is liquid, frozen or dehydrated.", "9.A container comprising a reagent as defined in claim 1.10.", "(cancelled) 11.A container as defined in claim 9, which is a closed vessel.", "12.A reagent, as defined in claim 1, wherein said furocoumarin is 8-MOP or Trioxsalen.", "13.A reagent or as defined in claim 12, wherein 8-MOP is used at a final concentration of about 0.015 μg/μL (or 0.07 mM) to about 0.12 μg/μL (or 0.56 mM).", "14.A reagent as defined in claim 12, wherein Trioxsalen is used at a final concentration of about 0.001 μg/μL (0.0044 mM) to 0.0075 μg/μL (0.033 mM).", "15.A reagent as defined in claim 1, which is for PCR.", "16.A method for rendering contaminating nucleic acids in a reagent unamplifiable in an amplification reaction of nucleic acids of interest, without substantially affecting the performance of the amplification reaction which comprises: a) providing a reagent to be contacted with said nucleic acids of interest; b) providing a furocoumarin compound; c) obtaining a mixture of said reagent and the furocoumarin compound; and d) treating said mixture with light energy of a wavelength in the UV range.", "17.A method as defined in claim 16, wherein the UV light treatment is equivalent to a treatment conducted in the presence of 8-MOP as the furocoumarin, with a Spectrolinker™XL-1000 apparatus, equipped with a UV sensor and a UV source of a wavelength spectrum of about 300 to 400 nm, and providing a total energy of about 750 to 4500 mJoules per square centimeter as measured by the UV sensor located at about 17.6 cm of the UV source while a reagent is disposed in 0.6 ml MaxyClear flip cap conical plastic tubes purchased from Axygen, located at about 10.8 cm from the UV source.", "18.A method as defined in claim 16, wherein said UV light treatment is equivalent to a treatment conducted in the presence of Trioxsalen as the furocoumarin, with a Spectrolinker™ XL-1000 apparatus, equipped with a UV sensor and a UV source of a wavelength spectrum of about 300 to 400 nm, and providing a total energy of about 500 to 1500 mJoules per square centimeter as measured by the UV sensor located at about 17.6 cm of the UV source while a reagent is disposed in 0.6 ml MaxyClear flip cap conical plastic tubes purchased from Axygen, located at about 10.8 cm from the UV source.", "19.A method as defined in claim 16, which further comprises a level of contaminating nucleic acids, the presence of which can be detected if its concentration is not below said level; said contaminating nucleic acids being used as a standard to monitor and optimize the conditions for nucleic acids inactivation.", "20.The method of claim 16, wherein the reagent comprises a protein, the function of which is not substantially affected by said treatment.", "21.The method of any one of claim 16, wherein the furocoumarin compound is a psoralen or an isopsoralen derivative.", "22.The method of claim 21, wherein the furocoumarin compound is 8-MOP or Trioxsalen.", "23.The method of claim 21, wherein the concentration of the furocoumarin compound is about 0.015 μg/μL (or 0.07 mM) to about 0.12 μg/μL (or 0.56 mM).", "24.The method of claim 21, wherein the concentration of the furocoumarin compound is about 0.001 μg/μL (or 0.0044 mM) to 0.0075 μg/μL (or 0.033 mM).", "25.The method of claim 16, wherein the reagent is involved in an amplification and/or detection reaction or in the test sample preparation.", "26.The method of claim 25, wherein the reagent comprises a component selected from the group consisting of: a nucleotide and/or nucleotide analog; an oligonucleotide primer and/or probe; a buffer solution; a monovalent and/or divalent ion; an enzyme selected from the group consisting of DNA polymerase, RNA polymerase, reverse transcriptase, DNA ligase, restriction enzyme, DNAase, RNAase, protease and any enzyme used for NAT or in test sample preparation for NAT; an amplification facilitator; a cryoprotector; a stabilizer; a solvent; and any suitable combination thereof.", "27.The method of claim 25, wherein the reagent is a PCR reagent.", "28.The method of claim 25, wherein the reagent is a RT-PCR reagent.", "29.The method of claim 16 wherein said mixture is treated with UV in a tubing.", "30.The method of claim 16, wherein said mixture is enclosed in a plastic vessel.", "31.The method of claim 30, wherein said mixture is treated with UV in immediate container into which a reaction with nucleic acids of interest is performed.", "32.The method of claim 16, wherein said UV light dose is applied and monitored by measurements with a radiometer equipped with a UV sensor or with an appropriate spectrometer.", "33.The method of claim 16, wherein said reaction mixture is treated using a suitable UV source including a laser, high intensity white light, an incandescent lamp and a diode.", "34.The method of claim 16, wherein the light treatment is performed using an apparatus consisting of a chamber equipped with UV lights and allowing to measure the UV dose." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The practical application of recombinant DNA technology in the field of infectious diseases was initially reported in 1980 by Moseley et al.", "(Moseley et al., 1980, J. Infect.", "Dis.", "142:892-898).", "Since those days, molecular biology technologies have undertaken a rapid evolution.", "Based on these technologies, a number of rapid and sensitive nucleic acid testing (NAT) methods have been developed for a variety of applications including diagnosis of infectious and genetic diseases in humans, animals and plants.", "Many of these NAT assays have been used in the field of microbiology to complement or replace the slower conventional culture-based identification systems (Picard and Bergeron, 2002, Drug Discovery Today 7:1092-1101; Boissinot and Bergeron, 2002, Curr.", "Opinion Microbiol.", "5:478482; Tang and Persing, 1999, Molecular detection and identification of microorganisms, p. 215-244, In Manual of Clinical Microbiology, Murray et al., American Society for Microbiology, Washington, D.C.; Lee et al.", "1997, Nucleic Acid Amplification Technologies: Application to Disease Diagnosis, Biotechniques Books, Eaton Publishing, Boston, Mass.", "; Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.).", "These assays have been designed for microbial detection and identification directly from clinical and/or environmental samples and are based on the use of a variety of NAT technologies including the widely used and powerful polymerase chain reaction (PCR).", "Other nucleic acid amplification technologies include among others the ligase chain reaction (LCR), the strand displacement amplification (SDA) as well as transcription-based amplifications such as the transcription mediated amplification (TMA) (Tang and Persing, 1999, Molecular detection and identification of microorganisms, p. 215-244, In Manual of Clinical Microbiology, Murray et al., American Society for Microbiology, Washington, D.C.; Lee et al., 1997, Nucleic Acid Amplification Technologies: Application to Disease Diagnosis, Biotechniques Books, Eaton Publishing, Boston, Mass.).", "Sensitive NAT technologies also include signal amplification methods such as the branched DNA (bDNA) probe technique.", "NAT can be used to detect the presence of any microbe in clinical samples.", "A number of PCR-based assays targeting highly conserved nucleotide sequences in microbes have been used by us and others to develop universal amplification assays for bacteria or fungi (Martineau et al., 2001, J. Clin.", "Microbiol.", "39:2541-2547; Schonhuber et al., 2001, BMC Microbiology 1:20; Ke et al., 1999, J. Clin.", "Microbiol.", "37:3497-3503; Loeffler et al, J. Clin.", "Microbiol.", "37:1200-1202; McCabe et al., 1999, Molecular Gen. Metabolism 66:205-211; Klausegger et al., 1999, J. Clin.", "Microbiol.", "37:464-466; Tanner et al.", "1998, Appl.", "Environ.", "Microbiol.", "64:3110-3113; Goh et al., 1996, J. Clin.", "Microbiol.", "34:818-823; Sandhu et al., 1995, J. Clin.", "Microbiol.", "33:2913-2919; Greisen et al., 1994, J. Clin.", "Microbiol.", "32:335-351; Schmidt et al., 1991, Biotechniques 11:176-177; Rand and Houck, 1990, Mol.", "Cell.", "Probes 4:445-450 and our co-pending patent application WO 01/23604 A2).", "However, because of the high sensitivity of NAT, the development of sensitive and broad-range (or universal) nucleic acid detection assays is hampered by the presence of microbial DNA and/or microbial cells that may be present in NAT reagents and which lead to false positive results.", "The most common source of false-positive results in NAT is associated with carry-over of previously amplified target nucleic acids.", "This type of contamination can be prevented by using proper laboratory procedures (Millar et al., 2002, J. Clin.", "Microbiol.", "40:1575-1580; Kwok and Higuchi, 1989, Nature, 239:237-238), or alternatively, by using techniques to inactivate amplification products such as the method using the uracil-N-glycosylase (UNG) (Longo et al., 1990, Gene 93:125-128).", "DNA inactivation using the photoreactive compounds psoralen or isopsoralen, which is used in the object of the present invention, may prevent amplification of contaminating target nucleic acids (Persing and Cimino, 1993, Amplification products inactivation methods p. 105-212, In Persing et al., Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.; Isaacs et al., 1991, Nucleic Acids Res.", "19:109-116; and U.S. Pat.", "No.", "5,221,608).", "Psoralens and isopsoralens are furocoumarin compounds representing a class of planar tricyclic photoreactive reagents that are known to form covalent monoadducts and crosslinks with nucleic acids upon activation with ultra-violet (UV) light.", "Examples of furocoumarin compounds are given in U.S. Pat.", "No.", "5,221,608, the contents of which are entirely incorporated by reference.", "These monoadducts can be formed between two adjacent pyrimidines on opposite strands of nucleic acids thereby creating interstrand crosslinks with both DNA and RNA.", "Such crosslinks prevent primer extension activities of polymerases.", "Psoralens and isopsoralens have the major advantage of allowing nucleic acid inactivation in closed vessels (such as PCR reaction vessels) thereby preventing carry-over contamination by nucleic acid aerosols.", "Another effective strategy to prevent carry-over contamination is to perform the nucleic acid amplification reactions in closed vessels such as in real-time PCR amplification and analysis (Foy and Parkes, 2001, Clin.", "Chem.", "47:990-1000).", "Another important source of false-positive results in NAT is extraneous nucleic acids introduced in reagents during the manufacturing process.", "For example, the Taq polymerase used in PCR has been shown by many investigators to be contaminated with bacterial DNA (Gale et al., 2003, Clin.", "Chem.", "49:415-424; Corless et al., 2000, J. Clin.", "Microbiol.", "38:1747-1752; Maiwald et al., 1994, Mol.", "Cell.", "Probes 8:11-14; Meier et al., 1993, J. Clin.", "Microbiol.", "31:646652; Schmidt et al., 1991, Biotechniques 11:176-177; Jinno et al., 1990, Nucleic Acids Res.", "18:6739; Rand and Houck, 1990, Mol.", "Cell.", "Probes 4:445-450; and U.S. Pat.", "No.", "5,532,145).", "Analysis of the conserved bacterial rRNA gene sequences contaminating different preparations of Taq DNA polymerase revealed that these nucleic acids were closely related to the genera Corynebacterium, Afthrobacter, Mycobacteiium, Pseudomonas, Alcaligenes and Azotobacter (Hughes et al., 1994, J. Clin.", "Microbiol., 32:2007-2008; Maiwald et al., 1994, Mol.", "Cell.", "Probes 8:11-14).", "Importantly, the contaminating DNA sequences did not match with that of the species Eschedchia coli and Thermus aquaticus which were the bacteria used to produce these ezymes.", "Because of the nature of this type of contamination, the use of UNG or of closed vessel assays as well as careful laboratory techniques cannot circumvent this important NAT reagents nucleic acid contamination problem.", "DNA inactivation using psoralens or isopsoralens combined with a UV treatment has been used to prevent amplification of microbial DNA contaminating PCR reagents (Corless et al., 2000, J. Clin.", "Microbiol.", "38:1747-1752; Klausegger et al., 1999, J. Clin.", "Microbiol.", "37:464-466; Hughes et al., 1994, J. Clin.", "Microbiol., 32:2007-2008; Meier et al., 1993, J. Clin.", "Microbiol.", "31:646-652; Jinno et al., 1990, Nucleic Acids Res.", "18:6739; and U.S. Pat.", "No.", "5,532,145).", "However, there is no standardized method for nucleic acid inactivation using these photoreactive compounds allowing efficient and reproducible nucleic acid inactivation without substantial reduction in the performance of the nucleic acid amplification and/or detection assay.", "The words “without substantial” or “not have a substantial” are used throughout the present invention to mean “without or with minimal”.", "Several investigators have reported an important reduction in the analytical sensitivity of NAT assays attributable to the UV treatment in the presence of psoralen or isopsoralen (Corless et al., 2000, J. Clin.", "Microbiol.", "38:1747-1752; Meier et al., 1993, J. Clin.", "Microbiol.", "31:646-652 and U.S. Pat.", "No.", "5,532,145).", "Corless et al.", "(2000, J. Clin.", "Microbiol.", "38:1747-1752) compared several methods to eliminate nucleic acid contamination from PCR reagents.", "They concluded that it was not possible to eliminate contaminating nucleic acids from the PCR reagents without significantly decreasing the analytical sensitivity of their real-time PCR assays.", "When they tested a combination of 8-methoxypsoralen (8-MOP) and UV irradiation, complete DNA decontamination of the PCR reagents was achieved after 5 minutes of UV exposure.", "They have not specified the UV dose (in mJoule/cm 2 ) nor did they described the reagent container and its distance from the UV source.", "They observed a 5 to 7 logs reduction in the analytical sensitivity of the real-time PCR assays using this non-standardized 8-MOP-based DNA inactivation method.", "In fact, their experimental procedure does not include proper control of key parameters such as those disclosed in the present invention which ensure that an optimal UV energy dose is administered to the reagents containing an optimal 8-MOP concentration.", "The present invention allows for efficient nucleic acids inactivation while reducing the performance of the assay by only about 1 log or less.", "This is achieved by (i) monitoring the energy dose with a UV sensor by measuring the UV dose in mJoules per square centimeters, (ii) maintaining a constant distance between the reagents and the UV source, (iii) testing the reagent container for its permeability to UV treatment and (iv) optimising the 8-MOP concentration.", "U.S. Pat.", "No.", "5,532,145 describes the use of degassing to remove oxygen from PCR reaction mixtures containing a furocoumarin prior to UV irradiation to preserve Taq DNA polymerase activity.", "However, the degassing process is not practical as it involves freezing the reaction mixture to be decontaminated in dry/ice ethanol, thawing and applying vacuum for 30 seconds three times.", "As revealed in the present invention it is simpler to control the parameters of the UV treatment.", "These parameters include the type of furocoumarin compound and its concentration, the UV exposure, the intensity of the UV source, the length of the UV treatment and the wavelengths spectrum of the UV source which are important factors in achieving an efficient and reproducible performance in DNA inactivation, and this, without substantial detrimental effect on the performance of NAT assays.", "Other methods to inactivate DNA contaminating NAT reagents have been used with very limited success.", "These methods include the use of UV irradiation alone, a treatment with DNAase and/or restriction endonucleases and a treatment with exonucleases (Corless et al., 2000, J. Clin.", "Microbiol.", "38:1747-1752; Zhu et al., 1991, Nucleic Acids Res.", "19:2511).", "Also, a pre-filtration step for the PCR mix prior to the addition of the test sample have been used to remove nucleic acids present in PCR reagents (Yang et al., 2002, J. Clin.", "Microbiol.", "40:3449-3454)." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to reagents submitted to an improved treatment using furocoumarin derivatives (e.g.", "psoralens and/or isopsoralens) and UV irradiation to inactivate contaminating nucleic acids from NAT reagents, without substantial hindering of the performance of the NAT methods, and this, without the need to remove oxygen in order to avoid the presence of damaging oxygen radical species (by degassing for example).", "This treatment includes careful control and monitoring of some experimental conditions including the quality of the vessel containing the reaction mixture to be treated as well as the UV dose and intensity of the light source in the UV wavelengths spectrum.", "The present method and resulting products (reagents and containers with reagents) ensure a reproducible and efficient nucleic acid inactivation.", "It is an object of the present invention to provide reagents useful in the obtention of samples which are to be submitted to amplification and/or detection of nucleic acids in which the concentration of amplifiable and/or detectable contaminating nucleic acids is low, if not totally absent, so as not to substantially interfere with the detection of the nucleic acids targeted in the reaction.", "These reagents may include a protein, the function of which should not be substantially affected by the treatment of this invention.", "Such a protein may be an enzyme.", "If a nucleic acid amplification reaction is to be performed, the enzyme may be a polymerase, a reverse transcriptase, a ligase or a restriction endonuclease.", "It may also be an enzyme useful in the test sample preparation steps for nucleic acid extraction preceding an amplification and/or detection reaction, for example a DNAase, a RNAase or a protease.", "These reagents include nucleotides and/or nucleotide analogs, oligonucleotides (primers and/or probes), buffer solutions, ions (monovalent and/or divalent), enzymes (DNA polymerase, RNA polymerase, reverse transcriptase, DNA ligase, restriction enzymes, DNAase, RNAase, protease or any other enzymes used for NAT or in test sample preparation for NAT), amplification facilitators (e.g.", "betaine, dimethyl sulfoxide, bovine serum albumin, tetramethylamonium chloride), cryoprotectors (e.g.", "glycerol), stabilizers (e.g.", "trehalose) and a solvent (usually water).", "In a particularly preferred embodiment, these reagents containing no or a low level of detectable contaminating DNA or RNA may be provided separately or as separate components of a kit, or mixed together, and may be liquid, frozen or dehydrated.", "Preferably, the reagents are any combination suitable for a nucleic acid amplification and/or detection reaction.", "It is another object of the present invention to provide for cleaner reagents and kits for the preparation of nucleic acids (sample preparation and nucleic acids extraction) for NAT assays as well as to provide an efficient method to inactivate nucleic acids contaminating said reagents and kits including purifying devices and columns.", "It is another object of this invention to provide a container, such as a closed vessel, which comprises the reagents treated in accordance with the present invention.", "The closed vessel could be submitted to the same treatment, simultaneously with the treatment of the reagents.", "Indeed, the reagents could be placed into the vessel and then submitted to the treatment of this invention.", "It is another object of this invention to provide an improved method using furocoumarin compounds and UV light for nucleic acid inactivation to treat reagents prior to NAT in order to prevent false-positive results, said improved method comprising: A.", "A reaction mixture which contains reagents and enzymes required for NAT per se or for one or more preparative steps prior to NAT, as well as one or more furocoumarin compound(s); and B.", "Said reaction mixture being treated with UV light under controlled conditions wherein the UV exposure as well as the intensity of the emission peaks of the light source in the UV spectrum are monitored to ensure a delivered UV dose sufficient to inactivate contaminating nucleic acids without substantial detrimental effect on the performance of the NAT assay; For NAT assay, the following steps would be added: C. Said UV treated reaction mixture being subsequently supplemented with the test sample and/or an internal control template; and D. Said reaction mixture supplemented with the test sample and/or internal control template being subjected to nucleic acid testing per se under appropriate conditions.", "The testing preferably involves nucleic acids amplification and/or detection.", "The furocoumarin compound is usually a psoralen or an isopsoralen derivative.", "In a preferred embodiment, the furocoumarin compound is 8-methoxypsoralen (8-MOP), trioxsalen, psoralen and/or FQ (1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one).", "In a particularly preferred embodiment, the furocoumarin compound is 8-MOP.", "In a preferred embodiment, NAT is performed by using target or probe amplification techniques or signal amplification techniques or any other NAT technologies performed in liquid phase or onto solid supports.", "In a particularly preferred embodiment, NAT is performed by using the PCR amplification technology performed in liquid phase or onto solid supports.", "In a preferred embodiment, the container wherein the NAT assay may take place is the immediate container in which the NAT is performed.", "It is usually a closed vessel.", "The closed vessel may also be a tubing or a tube.", "In a particularly preferred embodiment, the closed vessel is a plastic tube.", "The UV treatment is performed using an apparatus consisting of a chamber equipped with a UV source and a UV sensor to monitor the energy dose of the treatment.", "In a preferred embodiment, the intensity of the emission peaks of the light source in the UV spectrum is monitored using a UV sensor.", "In a particularly preferred embodiment, said UV sensor is used to monitor the intensity of the emission peaks of the light source in the UV spectrum inside the UV irradiation chamber of an apparatus.", "In a preferred embodiment, the intensity of the emission peaks of the light source in the UV spectrum generated is monitored using a suitable radiometer or spectrometer.", "In a particularly preferred embodiment, said radiometer or spectrometer is used to monitor the intensity of the emission peaks of the light source in the UV spectrum inside the UV irradiation chamber of an apparatus.", "The test sample may be of any origin, preferably of clinical or environmental source.", "In another preferred embodiment, an internal control is used to verify the efficiency of each NAT reaction.", "In another preferred embodiment, the detection method is based upon hybridization with a labelled probe.", "In a further preferred embodiment, the said probe is labelled with a fluorophore." ], [ "FIELD OF THE INVENTION The present invention relates to reagents submitted to an improved treatment using furocoumarin derivatives (e.g.", "psoralens and/or isopsoralens) and UV irradiation to inactivate contaminating DNA and/or RNA from nucleic acid testing (NAT) reagents, without or with minimal hindering of the performance of the NAT method.", "BACKGROUND OF THE INVENTION The practical application of recombinant DNA technology in the field of infectious diseases was initially reported in 1980 by Moseley et al.", "(Moseley et al., 1980, J. Infect.", "Dis.", "142:892-898).", "Since those days, molecular biology technologies have undertaken a rapid evolution.", "Based on these technologies, a number of rapid and sensitive nucleic acid testing (NAT) methods have been developed for a variety of applications including diagnosis of infectious and genetic diseases in humans, animals and plants.", "Many of these NAT assays have been used in the field of microbiology to complement or replace the slower conventional culture-based identification systems (Picard and Bergeron, 2002, Drug Discovery Today 7:1092-1101; Boissinot and Bergeron, 2002, Curr.", "Opinion Microbiol.", "5:478482; Tang and Persing, 1999, Molecular detection and identification of microorganisms, p. 215-244, In Manual of Clinical Microbiology, Murray et al., American Society for Microbiology, Washington, D.C.; Lee et al.", "1997, Nucleic Acid Amplification Technologies: Application to Disease Diagnosis, Biotechniques Books, Eaton Publishing, Boston, Mass.", "; Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.).", "These assays have been designed for microbial detection and identification directly from clinical and/or environmental samples and are based on the use of a variety of NAT technologies including the widely used and powerful polymerase chain reaction (PCR).", "Other nucleic acid amplification technologies include among others the ligase chain reaction (LCR), the strand displacement amplification (SDA) as well as transcription-based amplifications such as the transcription mediated amplification (TMA) (Tang and Persing, 1999, Molecular detection and identification of microorganisms, p. 215-244, In Manual of Clinical Microbiology, Murray et al., American Society for Microbiology, Washington, D.C.; Lee et al., 1997, Nucleic Acid Amplification Technologies: Application to Disease Diagnosis, Biotechniques Books, Eaton Publishing, Boston, Mass.).", "Sensitive NAT technologies also include signal amplification methods such as the branched DNA (bDNA) probe technique.", "NAT can be used to detect the presence of any microbe in clinical samples.", "A number of PCR-based assays targeting highly conserved nucleotide sequences in microbes have been used by us and others to develop universal amplification assays for bacteria or fungi (Martineau et al., 2001, J. Clin.", "Microbiol.", "39:2541-2547; Schonhuber et al., 2001, BMC Microbiology 1:20; Ke et al., 1999, J. Clin.", "Microbiol.", "37:3497-3503; Loeffler et al, J. Clin.", "Microbiol.", "37:1200-1202; McCabe et al., 1999, Molecular Gen. Metabolism 66:205-211; Klausegger et al., 1999, J. Clin.", "Microbiol.", "37:464-466; Tanner et al.", "1998, Appl.", "Environ.", "Microbiol.", "64:3110-3113; Goh et al., 1996, J. Clin.", "Microbiol.", "34:818-823; Sandhu et al., 1995, J. Clin.", "Microbiol.", "33:2913-2919; Greisen et al., 1994, J. Clin.", "Microbiol.", "32:335-351; Schmidt et al., 1991, Biotechniques 11:176-177; Rand and Houck, 1990, Mol.", "Cell.", "Probes 4:445-450 and our co-pending patent application WO 01/23604 A2).", "However, because of the high sensitivity of NAT, the development of sensitive and broad-range (or universal) nucleic acid detection assays is hampered by the presence of microbial DNA and/or microbial cells that may be present in NAT reagents and which lead to false positive results.", "The most common source of false-positive results in NAT is associated with carry-over of previously amplified target nucleic acids.", "This type of contamination can be prevented by using proper laboratory procedures (Millar et al., 2002, J. Clin.", "Microbiol.", "40:1575-1580; Kwok and Higuchi, 1989, Nature, 239:237-238), or alternatively, by using techniques to inactivate amplification products such as the method using the uracil-N-glycosylase (UNG) (Longo et al., 1990, Gene 93:125-128).", "DNA inactivation using the photoreactive compounds psoralen or isopsoralen, which is used in the object of the present invention, may prevent amplification of contaminating target nucleic acids (Persing and Cimino, 1993, Amplification products inactivation methods p. 105-212, In Persing et al., Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.; Isaacs et al., 1991, Nucleic Acids Res.", "19:109-116; and U.S. Pat.", "No.", "5,221,608).", "Psoralens and isopsoralens are furocoumarin compounds representing a class of planar tricyclic photoreactive reagents that are known to form covalent monoadducts and crosslinks with nucleic acids upon activation with ultra-violet (UV) light.", "Examples of furocoumarin compounds are given in U.S. Pat.", "No.", "5,221,608, the contents of which are entirely incorporated by reference.", "These monoadducts can be formed between two adjacent pyrimidines on opposite strands of nucleic acids thereby creating interstrand crosslinks with both DNA and RNA.", "Such crosslinks prevent primer extension activities of polymerases.", "Psoralens and isopsoralens have the major advantage of allowing nucleic acid inactivation in closed vessels (such as PCR reaction vessels) thereby preventing carry-over contamination by nucleic acid aerosols.", "Another effective strategy to prevent carry-over contamination is to perform the nucleic acid amplification reactions in closed vessels such as in real-time PCR amplification and analysis (Foy and Parkes, 2001, Clin.", "Chem.", "47:990-1000).", "Another important source of false-positive results in NAT is extraneous nucleic acids introduced in reagents during the manufacturing process.", "For example, the Taq polymerase used in PCR has been shown by many investigators to be contaminated with bacterial DNA (Gale et al., 2003, Clin.", "Chem.", "49:415-424; Corless et al., 2000, J. Clin.", "Microbiol.", "38:1747-1752; Maiwald et al., 1994, Mol.", "Cell.", "Probes 8:11-14; Meier et al., 1993, J. Clin.", "Microbiol.", "31:646652; Schmidt et al., 1991, Biotechniques 11:176-177; Jinno et al., 1990, Nucleic Acids Res.", "18:6739; Rand and Houck, 1990, Mol.", "Cell.", "Probes 4:445-450; and U.S. Pat.", "No.", "5,532,145).", "Analysis of the conserved bacterial rRNA gene sequences contaminating different preparations of Taq DNA polymerase revealed that these nucleic acids were closely related to the genera Corynebacterium, Afthrobacter, Mycobacteiium, Pseudomonas, Alcaligenes and Azotobacter (Hughes et al., 1994, J. Clin.", "Microbiol., 32:2007-2008; Maiwald et al., 1994, Mol.", "Cell.", "Probes 8:11-14).", "Importantly, the contaminating DNA sequences did not match with that of the species Eschedchia coli and Thermus aquaticus which were the bacteria used to produce these ezymes.", "Because of the nature of this type of contamination, the use of UNG or of closed vessel assays as well as careful laboratory techniques cannot circumvent this important NAT reagents nucleic acid contamination problem.", "DNA inactivation using psoralens or isopsoralens combined with a UV treatment has been used to prevent amplification of microbial DNA contaminating PCR reagents (Corless et al., 2000, J. Clin.", "Microbiol.", "38:1747-1752; Klausegger et al., 1999, J. Clin.", "Microbiol.", "37:464-466; Hughes et al., 1994, J. Clin.", "Microbiol., 32:2007-2008; Meier et al., 1993, J. Clin.", "Microbiol.", "31:646-652; Jinno et al., 1990, Nucleic Acids Res.", "18:6739; and U.S. Pat.", "No.", "5,532,145).", "However, there is no standardized method for nucleic acid inactivation using these photoreactive compounds allowing efficient and reproducible nucleic acid inactivation without substantial reduction in the performance of the nucleic acid amplification and/or detection assay.", "The words “without substantial” or “not have a substantial” are used throughout the present invention to mean “without or with minimal”.", "Several investigators have reported an important reduction in the analytical sensitivity of NAT assays attributable to the UV treatment in the presence of psoralen or isopsoralen (Corless et al., 2000, J. Clin.", "Microbiol.", "38:1747-1752; Meier et al., 1993, J. Clin.", "Microbiol.", "31:646-652 and U.S. Pat.", "No.", "5,532,145).", "Corless et al.", "(2000, J. Clin.", "Microbiol.", "38:1747-1752) compared several methods to eliminate nucleic acid contamination from PCR reagents.", "They concluded that it was not possible to eliminate contaminating nucleic acids from the PCR reagents without significantly decreasing the analytical sensitivity of their real-time PCR assays.", "When they tested a combination of 8-methoxypsoralen (8-MOP) and UV irradiation, complete DNA decontamination of the PCR reagents was achieved after 5 minutes of UV exposure.", "They have not specified the UV dose (in mJoule/cm2) nor did they described the reagent container and its distance from the UV source.", "They observed a 5 to 7 logs reduction in the analytical sensitivity of the real-time PCR assays using this non-standardized 8-MOP-based DNA inactivation method.", "In fact, their experimental procedure does not include proper control of key parameters such as those disclosed in the present invention which ensure that an optimal UV energy dose is administered to the reagents containing an optimal 8-MOP concentration.", "The present invention allows for efficient nucleic acids inactivation while reducing the performance of the assay by only about 1 log or less.", "This is achieved by (i) monitoring the energy dose with a UV sensor by measuring the UV dose in mJoules per square centimeters, (ii) maintaining a constant distance between the reagents and the UV source, (iii) testing the reagent container for its permeability to UV treatment and (iv) optimising the 8-MOP concentration.", "U.S. Pat.", "No.", "5,532,145 describes the use of degassing to remove oxygen from PCR reaction mixtures containing a furocoumarin prior to UV irradiation to preserve Taq DNA polymerase activity.", "However, the degassing process is not practical as it involves freezing the reaction mixture to be decontaminated in dry/ice ethanol, thawing and applying vacuum for 30 seconds three times.", "As revealed in the present invention it is simpler to control the parameters of the UV treatment.", "These parameters include the type of furocoumarin compound and its concentration, the UV exposure, the intensity of the UV source, the length of the UV treatment and the wavelengths spectrum of the UV source which are important factors in achieving an efficient and reproducible performance in DNA inactivation, and this, without substantial detrimental effect on the performance of NAT assays.", "Other methods to inactivate DNA contaminating NAT reagents have been used with very limited success.", "These methods include the use of UV irradiation alone, a treatment with DNAase and/or restriction endonucleases and a treatment with exonucleases (Corless et al., 2000, J. Clin.", "Microbiol.", "38:1747-1752; Zhu et al., 1991, Nucleic Acids Res.", "19:2511).", "Also, a pre-filtration step for the PCR mix prior to the addition of the test sample have been used to remove nucleic acids present in PCR reagents (Yang et al., 2002, J. Clin.", "Microbiol.", "40:3449-3454).", "SUMMARY OF THE INVENTION The present invention relates to reagents submitted to an improved treatment using furocoumarin derivatives (e.g.", "psoralens and/or isopsoralens) and UV irradiation to inactivate contaminating nucleic acids from NAT reagents, without substantial hindering of the performance of the NAT methods, and this, without the need to remove oxygen in order to avoid the presence of damaging oxygen radical species (by degassing for example).", "This treatment includes careful control and monitoring of some experimental conditions including the quality of the vessel containing the reaction mixture to be treated as well as the UV dose and intensity of the light source in the UV wavelengths spectrum.", "The present method and resulting products (reagents and containers with reagents) ensure a reproducible and efficient nucleic acid inactivation.", "It is an object of the present invention to provide reagents useful in the obtention of samples which are to be submitted to amplification and/or detection of nucleic acids in which the concentration of amplifiable and/or detectable contaminating nucleic acids is low, if not totally absent, so as not to substantially interfere with the detection of the nucleic acids targeted in the reaction.", "These reagents may include a protein, the function of which should not be substantially affected by the treatment of this invention.", "Such a protein may be an enzyme.", "If a nucleic acid amplification reaction is to be performed, the enzyme may be a polymerase, a reverse transcriptase, a ligase or a restriction endonuclease.", "It may also be an enzyme useful in the test sample preparation steps for nucleic acid extraction preceding an amplification and/or detection reaction, for example a DNAase, a RNAase or a protease.", "These reagents include nucleotides and/or nucleotide analogs, oligonucleotides (primers and/or probes), buffer solutions, ions (monovalent and/or divalent), enzymes (DNA polymerase, RNA polymerase, reverse transcriptase, DNA ligase, restriction enzymes, DNAase, RNAase, protease or any other enzymes used for NAT or in test sample preparation for NAT), amplification facilitators (e.g.", "betaine, dimethyl sulfoxide, bovine serum albumin, tetramethylamonium chloride), cryoprotectors (e.g.", "glycerol), stabilizers (e.g.", "trehalose) and a solvent (usually water).", "In a particularly preferred embodiment, these reagents containing no or a low level of detectable contaminating DNA or RNA may be provided separately or as separate components of a kit, or mixed together, and may be liquid, frozen or dehydrated.", "Preferably, the reagents are any combination suitable for a nucleic acid amplification and/or detection reaction.", "It is another object of the present invention to provide for cleaner reagents and kits for the preparation of nucleic acids (sample preparation and nucleic acids extraction) for NAT assays as well as to provide an efficient method to inactivate nucleic acids contaminating said reagents and kits including purifying devices and columns.", "It is another object of this invention to provide a container, such as a closed vessel, which comprises the reagents treated in accordance with the present invention.", "The closed vessel could be submitted to the same treatment, simultaneously with the treatment of the reagents.", "Indeed, the reagents could be placed into the vessel and then submitted to the treatment of this invention.", "It is another object of this invention to provide an improved method using furocoumarin compounds and UV light for nucleic acid inactivation to treat reagents prior to NAT in order to prevent false-positive results, said improved method comprising: A.", "A reaction mixture which contains reagents and enzymes required for NAT per se or for one or more preparative steps prior to NAT, as well as one or more furocoumarin compound(s); and B.", "Said reaction mixture being treated with UV light under controlled conditions wherein the UV exposure as well as the intensity of the emission peaks of the light source in the UV spectrum are monitored to ensure a delivered UV dose sufficient to inactivate contaminating nucleic acids without substantial detrimental effect on the performance of the NAT assay; For NAT assay, the following steps would be added: C. Said UV treated reaction mixture being subsequently supplemented with the test sample and/or an internal control template; and D. Said reaction mixture supplemented with the test sample and/or internal control template being subjected to nucleic acid testing per se under appropriate conditions.", "The testing preferably involves nucleic acids amplification and/or detection.", "The furocoumarin compound is usually a psoralen or an isopsoralen derivative.", "In a preferred embodiment, the furocoumarin compound is 8-methoxypsoralen (8-MOP), trioxsalen, psoralen and/or FQ (1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one).", "In a particularly preferred embodiment, the furocoumarin compound is 8-MOP.", "In a preferred embodiment, NAT is performed by using target or probe amplification techniques or signal amplification techniques or any other NAT technologies performed in liquid phase or onto solid supports.", "In a particularly preferred embodiment, NAT is performed by using the PCR amplification technology performed in liquid phase or onto solid supports.", "In a preferred embodiment, the container wherein the NAT assay may take place is the immediate container in which the NAT is performed.", "It is usually a closed vessel.", "The closed vessel may also be a tubing or a tube.", "In a particularly preferred embodiment, the closed vessel is a plastic tube.", "The UV treatment is performed using an apparatus consisting of a chamber equipped with a UV source and a UV sensor to monitor the energy dose of the treatment.", "In a preferred embodiment, the intensity of the emission peaks of the light source in the UV spectrum is monitored using a UV sensor.", "In a particularly preferred embodiment, said UV sensor is used to monitor the intensity of the emission peaks of the light source in the UV spectrum inside the UV irradiation chamber of an apparatus.", "In a preferred embodiment, the intensity of the emission peaks of the light source in the UV spectrum generated is monitored using a suitable radiometer or spectrometer.", "In a particularly preferred embodiment, said radiometer or spectrometer is used to monitor the intensity of the emission peaks of the light source in the UV spectrum inside the UV irradiation chamber of an apparatus.", "The test sample may be of any origin, preferably of clinical or environmental source.", "In another preferred embodiment, an internal control is used to verify the efficiency of each NAT reaction.", "In another preferred embodiment, the detection method is based upon hybridization with a labelled probe.", "In a further preferred embodiment, the said probe is labelled with a fluorophore.", "DETAILED DESCRIPTION OF THE INVENTION This invention will be described hereinbelow, with reference to specific or preferred embodiments and accompanying figures, the purpose of which is to illustrate the invention rather than to limit its scope.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1: Examples of automated systems for manufacturing processes allowing controlled UV treatments and aliquoting of the treated reagents.", "Panel A: Manufacturing process using a tubing in which the reagent flow is controlled by a pump.", "The treated reagents are subsequently aliquoted in the NAT reaction vessels.", "Panel B: Manufacturing process using the immediate container in which the NAT is performed.", "This panel shows an example with the Smart Cycler tubes from Cepheid.", "FIG.", "2: UV irradiation chamber of the Spectrolinker apparatus.", "Panel A: Top view.", "Panel B: Side view.", "FIG.", "3: Determination of the optimal UV exposure for psoralen-based DNA inactivation of PCR reagents.", "Melting curves of the PCR products amplified on a LightCycler with the Staphylococcus-specific PCR assay (Tm around 83° C.) showing the difference between different UV exposures.", "The peaks in the range of 70 to 82° C. correspond to the Tm of non-specific amplification products including primer dimers.", "Purified genomic DNA from Staphylococcus aureus ATCC 29737 (100 genome copies per reaction) was added to all reaction mixtures prior to DNA inactivation.", "Panel A: Melting curves after DNA inactivation with a UV dose of 1000 mJ/cm2, Panel B: DNA inactivation with a UV dose of 1500 mJ/cm2, Panel C: DNA inactivation with a UV dose-of 2000 mJ/cm2, Panel D: DNA inactivation with a UV dose of 2400 mJ/cm2 and Panel E: untreated reactions.", "FIG.", "4: Determination of the optimal psoralen concentration for DNA inactivation of PCR reagents.", "Real-time detection on a Smart Cycler using a Streptococcus agalactiae-specific PCR assay showing the difference between different 8-MOP concentrations.", "Purified genomic DNA from Streptococcus agalactiae ATCC 12973 (106 genome copies per reaction) was added to all reaction mixtures prior to DNA inactivation.", "Panel A: DNA inactivation with a 8-MOP concentration of 0.03 μg/μL, Panel B: DNA inactivation with a 8-MOP concentration of 0.06 μg/μL, Panel C: DNA inactivation with a 8-MOP concentration of 0.12 μg/μL and Panel D: DNA inactivation with a 8-MOP concentration of 0.24 μg/μL.", "The curve (Δ) of each panel corresponds to a control reaction not exposed to UV treatment.", "FIG.", "5: Effect of the volume on psoralen-based DNA inactivation with a real-time PCR assay based on detection with molecular beacon probes.", "Real-time detection on a Smart Cycler using a MRSA-specific PCR assay showing the effect of the volume of the PCR reaction mixture.", "Purified genomic DNA from Staphylococcus aureus ATCC 33592 (100 genome copies per reaction) was added to all reaction mixtures containing 8-MOP prior to UV treatment.", "Panel A: DNA inactivation in 0.6 mL plastic tubes of reaction mixture volumes ranging from 100 to 500 μL.", "Panel B: DNA inactivation in 1.5 mL plastic tubes of reaction mixture volumes ranging from 100 to 1000 μL.", "The curves (Δ) of each panel correspond to control reactions not exposed to UV treatment.", "FIG.", "6: Determination of the influence of psoralen-based DNA inactivation with two different concentrations of 8-MOP on the efficiency and analytical sensitivity of a PCR assay.", "Melting curves of the PCR products amplified on a LightCycler with the Staphylococcus-specific PCR assay (Tm around 83-84° C.) showing the difference on the analytical sensitivity of a PCR assay according to 8-MOP concentration and UV exposure.", "The peaks in the range of 74 to 82° C. correspond to the Tm of non-specific amplification products including primer dimers.", "Purified genomic DNA from Staphylococcus aureus ATCC 29737 was added after DNA inactivation at concentrations of 2 to 8 genome copies per reaction.", "Panel A: melting curves after DNA inactivation with 0.06 μg/μL of 8-MOP and UV dose of 2400 mJ/cm2, Panel B: DNA inactivation with 0.06 μg/μL of 8-MOP and UV dose of 1500 mJ/cm2, Panel C: DNA inactivation with 0.03 μg/μL of 8-MOP and UV dose of 2400 mJ/cm2 and Panel D: DNA inactivation with 0.03 μg/μL of 8-MOP and UV dose of 1500 mJ/cm2.The curves () of each panel correspond to control reactions to which no DNA was added.", "Curve (Δ) corresponds to 2 genome copies per reaction, curve (∘) corresponds to 4 genome copies per reaction and curve (□) corresponds to 8 genome copies per reaction.", "FIG.", "7: Efficiency of the psoralen-based DNA inactivation in a real-time PCR assay using molecular beacons.", "Real-time detection on a Smart Cycler using a Streptococcus agalactiae-specific PCR assay showing the effect of 8-MOP and UV on a PCR assay using molecular beacons.", "Purified genomic DNA from S. agalactiae ATCC 12973 was added after DNA inactivation at concentrations of 3 to 100 genome copies per reaction.", "Panel A: no 8-MOP and no UV exposure, Panel B: Addition of 0.06 μg/μL of 8-MOP and no UV exposure, Panel C: Addition of 0.06 μg/μL of 8-MOP and UV dose of 1500 mJ/cm2.The curves () of each panel correspond to control reactions to which no DNA was added.", "Curve (Δ) corresponds to 3 genome copies per reaction, curve (∘) corresponds to 6 genome copies per reaction, curve (□) corresponds to 12 genome copies per reaction, curve (Δ) corresponds to 25 genome copies per reaction, curve (∘) corresponds to 50 genome copies per reaction and curve (□) corresponds to 100 genome copies per reaction.", "FIG.", "8: Efficiency of psoralen to inactivate TEM DNA contaminating molecular biology grade enzymes.", "Conventional PCR amplification with the TEM PCR assay (790-bp amplicon) and the internal control (252-bp amplicon) showing the difference between non treated samples (lanes 1 to 6) and treated with 8-MOP and UV for DNA inactivation (lanes 7 to 12).", "Lanes 1, 2, 7 and 8 are control reactions to which no DNA sample was added after DNA inactivation.", "Purified genomic DNA from Eschelichia coli CCRI-9767 carrying the TEM-1 gene was added after DNA inactivation at concentrations of 1 (lanes 3, 4, 9 and 10) and 10 (lanes 5, 6, 11 and 12) genome copies per PCR reaction.", "A 100-bp molecular size ladder was used (lane M).", "FIG.", "9: Efficiency of psoralen to inactivate microbial DNA contaminating Taq polymerase preparations.", "Melting curves of the PCR products amplified on a LightCycler with a universal PCR assay for bacteria showing the difference between non treated samples and samples treated with 8-MOP and UV for DNA inactivation of microbial DNA naturally present in PCR reagents.", "Purified genomic DNA from Staphylococcus aureus ATCC 29737 was added after DNA inactivation at concentrations of 10 and 25 genome copies per reaction.", "The peak at around 83-84° C. corresponds to the specific PCR product amplified from the spiked S. aureus DNA.", "The peaks in the range of 72 to 82° C. correspond to the Tm of non-specific amplification products including primer dimers while those over 86° C. correspond to DNA contamination observed with the untreated reaction mixture.", "Panel A: Melting curves of untreated samples, Panel B: Melting curves after DNA inactivation with 0.06 μg/μL of 8-MOP and a UV dose of 1500 mJ/cm2.The curves () of each panel correspond to control reactions to which no DNA was added.", "Curve (Δ) corresponds to 10 genome copies per reaction and curve (∘) corresponds to 25 genome copies per reaction.", "FIG.", "10: Influence of the intensity of the UV source on the efficiency of DNA inactivation.", "Real-time detection on a Smart Cycler using a MRSA-specific PCR assay showing the effect of UV lamp generating intensities ranging from 1300 to 4200 μW/cm2.Purified genomic DNA from S. aureus ATCC 33592 was added after DNA inactivation at concentrations of 1 to 106 genome copies per reaction.", "Curve (Δ) corresponds to the untreated reactions (i.e.", "no 8-MOP, no UV treatment).", "Curve (∘) corresponds to reactions containing 8-MOP but not exposed to UV.", "Curve (□) corresponds to reactions exposed to a UV source generating an intensity of 4200 μW/cm2.Curve (Δ) corresponds to reactions exposed to a UV source generating an intensity of 3700 μW/cm2.Curve (□) corresponds to reactions exposed to a UV source generating an intensity of 3200 μW/cm2.Curve (⋄) corresponds to reactions exposed to a UV source generating an intensity of 2600 μW/cm2.Curve (x) corresponds to reactions exposed to a UV source generating an intensity of 1900 μW/cm2.Curve () corresponds to reactions exposed to a UV source generating an intensity of 1300 μW/cm2.FIG.", "11: Determination of the optimal psoralen concentration for DNA inactivation of PCR reagents.", "Real-time detection on a Smart Cycler using a MRSA-specific assay showing fluorescence curves for different 8-MOP concentrations.", "Purified genomic DNA from S. aureus ATCC 33592 (104 genome copies per reaction) was added to all reaction mixtures prior to DNA inactivation.", "Panel A: untreated reaction (i.e.", "no 8-MOP and no UV), Panel B: DNA inactivation with a 8-MOP concentration of 0.015 μg/μL, Panel C: DNA inactivation with a 8-MOP concentration of 0.03 μg/μL, Panel D: DNA inactivation with a 8-MOP concentration of 0.06 μg/μL and Panel E: DNA inactivation with a 8-MOP concentration of 0.12 μg/μL.", "The curves (Δ) of each panel correspond to control reactions not exposed to UV treatment.", "FIG.", "12: Determination of the influence of psoralen-based DNA inactivation on the efficiency and analytical sensitivity of a S. agalactiae-specific assay.", "Real-time detection on a Smart Cycler using a S. agalactiae-specific assay showing the effect of 8-MOP and UV.", "Purified genomic DNA from S. agalactiae ATCC 12973 was added after DNA inactivation at concentrations of 2.5 and 5 genome copies per reaction.", "Panel A: untreated reactions (no 8-MOP and no UV).", "Panel B: Addition of 0.06 μg/μL of 8-MOP and no UV exposure, Panel C: Addition of 0.06 μg/μL of 8-MOP and UV dose of 1500 mJ/cm2.The curves () of each panel correspond to control reactions to which no DNA was added.", "Curves (□) correspond to 2,5 genome copies per reaction and curves (Δ) correspond to 5 genome copies per reaction.", "FIG.", "13: Determination of the influence of psoralen-based DNA inactivation on the efficiency and analytical sensitivity of a Staphylococcus-specific assay.", "Melting curves of the PCR products amplified on a Smart Cycler with the Staphylococcus-specific PCR assay (Tm around 83° C.) showing the effect of 8-MOP and UV.", "Purified genomic DNA from S. aureus ATCC 33592 was added after DNA inactivation at concentrations of 2.5 and 10 genome copies per reaction.", "Panel A: untreated reactions (no 8-MOP and no UV).", "Panel B: Addition of 0.06 μg/μL of 8-MOP and no UV exposure.", "Panel C: Addition of 0.06 μg/μL of 8-MOP and UV dose of 1500 mJ/cm2.The curves () of each panel correspond to control reactions to which no DNA was added.", "Curves () correspond to 2,5 genome copies per reaction and curves () correspond to 10 genome copies per reaction.", "The Taq polymerase used in PCR as well as other commercially available enzymes have been shown to be contaminated with bacterial DNA as mentioned above.", "The use of furocoumarin-based DNA inactivation to prevent amplification of microbial DNA contaminating PCR reagents has been reported (Gale et al., 2003, Clin.", "Chem.", "49:415-424; Corless et al., 2000, J. Clin.", "Microbiol.", "38:1747-1752; Klausegger et al., 1999, J. Clin.", "Microbiol.", "37:464-466; Hughes et al., 1994, J. Clin.", "Microbiol., 32:2007-2008; Meier et al., 1993, J. Clin.", "Microbiol.", "31:646-652; Jinno et al., 1990, Nucleic Acids Res.", "18:6739; and U.S. Pat.", "No.", "5,221,608 and 5,532,145).", "However, none of the reported methods led to effective and reproducible nucleic acid inactivation without substantial reduction in the performance of the NAT assay mainly because these methods are not properly controlled and not standardized.", "We demonstrate hereinbelow with many PCR amplification assays that standardization and careful monitoring of the UV treatment is critical to achieve efficient and reproducible psoralen-based nucleic acids inactivation without substantial detrimental effect on the performance of each PCR assay.", "The present invention relates to reagents and vessels containing these same reagents for amplification and/or detection of nucleic acids in which the concentration of contaminating nucleic acids is so low, if any, that they do not interfere with the detection of the nucleic acids targeted in the reaction.", "These reagents include nucleotides and/or nucleotide analogs, oligonucleotides (primers and probes), buffer solution, ions (monovalent and divalent), enzymes (DNA polymerase, RNA polymerase, reverse transcriptase, DNA ligase or any other enzymes used for NAT), amplification facilitators (e.g.", "betaine, bovine serum albumine, dimethyl sulfoxide, amonium chloride), cryoprotectors (e.g.", "glycerol), stabilizers (e.g.", "trehalose) and a solvent (usually water).", "These reagents containing no or a low level of detectable contaminating DNA may be provided separately, or as separate components of a kit, or mixed together and may be liquid, frozen or dehydrated.", "Factors to be monitored are (i) the intensity of the UV source, (ii) the energy dose received by the reagent(s), (iii) the composition of the reagent(s), (iv) the nature of the container and its UV transparency, (v) the volume of the reagent(s), and (vi) the type and the concentration of the furocoumarin compound(s).", "All these factors should be optimized to inactivate at least 100 copies of spiked control nucleic acids without substantial reduction in the performance of the NAT assay.", "Commercially available reagents and kits for the preparation of nucleic acids are often contaminated with bacterial DNA and treatment with DNAase and gamma irradiation are not sufficient to eliminate these nucleic acids (Van der Zee et al., 2002.J.", "Clin.", "Microbiol.", "40:1126).", "It is therefore an object of the present invention to provide for cleaner reagents and kits for the preparation of nucleic acids for NAT assays as well as to provide an efficient method to inactivate nucleic acids contaminating said reagents and kits.", "Said nucleic acids amplification and/or detection reagents are preferably treated with an improved method using one or more furocoumarin compound(s) and UV light for nucleic acid inactivation prior to NAT in order to prevent false-positive results, said improved method comprising the following steps.", "1) A reaction mixture which contains reagents required for NAT as well as one or more furocoumarin compound(s).", "The furocoumarin compound used is preferentially a psoralen or isopsoralen derivative.", "The psoralen derivative is preferentially 8-methoxypsoralen (8-MOP) resuspended in DMSO at a concentration 2.5 mg/mL.", "The final concentration of 8-MOP in the reaction mixture is of 0.03 to 0.24 μg/μL and preferentially of 0.06 μg/μL (or 0.25 mM).", "See Example 13 for conditions with nucleic acid inactivation using furocoumarin compounds other than 8-MOP.", "As mentioned above, a number of sensitive NAT technologies are currently available of which the most widely used is nucleic acid amplification by PCR (see examples).", "The NAT assay may be performed in liquid phase or onto solid supports.", "The reaction mixture is preferentially placed into a closed vessel prior to the UV treatment.", "The closed vessel may be the immediate container in which the NAT is performed or, alternatively, a tubing or a tube.", "The vessel can be closed, and once closed, evaporation of reagents and/or solvent(s) is avoided.", "See FIG.", "1 for an illustration of a manufacturing process for furocoumarin-based nucleic acid inactivation using a tubing (panel A) or the immediate container (panel B).", "The closed vessel is preferentially a plastic tube.", "In a more particular embodiment the vessel is a 0.6 mL plastic tube (such as the MaxyClear flip cap conical tubes from Axygen).", "On the other hand, the reaction mixture to be treated may have a volume as low as 0.1 mL and as high as 1000 mL depending on the size of the vessel used.", "The UV treatment is preferentially performed on reaction mixtures placed in vessels which have been validated for furocoumarin-based nucleic acid inactivation because this process is influenced by the quality of the vessel.", "For example, the composition and thickness of the plastic must be kept constant in order to provide a uniform dosage of UV.", "Our experience demonstrates that validation for furocoumarin-based nucleic acid inactivation of different lots of vessels from the same manufacturer having identical specifications is important.", "The reaction mixture volume and the psoralen concentration are also important parameters to optimize.", "(2) Said reaction mixture being treated with UV light under controlled conditions wherein the UV exposure as well as the intensity and wavelenght spectrum of the UV source is monitored by using a UV sensor, a radiometer equipped with a UV sensor or a suitable spectrometer.", "This allowed to ensure that the UV dose was appropriate to inactivate efficiently contaminating nucleic acids without substantial detrimental effect on the performance of the NAT assay.", "Furthermore, the tight control of the UV treatment was required to achieve an effective and highly reproducible furocoumarin-based nucleic acid inactivation.", "The reaction mixture would ideally contain all components of the NAT reaction except for the test sample and/or the internal control template to prevent inactivation of target nucleic acids to be detected.", "The NAT is preferably performed using PCR.", "The NAT may also be reverse transcriptase PCR (RT-PCR) for RNA detection or any other NAT method.", "As an example, the following 124 μL PCR reaction mixture can be treated with a controlled UV dose in 0.6 mL plastic tubes.", "The treated PCR reaction components may include 0.4 μM of each PCR primers, 2.5 mM MgCl2, 3.3 mg/mL of bovine serum albumin (BSA), 200 μM of each of the four deoxynucleoside triphosphates (dNTPs) (Pharmacia), 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.5 X/μL of SYBR Green I (Molecular Probes), 0.5 unit of Taq DNA polymerase (Promega) coupled with TaqStart antibody (Clontech).", "The test sample is added to each PCR reaction after the UV treatment.", "If an internal control template is used, it must also be added to the PCR reaction mixture after the standardized UV treatment.", "Clearly, reagent concentrations other than those mentionned above may be used.", "Futhermore, other components such as fluorescent probes, detergents or other types of enzymes may also be used.", "The UV source may be positioned to allow an optimal UV treatment to achieve an efficient furocoumarin-based nucleic acid inactivation of a NAT reaction mixture enclosed into a tube, a tubing or the immediate container (FIG.", "1).", "Alternatively, the UV treatment may be performed by using an apparatus consisting of a chamber equipped with UV lights and a UV sensor to monitor the energy of the treatment in Joule per unit of surface.", "In a particularly preferred embodiment, said apparatus allowing to monitor the energy of the UV treatment is the Spectrolinker XL-1000 UV Crosslinker (Spectronics Corp.) equipped with UV lamps (wavelenghts spectrum of 320 to 400 nm with an emission peak at around 354 nm based on analysis with a Spectronics SLM-Aminco spectrometer from Thermo Galactic) and a UV sensor.", "In another particularly preferred embodiment, said reaction mixture is disposed in 0.6 mL plastic containers located at about 10.8 centimeters from the UV source of the Spectrolinker apparatus.", "FIG.", "2 shows the irradiation chamber of the Spectrolinker apparatus.", "The intensity of the emission peaks of the UV lamps in the UV spectrum may also be monitored by using a radiometer equipped with a UV sensor such as the UVX digital radiometer with a UVX-36 sensor for 365 nm (UVP) or a suitable spectrometer such as the Spectronic SLM-Aminco (Thermo Galactic).", "See the examples for more specifications on the UV treatment using the Spectrolinker apparatus.", "Suitable UV sources generating UV light in the wavelength spectrum of 320 to 400 nm include among others a laser, high intensity white light, an incandescent lamp and a diode.", "The optimal UV treatment is dependent on (i) the distance from the UV source, (ii) the composition and thickness of the used closed reagent vessel or tubing and (iii) the composition and the volume of the NAT reaction mixture.", "In order to ascertain the efficiency of the nucleic acid inactivation protocol and the absence of substantial detrimental effect on the performance of the NAT assay, reaction mixtures spiked with the target template as well as reaction mixtures not spiked with the target nucleic acids were used.", "As shown in the examples, the reaction mixtures were spiked with template nucleic acids targeted by the assay.", "At least 100 copies of spiked target nucleic acids per PCR reaction containing 0.5 unit of Taq polymerase was preferentially used to evaluate the furocoumarin-based nucleic acid inactivation protocol because it has been demonstrated by our group (data not shown) and others (Rand and Houck, 1990, Mol.", "Cell.", "Probes 4:445-450; Meier et al., 1993, J. Clin.", "Microbiol.", "31:646-652) that the most heavily contaminated commercial preparations of Taq polymerase contain approximately 100 to 500 bacterial genomes per unit of enzyme.", "3) Said UV treated reaction mixture being subsequently supplemented with the test sample and/or an internal control template.", "The test sample may be cells, purified nucleic acids or biological specimens preferentially of clinical or environmental source.", "The target nucleic acid is preferentially microbial DNA.", "An internal control template nucleic acid targeted by the assay and added to each NAT reaction may be used to verify the efficiency of the reaction and to ensure that there is no significant inhibition by the test sample.", "4) Said reaction mixture supplemented with the test sample and/or internal control template is subjected to NAT performed under appropriate conditions.", "Said nucleic acid amplification technologies include among others, PCR, RT-PCR, LCR, SDA as well as transcription-based amplifications such as TMA.", "Preferentially, the NAT assay is PCR.", "The PCR amplification is performed under optimized cycling conditions and amplicon detection can be based (i) on real-time hybridization with internal probes labeled with a fluorophore (e.g.", "molecular beacons) or, alternatively, (ii) on the incorporation of SYBR Green I and melting curve analysis of the amplification products.", "Standard agarose gel electrophoresis may also be used for amplicon detection.", "The examples will provide more details about PCR cycling and real-time or post-amplification amplicons detection.", "Preferentially, the nucleic acids inactivation process does not have any substantial detrimental effect on the performance of the assay.", "The performance of the fluorescence-based NAT assays and of the furocoumarin-based nucleic acids inactivation method was monitored by verifying and/or analysing the fluorescence curves, the amplicon melting curves, the analytical sensitivity, the cycle thresholds and/or the fluorescence end points.", "Standard agarose gel has also been used to verify the performance of the NAT assays and of nucleic acids inactivation.", "EXAMPLES EXAMPLE 1 Determination of the Optimal UV Dose for Psoralen-Based DNA Inactivation The goal of these experiments was to determine the optimal UV exposure to inactivate contaminating DNA in PCR reagents and this without substantial reduction in the performance of the assay.", "Method: This evaluation was performed using Staphylococcus-specific PCR primers that we have previously described (Martineau et al., 2001, J. Clin.", "Microbiol.", "39:2541-2547).", "These primers were used on the Roche LightCycler instrument.", "PCR amplifications were performed from purified DNA prepared by using the G NOME DNA extraction kit (Bio 101).", "A master mix containing the equivalent of around 100 S. aureus genome copies per PCR reaction and 0.06 μg/μL of 8-MOP (Sigma) was distributed into 4 aliquots of 124 μL in 0.6 mL plastic tubes (MaxyClear flip cap conical tubes from Axygen).", "Each aliquot was then treated with UV using a Spectrolinker XL-1000 UV Crosslinker (Spectronics Corp.) equipped with a UV sensor and with UV lamps having a wavelenghts spectrum of 320 to 400 nm with an emission peak at around 354 nm (FIG.", "2).", "The tubes containing the reaction mixture to be treated were placed onto a wire rack support in order to minimize shadowing or obstruction effects on the UV sensor.", "Up to 11 reaction mixture tubes were placed onto the wire rack positioned in the center of the UV irradiation chamber (FIG.", "2) so that the reagent tubes were located at about 10.8 centimeters from the UV source of the Spectrolinker apparatus.", "The tubes were placed in the middle of the rack when fewer tubes were treated.", "All tubes were placed on the rack at an angle of 15 to 20 degrees to prevent contact between the reaction mixture to be treated and the tube cap.", "The intensity of the Spectrolinker five UV lamps could also be measured by using a UVX digital radiometer equipped with a UVX-36 sensor for 365 nm UV (UVP) which was positioned in the middle of the floor of the irradiation chamber.", "The length of the UV treatment was automatically determined by the apparatus based on the intensity of the UV source as measured by its integrated UV sensor.", "Aliquots of the PCR master mix were treated with the following UV doses in mJ/cm2 measured by the UV sensor of the Spectrolinker apparatus: 1000, 1500, 2000 and 2400 mJ/cm2.A total of 7 identical PCR reactions was tested for each UV treated aliquot.", "Two PCR reactions not treated with UV served as negative controls.", "Fluorogenic detection of PCR products with the LightCycler was carried out using 0.4 μM of both Staphylococcus-specific PCR primers, 8.0 mM MgCl2, 0.55 mg/mL of BSA, 200 μM of each of the four dNTPs (Pharmacia), 50 mM Tris-HCl (pH 9.1), 16 mM (NH4)2SO4, 0.5 X/μL of SYBR Green I (Molecular Probes), 1.25 unit of KlenTaq1 (AB Peptides) DNA polymerase coupled with TaqStart antibody (Clontech) and 1 μL of test sample all in a final volume of 15 μL.", "The KlenTaq1 enzyme is missing the N-terminal portion of the wild-type full length Taq DNA polymerase.", "The optimal cycling conditions were 1 minute at 94° C. for initial denaturation, and then 45 cycles of three steps consisting of 0 second at 95° C., 5 seconds at 60° C. and 9 seconds at 72° C. Amplification was monitored at each cycle by measuring the level of fluorescence emited by the incorporated SYBR Green I.", "After the amplification process, melting curves of the amplification products were generated and analysed for each test sample.", "Results and discussion: The inactivation of the spiked S. aureus genomic DNA was complete with UV doses of 1500, 2000 and 2400 mJ/cm2 while the inactivation was partial with a dose of 1000 mJ/cm2 (FIG.", "3).", "We concluded that, with this system and with these reagents and plastic tubes, the optimal UV dose was 1500 mJ/cm2 because it is the lowest effective UV exposure.", "It should be mentionned that we have also tested a UV dose of 1500 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus) with other assays amplifying DNA contaminating reagents and found it effective as well (i.e.", "allowed complete DNA inactivation without substantial detrimental effect on the performance of the assay) (data not shown).", "Thus, any system capable of providing a UV dose to the treated reagent(s) which is equivalent or comparable to the range of 1500 to 2400 mJ/cm2 obtained with the above set-up, system or apparatus is within the scope of this invention.", "EXAMPLE 2 Determination of the Optimal Psoralen Concentration for Decontamination The objective of these experiments was to determine the optimal psoralen concentration to inactivate DNA in PCR reagents with a S. agalactiae-specific assay.", "Method: This evaluation was performed using PCR primers specific for S. agalactiae (also called group B streptococci (GBS)) that we have previously described (Ke et al., 2000, Clin.", "Chem.", "46:324-331).", "A molecular beacon (FAM-CCACGCCCCAGCAAATGGCTCAAAAGCGCGTGG-DABCYL hybridizing to S. agalactiae-specific amplicons) was synthesized and HPLC-purified by Biosearch Technologies Inc. Purified genomic DNA was prepared as described in Example 1.Amplification reactions were performed using a Smart Cycler thermal cycler (Cepheid) in a 25 μL reaction mixture containing 50 mM Tris-HCl (pH 9.1), 16 mM ammonium sulfate, 8 mM MgCl2, 0.4 μM of primer Sag59 (5′-TTTCACCAGCTGTATTAGMGTA-3′) and 0.8 μM of primer Sag190 (5′-GTTCCCTGAACATTATCTTTGAT-3′), 0.2 μM of the GBS-specific molecular beacon, 200 μM each of the four dNTPs, 450 μg/mL of BSA, 1.25 unit of KlenTaq1 DNA polymerase (AB Peptides) combined with TaqStart antibody (Clontech), 106 genome copies of S. agalactiae and 0.03 to 0.24 μg/μL of 8-MOP.", "The 8-MOP concentrations tested for decontaminating the spiked S. agalactiae genomic DNA were 0.03, 0.06, 0.12 and 0.24 μg/μL.", "For each psoralen concentration, one reaction was not treated with UV while the 7 other reactions were treated with a UV dose of 1500 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "All PCR reaction mixtures were then submitted to thermal cycling (3 min at 94° C., and then 45 cycles of 5 sec at 95° C. for the denaturation step, 14 sec at 56° C. for the annealing step, and 5 sec at 72° C. for the extension step).", "The GBS-specific amplifications were measured by the increase in fluorescence during the amplification process.", "Subsequently, 10 μL of each PCR-amplified reaction mixture was also analysed by electrophoresis at 170 V for 30 min, in a 2% agarose gel containing 0.25 μg/mL of ethidium bromide.", "For agarose gel analysis, the size of the amplification products was estimated by comparison with a 50-bp molecular size standard ladder.", "Results and discussion: It was found that the psoralen concentrations of 0.03 μg/μL (0.14 mM) and 0.06 μg/μL (0.28 mM) were the most effective to decontaminate the spiked 106 genome copies of S. agalactiae per PCR reaction (FIG.", "4).", "The cycle thresholds were reduced by about 10 cycles as compared to the control reaction without UV treatment.", "This corresponds to a decrease of approximately 3 logs in the load of amplifiable S. agalactiae genomic DNA.", "Regarding, the higher 8-MOP concentrations tested (i.e.", "0.12 and 0.24 μg/μL) the fluorescence end points were significantly lower (FIG.", "4).", "The almost perfect overlap of the fluorescence curves for the 7 treated reactions for each psoralen concentration tested demonstrates the excellent reproducibility of this system to inactivate DNA.", "Importantly, we have tested the 8-MOP concentration of 0.06 μg/μL (0.28 mM) with other PCR assays amplifying DNA contaminating reagents and found it effective as well (i.e.", "allowed complete DNA inactivation of around 100 spiked genomic bacterial DNA) (data not shown).", "As far as 8-MOP is concerned a concentration of 0.03 to about 0.09 μg per μL would be preferred, namely about 0.06 μg/μL.", "Any other compound of the furocoumarin class having the same or comparable potency as this concentration of 8-methoxypsolaren is within the scope of this invention (see Example 13).", "EXAMPLE 3 Determination of the Effect of the Volume on Psoralen-Based DNA Inactivation Using a Staphylococcus-Specific PCR Assay Based on SYBR Green I Detection The objective of these experiments was to determine if the volume of the reaction mixture had an effect on the efficiency of the process of DNA inactivation by psoralen and UV treatment.", "Method: This evaluation was performed using the Staphylococcus-specific PCR assay with purified DNA as described in Example 1.Reaction mixture (containing 0.06 μg/μL of 8-MOP and 100 genome copies of S. aureus per 15 uL of reaction mixture) volumes of 100, 200, 300, 400 and 500 μL were tested in the 0.6 mL plastic tubes described in Example 1.Each reaction volume was treated with a UV dose of 2400 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "Subsequently, each treated volume was used to prepare 6 identical PCR reaction.", "Two reactions not treated with UV served as negative controls.", "Results and discussion: It was found that for all 5 volumes tested, the inactivation of the spiked 100 genome copies of S. aureus was complete based on PCR amplification and detection results (data not shown).", "We concluded that DNA inactivation for these 5 volumes was effective.", "Moreover, we have noted that psoralen plus UV treatment is also influenced by the quality of the plastic tubes used.", "For example, thicker plastic tubes or plastic compounds absorbing more UV may require a stronger UV exposure.", "We routinely validate each lot of plastic tubes to ensure that they allow efficient psoralen-based DNA inactivation.", "EXAMPLE 4 Effect of the Volume on Psoralen-Based DNA Inactivation with a Real-Time PCR Assay Based Detection with Fluorescent Probes The objective of these experiments was to determine if the volume of the reaction mixture for a real-time PCR assay had an effect on the efficiency of the process of DNA inactivation by psoralen and UV treatment.", "Method: This evaluation was performed using a PCR assay for the specific detection of methicillin-resistant Staphylococcus aureus (MRSA).", "PCR amplifications were performed from purified DNA as described in Example 1.Reaction mixture (containing 0.06 μg/μL of 8-MOP and 100 genome copies of a MRSA strain per 15 uL of reaction mixture) volumes of 100, 200, 300, 400 and 500 μL were treated in the 0.6 mL plastic tubes described in Example 1.Also, volumes of 100, 200, 500 and 1000 μL of the same PCR reaction mixture containing 8-MOP and spiked MRSA genomic DNA were treated in 1,5 mL plastic tubes tubes (MaxyClear flip cap conical tubes from Axygen).", "Each reaction volume was treated with a UV dose of 1500 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "Subsequently, each treated volume was used to prepare 4 identical PCR reactions.", "Two reactions not treated with UV served as negative controls.", "Amplification reactions were performed using a Smart Cycler thermal cycler (Cepheid) in a 25 μL reaction mixture containing 100 genome copies of an MRSA strain added prior to the UV treatment, 0.8 μM of XSau325 primer (5′-GGATCMACGGCCTGCACA-3′), 0.4 μM of mec1V511 primer (5′-CAAATATTATCTCGTAATACCTTGTTC-3′), 0.2 μM of XSau-B5-A0 molecular beacon (FAM-CCCGCGCGTAGTTACTGCGTTGTMGACGTCCGCGGG-DABCYL), 3.45 mM MgCl2, 3.4 mg/mL of BSA, 330 μM of each of the four dNTPs, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.035 unit of Taq DNA polymerase (Promega) coupled with TaqStart antibody and 1 μL of test sample.", "The optimal cycling conditions were 3 minute at 95° C. for initial denaturation, and then 48 cycles of three steps consisting of 5 second at 95° C., 15 seconds at 60° C. and 15 seconds at 72° C. The MRSA-specific amplifications were measured by the increase in fluorescence during the amplification process.", "Subsequently, 10 μL of each PCR-amplified reaction mixture was also analysed by electrophoresis as described in Example 2.Results and discussion: It was found that for all 5 volumes tested in 0.6 mL tubes, the inactivation of the spiked genomic DNA of S. aureus was complete most of the times based on PCR amplification and detection results (FIG.", "5).", "One out of the four replicates for the tubes containing 200 μL or 400 μL of treated reaction mixture showed an almost complete DNA inactivation.", "Regarding inactivation in 1.5 mL tubes, all four replicates showed complete inactivation of the spiked genomic DNA for the tested volumes of 100, 200 and 500 μL (FIG.", "5).", "Two out of the four reactions in the tubes containing 1000 μL showed an almost complete DNA inactivation.", "This may be associated with an insufficient UV exposure for this larger volume.", "Comparison with a reaction mixture containing 8-MOP and not treated with UV revealed that there was no substantial reduction in the performance of the assay associated with the nucleic acid inactivation method (data not shown).", "These results demonstrate the versatility of this method to inactivate nucleic acids found in various volumes of PCR reaction mixtures enclosed in different types of plastic containers.", "EXAMPLE 5 Influence of Psoralen-Based DNA Inactivation with Two Different Concentrations of 8-MOP on the Analytical Sensitivity of a PCR Assay The objective of these experiments was to determine if DNA inactivation by using two different concentrations of 8-MOP and UV treatment has an influence on the efficiency of the process of DNA amplification by PCR.", "Method: This evaluation was performed using the Staphylococcus-specific PCR assay with purified DNA as described in Example 1.A volume of 132 μL containing no S. aureus DNA and 0.03 or 0.06 μg/μL of 8-MOP was treated with an energy dose of 1500 or 2400 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "Sensitivity assays were performed by adding to 15 μL aliquots two-fold dilutions of purified S. aureus genomic DNA after the UV treatment.", "The numbers of genome copies per PCR reaction tested were 2, 4, 8, 16 and 32.There was 2 negative control reactions to which no S. aureus DNA was added.", "The performance of the assay was monitored by verifying the analytical sensitivity of the assay based on amplicon melting curves analysis.", "Analysis of the fluorescence curves and of the amplicon melting curves was also performed.", "Results and discussion: It was demonstrated that there was no substantial decrease in the analytical sensitivity of the PCR assay with reaction mixtures submitted to the four different psoralen treatments (FIG.", "6) as compared with an untreated reaction mixture (i.e.", "no 8-MOP and no UV).", "Analysis of the fluorescence curves and of the amplicon melting curves did not revealed any substantial difference in the performance of the assay for the two 8-MOP concentrations and two UV doses tested.", "In conclusion, this optimized method for psoralen-based DNA inactivation does not interfere significantly with the PCR assay.", "This is crucial because apparent DNA inactivation may in fact be attributable to a reduction in the performance of the assay.", "EXAMPLE 6 Efficiency of the Psoralen-Based DNA Inactivation with Different Polymerases The objective of these experiments was to determine if DNA inactivation by psoralen and UV treatment has an influence on the efficiency of the Taq and KlenTaq1 polymerases.", "Method: This evaluation was performed using the Staphylococcus-specific PCR assay.", "The performance of this assay using either the Taq polymerase from Roche (as described in Example 9 except that the universal primers were not used) or the KlenTaq1 polymerase from AB Peptides (as described in Example 1) was compared.", "Both enzymes were coupled with the TaqStart antibody.", "The concentration of Taq polymerase was 0.025 unit/μL while that of KlenTaq1 was 0.125 unit/μL.", "A volume of 132 μL containing no S. aureus DNA and 0.06 μg/μL of 8-MOP was treated with UV lamps generating an energy of 1500 or 2400 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "Sensitivity assays were performed by adding two-fold dilutions of purified S. aureus genomic DNA to 15 μL aliquots of the treated PCR reaction mixtures.", "The numbers of genome copies per PCR reaction tested were 2, 4, 8, 16 and 32.There was two negative control reactions to which no S. aureus DNA was added.", "Results and discussion: It was demonstrated that there was no subtantial decrease in the analytical sensitivity of the PCR assay with reaction mixtures submitted to the two different psoralen treatments (i.e.", "(i) 0.06 μg/μL of 8-MOP with a UV dose of 1500 mJ/cm2 and (ii) 0.06 μg/μL of 8-MOP with a UV dose of 2400 mJ/cm2) as compared with an untreated reaction mixture (i.e.", "no 8-MOP and no UV treatment) (data not shown).", "Therefore, our optimized method for psoralen-based DNA inactivation does not interfere substantially with the PCR assay using these two polymerases.", "This is crucial because apparent DNA inactivation may in fact be attributable to a reduction in the performance of the assay.", "EXAMPLE 7 Efficiency of the Psoralen-Based DNA Inactivation in a Real-Time PCR Assay Using Fluorescent Probes The objective of these experiments was to determine if DNA inactivation by psoralen and UV treatment has an influence on the efficiency of a real-time PCR assay using fluorescent probes.", "Method: This evaluation was performed using the PCR assay specific for S. agalactiae described in Example 2 except that an additional molecular beacon (TET-CCACGCGAAAGGTGGAGCAATGTGMGGCGTGG-DABCYL) targeting the internal control template was used.", "The internal control was used to verify the efficiency of the PCR and to ensure that there was no significant PCR inhibition by the test sample.", "A volume of 132 μL of PCR reaction mixture containing no S. agalactiae DNA and 0.06 μg/μL of 8-MOP was treated with a UV dose of 1500 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "After the UV treatment, the equivalent of 100 copies per PCR reaction of the internal control template were added.", "Sensitivity assays were performed by adding two-fold dilutions of purified S. agalactiae genomic DNA to 15 μL aliquots of the treated PCR reaction mixture.", "The numbers of genome copies per PCR reaction tested were 3, 6, 12, 25, 50 and 100.There was 2 negative control reactions to which no S. agalactiae DNA was added.", "The performance of the assay was monitored by verifying three parameters including the analytical sensitivity of the assay, the cycle thresholds and the fluorescence end points.", "Results and discussion: There was no substantial decrease in the performance of the PCR assay associated with the psoralen-based DNA inactivation as revealed by a comparison with the untreated reaction mixtures (FIG.", "7).", "The internal control template was amplified normally for all PCR reactions thereby confirming the efficiency of each PCR amplification and detection using the molecular beacon specific to the internal control (data not shown).", "EXAMPLE 8 Efficiency of Psoralen to Inactivate TEM DNA Contaminating Molecular Biology Grade Enzymes The objective of these experiments was to determine if DNA inactivation by psoralen and UV treatment is effective to inactivate TEM DNA (coding for a beta-lactamase) which is frequently found in enzyme and other reagent preparations.", "Method: This evaluation was performed using a PCR assay specific for the beta-lactamase gene TEM described in our co-pending patent application WO 0123604 A (SEQ ID Nos.", "1907 and 1908).", "Internal control primers and template were used as previously described (Lansac et al., 2000, Eur.", "J. Clin.", "Microbiol.", "Infect.", "Dis.", "19:443-451).", "The internal control was used to verify the efficiency of the PCR and to ensure that there was no significant PCR inhibition by the test sample.", "Standard PCR amplifications were carried out on a PTC-200 thermocycler (MJ Research) using purified DNA prepared as described in Example 1.The PCR reaction mixture contained 0.06 μg/μL of 8-MOP, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 0.4 μM (each) of the TEM-specific primers, 200 μM (each) of the four dNTPs, 3.3 mg/mL of BSA and 0.5 unit of Taq polymerase (Promega) coupled with TaqStart antibody and 1 μL of test sample all in a final volume of 20 μL.", "This reaction mixture was treated with a UV exposure of 1500 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "Another identical reaction mixture without the 8-MOP and not treated with UV was also tested.", "The equivalent of 1 or 10 genome copies of Escherichia coli strain CCRI-9767 (strain RbK, TEM-1) carrying a TEM plasmid were added to two reactions after the UV treatment.", "The optimal cycling conditions were 3 minute at 94° C. for initial denaturation, and then 35 cycles of three steps consisting of 5 seconds at 95° C., 30 seconds at 55° C. and 30 seconds at 72° C. followed by a terminal extension of 5 minutes at 72° C. Detection of the PCR products was performed by electrophoresis as described in Example 2.Results and discussion: It was demonstrated that the psoralen plus UV treatment allowed complete inactivation of the TEM DNA present in Taq DNA polymerase preparations (FIG.", "8).", "The internal control was amplified normally for all PCR reactions thereby confirming their efficiency.", "We have tested many lots of Taq polymerase from various manufacturers and found that all of them were contaminated with TEM DNA.", "Surprisingly, this TEM contamination is also observed with native Taq polymerase purified from Thermus aquaticus.", "The source of TEM DNA is likely cloning vectors manipulated in the laboratories where the proteins are purified or in the laboratories in which NAT is performed.", "In conclusion, this psoralen-based DNA inactivation method performed prior to TEM DNA amplification and detection is effective to avoid false-positive results.", "EXAMPLE 9 Efficiency of Psoralen to Inactivate Microbial DNA Contaminating Taq Polymerase Preparations The objective of these experiments was to determine if DNA inactivation by the improved psoralen and UV treatment is effective to inactivate microbial DNA contaminating Taq DNA polymerase preparations in order to prevent false-positive results with a universal PCR assay for bacteria.", "Method: This evaluation was performed using a multiplex PCR assay targeting the tuf gene for the universal detection of bacteria.", "This PCR assay included universal primers that we have previously described (SEQ ID Nos 636 and 637 of our co-pending patent application PCT/CA00/01150) as well as the Staphylococcus-specific PCR primers (Martineau et al., 2001, J. Clin.", "Microbiol.", "39:2541-2547).", "Amplification reactions were performed using the Roche LightCycler plafform with purified DNA as described in Example 1.Each 15 μL reaction mixture contained 0.4 μM of both Staphylococcus-specific PCR primers, 1.0 μM of both universal primers, 2.5 mM MgCl2, 2.0 mg/mL of BSA, 200 μM of each of the four dNTPs, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.5 X/μL of SYBR Green I, 0.5 unit of Taq DNA polymerase (Roche) coupled with TaqStart antibody, 0.06 μg/μL of 8-MOP and 1 μL of test sample.", "This reaction mixture was treated with a UV exposure of 1500 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "Another identical reaction mixture without 8-MOP and not treated with UV was also tested.", "For each mixture, there was 2 positive control reactions to which the equivalent of 10 genome copies of S. aureus strain ATCC 29737 were added after the UV treatment.", "Two other positive control reactions to which the equivalent of 25 genome copies of S. aureus were added after the UV treatment were also used.", "The optimal cycling conditions were 1 minute at 94° C. for initial denaturation, and then 45 cycles of three steps consisting of 0 second at 95° C., 10 seconds at 60° C. and 20 seconds at 72° C. Amplification products analysis was performed as described in Example 1.Results and discussion: It was demonstrated that the psoralen plus UV treatment allowed complete inactivation of bacterial genomic DNA contaminating Taq DNA polymerase preparations and this without substantial detrimental effect on the performance of the PCR assay (FIG.", "9).", "On the other hand, the untreated PCR reaction mixture led to false-positive results.", "We have noted lot to lot variations in the load of contaminating DNA in Taq polymerase preparations and found that the most heavily contaminated preparations contained a maximum of about 100 microbial genome copies per unit of enzyme.", "In conclusion, this psoralen-based DNA inactivation method performed prior to universal (or broad-range) amplification and detection is effective to avoid false-positive results.", "EXAMPLE 10 Examples of Automated Systems for Manufacturing Processes Allowing Controlled UV Treatments and Aliquoting of the Treated Reagents.", "The process for furocoumarin-based nucleic acids inactivation may be automated for large-scale production.", "FIG.", "1 illustrates examples of automated systems for manufacturing processes using either a tubing (panel A) or the immediate container (panel B).", "These systems allow a controlled UV treatment and aliquoting of the treated reagents.", "The system using a UV transparent tubing is equipped with a pump allowing to control the flow of the NAT reaction mixture in such a way that the exposition to the controlled UV source is optimal for nucleic acid inactivation without substantial detrimental effect on the NAT reagents.", "The reagents are subsequently aliquoted in the NAT reaction vessels.", "The test sample and/or the internal control template are then added to each vessel.", "The system using the immediate container automates aliquoting in these vessels as well as the appropriate exposure to the UV source in order to achieve optimal nucleic acid inactivation without substantial detrimental effect on the NAT reagents.", "EXAMPLE 11 Determination of the Optimal UV Dose for Psoralen-Based DNA Inactivation The goal of these experiments was to determine the optimal UV energy dose to inactivate contaminating DNA in PCR reagents and this without substantial reduction in the performance of the assay.", "Method: This evaluation was performed using the MRSA-specific assay described in Example 4.Purified genomic DNA was prepared as described in Example 1.Amplifications were performed using a Smart Cycler in a 25 μL reaction mixture containing 105 genome copies of S. aureus added prior to UV treatment.", "The 8-MOP concentration used to inactivate the spiked S. aureus genomic DNA was 0.06 μg/μL.", "The UV energy doses tested were 750, 1500, 3000, 4500 and 6000 mJ/cm2.Inactivation treatments were achieved in 0.6 mL plastic tubes described in Example 1.Two reactions were not treated with UV while 6 reactions were treated for each of the UV doses tested (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "The performance of the MRSA-specific assay was verified for each UV exposure as follows and compared to untreated reaction (no 8-mop and no UV).", "A volume of 224 μL containing no S. aureus DNA and 0.06 μg/μL of 8-MOP was treated with an energy dose of 750 to 6000 mJ/cm2.Sensitivity assays were performed in duplicate by adding different amounts of purified S. aureus genomic DNA to 25.5 μL aliquots of each treated PCR reaction mixture.", "The numbers of genome copies per PCR reaction tested were 2.5, 5 and 10.There were 2 negative control reactions to which no S. aureus DNA was added.", "All PCR reaction mixtures were then submitted to thermal cycling as described in Example 4.The performance of the assay was monitored by verifying two parameters including the analytical sensitivity of the assay and the cycle thresholds.", "Results and discussion: All UV exposures tested allowed efficient inactivation of the spiked 105 genome copies of S. aureus per PCR reaction (Table 5).", "The DNA inactivation using the different UV exposures led to an increase in cycle thresholds ranging from about 7 cycles for the UV treatment of 750 mJ/cm2 to about 18 cycles for the UV tyreatment of 6000 mJ/cm2 as compared to the control reactions containing no 8-MOP and not exposed to UV (Table 5).", "This corresponds to a decrease of approximately 2 to 4 logs in the load of amplifiable S. aureus genomic DNA.", "Again, the almost perfect overlap of the fluorescence curves for the six treated reactions for each UV energy dose tested demonstrates the excellent reproducibility of this system to inactivate DNA (data not shown).", "There was no substantial variation in the analytical sensitivity as well as in the cycle thresholds with reaction mixtures submitted to a UV dose of 750, 1500 or 3000 mJ/cm2 as compared with an untreated reaction mixture (i.e.", "no 8-MOP and no UV) (Table 5).", "On the other hand, there was a more important variation in the analytical sensitivity and/or in the cycle thresholds with reaction mixtures submitted to a UV dose of 4500 or 6000 mJ/cm2 as compared with the untreated reaction mixture (i.e.", "no 8-MOP and no UV) (Table 5).", "The cycle tresholds were increased by about 5 cycles for the UV dose of 4500 mJ/cm2 but the assay still allowed the detection of 2.5 genome copies with this UV treatment.", "For a UV dose of 6000 mJ/cm2, there was an important reduction of the analytical sensitivity as revealed by the inability to detect 2.5 and 5 genome copies.", "Therefore, the apparent increase in DNA inactivation activity of a reaction mixture exposed to the UV doses of 4500 and 6000 mJ/cm2 was partly attributable to a detrimental effect on the PCR reagents.", "In conclusion, this optimized method for psoralen-based DNA inactivation did not reduce substantially the performance of the PCR assay with UV exposures ranging from 750 to 4500 mJ/cm2.EXAMPLE 12 Influence of the Intensity of the UV Source on the Efficiency of DNA Inactivation Method: This evaluation was performed using the MRSA-specific assay described in Example 4.PCR amplifications were performed from purified DNA as described in Example 1.Amplifications were performed using a Smart Cycler thermal cycler (Cepheid) in a 25 μL reaction mixture containing 105 genome copies of S. aureus added prior to UV treatment.", "The 8-MOP concentration was 0.06 μg per μL of reaction mixture.", "Nucleic acid inactivation treatment was achieved in 0.6 mL plastic tubes described in Example 1.For each UV source intensities tested, two reactions were not treated with UV while 6 other reactions were treated with a UV dose of 1500 mJ/cm2 using a UV source generating intensities ranging from 1300 to 4200 μW/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "Intensities of 4200, 3700 and 3200 μW/cm2 were generated by the five UV lamps of the apparatus.", "Lower intensities were generated using fewer lamps because the lamp intensities could not be reduced further even after prolonged usage: (i) intensities of 2600 were generated using 4 lamps (# 2 to 5); (ii) intensities of 1900 were generated using 3 lamps (# 3 to 5); and (iii) intensities of 1300 were generated using 2 lamps (# 4 and 5) (FIG.", "2).", "The performance of the MRSA-specific assay was verified for each set of lamps as follows.", "A volume of 224 μL containing no S. aureus DNA and 0.06 μg/μL of 8-MOP was treated with the UV lamps generating an intensity in the range of 1300 to 4200 μW/cm2 and an energy dose of 1500 mJ/cm2 (both measured by the UV sensor of the Spectrolinker apparatus).", "The UV source intensities tested were 4200, 3700, 3200, 2600, 1900 and 1300 μW/cm2.Sensitivity assays were performed by adding ten-fold dilutions of purified S. aureus genomic DNA to 25.5 μL aliquots of the treated PCR reaction mixture.", "The numbers of genome copies per PCR reaction tested were 1, 10, 102, 103, 104, 105 and 106 There was 2 negative control reactions to which no S. aureus DNA was added.", "All PCR reaction mixtures were then submitted to thermal cycling as described in Example 4.The performance of the assay was monitored by verifying two parameters including the analytical sensitivity of the assay and the cycle thresholds.", "Results and discussion: All UV source intensities tested allowed similar efficiencies of inactivation of the spiked 105 genome copies of S. aureus per PCR reaction (Table 1).", "The DNA inactivation using the different UV source intensities led to an increase in the cycle thresholds of about 10 to 13 cycles as compared to the control reactions containing 8-MOP but not exposed to UV treatment (Table 1).", "This corresponds to a decrease of approximately 3 logs in the load of amplifiable S. aureus genomic DNA.", "Again, the almost perfect overlap of the fluorescence curves for the six treated reactions for each UV source intensity tested demonstrates the excellent reproducibility of this system to inactivate DNA (data not shown).", "There was no substantial variation in the analytical sensitivity as well as in the cycle thresholds with reaction mixtures submitted to the different UV lamp intensities as compared with an untreated reaction mixture (i.e.", "no 8-MOP and no UV) (FIG.", "10).", "Therefore, this optimized method for psoralen-based DNA inactivation did not interfere significantly with the PCR assay in the range of UV source intensities tested with the same UV dose.", "EXAMPLE 13 Use of Different Furocoumarin Compounds for Nucleic Acid Inactivation Method: We have tested furocoumarin compounds other than 8-MOP including psoralen, angelicin, 4-aminomethyltrioxalen, trioxalen, HQ (1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one) and HFQ (4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one), using the MRSA-specific PCR assay described in Example 4.For each furocoumarin, a range of concentrations and of UV doses were tested in order to determine the optimal conditions for effective DNA inactivation without (if possible) substantial detrimental effect on the performance of the assay (Table 2).", "Results and discussion: The optimal concentration for each furocoumarin tested was initially determined by verifying the performance of the PCR assay in the presence of different concentrations of each compound in the absence of UV treatment.", "The optimal concentration varied widely depending on the furocoumarin (i.e.", "ranged from 0.003 to 0.06 μg/μL) (Table 2).", "This was attributable to the highly variable detrimental effect of these furocoumarins (without UV treatment) on the performance of the assay.", "For example, concentrations of trioxsalen higher than 0.003 μg/μL inhibited partially or completely the PCR assay as opposed to 8-MOP which was found to be optimal at around 0.06 μg/μL.", "Subsequently, the optimal UV dose in the range of 320 to 400 nm using the preestablished optimal concentration for each furocoumarin was determined.", "The optimal UV dose also varied depending on the furocoumarin (i.e.", "ranged from 500 to 1500 mJ/cm2 as measured by the UV sensor of the Spectrolinker apparatus as described in Example 1) (Table 2).", "The use of each compound in their respective optimal conditions revealed that the only furocoumarin not reducing the analytical sensitivity of the assay and allowing efficient DNA inactivation were 8-MOP and trioxsalen.", "Trioxalen was shown to be effective at concentrations in the range of 0.001 μg/μL (0.0044 mM) to 0.0075 μg/lμL (0.033 mM) with UV doses ranging from 500 to 1500 mJ/cm2.Psoralen and FQ reduced the sensitivity of the assay by about 1 log and 2 logs, respectively (Table 2).", "Angelicin, 4-aminomethyltrioxsalen and HFQ reduce by more than two-logs the analytical sensitivity of the PCR assay.", "It was concluded that the best furocoumarins for effective DNA inactivation without substantial detrimental effects on the performance of the assay were 8-MOP and trioxsalen.", "EXAMPLE 14 Determination of the Optimal Psoralen Concentration for DNA Inactivation of PCR Reagents The objective of these experiments was to determine the optimal psoralen concentration to inactivate DNA in PCR reagents with a MRSA-specific assay.", "Method: This evaluation was performed using the MRSA-specific assay described in Example 4.Purified genomic DNA was prepared as described in Example 1.Amplifications were performed using a Smart Cycler in a 25 μL reaction mixture containing 104 genome copies of S. aureus added prior to UV treatment.", "The 8-MOP concentrations tested to inactivate the spiked S. aureus genomic DNA were 0.015, 0.03, 0.06 and 0.12 μg per μL of reaction mixture.", "Inactivation treatments were achieved in 0.6 mL plastic tubes described in Example 1.For each psoralen concentration, two reactions were not treated with UV while 4 reactions were treated with a UV dose of 1500 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "The performance of the MRSA-specific assay was verified for each 8-MOP concentration as follows.", "A volume of 224 μL containing no S. aureus DNA and 0.015, 0.03, 0.06 or 0.12 μg/μL of 8-MOP was treated with an energy of 1500 mJ/cm2.Sensitivity assays were performed by adding different amounts of purified S. aureus genomic DNA to 25.5 μL aliquots of each treated PCR reaction mixture.", "The numbers of genome copies per PCR reaction tested were 2.5, 5 and 10.There were 2 negative control reactions to which no S. aureus DNA was added.", "All PCR reaction mixtures were then submitted to thermal cycling as described in Example 4.The performance of the assay was monitored by verifying two parameters including the analytical sensitivity of the assay and the cycle thresholds.", "Results and discussion: Cycle thresholds observed with the untreated reaction containing 0.015 to 0.12 μg/μL of 8-MOP were similar to the untreated reactions containing no 8-MOP (FIG.", "11, panel A).", "The fluorescence end points for the untreated reaction containing 0.015 to 0.12 μg/μL of 8-MOP were also comparable to the untreated reactions containing no 8-MOP.", "The most important reduction in the fluorescence end points (30 to 40% decrease) was observed with the highest psoralen concentration tested.", "These results demonstrate that all 8-MOP concentrations tested did not have a substantial detrimental effect on the performance of the assay.", "The cycle thresholds observed with the four different concentrations of 8-MOP exposed to UV treatment were increased by about 10 to 15 cycles as compared to control reactions not exposed to UV (FIG.", "11).", "This corresponds to a decrease of approximately 3 to 4 logs in the load of amplifiable S. aureus genomic DNA.", "Again, the almost perfect overlap of the fluorescence curves for the four treated reactions for each psoralen concentration tested demonstrates the excellent reproducibility of this system to inactivate DNA.", "The reaction mixtures submitted to UV treatment in the presence of the four different concentrations of 8-MOP showed no substantial decrease in terms of analytical sensitivity and cycle thresholds as compared with an untreated reaction mixture (i.e.", "no 8-MOP and no UV) (data not shown).", "The highest 8-MOP concentration tested (i.e.", "0.12 μg/μL) showed a more important increase in the cycle thresholds but the assay still allowed the detection of 2.5 copies of S. aureus genome per PCR reaction (Table 3).", "Therefore, this optimized method for psoralen-based DNA inactivation is effective to inactivate DNA in the range of 8-MOP concentrations tested and does not interfere substantially with the performance of the PCR assay.", "EXAMPLE 15 Determination of the Influence of Psoralen-Based DNA Inactivation on the Analytical Sensitivity of Three Different PCR Assays The objective of these experiments was to determine if DNA inactivation by psoralen and UV treatment has an influence on the efficiency of three PCR assays based on fluorescence detection targetting S. agalactiae, MRSA or the genus Staphylococcus.", "Method: This evaluation was performed using the SARM-specific assay described in Example 4, the S. agalactiae-specific assay described in Example 2, and the Staphylococcus-specific assay described in Example 9 except that the universal primers were not used.", "Purified genomic DNA was prepared as described in Example 1.A volume of 168 μL of PCR reaction mixture containing no target DNA and 0.06 μg/μL of 8-MOP was treated with UV lamps generating a wavelengths range of 320 to 400 nm and an energy of 1500 mJ/cm2 (measured by the UV sensor of the Spectrolinker apparatus as described in Example 1).", "Sensitivity assays were performed by adding purified target genomic DNA to 25.5 μL aliquots of the treated reaction mixture.", "The numbers of genome copies per PCR reaction tested were 2.5, 5 and 10.There was 2 negative control reactions to which no target DNA was added.", "The performance of the assay was monitored by verifying three parameters including the analytical sensitivity of the assay, the cycle thresholds and the fluorescence end points.", "Results and discussion: For all three fluorescence-based PCR assays evaluated there was no substantial decrease in their performance by the psoralen-based treatment as compared with an untreated reaction mixture (i.e.", "no 8-MOP and no UV) (FIGS.", "12 and 13; Table 4).", "More precisely, the DNA inactivation method (i) did not influenced at all the analytical sensitivity of the three assays, (ii) increased the cycle tresholds by about 0 to 3 depending on the assay, and (iii) decreased the fluorescence end points by up to about 50%.", "The negative effect of the psoralen-based treatment was more important on the MRSA-specific assay (average cycle treshold increase of 1.8 (or 4.9%) and average decrease in fluorescence end points of about 46%) as compared to the S.agalactiae-specific assay (no change in the average cycle treshold and average decrease in fluorescence end points of about 17%) or the Staphylococcus-specific assay (average cycle treshold increase of 0.9 (or 2.5%) and average decrease in fluorescence end points of about 28%) (Table 4).", "The different composition of the reaction mixture for each PCR assay may explain this variable detrimental effect by the nucleic acid inactivation method.", "In conclusion, the practice of this invention yielded an optimized method for psoralen-based DNA inactivation which do not interfere substantially with the overall performance of different PCR assays.", "The negative influence on the performance of different PCR assays varied but remained minimal.", "The conditions found above to be optimal for bNA inactivation provide for a standard to which any other system components (tubes and UV treatment components) may be compared to in order to find equivalently good inactivation.", "Therefore, any method, and any reagent or container comprising the reagent which result from such any method, should provide an equivalent or comparable decontamination to the following: a treatment conducted as described in Example 1 with a Spectrolinker™XL-1000 apparatus, equipped with a UV sensor and a UV source of a wavelength spectrum of about 300 to 400 nm, and providing a total energy of about 750 to 4500 mJoules per square centimeter as measured by the UV sensor located at about 17.6 cm of the UV source while a reagent is disposed in 0.6 ml MaxyClear flip cap conical plastic tubes purchased from Axygen, located at about 10.8 cm from the UV source.", "It is obvious for a person skilled in the art that the UV energy values mentionned in this invention are related to the relative disposition of the reaction mixture tubes to be treated, the UV lamps and the UV sensor.", "A redisposition of these three elements is possible and would also fall within the scope of this invention.", "The energy values would need to be readjusted in accordance with the well-known laws of physics.", "Ideally, the sensor and the reaction mixture would need to be as close as possible from each other so that the energy measured by the sensor is very close to the energy dose really administered to the reaction mixture.", "Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.", "TABLE 1 DNA inactivation performance with the MRSA-specific assay based on cycle treshhold analysis of UV treated versus untreated reaction mixtures containing 8-MOP Not treated with UV UV-treated UV lamp intensity Cycle Standard Cycle Standard (μW/cm2) threshold deviation threshold deviation 4200 26.2 0.2 35.4 0.1 3700 26.8 0.0 35.6 0.3 3200 26.4 0.3 37.9 0.2 2600 26.9 0.5 36.9 0.4 1900 26.3 0.1 36.5 0.2 1300 27.3 0.0 40.4 1.8 TABLE 2 Optimal conditions and performance for DNA inactivation using different furocoumarins Optimal Genome Range of Range of furocoumarin copies concentrations UV dose tested concentration Optimal UV detected after Furocoumarin tested (μg/μL)* (mJ/cm2) (μg/μL) dose (mJ/cm2) treatment 8-MOP 0.015 to 0.24 750 to 6000 0.06 1500 1 Angelicin 0.015 to 0.12 500 to 1500 0.03 1500 >100 4-aminomethyltrioxsalen 0.004 to 0.12 500 to 1500 0.004 750 >100 Trioxsalen 0.001 to 0.12 200 to 1500 0.003 500 1 Psoralen 0.004 to 0.12 500 to 1500 0.004 750 10 FQ (1,4,6,8-tetramethyl- 0.003 to 0.012 200 to 1500 0.006 1500 100 2H-furo[2,3-h]quinolin-2- one) HFQ (4,6,8,9-tetramethyl- 0.003 to 0.024 200 to 1500 0.012 1500 >100 2H-furo[2,3-h]quinolin-2- one) All furocoumarins compounds were resuspended and diluted in DMSO.", "The final concentration of DMSO in the PCR reactions was 2.4%.", "TABLE 3 Effect of DNA inactivation using various concentrations of 8-MOP on the performance of the MRSA-specific assay Number of S. aureus genome copies per PCR reaction 8-MOP 2.5 copies 5 copies 10 copies concentration Cycle Standard Cycle Standard Cycle Standard (μg/μL) threshold deviation threshold deviation threshold deviation 0 38.8 1.5 38.0 0.3 36.8 0.2 0.015 40.2 0.4 41.1 0.5 39.1 1.0 0.03 43.0 1.1 40.5 0.2 40.4 0.0 0.06 42.2 0.9 41.8 0.1 40.2 0.8 0.12 42.6 0.1 43.1 2.0 42.0 1.0 TABLE 4 Effect of DNA inactivation on the performance of three PCR assays Number of Average cycle threshold Average fluorescence end point genome copies No 8-MOP 8-MOP No 8-MOP 8-MOP PCR assay per PCR reaction no UV no UV 8-MOP + UV no UV no UV 8-MOP + UV MRSA- 2.5 copies 40.0 38.5 41.6 190.5 110.5 81.5 specific 5 copies 37.6 37.3 38.9 162.5 132.5 110.0 assay 10 copies 35.7 37.9 38.8 236.0 167.0 117.5 S. agalactiae- 2.5 copies 43.3 41.8 42.6 65.5 55.5 54.5 specific 5 copies 41.2 41.7 40.5 72.5 63.0 72.0 assay 10 copies 39.7 40.0 40.7 93.0 81.0 63.5 Staphylococcus- 2.5 copies 37.7 36.7 37.6 185.5 194.0 139.0 specific 5 copies 35.8 36.7 36.7 214.5 188.0 163.0 assay 10 copies 34.3 35.1 36.1 266.0 222.0 170.5 *The nucleic acid inactivation was performed with 0.06 μg/μL of 8-MOP and a UV treatment of 1500 mJ/cm2.TABLE 5 Effect of the UV energy dose on the performance of DNA inactivation using 8-MOP Sensitivity Number of genome copies per PCR reaction DNA inactivation UV dose (mJ/cm2) Cycle threshold 2.5 5 10 Untreated3 Treated 0 Cycle threshold average 41.8 40.1 40.0 25.6 NA4 Standard deviation 0.7 1.4 0.2 0.3 NA 750 Cycle threshold average 43.8 43.6 41.6 25.7 32.5 Standard deviation 1.7 1.5 0.4 0.2 0.3 1500 Cycle threshold average 45.4 42.6 41.6 26.3 34.5 Standard deviation 1.0 0.3 0.3 0.1 0.3 3000 Cycle threshold average 44.8 45.0 42.9 25.6 36.3 Standard deviation 0.7 0.3 1.0 0.3 0.1 4500 Cycle threshold average 47.21 45.6 44.9 25.9 38.4 Standard deviation NA 0.4 0.0 0.0 0.4 6000 Cycle threshold average —2 —2 47.51 25.9 43.3 Standard deviation NA NA NA 0.2 0.7 1One out of two PCR reactions was positive.", "2A dash means that both PCR reactions were negative.", "3The untreated reaction mixture for the UV dose of 0 mJ/cm2 contained no 8-MOP while the untreated reaction mixtures for the UV doses of 750 to 6000 mJ/cm2 contained 0.06 μg/μL of 8-MOP.", "4NA means not applicable." ] ]	Patent_10469419
[ [ "Brain electrode", "The present invention relates to an electrode (1), in particular a deep brain stimulating (DBS) electrode or a deep brain lesioning electrode.", "The present invention also relates to a method for manufacturing the electrode (1) of the present invention and the use of the electrode.", "The present invention also relates to a directional electrode." ], [ "1.An electrode comprising: (a) a core comprising one or more insulated wires having non-insulated ends; (b) an insulating sheath around the core, wherein the non-insulated ends of the one or more wires are exposed; and (c) one or more electrode areas formed by depositing electrically conducting material on the surface of the sheath, wherein the one or more electrode areas are in electrical contact with at least one of the non-insulated ends.", "2.The electrode of claim 1 wherein the core comprises a plurality of insulated wires having non-insulated ends.", "3.The electrode of claim 2, wherein each end of each insulated wire is in electrical contact with a separate electrode area.", "4.The electrode of claim 1, wherein the electrically conducting material is deposited by jet printing, etching, photolithography, plasma deposition, evaporation and electroplating.", "5.The electrode of claim 1, wherein the electrically conducting material is gold or platinum.", "6.The electrode of claim 1, wherein the electrode is a deep brain stimulating (DBS) or deep brain lesioning electrode.", "7.A method for constructing an electrode according to claim 1 comprising: (a) coating the core of one or more insulated wires with the electrically insulating sheath, wherein the non-insulated ends of the one or more wires are not coated by the sheath; and (b) depositing electrically conducting material on the surface of the sheath to form one or more electrode areas which are in electrical contact with at least one of the non-insulating ends.", "8.The method of claim 7, wherein the one or more insultated wires are wound around a supporting member.", "9.The method of claim 8, wherein the supporting member is a tungsten wire.", "10.The method of claim 7, wherein the electrically conducting material is deposited by jet printing, etching, photolithography, plasma deposition, evaporation and electroplating.", "11.A directional electrode comprising: (a) a core comprising one or more insulated wires having non-insulated ends; (b) an electrically insulating sheath around the core, wherein the non-insulating ends of the one or more wires are exposed; and (c) one or more electrode areas on the surface of the sheath in electrical contact with at least one of the non-insulated ends wherein each electrode area extends over less than half the circumference of the electrode.", "12.The directional electrode of claim 11, wherein each of the one or more electrode areas extends over less than a quarter of the circumference of the electrode.", "13.The directional electrode of claim 12, wherein each of the one or more electrode areas extends over about an eighth of the circumference of the electrode.", "14.The directional electrode claim 11, wherein the longitudinal axis of the one or more electrode areas are parallel to or perpendicular to the longitudinal axis of the electrode.", "15.The directional electrode of claim 11 wherein the core comprises a plurality of insulated wires having non-insulated ends.", "16.The directional electrode of claim 15, wherein each end of each insulated wire is in electrical contact with a separate electrode area.", "17.The directional electrode of claim 16, wherein the plurality of electrode areas are in a staggered arrangement.", "18.The directional electrode of claim 11, wherein the electrically conducting material is gold or platinum.", "19.The directional electrode of claim 11, wherein the electrode is a deep brain stimulating (DBS) or deep brain lesioning electrode.", "20.The directional electrode of claim 11, comprising a line along the length of the electrode in alignment with the electrode areas for orientating the position of the electrode areas.", "21.The directional electrode of claim 11, which produces a monopolar current.", "22.The directional electrode of claim 11, which produces a bipolar current.", "23.A method for constructing the directional electrode of claim 11, comprising: (a) coating the core of one or more insulated wires with the electrically insulating sheath, wherein the non-insulated ends of the one or more wires are not coated by the sheath; and (b) depositing electrically conducting material on the surface of the sheath to form the one or more electrode areas which are in electrical contact with at least one of the non-insulating ends.", "24.The method of claim 23, wherein the one or more insulated wires are wound around a supporting member.", "25.The method of claim 24, wherein the supporting member is a tungsten wire.", "26.The method of claim 23, wherein the electrically conducting material is deposited by jet printing, etching, photolithography, plasma deposition, evaporation and electroplating.", "27.Use of the directional electrode of claim 11 in therapy.", "28.A brain electrode arranged to produce an effective field which is offset to one side of the electrode and which has a plane of symmetry through a plane through the longitudinal axis of the electrode.", "29.A method of making a brain electrode comprising the steps of: arranging an elongate conductive electrode core in a mould cavity, arranging a conductor to contact the core and to extend outside the cavity of the mould, casting moulding material into the cavity of the mould to form a coating on the core so that the conductor creates a path to the core through the coating." ], [ "The present invention relates to an electrode, in particular a deep brain stimulating (DBS) electrode or a deep brain lesioning electrode.", "The present invention also relates to a method for manufacturing the electrode of the present invention and the use of the electrode.", "Stimulating and lesioning electrodes are used in a variety of surgical procedures, in particular, DBS electrodes are used in a variety of neurosurgical procedures.", "A surgeon wishing to stimulate or lesion a particular area of nervous tissue, can target the end of an electrode to the target site so that a desired electrical current can be delivered.", "Numerous methods are known for targeting the electrode to the desired site including stereotactic methods.", "Generally, deep brain stimulating electrodes are manufactured by forming a coil of one or more insulated wires having non-insulated ends on a support, welding electrode conducting areas on to the non-insulated ends of the wires and placing a sheath of non-conducting material over the non-conducting parts of the electrode.", "It is clear that such a method for producing an electrode is laborious and therefore expensive.", "Furthermore, as numerous parts are used in the construction of the electrode, it is possible that the overall diameter of the electrode will vary along its length.", "In particular, the electrode areas which are welded on to the electrode, especially to spot weld points, can be proud of the rest of the surface of the electrode leading to difficulties in inserting the electrode.", "A further problem with electrodes constructed in this manner is that the electrode has to be of a sufficient size for it to enable electrode conducting areas to be welded onto the non-insulated ends of the wires.", "There is therefore a need in the art for an electrode which can be constructed more efficiently and with greater accuracy.", "It is becoming increasingly common for patients with disorders of brain function, including disorders of movement, intractable pain, epilepsy and some psychiatric disorders to be treated with deep brain stimulation.", "DBS electrodes are chronically implanted into the fine targets in the brain where electrical stimulation will disrupt abnormal neural firing in these patients to alleviate their symptoms.", "Brain targets for treating functional disorders are usually deeply situated and of small volume.", "For example, the optimum target for treating Parkinson's disease is situated in the sub-thalamic nucleus (STN) and is a sphere of 3 to 4 mm in diameter or an ovoid of 3 to 4 mm in diameter and 4 to 5 mm in length.", "Other targets such as the globus pallidus (used for treating hyper- or hypo-kinetic disorders) or targets in the thalamus (used for treating tremor) are usually no more than 1 to 2 mm larger.", "Current DBS electrodes, for example those supplied by Medtronic Inc, Minneapolis, Minn., are of dimensions to accommodate such volumes.", "For example, such electrodes have a diameter of about 1.27 mm and have 4 ring electrodes of the same diameter positioned at their distal end.", "Each ring electrode has a length of 1.5 mm with a 1.5 or 0.5 mm separation.", "In use, the DBS electrode is connected to a battery driven pulse generator via a cable and the equipment implanted subcutaneously, generally with the pulse generator positioned below the clavicle.", "The frequency, amplitude and pulse width of the stimulating current delivered to the electrode contacts can be programmed using external induction.", "A problem with the use of such electrodes is the difficulty in accurately placing the electrode within the desired target.", "The accuracy of placement is key to the effectiveness of the treatment.", "For a small target such as the STN, misplacement of the electrode by no more than 1 mm will not only result in sub-optimal symptomatic control but may induce unwanted side effects such as weakness, altered sensation, worsened speech or double vision (see FIG.", "4).", "The established method to place an electrode into a functional brain target is first to localise the area of abnormal brain function.", "This is achieved by fixing a stereotactic reference frame to the patient's head, which can be seen on diagnostic images, and from which measurements can be made.", "The stereotactic frame then acts as a platform from which the electrode is guided to the target using a stereoguide that is set to the measured co-ordinates.", "However, functional neurosurgical targets are often difficult or impossible to visualise on diagnostic images and so their actual position may need to be inferred with reference to visible land marks in the brain and using a standard atlas of the brain to assist the process.", "Due to anatomical variation between an individual and the atlas and even between different sides of the same brain in an individual such differences can lead to error in target localisation.", "Errors in target localisation may also result from patient movement during image acquisition or geometric distortion of images which can be intrinsic to the imaging methods.", "Such errors may be further compounded at surgery by per-operative brain shift.", "This may result from the change in head position from that during image acquisition to the position on the operating table, from leakage of cerebrospinal fluid when a burr hole is made with subsequent sinking of the brain and/or from the passage of the electrode through the brain substance.", "Surgeons attempt to correct these errors by performing per-operative electrophysiological studies on the patients undergoing functional neurosurgery who are kept awake during the procedures.", "These studies include microelectrode recording of the neural firing in the planned target area and/or stimulation of the target area using a test electrode.", "A series of passes are made through the target area with microelectrodes and sample recordings taken.", "The target is defined by its characteristic patterns of firing.", "Because of the jelly-like consistency of the brain and the depth of the functional targets within it, there needs to be a space of about 2 mm between different microelectrode passes to prevent the electrode passing down a previously made track.", "Thus, for a small target such as the STN, it is possible for the recordings from two microelectrode passes, 2 mm apart, to both register location within the target structure but to find neither of them to be optimally located centrally within the target.", "Likewise, if a test stimulation electrode is passed just off the optimal target position, i.e.", "±1 millimetre, then a second pass to correct this error will almost inevitably result in the electrode passing down the same track.", "If an electrode is placed exactly in the centre of a target having a 3 mm diameter, then the distance from the electrode surface to the edge of the target is usually under 1 mm.", "If the current spreads beyond this, then side effects can be incurred.", "For these reasons, given the small chance that an electrode will be placed in the centre of a target and that a placement error of ±1 mm can result in sub-optimal treatment with side effects, which cannot readily be corrected with repositioning, there is a need for an electrode which overcomes at least some of these problems.", "U.S. Pat.", "No.", "5,843,148 discloses a high resolution brain stimulation lead, wherein the electrode comprises ring segments diagonally arranged along the circumference of the lead.", "Accordingly, in theory by passing a stimulation current between electrode contact areas (i.e.", "ring segment), off axis stimulation can be achieved.", "Off axis stimulation refers to the generation of an electric field that is displaced to one side of the electrode.", "Furthermore, by rotating the lead, different volumes of tissue around the lead circumference may be stimulated.", "The major problem with this device is that the diagonal geometry of the ring segment results in a complex electric field which will spiral around the portion of the diameter of the electrode and is necessarily elongated along the axis of said electrode.", "The proposed configuration would therefore not form an off axis electric field that is suitable for treating a desired target.", "Furthermore, this device does not enable one skilled in the art to adjust the volume of tissue being stimulated in both the axial plane and the horizontal plane independently.", "Instead, on rotating the electrode, the volume of tissue stimulated varies in both the horizontal and axial planes, making interpretation of patient's responses extremely difficult.", "Furthermore, the complex geometry of the proposed electrode would be difficult to construct and vulnerable to mechanical failure.", "There is therefore a need for an electrode which overcomes at least some of the problems associated with the prior art electrodes.", "In a first embodiment of the present invention there is provided an electrode having a proximal and distal end comprising a core comprising: (a) one or more insulated wires extending from the proximal end to the distal end wherein the one or more insulated wires have non-insulated ends, present at the proximal and distal ends; (b) an insulating sheath around the core, wherein the non-insulated ends of the one or more wires are not covered by the insulating sheath; and (c) one or more electrode conducting areas formed by depositing electrically conducting material on the surface of the sheath, wherein the one or more electrode conducting areas are in electrical contact with at least one of the a non-insulated ends of the one or more insulated wires.", "The term “electrode” refers to any electrical conducting lead for enabling the production of an electric field at a desired site.", "Preferably the electrode is a DBS or deep brain lesioning electrode.", "Such electrodes are well known to those skilled in the art.", "The one or more insulated wires are arranged so that an electric current can be passed from the proximal end of the electrode to the distal end of the electrode.", "Preferably a separate electrode conducting area is formed for the end of each one or more insulated wires at the distal end of the electrode.", "By making an electrical connection to the corresponding end of the wire at the proximal end, the electrode conducting area will be electrically charged.", "Preferably electrode conducting areas are present at one or both ends of the electrode.", "The term “insulated” as used herein means electrically insulated.", "The insulated wires used in the electrode of the present invention can be any insulated wires.", "Preferably the insulated wires are made from gold, a gold alloy or a platinum/iridium alloy.", "It is further preferred that the core of the electrode comprises a plurality of the insulated wires.", "It is particularly preferred that the core comprises 3 or 4 insulated wires.", "The insulating sheath can be made from any non-conductive material, preferably a plastics material.", "In particular, it is preferred that the insulating sheath is made from polyurethane.", "The purpose of the electrode of the present invention is to produce an electric field at a desired target site.", "The electrode has a proximal end which, in use, is connected to an electricity source.", "The proximal end is preferably connected to the electricity source by the one or more electrode conducting areas present at the proximal end of the electrode.", "Preferably each electrode conducting area at the proximal end is connected to an electrode conducting area at the distal end of the electrode via an insulated wire.", "Electrode conducting areas at the distal end are positioned, during use, at the target site and an electric field is produced.", "Depending on the electrical connections made at the proximal end, the electric field will be generated by corresponding electrode conducting areas present at the distal end.", "Accordingly, it is possible to produce an electric field with different electrode conducting areas and furthermore it is possible to generate either a mono-polar or bi-polar electric field.", "Altering the connections of an electrode to an electric source is well known to those skilled in the art.", "In particular, the technical manual for Medtronic's DBS leads 3389 and 3387 clearly discusses changing electrical connections at the proximal end of an electrode to change the electric field generated at the distal end of the electrode.", "The electrode of the first embodiment of the present invention is preferably less than 2 mm in diameter, more preferably less than 1.5 mm in diameter, most preferably 1.27 mm in diameter.", "The electrode can be of any length and is preferably between about 10 cm and 30 cm in length.", "The length of the electrode will vary depending on the distance of the desired target from an accessible surface of the patient.", "The electrode conducting areas formed on the electrode can be any desired shape.", "Preferably the electrode conducting areas are formed as annular rings around the electrodes.", "For producing a directional electric field areas such as squares or rectangles can be formed on a part of the circumference of the electrode.", "Preferably each electrode conducting area extends over less than half, more preferably less than a quarter and most preferably between about an eighth and a sixteenth of the circumference of the electrode by restricting the size of electrode conducting area.", "By restricting the area of the electrode conducting area it is possible to produce a directional electrical field as is discussed in greater detail below.", "Preferably the electrode conducting area is positioned on the electrode so that its longitudinal axis is parallel to or perpendicular to the longitudinal axis of the electrode.", "By ensuring that the electrode conducting area is so orientated it is possible for the surgeon to determine the effects of moving the electrode with greater ease.", "Preferably the electrode conducting areas are rectangular in shape and the longitudinal axis of the rectangle is parallel to the longitudinal axis of the electrode.", "It is further preferred that the rectangles are about 1.5 to 3 mm in length and about 0.2 to 0.5 mm in width.", "If there is more than one electrode conducting area present on the electrode the electrode conducting areas are preferably arranged in a line parallel to the longitudinal axis of the electrode (See FIG.", "5).", "Alternatively, it is preferred that each electrode conducting area is staggered along the length of the electrode (see FIG.", "6A).", "By ensuring that the electrode conducting areas are staggered, it again allows greater flexibility to the surgeon for producing the electric field at different positions along the length of the electrode on which the electrode conducting areas are positioned.", "The electrically conducting material can be any material suitable for forming an electrode conducting area including metals, polymers etc.", "Preferably the electrically conducting material is gold or platinum.", "The one or more electrode conducting areas can be formed by any method.", "Preferably, the electrode conducting areas are formed by depositing electrically conducting material of the surface of the sheath.", "There are numerous methods well known to those skilled in the art for depositing electrically conducting material on the surface of various materials.", "Preferably the electrically conducting material is deposited by jet printing, etching, photolithography, plasma deposition, evaporation, electroplating, or any other suitable technique.", "Jet printing techniques are well known to those skilled in the art.", "For example, in U.S. Pat.", "No.", "5,455,998, an ink jet head for depositing conductive ink onto a desired surface is disclosed.", "U.S. Pat.", "No.", "5,114,744 discloses a method for applying a conductive material to a substrate using an ink jet.", "Furthermore, WO 99/43031 discloses a method for depositing by ink jet printing an electrode layer onto a device.", "Etching methods for depositing electrically conducting material are also well known to those skilled in the art.", "In particular, such methods are described in Plasma Etching in Microtechnology, Universiteit Twente, Fluitman and Elwenspoek, ISBN: 103650810x.", "See also Jansen et al, Journal of Micromechanics and Microengineering, 14-28, 1996.Photolithography techniques are also well known to those skilled in the art and are described in Geiger et al, VLSI, Design Techniques for Analogue and Digital Circuits, Chapter 2, 1990.WO 90/33625 describes a process for depositing a conductive layer on a substrate comprising depositing ink on the substrate by means of lithographic printing to form a seeding layer and then depositing an electrically conducting layer.", "There are numerous deposition techniques including evaporation, sputtering and vapour deposition.", "All these methods are described in VLSI Design Techniques for Analogue and Digital Circuits (supra).", "Electroplating techniques are well known to those skilled in the art and have been used for depositing electrically conductive material at a desired site on numerous materials.", "A further method by which it is possible to deposit electrically conducting material is by using conductive spray paint.", "Conductive spray paint may be used in combination with an ink jet printing head.", "Furthermore, companies such as Precision Painting, Anaheim, Calif., have been applying electrically conductive coatings such as copper and nickel to a variety of objects.", "Accordingly, such methods can be used in order to provide an electrically conducting material to a desired substrate.", "By depositing electrically conducting material on the surface of the sheath, the electrode can be produced easily and inexpensively as it is no longer necessary to weld the electrically conducting parts to the non-insulated ends of the wires.", "The electrode of the first embodiment of the present invention is robust as it does not comprise welded contacts.", "Furthermore, by depositing the electrically conducting material using the methods described above, it is possible to produce the electrode conducting areas precisely and in virtually any size and shape.", "Furthermore, the electrically conducting material can be deposited as a thin coating ensuring that the diameter of the electrode does not increase significantly and therefore does not affect the insertion of the electrode.", "The electrodes can also be made very small (less than 1 mm in diameter) because it is not necessary to spot weld electrode conducting areas on to the electrode.", "In a further preferred embodiment the electrode according to the first embodiment of the present invention may have a flexible distal end allowing the distal end to be bent, using for example a J wire, so that it can be moved to a desired position.", "The present invention also relates to a method for constructing an electrode according to the first embodiment of the present invention comprising: coating a core comprising one or more insulated wires with an electrically insulating sheath, wherein the non-insulated ends of the one or more wires are not coated by the sheath; and depositing electrically conducting material on the surface of the sheath to form one or more electrode areas which are in electrical contact with at least one of the non-insulating ends of the one or more insulated wires.", "Preferably, the core is formed by winding the one or more insulated wires around a supporting member.", "Preferably the supporting member is a tungsten wire.", "The supporting member is removed one the electrode is formed.", "The electrically conducting material can be deposited by any method, including jet printing, etching photolithography, plasma deposition, evaporation and electroplating.", "The present method is a simple and efficient method for the production of electrodes and allows greater flexibility in the production of the electrode conducting area on the electrode.", "The method of the present invention can be automated to further reduce the cost of producing the electrode.", "In the second embodiment of the present invention, there is provided a directional electrode having a proximal end and a distal end comprising: (a) a core comprising one or more insulated wires extending from the proximal end to the distal wherein the one or more insulated wires have non-insulated ends present at the proximal and distal ends; (b) an electrically insulating sheath around the core, wherein the non-insulating ends of the one or more wires are not covered by the insulating sheath; and (c) one or more electrode areas in electrical contact with at least one of the non-insulated ends of the wire, wherein each electrode area extends over less than half the circumference of the electrode.", "The term “directional electrode” refers to an electrode which produces an electric field that it is not uniformly formed around the circumference of the electrode.", "Instead the electric field is displaced to one side of the electrode.", "By having an electric field displaced to one side of the electrode, it is possible to change the position of the electric field by rotating the electrode.", "This has the advantage that when the electrode is placed in a sub-optimal position, it is possible to rotate the electrode and thereby alter the position where the electric field is produced relative to the target tissue, resulting in increased flexibility of the system and enabling the production of an electric field at an optimal position relative to the desired tissue.", "By ensuring that the electrode conducting area extends over less than half the circumference of the electrode, it ensures that the electric field is displaced to one side of the electrode.", "Preferably, the electrode conducting area extends over less than a quarter, more preferably between about an eighth and a sixteenth of the circumference of the electrode.", "The smaller the electrode conducting area, the greater displacement of the electric field generated.", "However, if the electrode conducting area becomes too small (less than a sixteenth) it is possible that the electric field becomes toxic and causes tissue death using conventional current supply levels.", "Accordingly, the amount of displacement required can be altered by using different electrode areas having different sizes.", "The directional electrode of the present invention may therefore comprise different sized electrode conducting areas which can be used in order to displace the electric field to different degrees.", "It may be desirable to have a small electrode conducting area (e.g.", "less than a sixteenth of the circumference) when it is desired to cause tissue death in a defined area.", "As indicated above for the electrode of the first embodiment of the present invention, the electrode conducting area can be any shape.", "It is also preferred that the longitudinal axis of the one or more electrode conducting areas are parallel or perpendicular to the longitudinal axis of the electrode.", "Preferably the one or more electrode areas are rectangular in shape and are about 1.5 to 3 mm in length and 0.2 to 0.5 mm in width.", "The core, insulating wires and electrically insulating sheath of the directional electrode are as defined for the electrode according to the first embodiment of the present invention.", "As for the first embodiment of the present invention, it is preferred that the electrically conducting material, is gold or platinum.", "It is further preferred that the directional electrode of the present invention comprises a mark at the proximal end of the electrode in alignment with the electrode areas for orientating the position of the electrode areas.", "Preferably the mark is a line along the length of the electrode.", "The line does not have to be continuous along the length of the electrode and is used by the surgeon in order to be able to determine the position of the electrode conducting areas.", "It is preferred that the directional electrode according to the second embodiment of the present invention is a DBS electrode or a deep brain lesioning electrode.", "As for the electrode of the first embodiment of the present invention, the directional electrode can be used to produce mono-polar current or bi-polar current.", "The directional electrode of the second embodiment of the present invention can be constructed by any method, including the method used to construct the electrode according to the first embodiment of the present invention or via the prior art method comprising welding the electrode conducting areas into place on the electrode.", "The present invention further provides a method for constructing the directional electrode according to the second embodiment of the present invention comprising: coating a core of one or more insulated wires having non-insulated ends with an electrically insulating sheath, wherein the non-insulated ends of the one or more wires are not coated by the sheath; and depositing electrically conducting material on the surface of the sheath to form the one or more electrode areas which are in electrical contact with a non-insulating end of the one or more insulated wires.", "Preferably electrically conducting material is deposited by jet printing, etching, photolithography, plasma deposition, evaporation or electroplating according to the method described in respect of the electrode according to the first embodiment of the present invention.", "The present invention also provides the use of the directional electrode of the second embodiment of the present invention for use in therapy.", "Preferably the therapy is the surgical treatment of abnormalities of brain function, including abnormalities of movement such as Parkinson's disease, Chorea, tremor, multiple sclerosis and cerebral palsy; abnormalities of the mind including depression and obsessive compulsive states, chronic pain syndromes and epilepsy.", "The directional electrode can also be used to lesion brain tumours, especially in eloquent areas.", "In use, the electrode is usually inserted over a supporting wire to provide the required stiffness needed to insert the electrode into the brain of a patient.", "Alternatively, and provided a plug is not inserted into the end of the electrode, the electrode can be inserted over a guide wire and passed down the guide wire to the desired position.", "Embodiments of the present invention will now be described by way of example only and with reference to the accompanying drawings, in which: FIG.", "1 shows a core of an electrode comprising four insulated wires wound on a tungsten wire support.", "FIG.", "2 shows a mould half for producing an insulating sheath around a core.", "FIG.", "3 shows (A) an electrode with protruding non-insulated wire ends, (B) an electrode having electrode conducting areas at one end, (C) an electrode having electrode conducting areas at both ends, (D) a cross section of the end of an electrode having a bung inserted in the end of the electrode.", "FIG.", "4 shows a schematic view of a desired target site and shows optimal and sub-optimal positions of an electrode.", "FIG.", "5 shows the distal end of a directional electrode comprising four electrode conducting areas arranged in a line.", "FIG.", "6 shows (A) the distal end of a directional electrode comprising four electrode conducting areas in a staggered arrangement, (B) shows a sectional view of the electrode through line x-x.", "EXAMPLES Example 1 Constructing an Electrode An electrode (1) having a proximal end and a distal end is constructed by winding four platinum/iridium alloy insulated wires (diameter of 0.10 mm) onto a tungsten wire (5) in order to form the structure shown in FIG.", "1.Thus the ends of each platinum/iridium wire extend radially away from the tungsten wire and are spaced apart along the length of the tungsten wire.", "This structure forms the core (3) of the electrode (1).", "The core (3) is then inserted into a mould (7) and a polyurethane sheath (9) cast around the core (3).", "The tungsten wire (5) is held under tension in the mould (7).", "The ends (4) of the insulated wires protrude from the sheath (9) formed around the core (3) and are then cut flush to the surface of the sheath (9).", "By cutting the ends (4) of the wires so that they are flush to the surface of the sheath (9), the metallic core of the wires will be exposed on the surface of the sheath (see FIG.", "3A).", "Electrode conducting areas (11) are then formed on the sheath (9) and in contact with the metallic surface of each of the cut wires.", "The electrically conducting material used is platinum.", "The platinum is deposited as a ring around the electrode (1) on the sheath (9) of the electrode (1) to form an electrode conducting area (11) as a ring around the electrode (1).", "FIGS.", "3B and C clearly show the formation of the electrode conducting areas (11) on the proximal and distal ends of the electrode (1).", "In this example the platinum is deposited by depositing ink on the sheath (9) by lithographic printing thereby forming a seeding layer, and depositing platinum by electroless deposition (see WO 00/33262).", "Once the electrode conducting areas (11) are formed on the sheath (9), the tungsten wire (5) is removed and a plug (15) is inserted in the distal end of the electrode (1) (see FIG.", "3D).", "On inserting the electrode into the brain of a patient, a tungsten wire is inserted into the electrode to provide the electrode with sufficient rigidity for insertion.", "In use, the proximal end of the electrode (1) is connected to a pulse generator.", "The electrode (1) can then be used to produce a mono-polar electrical field or a bipolar electrical field (4) at the distal end of the electrode (1) depending on the electrical contacts made with the generator.", "The resulting electrode (1) can be used in a variety of surgical procedures, in particular in a variety of neurosurgical procedures.", "Example 2 Method of Constructing a DBS Directional Electrode An electrode (1) is constructed in accordance with the method described in Example 1 except that the platinum material deposited in order to form the electrode conducting areas (11) at the distal end of the electrode is deposited in four discrete rectangles on one side of the electrode (1) (see FIG.", "5).", "Each electrode conducting area (11) is approximately 1.5 mm long and 0.5 mm in width.", "The width constitutes 45° of the electrode's circumference as the electrode's diameter is 1.27 mm.", "A gap of 0.5 mm is formed between each electrode conducting area (11).", "The proximal end of the electrode (1) has electrode conducting areas (11) formed as rings in accordance with the method disclosed in Example 1.The electrode (1) also comprises a line (13) running along the length of the electrode (1) which is aligned with the electrode conducting areas (11) and serves as an indicator of the orientation of the electrode conducting areas (11).", "Example 3 Method of Constructing a DBS Directional Electrode with Staggered Electrode Conducting Areas In another example, the electrode conducting areas (11) are formed at the distal end of the electrode (1) in a staggered arrangement (see FIG.", "6A).", "The electrode conducting areas (11) are about 3 mm in length and 0.5 mm in width and each electrode conducting area (11) is separated from its neighbour by 0.2 mm.", "Use of the Directional Electrode A directional DBS electrode (1) made according to Example 2 or Example 3 is inserted into the brain of a patient so that the distal end of the electrode (1) is placed at the desired target.", "The target is stimulated to confirm accurate localisation and the electrode (1) is rotated in order to ensure that the optimum position of the electrode conducting areas (11) is obtained.", "The indicated line (13) on the electrode (1) will assist with this orientation.", "The DBS electrode (1) is now fixed to the patient's skull and connected to a generator that is implanted subcutaneously in the patient.", "Generally, the electrode of Example 2 will be used to produce a bipolar electric current and the electrode of Example 3 will be used to produce a monopolar electric current.", "If the electrode (1) position proves to be sub-optimal post operatively, then it is possible to try the alternative electrode conducting areas (11) in order to see if the position can be optimised by utilising one of the alternative electrode conducting areas (11).", "The directional electrode (1) enables the surgeon to be able to alter the position of producing an electrical current by simply rotating the electrode (1) by utilising other electrode conducting areas (11) formed on the distal end of the electrode (1)." ] ]	Patent_10469423
[ [ "Formulation and method for depositing a material on a substrate", "A formulation for depositing a material on a substrate, the formulation comprising the material to be deposited on the substrate dissolved in a solvent system comprising a first solvent component having a relatively high boiling point and which exhibits a relatively low solubility with respect to the material to be deposited, and a second solvent component having a relatively low boiling point and which exhibits a relatively high solubility with respect to the material to be deposited." ], [ "1.A formulation for depositing a material on a substrate according to an ink-jet technique, the formulation comprising the material to be deposited on the substrate dissolved in a solvent system comprising a first solvent component having a relatively high boiling point and which exhibits a relatively low solubility with respect to the material to be deposited, and a second solvent component having a relatively low boiling point and which exhibits a relatively high solubility with respect to the material to be deposited.", "2.A formulation according to claim 1 wherein the second solvent component has a boiling point in the range of 100 to 200° C. 3.A formulation according to claim 1 or claim 2 wherein the first solvent component has a boiling point in the range of 130 to 300° C. 4.A formulation according to claim 1 wherein the difference in boiling point between the first and second solvent components is in the range of 30 to 250° C. 5.A formulation according to claim 4 wherein the difference in boiling point between the first and second solvent components is in the range of 70 to 150° C. 6.A formulation according to any preceding claim wherein the solubility of the material to be deposited in the first solvent component is up to 0.5% weight per volume.", "7.A formulation according to claim 6 wherein the solubility of the material to be deposited in the first solvent component is in the range of 0.03 to 0.3% weight per volume.", "8.A formulation according to any preceding claim wherein the solubility of the material to be deposited in the second solvent component is greater than 0.5% weight per volume.", "9.A formulation according to claim 8 wherein the wherein the solubility of the material to be deposited in the second solvent component is greater than 1.5% weight per volume.", "10.A formulation according to any preceding claim wherein the amount of material in the formulation and the proportion of the first solvent component are selected such that upon removal of the second solvent component the remaining solution of the material in the first solvent component would be at or above saturation.", "11.A formulation according to claim 1 wherein the first solvent component comprises α-tetralone and the second solvent component comprises 1,2-dimethylbenzene.", "12.A formulation according to claim 1 wherein the first solvent component comprises cyclohexylbenzene and the second solvent component comprises 1,2-dimethylbenzene.", "13.A formulation according to claim 1 wherein the first solvent component comprises xylene and 1,2,4-trimethylbenzene and the second solvent component comprises isopropylbiphenyl.", "14.A method of depositing a material on a substrate comprising depositing one or more drops of a solution of the material onto the substrate through a nozzle according to an ink-jet technique and drying the drops, wherein the solution of the material comprises a formulation according to any preceding claim.", "15.A method according to claim 14 wherein the variation in thickness of the dried drop is less than 30% of the maximum thickness.", "16.A method of producing a light-emitting device comprising a layer of an electroluminescent material sandwiched between two electrodes such that charge carriers can move between the electrodes and the layer of electroluminescent material, wherein the layer of electroluminescent material is produced by a method according to claim 14 or claim 15.17.A use of a formulation according to any of claims 1 to 13 for reducing or avoiding a ring deposition effect.", "18.A formulation according to claim 1 excluding 1% w/v of a triblend containing approximately 14 wt.", "% of a ternary polymer containing fluorene (FB8) units, benzothiadiazole (BT) units and triarylene units, 56 wt.", "% of F8BT and 30 wt.", "% TFB in a solvent mixture containing 20 vol.", "% 1,2,4-trimethylbenzene and 80 vol.", "% xylene or a solvent mixture containing 50 vol.", "% 1,2,4-trimethylbenzene and 50 vol.", "% xylene." ], [ "The present invention relates to a formulation for depositing a material on a substrate, to a method of depositing a material on a substrate particularly by an ink-jet deposition technique, and to a method of producing a light-emitting device by such a deposition method.", "As shown schematically in FIG.", "1, a light-emitting device typically comprises a layer of an electroluminescent polymer 3 sandwiched between a cathode 2 and an anode 1 such that charge carriers can move between the electrodes and the layer of electroluminescent polymer.", "It is typically produced by depositing a layer of electroluminescent polymer 3 on a glass substrate 5 coated with an anode layer 1 such as a layer of indium-tin oxide (ITO) and depositing a cathode layer 2 such as a calcium layer over the layer of electroluminescent polymer 3.The device may comprise further layers, such as a hole transport layer 4 (such as a layer of doped polyethylene dioxythiophene as described in EP0686662) provided between the anode 1 and the electroluminescent polymer layer 3 and an electron transport layer provided between the cathode and the electroluminescent layer (not provided in the device shown in FIG.", "1).", "An ink-jet technique may be used for depositing the layer of electroluminescent polymer.", "Such a technique is described in EP0880303A1, whose content is incorporated herein by reference.", "This technique basically involves the controlled deposition of drops of a solution of the electroluminescent polymer through a nozzle followed by evaporation of the solvent.", "This technique is particularly suited to the deposition of patterned layers of the electroluminescent polymer.", "For example, in some applications, it may be required to have a layer of electroluminescent polymer comprising an ordered array of pixels, wherein each pixel is produced by the deposition of a single drop of solution of the electroluminescent polymer.", "It is desirable in such cases that the polymer is distributed uniformly in the spot remaining after evaporation of the solvent.", "Solutions of the electroluminescent polymer in solvents such as isodurene have conventionally been used in this technique.", "However, there has been noticed the problem with conventional solvents that after a drop of the deposited solution has dried most of the electroluminescent polymer is deposited as a ring around the edge of the remaining spot leaving only a very thin film of the polymer at the centre of the spot.", "This can result in relatively poor device efficiency.", "It is an aim of the present invention to provide a formulation with which a film of the desired thickness profile can be deposited on a substrate according to a technique involving controlled drop deposition.", "According to a first aspect of the present invention, there is provided a formulation for depositing a material on a substrate, the formulation comprising the material to be deposited on the substrate dissolved in a solvent system comprising a first solvent component having a relatively high boiling point and which exhibits a relatively low solubility with respect to the material to be deposited, and a second solvent component having a relatively low boiling point and which exhibits a relatively high solubility with respect to the material to be deposited.", "References to the solubility of each solvent component with respect to the material are references to the solubility of the material in each solvent component.", "Ink-jet techniques typically, require formulations having a viscosity of up to about 20 cps.", "A typical viscosity for an ink-jet formulation is about 10 cps.", "The first solvent component may comprise one or more solvents that have a relatively high boiling point and exhibit a relatively low solubility with respect to the material to be deposited (in relation to the second solvent component), and the second solvent component may likewise comprise one or more solvents that have a relatively low boiling point and exhibit a relatively high solubility with respect to the material to be deposited (in relation to the first solvent component).", "In one embodiment, the second solvent component has a boiling point in the range of 100 to 200° C. and the first solvent component has a boiling point in the range of 130 to 300° C. The difference in boiling point between the first and second solvent components is preferably in the range of 30 to 250° C., further preferably in the range of 70 to 150° C. The solubility of the material to be deposited in the first solvent component is preferably up to 0.5% weight per volume, further preferably in the range of 0.03 to 0.3% weight per volume, and the solubility of the material to be deposited in the second solvent is preferably greater than 0.5% weight per volume, further preferably greater than 1.5% weight per volume.", "The proportion of the first solvent component should preferably be selected such that upon removal of the second solvent component the remaining solution of the material in the first solvent component would be substantially at or above saturation.", "In one embodiment, the proportion of the first solvent component is in the range of 10 to 60 volume percent, further preferably 20 to 50 volume percent.", "In one embodiment, the proportion of the second solvent component is in the range of 40 to 90 volume percent, preferably 50 to 80 volume percent.", "According to another aspect of the invention, there is provided a method of depositing a material on a substrate comprising depositing one or more drops of a solution of the material onto the substrate through a nozzle according to an ink-jet technique and drying each deposited drop, wherein the solution of the material comprises a formulation as described above.", "The solvent system is selected such that the material begins to precipitate at a relatively early stage in the drying of the drop, i.e.", "such that precipitation occurs whilst a substantial amount of solvent remains unevaporated.", "The solvent system is selected such that the variation in thickness of the dried drop of material on the substrate is less than 80% of the maximum thickness, preferably less than 50%, and further preferably less than 30%.", "It is most preferred that the thickness variation is less than 15%.", "In one embodiment, the solvent system is selected such that in a formulation having 0.5% w/v of the material to be deposited, the dried drop has a thickness at its centre (i.e.", "the region enclosed by the ring of increased thickness, if present) of 60 to 140 nm, preferably about 70 nm to 100 nm, which is desirable for EL efficiency and lifetime of an organic light-emitting device.", "In the above, the material for deposition on the substrate may, for example, be an organic material such as a polymer or a blend of polymers.", "In one application, it comprises one or more semiconducting conjugated polymers such as a charge transport polymer or a light-emissive polymer, or a blend of the two.", "According to yet another aspect of the present invention, there is provided a method of producing a light-emitting device comprising a layer of an electroluminescent material sandwiched between two electrodes such that charge carriers can move between the electrodes and the layer of electroluminescent material, wherein the layer of electroluminescent material is produced by a method as described above.", "According to another aspect of the present invention, there is provided a use of a formulation as described above for reducing or avoiding a ring deposition effect.", "Embodiments of the present invention shall be described hereunder, by way of example only, with reference to the accompanying drawings in which: FIG.", "1 is a schematic view of a light-emissive device; FIGS.", "2 and 3 are graphs showing the thickness profile for a spot obtained by an ink-jet technique using a formulation according to embodiments of the present invention; FIG.", "4 is a graph showing the thickness profile for a spot of an electroluminescent polymer deposited using a solvent consisting entirely of 1,2-dimethylbenzene; FIG.", "5 is a graph showing the thickness profile, for a spot obtained by an ink-jet technique using a formulation according to another embodiment of the present invention; and FIG.", "6 illustrates some recurring units and polymers.", "EXAMPLE 1 A solvent blend was prepared from 60 vol.", "% 1,2-dimethylbenzene (b.p.", ": 144.4° C.) and 40 vol.", "% α-tetralone (b.p.", ": 255° C.).", "A 0.5% w/v solution of an alternating polymer of 9,9-dioctylfluorene units and benzothiadiazole units (F8BT) having a peak molecular weight of about 266,000 was prepared using this solvent blend.", "Drops of this solution were deposited by an ink-jet method on the surface of a polyimide substrate that had been modified to lower its surface energy.", "The drops were allowed to dry at room temperature and humidity (20° C.±1.0° C. and 30-40% R.H.), and Dektak measurements of the profile of the dried drops were taken.", "The results of the measurements are shown in FIG.", "2.EXAMPLE 2 A solvent blend was prepared from 60 vol.", "% 1,2-dimethylbenzene and 40 vol.", "% cyclohexylbenzene.", "Drops of a 0.5% w/v solution of the same F8BT polymer as Example 1 were deposited on a surface-modified polyimide substrate by an ink-jet method.", "Dektak measurements of the profile of the dried drops were taken.", "The results of the measurements are shown in FIG.", "3.COMPARATIVE EXAMPLE Drops of a 0.5% w/v solution of the same F8BT polymer as Examples 1 and 2 in 1,2-dimethylbenzene were deposited on a surface-modified polyimide substrate by an ink-jet method.", "Dektak measurements of the profile of the dried drops were taken.", "The results of the measurements are shown in FIG.", "4.As can be seen from a comparison of FIGS.", "2, 3 and 4, the use of the formulations according to the present invention produced dried drops that were significantly improved in terms of uniformity of thickness compared to the comparative example.", "It is thought that the improved thickness uniformity is a result of the following mechanism.", "During drying of the drop, the volatile low boiling point solvent evaporates quickly leaving behind a saturated solution in the high boiling point solvent; this in turn causes the polymer to be precipitated rapidly preventing radial flow within the drop and thus creating a relatively uniform distribution of the polymer in the drop.", "Furthermore, the improved uniformity of thickness results in an increased thickness at the centre of the drop for the same concentration of material in the formulation.", "Being able to control the thickness at the centre of the drop is considered to be an important factor in improving the efficiency of a light-emitting device produced by an ink-jet technique.", "The present invention thus allows an increase in centre thickness without increasing the concentration in the formulation of the material to be deposited.", "This can have advantages in producing a device having pixels of the desired size at the desired resolution.", "Good results have also been achieved using a solvent blend consisting of 40 vol.", "% mixed isomers of xylene (b.p.", ":138° C.); 40 vol.", "% 1,2,4-trimethylbenzene (b.p.", ":168° C.) and 20 vol.", "% 3-isopropylbiphenyl (b.p.", ":295° C.).", "FIG.", "5 shows a Dektak measurement profile of a dried drop deposited by an ink-jet technique using a 0.5% w/v solution of a polymer blend in this three component solvent system.", "The polymer blend is relatively highly soluble in the first two, low boiling point solvents relative to the third high boiling point solvent." ] ]	Patent_10469443
[ [ "Thermally insulating structural components resistant to high temperature corrosive media", "The invention relates to a thermally insulating structural component (10) such as a cover of a container (5), in particular a molten salt electrolytic cell which component (10) is inert and resistant to corrosive media (1) at high temperature in the form of liquids, vapours and/or gases, in particular NaAlF4, AlF3, HF or O2, and which during use is exposed to such corrosive media (1).", "The component (10) comprises thermal insulating material (30) shielded from the corrosive media (1) by an openly porous or reticulated alumina structure (20) which is made impermeable by a compact filler material (15) resistant and inert to the corrosive media and comprising compacted particles of refractory material, in particular alumina cement.", "The structural component (10) can have a top metallic shell (40) which extends over the thermal insulating material (30) and downwards along lateral sides of the component (10).", "The insulating material (30) can be secured to the alumina structure (20) through nuts (50) and bolts (60)." ], [ "1.A thermally insulating cover of a cell for the electrowinning of aluminium from alumina dissolved in a fluoride-based molten electrolyte, which cover is inert and resistant to high temperature corrosive media in the form of liquids, vapours and/or gases that are contained in the cell and that comprise vapours from the electrolyte, and which cover during use is exposed to such corrosive media, said cover comprising thermal insulating material shielded from the corrosive media by an openly porous or reticulated alumina structure which is made impermeable by a compact filler made of material resistant and inert to said corrosive media, the filler material comprising compacted particles of refractory material.", "2.The thermally insulating cover of claim 1, wherein said filler completely fills an outermost part of the structure, in particular a outermost bottom part.", "3.The thermally insulating cover of claim 2, wherein the filler extends throughout the alumina structure.", "4.The thermally insulating cover of claim 1 or 2, wherein the alumina structure is partly filled by the filler, leaving a filler-free part of the alumina structure, in particular a filler-free top part.", "5.The thermally insulating cover of claim 4, wherein the filled part forms a layer in the alumina structure.", "6.The thermally insulating cover of any preceding claim, wherein the alumina structure is a plate having a thickness in the range of 20 to 150 mm.", "7.The thermally insulating cover of claim 6 when depending on claim 5, wherein the filler forms a layer in the plate, the layer having a thickness in the range of 10 to 100 mm.", "8.The thermally insulating cover of any preceding claim, wherein the alumina structure is covered with a layer made of the filler material.", "9.The thermally insulating cover of claim 8, wherein the layer covering the alumina structure has a thickness in the range of 2 to 10 mm.", "10.The thermally insulating cover of any preceding claim, wherein the refractory material of the filler comprises at least one compound selected from metal oxides, carbides and nitrides.", "11.The thermally insulating cover of claim 10, wherein the filler comprises a mixture of metal oxide particles with particles of at least one compound selected from carbides and nitrides.", "12.The thermally insulating cover of any preceding claim, wherein the filler is a slurry-applied filler comprising dried colloidal and/or non-colloidal particles of at least one compound selected from metal oxides, carbides and nitrides and precursors thereof.", "13.The thermally insulating cover of claim 12, wherein the filler comprises a cement that consists predominantly of at least one of alumina, silica and titania particles.", "14.The thermally insulating cover of claims 13, wherein the cement comprises a mixture of silica an alumina, preferably containing at least 80 weight % alumina.", "15.The thermally insulated cover of claim 12, wherein the filler comprises a mixture of alumina and titania.", "16.The thermally insulating cover of claim 12, wherein the filler comprises particles of at least one compound selected from carbides and nitrides in a dried colloidal metal oxide carrier.", "17.The thermally insulating cover of any preceding claim, which comprises one or more insulating layers and a protective layer that shields the insulating layer(s) from said corrosive media, the protective layer being made of said openly porous or reticulated filled alumina structure.", "18.The thermally insulating cover of claim 17, wherein the insulating layer(s) and the protective layer are mechanically secured together.", "19.The thermally insulating cover of claim 18, wherein the insulating layer(s) and the protective layer are mechanically secured together by means of one or more metallic attachment members extending through vertical holes of the protective layer and the insulating layer.", "20.The thermally insulating cover of claim 19, wherein the or each vertical hole in the insulating layer(s) extends into a recess located in a bottom face of the alumina structure, said recess being arranged to embed a head of one or more of the attachment members, said recess being filled with said filler to protect the attachment member from said corrosive media.", "21.The thermally insulating cover of claim 17, 18, 19 or 20, which comprises a top metallic shell which extends over the insulating layer(s) and downwards along lateral sides of the cover.", "22.A cell for the electrowinning of aluminium from alumina dissolved in a fluoride-based molten electrolyte, comprising a thermally insulating cover as defined in any preceding claim." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The technology for the production of aluminium by the electrolysis of alumina, dissolved in molten cryolite containing salts, at temperatures around 950° C. is more than one hundred years old.", "Conventional aluminium production cells are constructed so that in operation a crust of solidified molten electrolyte forms around the inside of the cell sidewalls.", "At the top of the cell sidewalls, this crust is extended by a ledge of solidified electrolyte which projects inwards over the top of the molten electrolyte.", "The solid crust in fact extends over the top of the molten electrolyte between the carbon anodes.", "To replenish the molten electrolyte with alumina in order to compensate for depletion during electrolysis, this crust is broken periodically at selected locations by means of a crust breaker, fresh alumina being fed through the hole in the crust.", "This crust/ledge of solidified electrolyte forms part of the cell's heat dissipation system in view of the need to keep the cell in operation at constant temperature despite changes in operating conditions, as when anodes are replaced, or due to damage/wear to the sidewalls, or due to over-heating or cooling as a result of great fluctuations in the operating conditions.", "In conventional cells, the crust is used as a means for automatically maintaining a satisfactory thermal balance, because the crust/ledge thickness self-adjusts to compensate for thermic unbalances.", "If the cell overheats, the crust dissolves partly thereby reducing the thermic insulation, so that more heat is dissipated through the sidewalls leading to cooling of the cell contents.", "On the other hand, if the cell cools the crust thickens which increases the thermic insulation, so that less heat is dissipated, leading to heating of the cell contents.", "The presence of a crust of solidified electrolyte is considered to be important to achieve satisfactory operation of commercial cells for the production of aluminium on a large scale.", "In fact, the heat balance is one of the major concerns of cell design and energy consumption, since only about 25% of such energy is used for the production of aluminium.", "Optimization of the heat balance is needed to keep the proper bath temperature and heat flow to maintain a frozen electrolyte layer (side ledge) with a proper thickness.", "In conventional cells, the major heat losses occur at the sidewalls, the current collector bars and the cathode bottom, which account for about 35%, 8% and 7% of the total heat losses respectively, and considerable attention is paid to providing a correct balance of these losses.", "Further losses of 33% occur via the carbon anodes, 10% via the crust and 7% via the deck on the cell sides.", "This high loss via the anodes is considered inherent in providing the required thermal gradient through the anodes.", "In the literature, there have been suggestions for cells operating without a crust of solidified electrolyte.", "U.S. Pat.", "No.", "5,368,702 (de Nora) discloses a multimonopolar aluminium production cell operating with tubular anodes in a crustless molten electrolyte which is thermally insulated by a cover.", "The cover is lined underneath with a layer of thermally insulating material.", "U.S. Pat.", "No.", "5,415,742 (La Camera/Tomaswick/Ray/Ziegler) disclose another aluminium production cell operating with a crustless molten electrolyte which is thermally insulated by a cover.", "Despite previous efforts to develop a cell design for operation with a crustless molten electrolyte, there is still a need to provide a thermic insulating material for cell covers which is resistant to electrolyte vapours and gases evolved during electrolysis and which is sufficiently lightweight but mechanically resistant to be used for removable covers." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention proposes a thermally insulating cover of a container, in particular a molten salt electrolytic cell, which cover is inert and resistant to corrosive media at high temperature in the form of liquids, vapours and/or gases contained in the container, in particular NaAlF 4 , AlF 3 , HF or O 2 , and which during use is exposed to such corrosive media.", "The cover comprises thermal insulating material shielded from the corrosive media by an openly porous or reticulated alumina structure which is made impermeable by a compact filler made of material resistant and inert to said corrosive media.", "The filler material comprises compacted particles of refractory material.", "The thermal insulating material may consist of or include a part of the openly porous or reticulated alumina structure that contains no filler.", "The thermal insulting material may also include or be formed of an entirely different body above the openly porous or reticulated filled alumina structure.", "The filler may extend throughout the alumina structure.", "Alternatively, the alumina structure may be only partly filled by the filler, leaving a filler-free part of the alumina structure, in particular a filler-free top part.", "For example, the filled part forms a layer which may form an outer surface of the cover, in particular an outer surface of a bottom part of the cover.", "In addition, the alumina structure may be covered with an outside layer made of the same filler material, the outside layer forming an outer surface of the cover.", "Usually, the alumina structure is a plate having a thickness in the range of 20 to 150 mm.", "A layer inside the alumina structure may have a thickness in the range of 10 to 100 mm.", "An outside layer covering the alumina structure can be 2 to 10 mm thick.", "Usually, the refractory material of the filler comprises at least one compound selected from metal oxides, carbides and nitrides.", "For instance, the filler comprises a mixture of metal oxide particles with particles of at least one compound selected from carbides and nitrides.", "These particles may be applied in a colloidal metal oxide carrier.", "Advantageously, the filler is a slurry-applied filler comprising dried colloidal and/or non-colloidal particles of at least one compound selected from metal oxides, carbides and nitrides, in particular selected from oxides, carbides and nitrides of titanium, zirconium, hafnium, vanadium, silicon, niobium, tantalum, nickel, molybdenum and iron.", "Suitable colloids may be selected from colloidal alumina, ceria, lithia, magnesia, silica, thoria, yttria, zirconia, tin oxide, zinc oxide and mixtures thereof.", "Colloidal precursors of such oxides, in particular hydroxides, may also be used.", "Further colloidal slurries which may be used as a filler material are disclosed in U.S. Pat.", "Nos.", "5,310,476 and 5,364,513 (both in the name of Sekhar/de Nora).", "For instance, the filler comprises a cement that consists predominantly of at least one of alumina, silica and titania particles.", "The filler may be made of alumina and silica, in particular with an alumina content of at least 80 weight %, in particular at least 90 or even 95 weight %.", "The filler may comprise a mixture of alumina and titania.", "In one embodiment, the thermally insulating cover comprises an insulating layer and a protective layer that shields the insulating layer(s) from the corrosive media, the protective layer being made of the openly porous or reticulated filled alumina structure.", "Preferably, the insulating layer(s) and the protective layer are mechanically secured together, in particular by means of one or more metallic attachment members extending through vertical holes of the protective layer and the insulating layer.", "The hole(s) in the insulating layer(s) may extend into recess(es) located in a bottom face of the alumina structure.", "Each recess can be arranged to embed a head of one or more of the attachment members and can be filled with the filler to protect the attachment member from the corrosive media.", "Usually, a top metallic shell extends over the insulating layer(s) and downwards along lateral sides of the cover.", "The thermally insulating cover of the invention may be used on any container containing high temperature oxidising and/or corrosive media, in particular vapours and/or gases.", "In particular, the cover is used for aluminium electrowinning cells.", "The cover can also be used for other molten salt electrolytic cells, for example for the production of magnesium or other metals produced electrolytically.", "The cover may also be used in furnaces, such as arc furnaces for the production of steel or molten metal treatment apparatus, such as metal degassing apparatus.", "Further details of such apparatus may be found in WO00/63630 (Holz/Duruz), WO01/42168 (de Nora/Duruz) and WO01/42531 (Nguyen/Duruz/de Nora).", "More generally, the invention relates to a thermally insulating structural component which is inert and resistant to corrosive media at high temperature in the form of liquids, vapours and/or gases, in particular NaAlF 4 , AlF 3 , HF or O 2 , and which during use is exposed to such corrosive media.", "In accordance with the invention, the component is made an openly porous or reticulated alumina structure which is made impermeable by a compact filler made of material resistant and inert to said corrosive media.", "The component may comprise any of the above described features or combination thereof.", "The component may be a rigid, fire-resistant, lightweight panel, wall, door, lid, beam, balk or girder, housing or other structural component that can be utilised in the construction of containers, pressure vessels, reservoirs, ovens or furnaces etc.", "The invention will be further described in the following Example." ], [ "FIELD OF THE INVENTION The invention relates to thermally insulating structural components such as a cover of a container, in particular of a molten salt electrolytic cell.", "The structural components are inert and resistant to high temperature corrosive media in the form of liquids, vapours and/or gases, such as NaAlF4, AlF3, HF or O2.BACKGROUND OF THE INVENTION The technology for the production of aluminium by the electrolysis of alumina, dissolved in molten cryolite containing salts, at temperatures around 950° C. is more than one hundred years old.", "Conventional aluminium production cells are constructed so that in operation a crust of solidified molten electrolyte forms around the inside of the cell sidewalls.", "At the top of the cell sidewalls, this crust is extended by a ledge of solidified electrolyte which projects inwards over the top of the molten electrolyte.", "The solid crust in fact extends over the top of the molten electrolyte between the carbon anodes.", "To replenish the molten electrolyte with alumina in order to compensate for depletion during electrolysis, this crust is broken periodically at selected locations by means of a crust breaker, fresh alumina being fed through the hole in the crust.", "This crust/ledge of solidified electrolyte forms part of the cell's heat dissipation system in view of the need to keep the cell in operation at constant temperature despite changes in operating conditions, as when anodes are replaced, or due to damage/wear to the sidewalls, or due to over-heating or cooling as a result of great fluctuations in the operating conditions.", "In conventional cells, the crust is used as a means for automatically maintaining a satisfactory thermal balance, because the crust/ledge thickness self-adjusts to compensate for thermic unbalances.", "If the cell overheats, the crust dissolves partly thereby reducing the thermic insulation, so that more heat is dissipated through the sidewalls leading to cooling of the cell contents.", "On the other hand, if the cell cools the crust thickens which increases the thermic insulation, so that less heat is dissipated, leading to heating of the cell contents.", "The presence of a crust of solidified electrolyte is considered to be important to achieve satisfactory operation of commercial cells for the production of aluminium on a large scale.", "In fact, the heat balance is one of the major concerns of cell design and energy consumption, since only about 25% of such energy is used for the production of aluminium.", "Optimization of the heat balance is needed to keep the proper bath temperature and heat flow to maintain a frozen electrolyte layer (side ledge) with a proper thickness.", "In conventional cells, the major heat losses occur at the sidewalls, the current collector bars and the cathode bottom, which account for about 35%, 8% and 7% of the total heat losses respectively, and considerable attention is paid to providing a correct balance of these losses.", "Further losses of 33% occur via the carbon anodes, 10% via the crust and 7% via the deck on the cell sides.", "This high loss via the anodes is considered inherent in providing the required thermal gradient through the anodes.", "In the literature, there have been suggestions for cells operating without a crust of solidified electrolyte.", "U.S. Pat.", "No.", "5,368,702 (de Nora) discloses a multimonopolar aluminium production cell operating with tubular anodes in a crustless molten electrolyte which is thermally insulated by a cover.", "The cover is lined underneath with a layer of thermally insulating material.", "U.S. Pat.", "No.", "5,415,742 (La Camera/Tomaswick/Ray/Ziegler) disclose another aluminium production cell operating with a crustless molten electrolyte which is thermally insulated by a cover.", "Despite previous efforts to develop a cell design for operation with a crustless molten electrolyte, there is still a need to provide a thermic insulating material for cell covers which is resistant to electrolyte vapours and gases evolved during electrolysis and which is sufficiently lightweight but mechanically resistant to be used for removable covers.", "SUMMARY OF THE INVENTION The invention proposes a thermally insulating cover of a container, in particular a molten salt electrolytic cell, which cover is inert and resistant to corrosive media at high temperature in the form of liquids, vapours and/or gases contained in the container, in particular NaAlF4, AlF3, HF or O2, and which during use is exposed to such corrosive media.", "The cover comprises thermal insulating material shielded from the corrosive media by an openly porous or reticulated alumina structure which is made impermeable by a compact filler made of material resistant and inert to said corrosive media.", "The filler material comprises compacted particles of refractory material.", "The thermal insulating material may consist of or include a part of the openly porous or reticulated alumina structure that contains no filler.", "The thermal insulting material may also include or be formed of an entirely different body above the openly porous or reticulated filled alumina structure.", "The filler may extend throughout the alumina structure.", "Alternatively, the alumina structure may be only partly filled by the filler, leaving a filler-free part of the alumina structure, in particular a filler-free top part.", "For example, the filled part forms a layer which may form an outer surface of the cover, in particular an outer surface of a bottom part of the cover.", "In addition, the alumina structure may be covered with an outside layer made of the same filler material, the outside layer forming an outer surface of the cover.", "Usually, the alumina structure is a plate having a thickness in the range of 20 to 150 mm.", "A layer inside the alumina structure may have a thickness in the range of 10 to 100 mm.", "An outside layer covering the alumina structure can be 2 to 10 mm thick.", "Usually, the refractory material of the filler comprises at least one compound selected from metal oxides, carbides and nitrides.", "For instance, the filler comprises a mixture of metal oxide particles with particles of at least one compound selected from carbides and nitrides.", "These particles may be applied in a colloidal metal oxide carrier.", "Advantageously, the filler is a slurry-applied filler comprising dried colloidal and/or non-colloidal particles of at least one compound selected from metal oxides, carbides and nitrides, in particular selected from oxides, carbides and nitrides of titanium, zirconium, hafnium, vanadium, silicon, niobium, tantalum, nickel, molybdenum and iron.", "Suitable colloids may be selected from colloidal alumina, ceria, lithia, magnesia, silica, thoria, yttria, zirconia, tin oxide, zinc oxide and mixtures thereof.", "Colloidal precursors of such oxides, in particular hydroxides, may also be used.", "Further colloidal slurries which may be used as a filler material are disclosed in U.S. Pat.", "Nos.", "5,310,476 and 5,364,513 (both in the name of Sekhar/de Nora).", "For instance, the filler comprises a cement that consists predominantly of at least one of alumina, silica and titania particles.", "The filler may be made of alumina and silica, in particular with an alumina content of at least 80 weight %, in particular at least 90 or even 95 weight %.", "The filler may comprise a mixture of alumina and titania.", "In one embodiment, the thermally insulating cover comprises an insulating layer and a protective layer that shields the insulating layer(s) from the corrosive media, the protective layer being made of the openly porous or reticulated filled alumina structure.", "Preferably, the insulating layer(s) and the protective layer are mechanically secured together, in particular by means of one or more metallic attachment members extending through vertical holes of the protective layer and the insulating layer.", "The hole(s) in the insulating layer(s) may extend into recess(es) located in a bottom face of the alumina structure.", "Each recess can be arranged to embed a head of one or more of the attachment members and can be filled with the filler to protect the attachment member from the corrosive media.", "Usually, a top metallic shell extends over the insulating layer(s) and downwards along lateral sides of the cover.", "The thermally insulating cover of the invention may be used on any container containing high temperature oxidising and/or corrosive media, in particular vapours and/or gases.", "In particular, the cover is used for aluminium electrowinning cells.", "The cover can also be used for other molten salt electrolytic cells, for example for the production of magnesium or other metals produced electrolytically.", "The cover may also be used in furnaces, such as arc furnaces for the production of steel or molten metal treatment apparatus, such as metal degassing apparatus.", "Further details of such apparatus may be found in WO00/63630 (Holz/Duruz), WO01/42168 (de Nora/Duruz) and WO01/42531 (Nguyen/Duruz/de Nora).", "More generally, the invention relates to a thermally insulating structural component which is inert and resistant to corrosive media at high temperature in the form of liquids, vapours and/or gases, in particular NaAlF4, AlF3, HF or O2, and which during use is exposed to such corrosive media.", "In accordance with the invention, the component is made an openly porous or reticulated alumina structure which is made impermeable by a compact filler made of material resistant and inert to said corrosive media.", "The component may comprise any of the above described features or combination thereof.", "The component may be a rigid, fire-resistant, lightweight panel, wall, door, lid, beam, balk or girder, housing or other structural component that can be utilised in the construction of containers, pressure vessels, reservoirs, ovens or furnaces etc.", "The invention will be further described in the following Example.", "BRIEF DESCRIPTION OF THE DRAWING The invention will be further described with reference to the accompanying schematic drawing which shows a section of a composite thermally insulating cell cover in accordance with the invention.", "DETAILED DESCRIPTION FIG.", "1 shows part of a composite thermally insulating cover 10 according to the invention which during use can be placed above a molten salt electrolyte of an electrolytic cell, in particular a cell for the electrowinning of aluminium from alumina dissolved in a fluoride-based crustless molten electrolyte.", "The composite thermally insulating cover 10 comprises a plurality of superimposed layers 20,30 secured in a steel outer shell 40 which extends over the superimposed layers 20,30 and downwards along lateral sides of the cover 10.As shown in FIG.", "1, the superimposed layers consist of three upper insulating layers 30 which can be made of known insulating material, for example CERABOARD™ material, and a lower protective layer 20.The protective layer 20 is made of the openly porous or reticulated filled alumina structure according to the invention and shields the insulating layers 30 from corrosive media, such as NaAlF4, AlF3, HF or O2, present as vapours in an aluminium production cell above the molten electrolyte.", "Superimposed layers 20,30 are secured in the steel outer shell 40 by pairs of nuts 50 and bolts 60, one pair of which is shown in FIG.", "1.The nuts 50 and bolts 60 can be made of ceramic material resistant to the corrosive media 1, such as fused alumina, or steel preferably coated with this ceramic material.", "Each bolt 60 extends through vertically aligned holes 25,35,45 of steel shell 40, insulating layers 30 and protective layer 20 with the bolt 60's head 65 anchored in a recess 23 located in the bottom face of protective layer 20.In accordance with the invention, the protective layer 20 is made of an openly porous or reticulated alumina structure which is impermeabilized by a compact filler 15 made of material resistant and inert to corrosive media 1 and comprising packed particles of at least one compound selected from metal oxides, carbides and nitrides, for instance an alumina cement.", "The alumina structure of protective layer 20 is partly filled with filler 15, leaving on top a filler-free part 22 of the alumina structure.", "As shown in FIG.", "1, the filled part 21 of the alumina structure forms the outer bottom surface of cover 10.Filler 15 is also used to fill the recess 23, so the bolt's heads 65 are completely embedded in filler 15 and protected from corrosive media 1 contained in the aluminium production cell.", "In a variation, the filler material of the protective layer 20 may extend outside the openly porous or reticulated structure, forming a surface layer on the bottom of cover 10.In another variation, the filler material extends throughout protective layer 20.In a further variation, the head 65 of the bolt 60 can be anchored with cement in a recess (having similar dimensions as recess 23) located in the upper face of the protective layer 20.In this case, the bolt 60 does not extend through the protective layer 20 and no through hole is needed in the protective layer 20 whose bottom face is continuous.", "The invention will be further described in the following Example.", "EXAMPLE 1 An openly porous alumina plate with a porosity of 20 ppi (equivalent to about 8 pores per centimetre) having a thickness of 5 cm and a surface of 25×25 cm was made impermeable to corrosive vapours by impregnating and coating its bottom face with an alumina slurry.", "The slurry used to impermeabilize the porous alumina plate was KERATHIN HA™ produced by RATH GmbH.", "This slurry is made of an aqueous binder containing ceramic particles with a liquid weight content between 30 and 50%.", "The ceramic particles consist essentially of particles of alumina (98 wt %) and of silica (2 wt %) having sizes below 0.5 mm.", "Layers of the slurry were successively applied to the plate's bottom face.", "Each layer of the slurry was allowed to dry for several minutes before applying the next.", "After several layers of the slurry had been applied, the dried and compacted slurry formed a filler layer of 5 to 10 mm inside the plate and a coating of about 3 mm outside the plate leaving the impregnated and coated plate's bottom face with no surface porosity.", "Typically, this can be achieved with three to six applied layers of the slurry depending on its rheology.", "EXAMPLE 2 An openly porous alumina plate with a porosity of 80 ppi (equivalent to about 32 pores per centimetre) having a thickness of 5 cm and a surface of 25×25 cm was made impervious by filling it throughout with a titania-alumina filler and then sealing it as in Example 1 by applying a coating on its bottom.", "The titania-alumina filling was produced from a slurry made of 40 g TiO2 particles (−325 mesh or <42 micrometer) in colloidal aluminium hydroxide consisting of 200 ml Nyacol® (Al-20, a milky liquid with a colloidal particle size grade of about 40 to 60 nanometer) and 20 ml CONDEA® (10/2 Sol, a clear, opalescent liquid with a colloidal particle size grade of about 10 to 30 nanometer), the aluminium hydroxide forming alumina upon heat treatment.", "The porous alumina plate was immersed into the titania slurry to infiltrate it with the slurry and then dried for 20 minutes as 60° C. The infiltration was repeated followed by drying for 10 hours at 60° C. (alternatively it can be dried for 24 hours at room temperature) and heat treating for 10 hours between about 780° and 800° C. During heat treatment, the titania reacted with the aluminium hydroxide to form a stable titanium-aluminium mixed oxide which increase the stability of the alumina plate.", "The filled alumina plate was then sealed off by coating its bottom face with an alumina slurry (KERATHIN HA™) as in Example 1.EXAMPLE 3 The impervious plates of Example 1 and 2 were tested as lids on top of crucibles containing a molten cryolite-based electrolyte at 870° C. The electrolyte comprised, in addition to cryolite, an excess of aluminium fluoride in an amount of 28% of the cryolite weight.", "During the test, the impermeabilized plates inhibited evaporation of the electrolyte and dissipation of heat.", "After 3 weeks, the impermeabilized plates were removed from the crucibles.", "Visual examination of the impermeabilized plates showed that they had not been damaged by chemical attack or otherwise." ] ]	Patent_10469454
[ [ "Security system with an intelligent dma controller", "A security subsystem is provided with at least a first security engine, a first set of registers and a control portion to perform a first security operation for each of a first number of data blocks of each of a first number of data segments of a first data object.", "In one embodiment, the security subsystem is provided with two security engines and two sets of registers to respectively perform the first security operation and a second security operation for the first data object and a similarly constituted second data object.", "In one embodiment, the first and second security operations are DES and hashing operations.", "In one embodiment, the multi-method security subsystem is embodied in a multi-service system-on-chip." ], [ "1.A security subsystem comprising: a first security engine to perform a first security operation on a block of data bits; a first plurality of registers to collectively store a first descriptor of a first data object having first one or more data segments, with each of said first one or more data segments having a plurality data bits; and a control portion coupled to said first registers and the first security engine to cause (a) said first descriptor of said first data object to be loaded into said first registers, first describing a first data segment of said first data object, and said first descriptor to be successively updated to correspondingly describe first additional data segments of said first data object, if any, one data segment at a time, and (b) data bits of each currently described one of said first data segments to be successively fetched, organized into blocks of data bits, and provided to said first security engine to have said first security operation to be successively performed on the provided blocks of data bits.", "2.The security subsystem of claim 1, where said first descriptor of said first data object includes, at a first instance in time, first storage location descriptions that describe first storage locations of data bits of a first of said first data segments of said first data object.", "3.The security subsystem of claim 2, where said first storage location descriptions comprise a starting storage location address and a size of the data bits of said first data segments of said first data object.", "4.The security subsystem of claim 2, where said first descriptor of said first data object includes, at a second instance in time, second storage location descriptions that describe second storage locations of data bits of a second of said first data segments of said first data object.", "5.The security subsystem of claim 4, where said first storage locations and said second storage locations are contiguous storage locations.", "6.The security subsystem of claim 4, where said first storage locations and said second storage locations are discontiguous storage locations.", "7.The security subsystem of claim 1, where said control portion further causes the results of said first security operations performed for the provided blocks of data bits to be successively returned.", "8.The security subsystem of claim 7, where said first descriptor of said first data object includes, at a first instance in time, first storage location descriptions that describe first storage locations for returning first results of said first security operations performed on the provided data bits of a first of said first data segments of said first data object.", "9.The security subsystem of claim 8, where said first storage location descriptions comprise a starting storage location address.", "10.The security subsystem of claim 8, where said first descriptor of said first data object includes, at a second instance in time, second storage location descriptions that describe second storage locations for returning second results of said second security operations performed on the provided data bits of a second of said first data segments of said first data object.", "11.The security subsystem of claim 10, where said first storage locations and said second storage locations are contiguous storage locations.", "12.The security subsystem of claim 10, where said first storage locations and said second storage locations are discontiguous storage locations.", "13.The security subsystem of claim 1, where said first descriptor of said first data object also describes operating parameters to be employed to perform said first security operation on each of said provided blocks of data bits of said first data object, and said control portion further causes said described operating parameters to be provided to said first security engine.", "14.The security subsystem of claim 1, wherein said first security operation is a DES operation.", "15.The security subsystem of claim 14, wherein said DES operation is a selected one of a DES cipher operation and a DES decipher operation.", "16.The security subsystem of claim 14, wherein said DES operation is a selected one of a DES ECB operation, a DES CBC operation and a DES CFB operation.", "17.The security subsystem of claim 14, wherein said first descriptor of said first data object also describes operating parameters including a first and a second key of to be employed to perform said DES operation on each of said provided blocks of data bits of said first data object, and said first control portion further causes said described operating parameters including said first and second keys of said DES operation to be provided to said first security engine.", "18.The security subsystem of claim 17, wherein said operating parameters further include a third key of said DES operation.", "19.The security subsystem of claim 14, wherein said DES operation is a selected one of a DES CBC operation and a DES CFB operation; said security subsystem further comprises a data router coupled to said security engine to selectively route a current block of data bits of said first data object and a result of the selected DES security operation for a prior block of data bits to said security engine; and said control portion is further coupled to said data router to control its operation.", "20.The security subsystem of claim 19, wherein <additional details on the data router>.", "21.The security subsystem of claim 1, wherein said security operation is a hashing operation.", "22.The security subsystem of claim 21, wherein said hashing operation is a selected one of a MD5 operation and a SHA-1 operation.", "23.The security subsystem of claim 21, wherein said first descriptor of said first data object also describes operating parameters including a plurality of chaining variables to be employed to perform said hashing operation on each of said blocks of data bits of said first data object, and said first control portion further causes said described operating parameters including said chaining variables to be provided to said first security engine.", "24.The security subsystem of claim 1, wherein said security subsystem further comprises a control register to facilitate a subsystem external to said security subsystem in providing one more control instructions to said control portion of said security subsystem.", "25.The security subsystem of claim 24, wherein at least one of said control instructions is a selected one of instructing said control portion to start said first security operation, to interrupt said external subsystem upon completing said first security operation for all blocks of data bits of said first data segments of said first data object, to interrupt said external subsystem upon completing said first security operation for all blocks of data bits of said first data object, and to stop said security subsystem upon completing said first security operation for all blocks of data bits of said first data segments of said first data object.", "26.The security subsystem of claim 1, wherein said security subsystem further comprises a status register to facilitate said control portion of said security subsystem in providing one or more status to a subsystem external to said security subsystem.", "27.The security subsystem of claim 26, wherein at least one of said status is a selected one of a pending interrupt issued on completion of said first security operation for all blocks of data bits of said first data segments of said first data object, a pending interrupt issued on completion of said first security operation for all blocks of data bits of said first data object, completion of said first security operation for all blocks of data bits of said first data segments of said first data object, completion of said first security operation for all blocks of data bits of said first data object and said security subsystem being in a busy state.", "28.The security subsystem of claim 1, wherein said security subsystem further comprises a second security engine to perform a second security operation on a block of data bits; a second plurality of registers to collectively store a second descriptor of a second data object having second one or more data segments, with each of said second one or more data segments having a plurality of data bits; and said control portion is further coupled to said second registers and the second security engine to cause (a) said second descriptor of said second data object to be loaded into said second registers, first describing a second data segment of said second data object, and said second descriptor to be successively updated to correspondingly describe second additional data segments of said second data object, if any, one data segment at a time, and (b) data bits of each currently described one of said second data segments to be successively fetched, organized into blocks of data bits, and provided to said second security engine to have said second security operation to be successively performed on the provided blocks of data bits.", "29.The security subsystem of claim 28, where said control portion further causes the results of said second security operations performed for the provided blocks of data bits to be successively returned.", "30.The security subsystem of claim 28, where said second descriptor of said second data object also describes operating parameters to be employed to perform said second security operation for each of said blocks of data bits of said second data object, and said control portion further causes said described operating parameters to be provided to said second security engine.", "31.The security subsystem of claim 28, wherein said first security operation is a DES operation and said second security operation is a hashing operation.", "32.The security subsystem of claim 1, wherein said security subsystem further comprises a data transfer unit coupled to said first security engine and said control portion to retrieve and provide said data bits of said first data object for said first security engine, and return the results of said first security operations performed for said data bits of said first data object, under the control of said control portion.", "33.In a security subsystem, a method of operation comprising: retrieving and storing a first descriptor describing a first data segment of a first data object; and causing first data bits of said described first data segment of the first data object to be successively retrieved, organized into blocks of data bits, provided to a first security engine of the security subsystem, have a first security operation performed by the first security engine on each of the provided blocks of data bits, and the results of the first security operations performed on the provided blocks of data bits to be returned.", "34.The method of claim 33, wherein the method further comprises accepting and storing a plurality of control instructions instructing said security subsystem in its manner of operation; and stopping said security subsystem, if so instructed, upon causing said first security operation to be performed on each of said provided blocks of data bits of said first data segment of said first data object.", "35.The method of claim 33, wherein the method further comprises accepting and storing a plurality of control instructions instructing said security subsystem in its manner of operation; and interrupting a subsystem external to said security subsystem, if so instructed, upon causing said first security operation to be performed on each of the provided blocks of data bits of said first data segment of said first data object.", "36.The method of claim 33, wherein the method further comprises updating said first descriptor to describe a second segment of said first data object; and causing second data bits of said described second segment of the first data object to be successively retrieved, organized into blocks of data bits, provided to said first security engine of the security subsystem, have said first security operation performed by the first security engine on each of the provided blocks of data bits, and the results of the first security operations performed on the provided blocks of data bits to be returned.", "37.The method of claim 36, wherein the method further comprises accepting and storing a plurality of control instructions instructing said security subsystem in its manner of operation; and interrupting a subsystem external to said security subsystem, if so instructed, upon causing said first security operation to be performed on all provided blocks of data bis of all data segments of said first data object.", "38.The method of claim 36, wherein said first data blocks of said first data segment of said first data object and said second data blocks of said second data segment of said first data object are stored in contiguous storage locations.", "39.The method of claim 36, wherein said first data blocks of said first data segment of said first data object and said second data blocks of said second data segment of said first data object are stored in discontiguous storage locations.", "40.The method of claim 36, wherein the results of said first security operations performed on said data bits of said first data segment of said first data object and the results of said first security operations performed on said data bits of said second data segment of said first data object are returned to contiguous storage locations.", "41.The method of claim 36, wherein the results of said first security operations performed on said data bits of said first data segment of said first data object and the results of said first security operations performed on said data bits of said second data segment of said first data object are stored in discontiguous storage locations.", "42.The method of claim 33, wherein said first descriptor of said first data object also describes operating parameters to be employed to perform said first security operation on each of said organized blocks of data bits of said first data segment of said first data object, and the method further comprises providing the described operating parameters to said first security engine.", "43.The method of claim 33, wherein said first security operation is a DES operation.", "44.The method of claim 43, wherein said DES operation is a selected one of a DES cipher operation and a DES decipher operation.", "45.The method of claim 43, wherein said DES operation is a selected one of a DES ECB operation, a DES CBC operation and a DES CFB operation.", "46.The method of claim 43, wherein said first descriptor of said first data object also describes operating parameters including a first and a second key of to be employed to perform said DES operation on each of said first data blocks of said first data segment of said first data object, and the method further comprises providing said first and second keys of said DES operation to said first security engine.", "47.The method of claim 46, wherein said operating parameters further include a third key of said DES operation.", "48.The method of claim 43, wherein said DES operation is a selected one of a DES CBC operation and a DES CFB operation; and said method further comprises causing a selected one of a current block of data bits of said first data segment and a result of the selected DES security operation for a prior block of data bits to be provided to said security engine.", "49.The method of claim 48, wherein <additional details on the data router>.", "50.The method of claim 33, wherein said security operation is a hashing operation.", "51.The method of claim 50, wherein said hashing operation is a selected one of a MD5 operation and a SHA-1 operation.", "52.The method of claim 50, wherein said first descriptor of said first data object also describes operating parameters including a plurality of chaining variables to be employed to perform said hashing operation on each of said blocks of data bits of said first data segment of said first data object, and the method further comprises providing said chaining variables to said first security engine.", "53.The method of claim 33, wherein the method further comprises providing one or more status to a subsystem external to said security subsystem.", "54.The method of claim 53, wherein at least one of said status is a selected one of a pending interrupt issued on completion of said first security operation for all data bits of said first data segment of said first data object, a pending interrupt issued on completion of said first security operation for all data bits of said first data object, completion of said first security operation for all data bits of said first data segment of said first data object, completion of said security operation for all data bits of said first data object and said security subsystem being in a busy state.", "55.The method of claim 33, wherein the method comprises retrieving and storing a second descriptor describing a second segment of a second data object; and causing second data bits of said described second segment of the second data object to be successively retrieved, organized into blocks of data bits, and provided to a second security engine of the security subsystem, have a second security operation performed by the second security engine on each of the provided blocks of data bits, and the results of the second security operations performed on the blocks of data bits to be returned.", "56.The method of claim 55, wherein the method further comprises successively returning the results of said second security operations performed for the provided blocks of data bits.", "57.The method of claim 55, wherein said second descriptor of said second data object also describes operating parameters to be employed to perform said second security operation for each of said provided blocks of data bits of said second data segment of said second data object, and the method further comprises providing said described operating parameters to said second security engine.", "58.The method of claim 55, wherein said first security operation is a DES operation and said second security operation is a hashing operation.", "59.An apparatus comprising: a memory to store data and descriptive information of said data; a processor coupled to said memory to set up in said memory a first descriptor having first one or more parts, describing a first data object having first one or more data segments, with each of said first one or more data segments having a plurality of data bits; and a security subsystem coupled to said memory and said processor to perform a first security operation on each of a plurality of blocks of data bits of said first one or more data segments of said first data object, responsive to a request of said processor, wherein the security subsystem is equipped to (a) first retrieve a first part of said first descriptor, and then successively updates said first descriptor with its additional parts, if applicable, (b) successively fetch the data bits of said first one or more data segments of said first data object in accordance with the successive current descriptions of the first descriptor, (c) successively organize the fetched data bits into blocks of data bits, (d) successively perform said first security operation on said organized data blocks, and (e) successively return the results of said successive first security operations.", "60.The apparatus of claim 59, wherein the security subsystem comprises a first security engine to perform said first security operation for a block of data bits; a first plurality of registers to collectively store the currently retrieved part of a data object descriptor; and a control portion coupled to said first registers and the first security engine to cause (a) said first part of said first descriptor of said first data object to be loaded into said first registers, and then successively updated to successively describe said first one or more data segments of said first data object, (b) data bits of each currently described one of said first data segments to be successively fetched, organized into blocks of data bits, and provided to said first security engine to have said first security operation to be successively performed on the provided data blocks, and (c) the results of said successively performed first security operations to be returned.", "61.The apparatus of claim 59, wherein each of said first one or more parts of said first descriptor describes storage locations of data bits of a corresponding one of said first one or more data segments of said first data object.", "62.The apparatus of claim 61, wherein said first one or more data segments of said first data object comprise two or more data segments, and the storage locations of the data blocks of at least one of the data segments are discontiguous from the storage location of the data blocks of the other data segments of said first data object.", "63.The apparatus of claim 59, wherein each of said first one or more parts of said first descriptor describes storage locations for returning the results of said first security operations for the data bits of a corresponding one of said first one or more data segments of said first data object.", "64.The apparatus of claim 63, wherein said first one or more data segments of said first data object comprise two or more data segments, and the storage locations for returning the results of said first security operations performed for the data bits of at least one of the data segments are discontiguous from the storage location for returning the results of said first security operations performed for the data bits of the other data segments of said first data object.", "65.The apparatus of claim 59, wherein at least a first part of said first descriptor of said first data object also describes operating parameters to be employed to perform said first security operation for each of said blocks of data bits of said first data object.", "66.The apparatus of claim 59, wherein said first security operation is a DES operation.", "67.The apparatus of claim 66, wherein said DES operation is a selected one of a DES cipher operation and a DES decipher operation.", "68.The apparatus of claim 66, wherein said DES operation is a selected one of a DES ECB operation, a DES CBC operation and a DES CFB operation.", "69.The apparatus of claim 66, wherein at least a first part of said first descriptor of said first data object also describes operating parameters including a first and a second key of to be employed to perform said DES operation on each of said blocks of data bits of said first data object.", "70.The apparatus of claim 69, wherein said operating parameters further include a third key of said DES operation.", "71.The apparatus of claim 66, wherein said DES operation is a selected one of a DES CBC operation and a DES CFB operation; and said security subsystem is further equipped to selectively employ a current block of data bits of said first data object and a result of the selected DES security operation for a prior block of data bits to perform the selected DES operation.", "72.The apparatus of claim 59, wherein said security operation is a hashing operation.", "73.The apparatus of claim 72, wherein said hashing operation is a selected one of a MD5 operation and a SHA-1 operation.", "74.The apparatus of claim 72, wherein at least a first part of said first descriptor of said first data object also describes operating parameters including a plurality of chaining variables to be employed to perform said hashing operation for each of said blocks of data bits of said first data object.", "75.The apparatus of claim 59 wherein said security subsystem further comprises a control register to facilitate said processor in providing one more control instructions to said security subsystem.", "76.The apparatus of claim 75, wherein at least one of said control instructions is a selected one of instructing said security subsystem to start said first security operation, to interrupt said processor upon completing said first security operation for all blocks of data bits of said first data segments of said first data object, to interrupt said processor upon completing said first security operation for all blocks of data bits of said first data object, and to stop said security subsystem upon completing said first security operation for all blocks of data bits of said first data segments of said first data object.", "77.The apparatus of claim 59, wherein said security subsystem further comprises a status register to facilitate said security subsystem in providing one or more status to said processor.", "78.The apparatus of claim 77, wherein at least one of said status is a selected one of a pending interrupt issued on completion of said first security operation for all blocks of data bits of said first data segments of said first data object, a pending interrupt issued on completion of said first security operation for all blocks of data bits of said first data object, completion of said first security operation for all blocks of data bits of said first data segments of said first data object, completion of said first security operation for all blocks of data bits of said first data object and said security subsystem being in a busy state.", "79.The apparatus of claim 59, wherein said processor is also to set up in said memory a second descriptor having second one or more parts, describing a second data object having second one or more data segments, with each of said second one or more data segments having a plurality of data bits; and said security subsystem is also to perform a second security operation for data bits of said second one or more data segments of said second data object, responsive to a request of said processor, wherein the security subsystem is also equipped to (a) first retrieve a first part of said second descriptor, and then successively updates said second descriptor with its additional parts, if applicable, (b) successively fetch the data bits of said second one or more data segments of said second data object in accordance with the successive current descriptions of the second descriptor, (c) successively organized the successively fetched data bits into blocks of data bits, (d) successively perform said second security operation on said successively organized blocks of data bits, and (d) successively return the results of said successive second security operations.", "80.The apparatus of claim 79, wherein said first security operation is a DES operation and said second security operation is a hashing operation.", "81.The apparatus of claim 59, wherein said apparatus is disposed on a single integrated circuit.", "82.A method comprising: a processor setting up in a memory a first descriptor having first one or more parts, describing a first data object having first one or more data segments, with each of said first one or more data segments having a plurality of data bits; and a security subsystem performing a first security operation on the data bits of said first one or more data segments of said first data object, responsive to a request of said processor, by (a) first retrieving a first part of said first descriptor, and then successively updating said first descriptor with its additional parts, if applicable, (b) successively fetching the data bits of said first one or more data segments of said first data object in accordance with the successive current descriptions of the first descriptor, (c) successively organizing the fetched data bits into blocks of data bits, (d) successively performing said first security operation on said successively organized data blocks, and (d) successively returning the results of said successive first security operations.", "83.The method of claim 82, wherein each of said first one or more parts of said first descriptor describes storage locations of data bits of a corresponding one of said first one or more data segments of said first data object.", "84.The method of claim 83, wherein said first one or more data segments of said first data object comprise two or more data segments, and the storage locations of the data blocks of at least one of the data segments are discontiguous from the storage location of the data blocks of the other data segments of said first data object.", "85.The method of claim 82, wherein each of said first one or more parts of said first descriptor describes storage locations for returning the results of said first security operations for data bits of a corresponding one of said first one or more data segments of said first data object.", "86.The method of claim 85, wherein said first one or more data segments of said first data object comprise two or more data segments, and the storage locations for returning the results of said first security operations performed for the data bits of at least one of the data segments are discontiguous from the storage location for returning the results of said first security operations performed for the data bits of the other data segments of said first data object.", "87.The method of claim 82, wherein at least a first part of said first descriptor of said first data object also describes operating parameters to be employed to perform said first security operation for data bits of said first data object.", "88.The method of claim 82, wherein said first security operation is a DES operation.", "89.The method of claim 88, wherein said DES operation is a selected one of a DES cipher operation and a DES decipher operation.", "90.The method of claim 88, wherein said DES operation is a selected one of a DES ECB operation, a DES CBC operation and a DES CFB operation.", "91.The method of claim 88, wherein at least a first part of said first descriptor of said first data object also describes operating parameters including a first and a second key of to be employed to perform said DES operation on each of said data blocks of said first data object.", "92.The method of claim 91, wherein said operating parameters further include a third key of said DES operation.", "93.The method of claim 88, wherein said DES operation is a selected one of a DES CBC operation and a DES CFB operation; and said method further comprises said security subsystem selectively employing a current block of data bits of said first data object and a result of the selected DES security operation for a prior block of data bits to perform the selected DES operation.", "94.The method of claim 82, wherein said security operation is a hashing operation.", "95.The method of claim 94, wherein said hashing operation is a selected one of a MD5 operation and a SHA-1 operation.", "96.The method of claim 94, wherein at least a first part of said first descriptor of said first data object also describes operating parameters including a plurality of chaining variables to be employed to perform said hashing operation for each of said blocks of data bits of said first data object.", "97.The method of claim 82 wherein said method further comprises said processor providing one more control instructions to said security subsystem.", "98.The method of claim 97, wherein at least one of said control instructions is a selected one of instructing said security subsystem to start said first security operation, to interrupt said processor upon completing said first security operation for all data bits of one of said first data segments of said first data object, to interrupt said processor upon completing said first security operation for all data bits of said first data object, and to stop said security subsystem upon completing said first security operation for all data bits of one of said first data segments of said first data object.", "99.The method of claim 82, wherein said method further comprises said security providing one or more status to said processor.", "100.The method of claim 99, wherein at least one of said status is a selected one of a pending interrupt issued on completion of said first security operation for all data bits of one of said first data segments of said first data object, a pending interrupt issued on completion of said first security operation for all data bits of said first data object, completion of said first security operation for all data bits of one of said first data segments of said first data object, completion of said first security operation for all data bits of said first data object and said security subsystem being in a busy state.", "101.The method of claim 82, wherein the method further comprises said processor setting up in said memory a second descriptor having second one or more parts, describing a second data object having second one or more data segments, with each of said second one or more data segments having a plurality of data bits; and said security subsystem performing a second security operation on data bits of said second one or more data segments of said second data object, responsive to a request of said processor, by (a) first retrieving a first part of said second descriptor, and then successively updating said second descriptor with its additional parts, if applicable, (b) successively fetching the data blocks of said second one or more data segments of said second data object in accordance with the successive current descriptions of the second descriptor, (c) successively organizing the fetched data bits into blocks of data bits, (d) successively performing said second security operation for said successively organized blocks of data bits, and (e) successively returning the results of said successive second security operations.", "102.The method of claim 101, wherein said first security operation is a DES operation and said second security operation is a hashing operation." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to the field of security.", "More specifically, the present invention relates to the provision of a security subsystem having an intelligent direct memory access (DMA) controller in a multi-service system-on-chip to improve operational efficiency.", "2.Background Information Advances in integrated circuit technology have led to the birth and proliferation of a wide variety of integrated circuits, including but not limited to application specific integrated circuits, micro-controllers, digital signal processors, general purpose microprocessors, and network processors.", "Recent advances have also led to the birth of what's known as “system on a chip” or SOC.", "In various SOC applications, such as telecommunications, networking and content handling, it is often necessary to perform security operations of one or more types of security methods.", "The terms “security operations” and “security methods” as used in the present application include all known security operations/methods, as well as to be discovered security operations/methods that are compatible with the present invention.", "Examples of known security operations/methods include but are not limited to Data Encryption Standard (DES) methods and operations of all types, Electronic Codebook (ECB), Cipher Block Chaining (CBC), Cipher Feedback (CFB), and so forth, and hashing operations of all types, Message Digest (MD5), Secure HASH Algorithm (SHA-1) and so forth.", "Further, the security methods or operations often have to be performed for data of various types, including audio, video and other data, and of various subsystems, such as the subsystem responsible for interfacing the SOC to a network, the subsystem responsible for interfacing the SOC to a telecommunication line and so forth.", "Thus, a need exists to provide or support security operations of multiple security methods or operations in an efficient manner." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>The present invention will be described by way of exemplary embodiments, but not limitations, illustrated in the accompanying drawings in which like references denote similar elements, and in which: FIG.", "1 illustrates an overview of a system-on-chip including a security subsystem incorporated with the teachings of the present invention, in accordance with one embodiment; FIG.", "2 illustrates the method of the present invention, in accordance with one embodiment; FIG.", "3 illustrates the data descriptor of the present invention in further details, in accordance with one embodiment; FIGS.", "4 a - 4 d illustrate the base and continuation portion of a data descriptor in further details, in accordance with one embodiment; FIG.", "5 illustrates the security subsystem of the present invention in further details, in accordance with one embodiment; FIGS.", "6 a - 6 b illustrate the control and status registers of the security subsystem of FIG.", "5 in further details, in accordance with one embodiment; FIG.", "7 illustrates the further provision of a data traffic router for a DES security engine to support multiple variants of DES operations, in accordance with one embodiment; FIG.", "8 illustrates the data traffic router of FIG.", "7 in further details, in accordance with one embodiment; and FIG.", "9 illustrates the operational flow of the relevant aspects of the controller of the security subsystem of FIG.", "5 in further details, in accordance with one embodiment.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "This application claims priority to U.S.", "Provisional Application No.", "60/272,439, entitled “MULTI-SERVICE PROCESSOR INCLUDING A MULTI-SERVICE BUS”, filed Feb. 28, 2001, the specification of which is hereby fully incorporated by reference.", "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to the field of security.", "More specifically, the present invention relates to the provision of a security subsystem having an intelligent direct memory access (DMA) controller in a multi-service system-on-chip to improve operational efficiency.", "2.Background Information Advances in integrated circuit technology have led to the birth and proliferation of a wide variety of integrated circuits, including but not limited to application specific integrated circuits, micro-controllers, digital signal processors, general purpose microprocessors, and network processors.", "Recent advances have also led to the birth of what's known as “system on a chip” or SOC.", "In various SOC applications, such as telecommunications, networking and content handling, it is often necessary to perform security operations of one or more types of security methods.", "The terms “security operations” and “security methods” as used in the present application include all known security operations/methods, as well as to be discovered security operations/methods that are compatible with the present invention.", "Examples of known security operations/methods include but are not limited to Data Encryption Standard (DES) methods and operations of all types, Electronic Codebook (ECB), Cipher Block Chaining (CBC), Cipher Feedback (CFB), and so forth, and hashing operations of all types, Message Digest (MD5), Secure HASH Algorithm (SHA-1) and so forth.", "Further, the security methods or operations often have to be performed for data of various types, including audio, video and other data, and of various subsystems, such as the subsystem responsible for interfacing the SOC to a network, the subsystem responsible for interfacing the SOC to a telecommunication line and so forth.", "Thus, a need exists to provide or support security operations of multiple security methods or operations in an efficient manner.", "BRIEF DESCRIPTION OF DRAWINGS The present invention will be described by way of exemplary embodiments, but not limitations, illustrated in the accompanying drawings in which like references denote similar elements, and in which: FIG.", "1 illustrates an overview of a system-on-chip including a security subsystem incorporated with the teachings of the present invention, in accordance with one embodiment; FIG.", "2 illustrates the method of the present invention, in accordance with one embodiment; FIG.", "3 illustrates the data descriptor of the present invention in further details, in accordance with one embodiment; FIGS.", "4a-4d illustrate the base and continuation portion of a data descriptor in further details, in accordance with one embodiment; FIG.", "5 illustrates the security subsystem of the present invention in further details, in accordance with one embodiment; FIGS.", "6a-6b illustrate the control and status registers of the security subsystem of FIG.", "5 in further details, in accordance with one embodiment; FIG.", "7 illustrates the further provision of a data traffic router for a DES security engine to support multiple variants of DES operations, in accordance with one embodiment; FIG.", "8 illustrates the data traffic router of FIG.", "7 in further details, in accordance with one embodiment; and FIG.", "9 illustrates the operational flow of the relevant aspects of the controller of the security subsystem of FIG.", "5 in further details, in accordance with one embodiment.", "DETAILED DESCRIPTION OF THE INVENTION The present invention includes a security subsystem equipped with an intelligent DMA controller having particular application to system-on-chips with subsystems requiring security services.", "The security services may include encryption/decryption services/operations, such as DES based encryptions/decryptions, and/or hashing operations, such as MD5 and SHA-1.The present invention advantageously improves the operational efficiency of the system-on-chip, in particular, offloading the controller processor of a system-on-chip.", "In the following description, various features and arrangements will be described, to provide a thorough understanding of the present invention.", "However, the present invention may be practiced without some of the specific details or with alternate features/arrangement.", "In other instances, well-known features are omitted or simplified in order not to obscure the present invention.", "The description to follow repeatedly uses the phrase “in one embodiment”, which ordinarily does not refer to the same embodiment, although it may.", "The terms “comprising”, “having”, “including” and the like, as used in the present application, including in the claims, are synonymous.", "Overview Referring now to FIG.", "1, wherein a block diagram illustrating an overview of a SOC 100 including control processor 102, memory 104, security subsystem 106 incorporated with the teachings of the present invention, and other subsystems 108, in accordance with one embodiment, is shown.", "As illustrated, for the embodiment, control processor 102, memory 104, security subsystem 106 and other subsystems 108 are coupled to each other via on-chip bus 110, and communicate with each other in accordance with a predetermined bus protocol.", "In one embodiment, the on-chip bus and the bus protocol is the on-chip bus described in co-pending U.S. application Ser.", "No.", "10/______, contemporaneously filed, entitled “A Multi-Service System On-Chip Including On-Chip Memory with Multiple Access Paths”, which specification is hereby fully incorporated by reference.", "In other embodiments, other bus architectures and other bus communication protocols may be employed instead.", "Security subsystem 106 equipped with the teachings of present invention, is employed to provide security services/operations to meet the security service/operation needs of subsystems 108.As will be described in more details below, in addition to security engines 122 in support of various security methods, DES operations, hashing operations, and so forth, security subsystem 106 includes intelligent DMA 120 of the present invention.", "Resultantly, unless so desired, upon requested, security subsystem 106 may service a security need of one of subsystems 108 substantially without further interactions with control processor 102 and the requesting subsystem 108, thereby improving the overall operational efficiency of SOC 100.The terms “security service” and “security operation” are used interchangeably in the present application, depending on which term is more instrumental in assisting in understanding the present invention.", "Their core meanings or the essence of their meanings are synonymous.", "Except for the teachings of the present invention incorporated in subsystems 108, to allow subsystems 108 to have their security service needs met by security subsystem 106 in the aforementioned advantageous manner, subsystems 108 may otherwise be any one of a broad range of subsystems known in the art or to be developed.", "Examples of such subsystems include but are not limited to voice processors, peripheral device controllers, framer processors, network media access controllers, and the like.", "The exact mix is application dependent and non-essential to the practice of the present invention.", "Except for its use for its conventional function of storing data, in particular data objects 116 to have security operations performed and data descriptors 118 of the present invention describing data objects 116 and the security operations to be performed, memory 104 may otherwise be any one of a broad range of volatile or non-volatile storage units known in the art or to be developed.", "In one embodiment, the memory 104 is a storage unit with multiple access paths, which is the subject matter of the aforementioned co-pending and incorporated by reference U.S. patent application Ser.", "No.", "______.", "Control processor 102 controls the overall operation of SOC 100.In particular, for the embodiment, the control includes instructing security system 106 to perform a security operation on a data object 116 on behalf of one of subsystems 108, which instruction may be responsive to the request of the subsystem.", "The exact nature of the remaining control performed by control processor 102 is application dependent, and is not essential to the practice of the present invention.", "As alluded to earlier, control processor 102 is one of primary beneficiaries of the present invention.", "Further, for the illustrated embodiment, control processor 102 includes instruction cache 112 and data cache 114, to facilitate performance of its control operations.", "Method Referring now to FIG.", "2, wherein a flow chart illustrating a method of the present invention, in accordance with one embodiment, is shown.", "As illustrated, in accordance with the present invention, a subsystem 108 having a security service need for a data object, first sets up in memory 104 the data object, and a descriptor describing the data object, including the security operation to be performed and the operational parameters of the security operation, block 202.Referring now briefly to FIG.", "3, under the present invention, a data object 116 to have a security operation performed may comprise a number of data segments 116a-116n, with each data segments having a number of data bits.", "The number of data bits in each data segment may be greater than, equal to, or less than the data bit size of an atomic block of data on which the request security operation operates.", "For example, a DES operation operates on 64-bit data blocks, accordingly, a data segment of a data object to have a DES operation performed may be greater than, equal to, or less than 64 bits.", "Similarly, a MD5/SHA-1 operation operates on 512-bit data blocks, a data segment of a data object to have a MD5/SHA-1 operation performed may be greater than, equal to, or less than 512 bits.", "Further, the various data segments may be stored in contiguous or discontiguous memory locations, and need not be aligned to any word boundaries.", "A descriptor 118 describing a data object 116, the security operation to be performed, and the operation parameters, may include one or more parts, i.e.", "a base part 118a and zero or more continuation parts 118n, with the base part 118a describing the first data segment 116a, the security operation to be performed for all data segments 116a-116n and the operation parameters, and the continuation parts 118n correspondingly describing the additional data segments 116n, to be described more fully below.", "Returning now to FIG.", "2, upon setting up the data object 116 to have a security operation performed, and its descriptor 118, for the embodiment, the subsystem 108 requests control processor 102 to cause the desired security operation to be performed, block 204.Since, for the embodiment, the security operation to be performed, including the operation parameters, are described by the data descriptor 118 of the data object 116, accordingly only the location of the descriptor 118 needs to be made available to control processor 102.The information may be made available in any one of a number of manners known in the art.", "For example, the starting location of the descriptor may be place in a predetermined location associated with a particular interrupt, and the subsystem 108 interrupts control processor 102 accordingly, upon setting up the data object 116, its descriptor 118, and placement of the starting location of the descriptor 118 in the predetermined location.", "As a further example, the starting location of the descriptor 108 may be included as part of the security service request, and the security service request may be communicated to control processor 102 via a communication packet.", "Still referring to FIG.", "2, in response to the request, control processor 102 instructs security subsystem 106 to perform the requested security operation for the data object 116, including with the instruction, the starting location of the descriptor 118 of the data object 116, block 206.In one embodiment, the instruction is provided to security subsystem 106 in the form of a communication packet over bus 110.In response, as will be described in more detail below, security subsystem 106 first loads the base part 118a of the descriptor 118 of the data object 116, and thereafter successively updates the descriptor 118 with its continuation parts 118n, and in parallel, based on the descriptive information provided therein over time, successively fetches the data bits of the data segment 116a, organizes the data bits into the atomic data blocks of the requested security operation, provides the organized data blocks to the appropriate security engine for the requested security operation, causes the security engine to perform the security operation on the provided data blocks, and writes back the results of the security operation, block 208.Data Descriptor FIGS.", "4a-4d illustrate descriptor 118 of a data object 116, in accordance with one embodiment.", "More specifically, FIG.", "4a illustrates the base part 118a of a descriptor 118 for a DES operation for a data object 116, in accordance with one embodiment; FIG.", "4b illustrates a continuation part 118b of a descriptor 118 for a DES operation for a data object 116, in accordance with one embodiment; FIG.", "4c illustrates the base part 118a of a descriptor 118 for a hashing operation for a data object 116, in accordance with one embodiment; and FIG.", "4d illustrates a continuation part 118b of a descriptor 118 for a hashing operation for a data object 116, in accordance with one embodiment.", "As illustrated in FIG.", "4a, for the embodiment, the base part 118a of a descriptor 118 for a DES operation includes a next descriptor/part address 402 identifying the starting word location in memory 104 where the next part 118n of the descriptor 118 or the base part 118a of a next descriptor 118 is stored.", "The residual unused least significant bits are employed to facilitate identification of the part as being a base part 118a of a descriptor 118, and the next descriptor/part address information is valid, and may be acted on by the security subsystem 106.Base part 118a for a DES operation also includes a buffer size 404 and a starting address 406 (in memory 104) of the source buffer holding the base data segment 116a being described.", "Base part 118a also includes the starting address 408 (in memory 104) for the destination buffer for writing back the results of the security operation for the corresponding data bits of the base data segment 116a.", "Additionally, base part 118a for a DES operation also includes mode 410 specifying the type of DES operation, i.e.", "ECB, CBC or CFB, to be performed, and descriptor identifier 412 of the descriptor.", "Further, base part 118a of a DES operation also describes up to three keys 418-420, 422-424 and 426-428 for the DES operation, and for CBC or CFB mode of operation, base part 118a also describes the initial vector 414-416 of the DES operation.", "As illustrated in FIG.", "4b, for the embodiment, the continuation part 118n of a descriptor 118 for a DES operation also includes a next descriptor/part address 432 identifying the starting word location of memory 104 where the next part 118n of the descriptor 118 or the base part 118a of a next descriptor 118 is stored.", "Similarly, the residual least significant bits are employed to facilitate identification of the part as being a continuation of a descriptor 118, and the next descriptor/part address information is valid, and may be acted on by the security subsystem 106.Similar to the base part 118a of a descriptor 118 for a DES operation, a continuation part 118n of a descriptor 118 of a DES operation also includes a buffer size 434 and a starting address 436 (in memory 104) of the source buffer holding the continuation data segment 116n being described.", "Continuation part 118n also includes the starting address 438 (in memory 104) for the destination buffer for writing back the results of the security operation for the corresponding data bits of the continuation data segment 116n.", "As illustrated in FIG.", "4c, for the embodiment, the base part 118a of a descriptor 118 for a hashing operation includes a next descriptor/part address 442 identifying the starting word location in memory 104 where the next part 118n of the descriptor 118 or the base part 118a of a next descriptor 118 is stored.", "The residual unused least significant bits are employed to facilitate identification of the part as being a base part 118a of a descriptor 118, and the next descriptor/part address information is valid, and may be acted on by the security subsystem 106.Base part 118a for a hashing operation also includes a buffer size 444 and a starting address 446 (in memory 104) of the source buffer holding the base data segment 116a being described.", "Base part 118a also includes the starting address 448 (in memory 104) for the destination buffer for writing back the results of the security operation for the corresponding data bits of the base data segment 116a.", "Additionally, base part 118a for a hashing operation also includes mode 450 specifying the type of hashing operation, e.g.", "MD5 or SHA-1, to be performed, and descriptor identifier 452 of the descriptor.", "Further, base part 118a of a hashing operation also describes at least four chaining variable 454-460, for the hashing operation, and for the SHA-1 mode of operation, a fifth chaining variable 462.For a MD5 hashing operation, base part 118a also describes the “must write filer data” 462-464 of the hashing operation.", "As illustrated in FIG.", "4d, for the embodiment, the constitution of a continuation part 118n of a descriptor 118 for a hashing operation is the same as a continuation part 118n of a descriptor 118 for a hashing operation.", "Continuation part 118n of a descriptor 118 for a hashing operation includes a next descriptor/part address 472 identifying the starting word location of memory 104 where the next part 118n of the descriptor 118 or the base part 118a of a next descriptor 118 is stored.", "The residual unused least significant bits are employed to facilitate identification of the part as being a continuation of a descriptor 118, and the next descriptor/part address information is valid, and may be acted on by the security subsystem 106.Continuation part 118n of a descriptor 118 of a hashing operation also includes a buffer size 474 and a starting address 476 (in memory 104) of the source buffer holding the continuation data segment 116n being described.", "Continuation part 118n also includes the starting address 478 (in memory 104) for the destination buffer for writing back the results of the security operation for the corresponding data bits of the continuation data segment 116n.", "Security Subsystem FIG.", "5 illustrates security subsystem 106 of the present invention in further details, in accordance with one embodiment.", "As illustrated, for the embodiment, security subsystem 106 includes controller 502, registers 504, data transfer unit 506 and security engines 122, coupled to each other as shown.", "Data transfer unit 506 is employed to facilitate receipt of instructions from control processor 102 to perform security operations for various data objects 116, access and receipt of the various parts of the descriptors 118 of the various data objects 116, access and receipt of the various data segments of the various data objects 116, and write back of the results of the various security operations.", "One embodiment of data transfer unit 506 is described in the aforementioned Ser.", "No.", "______ copending and incorporated by reference U.S. patent application.", "In alternate embodiments, other data interfaces may be employed instead.", "Registers 504 include a number of collections, with each collection employed to store a fetched descriptor, e.g.", "one collection to store the descriptor of a DES operation to be or being performed, and another collection to store the descriptor of a hashing operation to be or being performed.", "In one embodiment, two collections of registers, with one collection dedicated to support a DES operation, and another collection dedicated to support a hashing operation, are provided.", "For the embodiment, registers 504 also include a number of collections of control registers, one collection each for each security operation concurrently supported, to facilitate control processor 102 in specifying for security subsystem 106 a number of general operation parameters for performing the corresponding security operation.", "In one embodiment, two such collections, one for a DES operation and another for a hashing operation, are supported.", "The content and meaning of these control parameters for one embodiment is described in further detail below referencing FIG.", "6a.", "For the embodiment, registers 504 also include a number of collections of status registers, one collection each for each security operation concurrently supported, to facilitate appraising control processor 102 of the current status of security subsystem 106 for the corresponding security operation.", "In one embodiment, two such collections, one for DES operation and another for hashing operation are supported.", "The content and meaning of these status for one embodiment is described in further detail below referencing FIG.", "6b.", "Registers 504 may be implemented via any one of a number of techniques known in the art.", "In one embodiment, a multi-port addressable memory unit is employed to implement all registers 504 in a single storage unit.", "Security engines 106 are employed to perform security operations of corresponding types.", "In one embodiment, one security engine coupled with a data traffic router for performing various types of DES operations, ECB, CBC and CFB (see FIG.", "7 where data traffic router is shown as element 702), one security engine for performing MD5 hashing operations, and one security engine for performing SHA-1 hashing operations are provided.", "Any one of a number of implementations known in the art may be employed to implement the various security engine cores, i.e.", "the security engine core for performing DEA ECB, CBC and CFB operations, the security engine core for performing MD5 hashing operations, and the security engine core for performing SHA-1 hashing operations.", "Their exact implementations are not essential aspects of the present invention.", "One embodiment of the data traffic router enabling a single DES security engine core to be provided for multiple modes of DES security operations will be further described below, referencing FIG.", "8.Controller 502 controls the operation of data transfer unit 506, registers 504 and security engines 106.The relevant operational flow for one embodiment will be described in further details below, referencing FIG.", "9.Control and Status Registers of the Security Subsystem As alluded to earlier, FIG.", "6a-6b illustrate one each of a collection of control registers 600 and a collection of status registers 620 for a security operation concurrently supported, e.g.", "a DES operation, or a hashing operation.", "Control registers 600 include register 602 for control processor 102 to globally disable or enable interrupt mode of operation for the security operation.", "Control registers 600 also include registers 604-608 for control processor 102 to instruct security subsystem 106 to interrupt control processor 102 upon completion of a data segment, upon completion of a data object or upon encountering an operation error while performing the security operation.", "Further, control registers 600 include registers 610-616 for control processor 102 to instruct security subsystem 106 to stop the security operation on completion of a data segment (including completion of a data object), halt operation altogether, to reset, or to start/continue for the security operation.", "Status registers 620 include registers 622-623 for conveying to control processor 102 a bad write address was encountered by security subsystem 106, and the remaining byte counts of the results of the security operation.", "Status registers 620 also include registers 624-628 for conveying to control processor 102 an interrupt is pending, where the interrupt is issued by security system 106 upon completion of a data segment, completion of a data object or encountering an error, for the security operation.", "Status registers 620 also include registers 630-632 for conveying to control processor 102 that processing for a data segment or a data object has been completed for the security operation.", "Further, status registers 620 include registers 634-638 for conveying to control processor 102 that the subsystem is “on”, its outputs are valid, or it is busy.", "Data Traffic Router FIG.", "8 illustrates data traffic router 702 of FIG.", "7 in further details, in accordance with one embodiment.", "As illustrated, data traffic router 702 includes a number of AND gates 802a-802c, a number of multiplexors 804a-804b, and a number of XOR gates 806a-806b, coupled to each other as shown.", "More specifically, AND gate 802a receives the data block (data_in) of the security operation and control variable A as inputs, and perform a logical AND operation on the inputs.", "The result of the logical AND operation is provided to XOR gate 806a, which also receives the output of AND gate 802b as its other input.", "XOR gate 806a performs the logical XOR operation on its inputs, and the output is provided as input to the DES security engine core.", "The output of AND gate 802b is generated based on the output of multiplexor 804a and control variable B.", "The output of multiplexor 804 is either the initial vector outputted from the earlier described initial vector registers, or the result of the DES operation on a prior data block, depending on the control variable C. In like manner, XOR gate 806b receives the output from the DES security engine core and the output of AND gate 802c as inputs, and performs a logical XOR operation on the inputs to produce the current result of the DES security operation (data_out).", "The output of AND gate 802c is generated based on the output of multiplexor 804c and control variable D. The output of multiplexor 804 is either the initial vector outputted from the earlier described initial vector registers, or the current input data block of the DES operation, depending on the control variable E. The setting of the control variables A through E, for the various modes of DES operations are given in the following table: Modes A B C D E ECB Encrypt/Decrypt 1 0 X 0 X CBC Encrypt 1 1 1/0 0 X CBC Decrypt 1 0 X 1 1/0 CFB Encrypt 0 1 1/0 1 0 CFB Decrypt 0/1 1/0 1/X 1 0 where X stands for “don't care”.", "Controller FIG.", "9 illustrates the relevant operational flow of controller 502 of FIG.", "5 in further details, in accordance with one embodiment.", "As shown, upon instructed by control processor 102, controller 502 of security subsystem 106 causes the base portion 118a of the addressed descriptor 118 to be loaded in the appropriate descriptor registers 504 for the specified security operation, e.g.", "the descriptor registers 504 for a DES operation or the descriptor registers 504 for a hashing operation, block 902.Next in accordance with the starting address location of the base data segment (and its size), controller 502 causes the data bits of the base data segment to be fetched via one or more fetches (for the embodiment of FIG.", "5, through data transfer unit 506, block 904.The number of fetches required depends on the size of the data bits of the data segment and the width of the data bus between memory 104 and security subsystem 106.As the data bits successively arrive, controller 502 determines if sufficient amount of data bits to form an atomic block of data bits for the security operation has been accumulated, block 906.For as long as there are insufficient amount of data bits to form an atomic block of data bits for the security operation has been accumulated, and the end of the currently described data segment has not been reached, block 912, fetching, i.e.", "block 904, continues.", "Once sufficient amount of data bits to form an atomic block of data bits for the security operation has been accumulated, controller 502 causes the data bits to be organized into a data block, and forwarded to the appropriate security engine, block 908.In due course (typically after a predetermined number of clock cycles), the result of the security operation on the provided data block becomes available.", "At such time, controller 502 causes the result to be written back to the storage locations of memory 104 as specified by the corresponding part of the descriptor 118, block 910.Concurrently, once an atomic data block is provided to the security engine for operation, controller 502 also continues operation back at block 912 to determine if the end of the currently described data segment has been reached.", "As described earlier, if the end of the currently described data segment has not been reached, controller 502 continues operation at block 904.If the end of the currently described data segment has been reached, controller 502 further determines if all data segments of the data object has been processed or if all processed, whether security operation for a next data object is to be started, block 914.For the embodiment, controller 502 makes the determination based on the earlier described next descriptor/part address and its associated valid bit denoting whether the next descriptor/part address is valid.", "If not all data segments of the data object has been processed or processing for a new data object is to be started, controller 502 causes a continuation part of the current descriptor or the base part of the next descriptor to be loaded into descriptor registers 504, block 916.In the former case, the descriptor of the data object is updated with the data segment related information describing a new data segment.", "Upon updating or reloading descriptor registers 504, controller 502 continues operation at block 904.Recall that the number of data bits of a data segment may be less than, equal to or greater than the size of the atomic block of the security operation, thus in the course of operation, at times, at block 912, after fetching all the data bits of a data segment in accordance to the starting address and the buffer size currently stored in descriptor registers 504, a quantity of data bits less than the size of the atomic data block of the security operation may remain.", "At such time, as described earlier, controller 502 determines if all data segments of the data object has been processed, block 914.If not, controller 504 causes a continuation part of the descriptor to loaded, updating the descriptor.", "For the embodiment, controller 504 also saves the address and size information of the previous part of the descriptor, e.g.", "in corresponding shadow registers (not shown).", "In one embodiment, controller 504 is equipped with 8 sets of shadow registers, enabling it to fetch as many as 8 data segments to form one atomic data block of the security operation.", "In other embodiments, more or less sets of shadow registers may be employed instead.", "Back at block 914, if indeed all data segments of the data object has been processed, the residual data bits are indicative of the fact that the data object has a size that is not modulo the size of the atomic data block of the security operation (64 bits in the case of a DES operation, and 512 bits in the case of hashing operation).", "For the embodiment, an error is returned, block 918.CONCLUSION AND EPILOGUE Thus, it can be seen from the above descriptions, an improved method and apparatus for performing security services/operations for subsystems of a SOC has been described.", "The novel scheme advantageously offloads the control processor of the SOC and enables the SOC to operate more efficiently.", "While the present invention has been described in terms of the foregoing embodiments, those skilled in the art will recognize that the invention is not limited to these embodiments.", "The present invention may be practiced with modification and alteration within the spirit and scope of the appended claims.", "Thus, the description is to be regarded as illustrative instead of restrictive on the present invention." ] ]	Patent_10469467
[ [ "Animal trap", "An animal trap (1) comprising a containment body (2) with at least two entrances, each entrance having a door (3) operable to close and contain an animal within the containment body, the entrances to the containment body being aligned so as to provide an animal to be trapped with a view through the containment body.", "There is also disclosed an animal trap system incorporating an animal trap having a transmitter unit (10) operable to transmit a first signal indicative of the presence of the trap." ], [ "1-14.", "(canceled) 15.An animal trap, comprising: a containment body having at least two entrances, each entrance having a door that closes to contain an animal within said containment body, said entrances aligned so as to provide an animal to be trapped with a view through said containment body; a release mechanism that initiates simultaneous closure of said doors; and a transmitter unit that transmits one or more signals.", "16.The animal trap of claim 15, further comprising an indicator unit that receives said one or more signals and provides one or more alerts.", "17.An animal containment system, comprising: a containment body having at least two entrances, each entrance having a door that closes to contain an animal within said containment body, said entrances aligned so as to provide an animal to be trapped with a view through said containment body; a release mechanism that initiates simultaneous closure of said doors; and a transmitter unit that transmits one or more signals.", "18.The containment system of claim 17, further comprising an indicator unit that receives said one or more signals and provides one or more alerts.", "19.A method that makes an animal trap, comprising: providing a containment body having at least two entrances, each entrance having a door that closes to contain an animal within said containment body, said entrances aligned so as to provide an animal to be trapped with a view through said containment body; coupling a release mechanism to said containment body, said release mechanism initiates simultaneous closure of said doors; and coupling a transmitter unit to said containment body, said transmitter unit transmits one or more signals.", "20.The method of claim 19, further comprising providing an indicator unit that receives said one or more signals and provides one or more alerts.", "21.A method that traps an animal, comprising: providing a containment body having at least two entrances, each entrance having a door that closes to contain an animal within said containment body, said entrances aligned so as to provide an animal to be trapped with a view through said containment body; closing said doors simultaneously using a release mechanism; and transmitting one or more signals using a transmitter unit.", "22.The method of claim 21, further comprising receiving said one or more signals using an indicator unit that provides one or more alerts.", "23.A dependent claim according to claim 15, 17, 19, or 21, wherein said transmitter transmits a first signal when said doors are open that indicates the presence of a set trap.", "24.A dependent claim according to claim 23, wherein said transmitter unit transmits a second signal when said doors are closed that indicates that a trap has been activated.", "25.A dependent claim according to claim 23, wherein said transmitter unit disables said first signal when said doors are closed.", "26.A dependent claim according to claim 24, wherein said transmitter unit disables said first signal and enables said second signal when said doors are closed.", "27.A dependent claim according to claim 15, 17, 19, or 21, wherein said transmitter unit transmits a unique identification code that identifies said transmitter unit in said one or more signals.", "28.A dependent claim according to claim 15, 17, 19, or 21, wherein said transmitter unit transmits said one or more signals at least once during a pre-determined time period.", "29.A dependent claim according to claim 16, 18, 20, or 22, wherein said indicator unit provides said one or more alerts under one or more of the following conditions: when said indicator unit does not receive a signal from said transmitter unit within a predetermined time period, or when said indicator unit receives a first signal from said transmitter unit indicative of the presence of a trap followed within a predetermined time period by a second signal indicative of the closure of said doors.", "30.A dependent claim according to claim 29, wherein said transmitter unit transmits a unique identification code that identifies said transmitter unit in said one or more signals and said one or more alerts further indicates the identity of said transmitter unit." ], [ "relates to an animal trap and more particularly to a humane animal trap.", "Animal traps are well known devices which may comprise a containment system, a closeable element and a trigger device operable to close the closeable element and secure an animal within the containment system.", "Poison or a device for killing the animal may also be included within the animal trap.", "Bait may be provided to entice the animal to go into or onto the trap.", "Conventional animal traps have only a single entrance and therefore present an animal with a “dead end”.", "Such traps are not attractive to animals such as rodents which like to have a free run through an obstacle and can be deterred from entering such traps because of the blind nature of the entrance.", "Animal traps are routinely checked to identify whether an animal has been trapped, to dispense with any trapped animals, to refresh a supply of bait within the trap and to check that the trap is not malfunctioning or has been triggered accidentally.", "Although such checks must be made routinely, a substantial period of time may elapse between checks, especially on large sites.", "If the trap is a humane trap—i.e.", "intended not to kill the animal upon containment, then there is a risk that the animal may die from starvation or under other stressful conditions a substantial period of time before the trap is due to be checked again.", "The containment of a dead animal is an invitation to other pests and diseases and comprises a further source of contamination for the surrounding environment.", "It is necessary, therefore, to check traps very frequently.", "The provision of an animal trap which is more readily accessible to animals and which results in a greater capture rate presents related problems in that the traps must, therefore, be checked even more regularly to ensure that any trapped animals are dealt with promptly.", "It is an object of the present invention to provide an animal trap system which seeks to overcome the problems associated with conventional animal traps such as those described above.", "Accordingly, one aspect of the present invention provides an animal trap comprising a containment body with at least two entrances, each entrance having a door operable to close and contain an animal within the containment body, the entrances to the containment body being aligned so as to provide an animal to be trapped with a view through the containment body.", "Preferably, a release mechanism is provided within the containment body to initiate closure of the doors.", "Conveniently, the release mechanism is a release mechanism common to all the doors and the doors are operable to close, upon release, simultaneously.", "The present invention also provides an animal trap system incorporating an animal trap having a transmitter unit operable to transmit a first signal indicative of the presence of the trap.", "Preferably, the transmitter unit is operable to transmit a second signal indicative of the activation of the trap.", "Conveniently, the transmitter unit includes an identification code generator to produce an indication code for transmission in the or each signal transmitted by the transmitter unit.", "Advantageously, the transmitter unit includes a switch to disable the first signal upon activation of the trap.", "Alternatively, the transmitter unit includes a switch to disable the first signal and enable the second signal upon activation of the trap.", "Preferably, the transmitter unit is operable to transmit periodically the or each signal.", "Conveniently, the period is at least once within a pre-determined time period.", "Advantageously, the predetermined time period is in the region of half an hour or less.", "Preferably, an indicator unit is provided having a receiver operable to receive signals from the or each transmitter unit, the indicator unit providing an alert signal when a signal is not received from a transmitter unit within a predetermined time period.", "Conveniently, an indication of the identity of a transmitter unit which has failed to transmit.", "Advantageously, the alert signal provides an indication of the identity of a transmitter unit upon receipt by the indicator unit of a transmission by that transmitter unit of a second signal after receipt of a different first signal.", "In order that the present invention may be more readily understood, embodiments thereof will now be described, by way of example, with reference to the accompanying drawings in which: FIG.", "1 is a perspective view of an animal trap embodying the present invention; FIG.", "2 is a cross-sectional side view of the animal trap in FIG.", "1 in an armed configuration; FIG.", "3 is a cross-sectional side view of the animal trap of FIG.", "1, a rodent having triggered the trap and the doors of the trap being partially closed; and FIG.", "4 is a cross-sectional side view of the animal trap of FIG.", "1, the trap being fully closed with a rodent contained inside.", "Referring to the Figures, an animal trap 1 embodying the present invention comprises a containment body 2 comprising a substantially rectangular tube, having two entrances at opposite ends of the tube.", "The containment body 2 and the doors 3 are preferably manufactured from a durable material resistant to wear and escape attempts by contained animals.", "A preferred material is sheet metal.", "The containment body has a hinged door 3 at either end.", "The doors 3 hinge about the top of each entrance.", "A release mechanism 4 and a platform 5 are located within the containment body 2—midway along the length of the containment body 2.As shown in FIG.", "2, when the doors 3 are flully hinged open, the bottom edge 6 of each door 3 rests on the release mechanism 4 within the containment body 2.The platform 5 depends from and is mechanically linked to the release mechanism 4 such that any movement of the platform 5 or pressure on the platform 5 is transmitted to the release mechanism and removes the support for the bottom edges 6 of the doors 3 which swing down under gravity to close the entrances thereby enclosing any animal within the containment body 2.Preferably, the hinges of the doors include an over-dead-centre or key-way locking mechanism so that the doors are automatically locked in the vertical position fully closing the containment body as shown in FIG.", "4.Appropriate release mechanisms, hinging mechanisms and locking mechanisms are well known to those of ordinary skill in the art.", "To release an animal, one of the doors is unlocked, the unlocking mechanism depending upon the locking mechanism implemented.", "If the doors are locked by a key-way mechanism, then the door is simply lifted such that a key protruding from the door is removed from a key-way slot into which the key has dropped upon closure of the door and rotated through 90° to the open condition.", "Bait can be provided on the platform 5 to entice rodents such as rats or mice into the animal trap 1 and onto or in proximity with the platform 5.The animal trap 1 embodying the present invention is specifically designed such that the animal is also enticed into the containment body 2 by the provision of a view of the environment beyond the trap.", "Since the animal can see through the trap, the animal is provided with confidence to approach the trap and move through the trap and into or onto the platform 5.Both entrances to the trap are not “blind”.", "This arrangement significantly increases the effectiveness of the animal trap.", "As a consequence of the increased effectiveness of the animal trap, such traps would need to be manually checked more regularly than conventional traps with blind entrances.", "However, such manual checking is time consuniing and expensive.", "Another aspect of the present invention therefore provides a simple and non-expensive alerting system for advising when an animal has been caught or when a trap or the system has malfunctioned or been accidentally triggered.", "A preferred embodiment of the alerting system comprises a transmitter unit 10 fixed to each of the animal traps 1 and a central indicator unit (not shown) located, for example, in a caretaker's or a security guard's office.", "The indicator unit incorporates a radio receiver but no transmitter.", "The transmitter units 10 are mounted on the animal traps 2 by a pair of electrically conductive lugs (not shown) which protrude from the top surface of the containment body.", "Corresponding holes are provided in the base of the transmitter unit 10 which registers on the lugs.", "Preferably, the lugs are provided in an asymmetric arrangement such that the transmitter unit 10 can only be located on the lugs in one orientation.", "Each transmitter unit 10 incorporates a power source such as a battery, a transmitter, an identification code generator, a modulator and a switch to alter the state of the modulator or the identification code generator.", "When the transmitter unit 10 is fixed by the lugs onto the containment body 2 (which is electrically conductive), this completes an electrical circuit activating the transmitter causing the transmitter to send a first signal which is indicative of the presence of a trap.", "If the containment body is manufactured from an electrically non-conductive material, then a conductive track or wire is connected between the lugs to allow the power circuit of the transmitter unit 10 to be closed.", "Each transmitter unit 10 has a unique code set by the identification code generator which preferably comprises one or more DIL switches.", "The settings of the DIL switches determine the identification code.", "The transmitted signals are modulated by the modulator to include the identification.", "code for each transmitter unit thereby enabling the identification of a transmitter unit upon receipt of the signal.", "When an animal enters a trap 1 and the doors 3 close behind it, a magnetic element 11 mounted on the top edge 12 of the door 3 adjacent the transmitter unit 10 moves into close proximity with the transmitter unit 10 and triggers a reed switch or the like within the transmitter which causes the modulator to change from transmitting the first signal which is indicative of the presence of the trap to transmit a second signal which is indicative that the trap has been activated (whether inadvertently or correctly by trapping an animal).", "The second signal still contains the identification code thereby allowing identification of the triggered trap.", "Each transmitter unit transmits the appropriate first or second signals periodically, preferably on a continuous basis at pseudo-random time intervals so that signals from other transmitter units are unlikely to collide with one another.", "The maximum time period between transmissions for each transmitter unit is set to be less than approximately half an hour.", "The radio receiver in the indicator unit receives radio signals transmitted by the respective transmitter units 10 in the vicinity.", "The indicator unit issues an error signal if no transmission is received from a particular transmitter unit 10 for a set period of time, for example half an hour.", "The indicator unit 10 continues to receive the first signals transmitted by the transmitter units 10 and only indicates that a trap 1 has been activated or a rodent caught when a transmitter unit stops transmitting the first signal and starts transmitting the second signal.", "Preferably, the indicator unit includes a digital display or the like which displays: the identity of the particularly transmitter associated with the trap which has been activated; an error indication; and a trap-activated indication.", "Thus, by viewing the indicator unit, an operator can determine which trap needs attention and what form of attention is required.", "The indicator unit also includes an audible warning signal generator to provide an audible warning that attention is required.", "It should be appreciated that this simple approach for checking that the animal traps and their transmitters are working correctly removes the need to interrogate individual traps for information and allows the design of the system to be greatly simplified.", "By using a combination of the above-described alerting system and an animal trap offering an animal a view of the environment beyond and through the trap a complete animal trap system is provided which operates effectively and which only requires maintenance or attention when an error is detected or an animal has been trapped thereby saving valuable man hours.", "The simplicity of the system also provides advantages over more complex systems which require interrogation of animal traps or their transmitters by a central transmitter or control unit.", "The provision of a transmitter unit 10 which is switchable to issue a first signal whilst the trap is armed and then start transmitting a second signal when the trap has been activated can be simplified further.", "Rather than switching to transmitting a second signal, the transmitter can be configured simply to stop transmitting on a continuous pseudo-random basis once the trap has been activated.", "The indicator unit would then be alerted that a particular transmitter is no longer transmitting.", "Whilst this would provide an indication that the trap has been activated, the switch turning the transmitter off, it could also be an indication that the transmitter has failed or the power source within the transmitter has run out.", "The disadvantage of this system is that it does not present the operator at the indicator unit with any information as to whether the transmitter has failed or whether the trap has been activated.", "In any event, the trap would need to be inspected and action taken either to restore the power supply, rearm the trap or deal with a trapped animal.", "In the present specification “comprise” means “includes or consists of” and “comprising” means “including or consisting of”.", "The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilized for realizing the invention in diverse forms thereof." ] ]	Patent_10469480
[ [ "Cloning vectors and method for molecular cloning", "The invention discloses a family of cloning vectors capable of cloning nucleic acid inserts of interest of long sizes, with low or reduced background and high efficiency of excision and method for preparing these vectors and library thereof.", "As example, it is disclosed a cloning vector comprising a construction vector segment (CS) and a replaceable segment (RS), wherein the size of CS is: 36.5 kb≦CS<38 kb, preferably CS is 37.5 kb, comprising lox recombination sites for Cre-recombination and/or att recombination sites for Gateway-like recombination, preferably also a background-reducing system selected from the group of: the ccdB gene, a lox sequence, the lacZ gene, and asymmetric site sequences recognized by restriction endonucleases." ], [ "1.A cloning bacteriophage vector comprising a construction segment (CS) and a replaceable segment (RS), wherein the size of CS is: X−1.2 kb≦CS<X; wherein X corresponding to the minimum size necessary to the vector for undergoing packaging.", "2.The cloning vector of claim 1, wherein the size of CS is: X−0.2 kb.", "3.A cloning bacteriophage vector comprising a construction segment (CS) and a replaceable segment (RS), wherein the size of CS is: 36.5 kb≦CS<38 kb.", "4.The cloning vector of claim 3, wherein CS is 37.5 kb.", "5.The cloning vector of claim 4, wherein CS is or comprises a foreign segment of 5.5 kb.", "6.The cloning vector of claims 1-5, wherein said bacteriophage is λ.", "7.The cloning vector of claim 1, wherein CS is a bacteriophage vector segment modified by comprising a plasmid segment at least comprising a ori.", "8.The cloning vector of claim 7, wherein said plasmid segment comprising a ori is selected from the group of: pBluescript (+), pUC, pBR322, and pBAC.", "9.The cloning vector of claim 1, wherein CS further comprises at least a selectable marker selected from the group consisting of: a DNA segment that encodes a product that provides resistance against otherwise toxic compounds; a DNA segment that encodes a product that suppresses the activity of a gene product; a DNA segment that encodes a product that is identifiable; a DNA segment that encodes a product that inhibits a cell function; a DNA segment that provides for the isolation of a desired molecule; a DNA segment that encodes a specific nucleotide recognition sequence which is recognized by an enzyme.", "10.The cloning vector of claim 9, wherein said selectable marker comprises at least a marker selected from the group consisting of an antibiotic resistance gene, an auxotrophic marker, a toxic gene, a phenotypic marker, an enzyme cleavage site, a protein binding site; and a sequence complementary to a PCR primer sequence.", "11.The cloning vector of claim 1, wherein said RS is flanked by two recombination sites, and said two recombination sites do not recombine with each other.", "12.The cloning vector of claim 11, wherein said two recombination sites are selected from the group consisting of attB, attP, attL, attR and derivatives thereof.", "13.The cloning vector of claim 11, wherein said two recombination sites flanking RS are lox recombination sites, which do not recombine with each other.", "14.The cloning vector of claims claim 1, wherein CS further comprising two lox recombinant sites, said two lox recombination sites being capable of recombine with each other.", "15.The cloning vector of claims 13-14, wherein the recombinant sites are loxP sites or derivatives thereof.", "16.The cloning vector of claims claim 1, wherein RS further comprising at least a background-reducing sequence.", "17.The cloning vector of claim 16, wherein said at least a background-reducing sequence is selected from the group consisting of: i) the ccdB gene, ii) the lacZ gene, iii) a lox sequence.", "18.The cloning vector of claim 17, wherein said iii) lox sequence is loxP or a derivative thereof.", "19.The cloning vector of claims claim 1, wherein RS is flanked by i) two homing endonuclease asymmetric recognition site sequences, which do not ligate with each other; or ii) two restriction asymmetric endonuclease cleavage sites sequences, which do not ligate with each other, recognizable by class IIS restriction enzymes.", "20.The cloning vector of claim 19, wherein said homing endonuclease is selected from the group consisting of: I-CeuI, PI-SceI, PI-PspI, and I-SceI.", "21.The cloning vector of claim 20, wherein said homing endonuclease asymmetric recognition site sequences are sequences from 18 to 39 bp.", "22.The cloning vector of claims claim 1, which is linear.", "23.The cloning vector of claim claim 1, wherein RS is replaced by a nucleic acid insert of interest.", "24.The cloning vector of claim 23, wherein said insert is selected from the group consisting of DNA, cDNA and RNA/DNA hybrid.", "25.The cloning vector of claim 23, wherein said insert is a long cDNA.", "26.The cloning vector of claim 23, wherein said insert is a full-length cDNA.", "27.The cloning vector of claim 26, wherein said full-length cDNA is a normalized and/or subtracted full-length cDNA.", "28.A method for cloning a nucleic acid insert of interest or for preparing a bulk nucleic acid library of interest, comprising the steps of: (a) preparing at least a cloning bacteriophage vector comprising a construction segment (CS) and a replaceable segment (RS), wherein the size of CS is: X−1.2 kb≦CS<X; wherein X corresponding to the minimum size necessary to the vector for undergoing packaging.", "(b) replacing RS with a nucleic acid insert of interest into the cloning vector obtaining the product according to claim 23; (c) allowing the in vivo or in vitro excision of the nucleic acid insert of interest or of the plasmid comprising the nucleic acid insert of interest; (d) recovering the (recombinant) plasmid carrying the nucleic acid insert of interest or a library of these plasmids.", "29.The method of claim 28, wherein between step b) and c) a step of amplification of the cloning vector is carried out.", "30.A bacteriophage cloning vector comprising a construction segment (CS) and a replaceable segment (RS), wherein said RS comprises at least the ccdB gene.", "31.A bacteriophage or plasmid cloning vector comprising a construction segment (CS) and a replaceable segment (RS), wherein said RS comprises at least a recombination site or a derivative thereof; or RS is flanked by two asymmetric site sequences, which do not ligate with each other, and are recognized by restriction endonucleases.", "32.The cloning vector of claims 30-31, wherein said bacteriophage is λ.", "33.The cloning vector of claim 30, wherein the size of the bacteriophage vector CS is: 32 kb≦CS≦45 kb.", "34.The cloning vector of claim 30, wherein CS is: 36.5 kb≦CS<38 kb.", "35.The cloning vector of claim 34, wherein CS is 37.5 kb.", "36.The cloning vector of claim 31, wherein said recombination site is lox recombination site or a derivative thereof.", "37.The cloning vector of claim 36, wherein said lox site is a loxP site or derivatives thereof.", "38.The cloning vector of claim 30, wherein the CS of said vector comprises a plasmid segment at least comprising an ori.", "39.The cloning vector of claim 38, wherein said plasmid segment comprising an ori is selected from the group consisting of :pBluescript(+), pUC, pBR322 and pBAC.", "40.The cloning vector of claim 30, wherein CS further comprises at least a selectable marker selected from the group consisting of: a DNA segment that encodes a product that provides resistance against otherwise toxic compounds; a DNA segment that encodes a product that suppresses the activity of a gene product; a DNA segment that encodes a product that is identifiable; a DNA segment that encodes a product that inhibits a cell function; a DNA segment that provides for the isolation of a desired molecule; a DNA segment that encodes a specific nucleotide recognition sequence which is recognized by an enzyme.", "41.The cloning vector of claim 40, wherein said selectable marker comprises at least a marker selected from the group consisting of an antibiotic resistance gene, an auxotrophic marker, a toxic gene, a phenotypic marker, an enzyme cleavage site, a protein binding site; and a sequence complementary to a PCR primer sequence.", "42.The cloning vector of claim 30, wherein said RS is flanked by two recombination sites, and said recombination sites do not recombine with each other.", "43.The cloning vector of claim 42, wherein said recombination sites are selected from the group consisting of attB, attP, attL, attR, and derivatives thereof.", "44.The cloning vector of claim 42, wherein said two recombination sites flanking RS are lox recombination sites or derivatives thereof and do not recombine with each other.", "45.The cloning vector of claim 44, wherein the lox recombination site is loxP or a derivative thereof.", "46.The cloning vector of claim 30, wherein CS further comprising two recombinant sites or derivatives thereof, these two recombination sites being capable of recombine with each other.", "47.The cloning vector of claim 46, wherein said two recombination sites are lox recombination sites or derivatives thereof.", "48.The cloning vector of claim 47, wherein said lox recombination site is loxP or a derivative thereof.", "49.The cloning vector of claim 30, wherein said RS further comprises the lacZ gene.", "50.The cloning vector of claim 30, wherein said asymmetric site sequences are i) two homing endonuclease asymmetric site sequences or ii) two restriction endonuclease cleavage sites sequences recognizable by class IIS restriction enzymes.", "51.The cloning vector of claim 50, wherein said restriction homing endonuclease capable of cutting said asymmetric site sequences is selected from the group consisting of: I-CeuI, PI-SceI, PI-PspI and I-SceI.", "52.The cloning vector of claims 50-51, wherein said homing endonuclease asymmetric recognition site sequences are sequences from 18 to 39 bp.", "53.The cloning vector of claim 30, which is linear.", "54.The cloning vector of claim 30, wherein RS is replaced by a nucleic acid insert of interest.", "55.The cloning vector of claim 54, wherein said insert is selected from the group consisting of DNA, cDNA and RNA/DNA hybrid.", "56.The cloning vector of claim 54, wherein said insert is a long cDNA.", "57.The cloning vector of claim 54, wherein said insert is a full-length cDNA.", "58.The cloning vector of claim 57, wherein said full-length cDNA is a normalized and/or subtracted full-length cDNA.", "59.A method for cloning a nucleic acid insert of interest or for preparing a bulk nucleic acid library of interest, comprising the steps of: (a) preparing at least a bacteriophage cloning vector comprising a construction segment (CS) and a replaceable segment (RS), said RS comprising the ccdB gene; (a) replacing RS with a nucleic acid insert of interest into the cloning vector; (c) allowing the in vivo or in vitro excision of the nucleic acid insert of interest or of the plasmid comprising the nucleic acid insert of interest; (d) recovering the (recombinant) plasmid carrying the nucleic acid insert of interest and lacking the ccdB gene or a library of these plasmids.", "60.The method of claim 59, wherein between the steps b) and c) an amplification step of the at least a cloning vector is carried out.", "61.A method for cloning a nucleic acid of interest or a bulk nucleic acid library of interest, comprising the step of: (a) preparing at least bacteriophage cloning vector comprising a construction segment (CS) and a replaceable segment (RS), wherein said RS comprises at least the ccdB gene; wherein RS is flanked by two recombination sites, and said two recombination sites do not recombine with each other; (b) replacing RS with a nucleic acid insert of interest into the cloning vector obtaining a product according to claims 54-58; (c) allowing the in vitro excision of the nucleic acid insert of interest by providing to the cloning vector of step b) at least a destination vector comprising a destination replaceable segment (RS) flanked by two recombination sites, said two recombination sites do not recombine with each other, and said destination RS comprises at least the ccdB gene; (d) recovering a recombinant plasmid carrying the nucleic acid insert of interest and lacking of the ccdB gene or a library of said plasmids.", "62.", "(The method of claim 61, wherein between the steps b) and c) an amplification step of the at least a plasmid is carried out.", "63.The method of claim 61, wherein said two recombination sites of both the cloning vector of step a) and the destination vector of step d) are derived from recombination site selected from the group consisting of attB, attP, attL, and attR or derivatives thereof.", "64.The method of claim 61, wherein said recombination sites flanking RS are lox recombination sites or derivatives thereof, and do not recombine with each other.", "65.The method of claim 64, wherein said lox recombination sites are loxP or derivatives thereof.", "66.A method for cloning a nucleic acid insert of interest or for preparing a bulk nucleic acid library of interest, comprising the steps of: (a) preparing at least a cloning vector comprising a construction segment (CS) and a replaceable segment (RS), said CS comprising two recombination sites which recombine with each other, and said RS comprising a recombination site capable of recombining with one of the two sites placed into CS; (b) replacing RS with a nucleic acid insert of interest into the cloning vector of step a); (c) allowing the in vivo or in vitro excision of the nucleic acid insert of interest or of the plasmid comprising the nucleic acid insert of interest; (d) recovering the (recombinant) plasmid carrying the nucleic acid insert of interest or a library of said plasmids.", "67.The method of claim 66, wherein said RS and CS recombination sites are lox recombination site or derivatives thereof 68.The method of claim 67, wherein said lox site is a loxP site or derivatives thereof.", "69.A method for cloning a nucleic acid insert of interest or for preparing a bulk nucleic acid library of interest, comprising the steps of: (a) preparing at least a cloning vector comprising a construction segment (CS) and a replaceable segment (RS), said RS being flanked by two endonuclease asymmetric recognition site sequences, which do not ligate with each other; (b) replacing RS with a nucleic acid insert of interest comprising two endonuclease asymmetric recognition site sequences flanking said insert of interest, said sequences being capable of ligating with the two sequences placed into the vector of step a), and obtaining a vector comprising the nucleic acid insert of interest; (c) allowing the in vivo or in vitro excision of the nucleic acid insert of interest or of the plasmid comprising the nucleic acid insert of interest; (d) recovering the (recombinant) excised plasmid or destination plasmid carrying the nucleic acid insert of interest or a library of said plasmids.", "70.The method of claim 69, wherein said endonuclease asymmetric recognition site sequences are: i) two homing endonuclease asymmetric recognition site sequences; or ii) two asymmetric restriction endonuclease cleavage site sequences recognizable by class IIS restriction enzymes.", "71.The method of claim 70, wherein said restriction homing endonucleases capable of cutting said asymmetric site sequences are selected from the group consisting of: I-CeuI, PI-Scei, PI-PspI and I-SceI.", "72.The method of claims 70, wherein said homing endonuclease asymmetric site sequences are from 18 to 39 bp.", "73.A method for cloning a nucleic acid insert of interest or preparing a bulk nucleic acid library of interest comprising the steps of: (a) preparing at least a cloning vector, comprising a construction segment (CS) and a replaceable segment (RS), wherein said CS is a bacteriophage vector comprising two lox recombination sites or derivatives thereof; (b) replacing RS with a nucleic acid insert of interest into the cloning vector; (c) packaging of the vector; (d) in vivo in liquid-phase infection of at least a cell expressing Cre-recombinase; (e) allowing the in vivo in liquid-phase excision of at least a plasmid comprising the nucleic acid insert of interest under condition of short-time growth or no growth of the excised plasmid; (ii.", ")(f) carrying out cellular lysis and recovery of the plasmid carrying the insert or of a library of said plasmids.", "74.The method of claim 63, further comprising the step of: (g) electroporating or transforming at least a cell, not expressing Cre-recombinase, making the plasmid(s) of step f) penetrating into said cell(s); (h) plating of cell(s) infected as at step g) and recovering the plasmid carrying the nucleic acid insert of interest or a library of said plasmids.", "75.The method of claim 72, wherein said bacteriophage is λ.", "76.The method of claim 73, wherein said lox recombination sites are loxP or derivatives thereof.", "77.The method of claim 73, wherein between the steps c) and d) an amplification of the packaged vector(s) is carried out.", "78.The method of claims 73-77, wherein the cloning vector of step a) is a cloning vector according to claims 1-22 or 30-53, and the product of step b) is a vector comprising the insert of interest according to claims 23-27 or 54-58.79.The method of claim 73, wherein the step e) is carried out in 0-3 hours at the temperature 20-45° C. 80.A method for cloning a nucleic acid insert of interest or for preparing a bulk nucleic acid library of interest comprising the step of: (a) preparing at least a cloning vector, comprising a construction segment (CS) and a replaceable segment (RS), wherein said CS is a bacteriophage vector segment comprising two lox recombination sites or derivatives thereof positioned at left and right side of said RS; (b) replacing RS with a nucleic acid insert of interest into the cloning vector; (c) in vitro packaging of the at least a bacteriophage cloning vector of step b) in presence of packaging extract; (d) extraction of bacteriophage cloning vector from the capside; (e) in vitro excision of the plasmid comprising the nucleic acid insert of interest from the vector in presence of Cre-recombinase; (f) recovery of said plasmid or library of plasmids.", "81.The method of claim 80, further comprising the step: (g) electroporating or transforming at least a cell, not expressing Cre-recombinase, making said plasmid entering into said cell; (h) plating the cell of step g) and recovering plasmid carrying the nucleic acid insert of interest or a library of said plasmids.", "82.The method of claims 80-81, wherein between the steps c) and d), an amplification step on plate of the bacteriophage is carried out.", "83.The method of claim 80, wherein the lox recombination sites are loxP or derivatives thereof.", "84.The method of claim 80, wherein said bacteriophage is λ.", "85.The method of claims 80-84, wherein the cloning vector of step a) is a cloning vector according to claims 1-22 or 30-53 and the insert of interest of step b) is according to claims 23-27 or 54-58.86.A bacteriophage cloning vector comprising a construction segment (CS) and a replaceable segment (RS), wherein said RS is flanked by two recombination sites, and said two recombinant sites do not recombine with each other.", "87.The cloning bacteriophage vector of claim 86, wherein said bacteriophage is λ.", "88.The cloning vector of claims 86-87, wherein said recombination sites are selected from the group consisting of attB, attP, attL, attR and derivatives thereof.", "89.The cloning vector of claim 86, wherein CS further comprises two lox recombination sites or derivatives thereof, said lox sites being capable of recombining with each other.", "90.The cloning vector of claim 89, wherein said lox recombination sites are loxP or derivatives thereof.", "91.The cloning vector of claim 86, wherein the size of the bacteriophage λ vector segment (CS) is: 32 kb≦CS≦45 kb.", "92.The cloning vector of claim 91, wherein CS is: 36.5 kb≦CS<38 kb.", "93.The cloning vector of claim 91, wherein CS is 37.5 kb.", "94.The cloning vector of claim 86, wherein the bacteriophage CS comprises a plasmid segment at least comprising an ori.", "95.The cloning vector of claim 94, wherein said plasmid segment comprising an ori is selected from the group consisting of: pBluescript(+), pUC, pBR322 and pBAC.", "96.The cloning vector of claim 86, wherein CS further comprises at least a selectable marker selected from the group consisting of: a DNA segment that encodes a product that provides resistance against otherwise toxic compounds; a DNA segment that encodes a product that suppresses the activity of a gene product; a DNA segment that encodes a product that is identifiable; a DNA segment that binds a product that modifies a substrate; a DNA segment that provides for the isolation of a desired molecule; a DNA segment that encodes a specific nucleotide recognition sequence which is recognized by an enzyme.", "97.The cloning vector of claim 96, wherein said selectable marker comprises at least a marker selected from the group consisting of an antibiotic resistance gene, an auxotrophic marker, a toxic gene, a phenotypic marker, an enzyme cleavage site, a protein binding site; and a sequence complementary to a PCR primer sequence.", "98.The cloning vector of claim 86, wherein RS further comprising at least a background-reducing sequence selected from the group consisting of: i) the ccdB gene, ii) the lacZ gene, iii) a lox sequence.", "99.The cloning vector of claim 98, wherein said lox sequence is loxP.", "100.The cloning vector of claim 86, wherein RS is flanked by i) two homing endonuclease asymmetric recognition site sequences, which do not ligate with each other; or ii) two asymmetric restriction endonuclease cleavage sites sequences recognizable by class IIS restriction enzymes.", "101.The cloning vector of claim 100, wherein said homing endonucleases capable of cutting said asymmetric site sequences are selected from the group consisting of: I-CeuI, PI-SceI, PI-PspI and I-SceI.", "102.The cloning vector of claims 100-101, wherein said homing endonuclease asymmetric site sequences are sequences from 18 to 39 bp.", "103.The cloning vector of claim 86, which is linear.", "104.The cloning vector of claim 86, wherein RS is replaced by a nucleic acid insert of interest.", "105.The cloning vector of claim 10, wherein said insert is selected from the group consisting of DNA, cDNA, RNA/DNA hybrid.", "106.The cloning vector of claim 104, wherein said insert is a long cDNA.", "107.The cloning vector of claim 104, wherein said insert is a full-length cDNA.", "108.The cloning vector of claim 107, wherein said full-length cDNA is a normalized and/or subtracted full-length cDNA.", "109.A method for cloning a nucleic acid insert of interest or for preparing a bulk nucleic acid library of interest, comprising the steps of: (a) preparing at least a cloning vector comprising a construction segment (CS) and a replaceable segment (RS), wherein said CS is a bacteriophage vector segment and RS is flanked by two recombination sites, and said two recombinant sites do not recombine with each other; (b) replacing said RS with a nucleic acid insert and obtaining the product of claims 105-108; (c) in vitro packaging the at least a bacteriophage cloning vector of step b); (d) allowing the in vitro excision of the nucleic acid insert(s) of interest by providing to the at least a cloning vector of step c) an at least a destination vector comprising a destination replaceable segment (RS) flanked by two recombination sites, and said two recombination sites do not recombine with each other; (e) recovering a recombinant plasmid carrying the nucleic acid insert of interest or a library of said plasmids.", "110.The method of claim 109, wherein said bacteriophage is λ.", "111.The method of claims 109-110, wherein said two recombination sites of both the cloning vector of step a) and the destination vector of step d) are derived from recombination sites selected from the group consisting of attB, attP, attL, attR and derivatives thereof.", "112.The method of claim 109, wherein said two recombinant sites of both step a) and step d) are lox recombination sites or derivatives thereof, which do not recombine each other.", "113.The method of claim 112, wherein said lox recombination site is loxP or derivative thereof.", "114.The method of claim 109, wherein said RS of the destination vector of step d) further comprises at least the ccdB gene 115.The method of claim 109, wherein the CS of the vector cloning further comprises a selectable marker.", "116.The method of claim 109, further comprising the steps of: (f) providing an at least a second destination vector comprising a destination replaceable segment (RS) flanked by two recombination sites, and said two recombination sites do not recombine with each other, in contact with the plasmid(s) of step (e).", "117.The method of claim 109, further comprising a step of 1) electroporating at least a cell making the plasmid obtained in step e) or f) entering said cell; and 2) plating the cell of step 1) and recovering of the plasmid or plasmids carrying the insert 118.A kit comprising at least a cloning vector or at least a library of vectors according to claim 1.119.A method for preparing at least one normalized and/or subtracted library comprising the steps of: (f) providing at least an excised plasmid or a destination plasmid prepared according to claim 28; (g) providing the plasmid of step b) to a pool of nucleic acid targets; (h) removing the hybrids; (i) collected the normalized and/or subtracted nucleic acid targets.", "120.The method of claim 119, wherein the plasmid of step b) is treating by 1) making at least a nick into only one strand of the double stranded plasmid(s); 2) removing the plasmid fragments which have been nicked; 3) collecting the single strand(s) which has not been nicked; 4) applying the steps (c)-(d).", "121.The method of claim 120, wherein the nick is introduced by using the GeneII protein.", "122.The method of claim 120, wherein the strand which has been nicked is removed by an esonuclease.", "123.The method of claim 122, wherein the esonuclease is ExoIII.", "124.A method for preparing at least a normalized and/or subtracted library comprising the steps of: (a) providing at least a cloning bacteriophage vector comprising a construction segment (CS) and a replaceable segment (RS), wherein the size of CS is: X−1.2 kb≦CS<X; wherein X corresponding to the minimum size necessary to the vector for undergoing packaging; wherein the CS of the vector comprises a F1 ori (b) replacing RS with a nucleic acid insert of interest according to claims 23-27; (c) adding an helper phage and producing a number of a single strand plasmid vector copies; (d) providing the copies of step c) to a pool of nucleic acids targets; (e) removing the hybrids; (f) collected the normalized and/or subtracted nucleic acid targets.", "125.A bacteriophage vector comprising a bacterial artificial chromosome (pBAC) or a segment thereof comprising at least an origin of replication (ori).", "126.The bacteriophage of claim 125, wherein the bacteriophage is □.bacteriophage.", "127.The bacteriophage of claim 125-126, wherein the pBAC or segment thereof further comprises: a site into which an DNA fragment can be cloned; at least one pair of inducible excision-mediating sites flanking the site into which the DNA fragment can be cloned, the excision-mediating sites defining an excisable fragment that comprises the site into which the DNA fragment can be cloned.", "128.The bacteriophage of claim 127, wherein the pair of excision-mediating sites are FRT sites.", "129.The bacteriophage of claim 127, wherein the pair of excision-mediating sites comprise a sequence as shown in SEQ ID NO:45.130.The bacteriophage of claim 125, wherein the ori is an ori capable of maintaining the plasmid at single copy.", "131.The bacteriophage of claim 125, wherein the pBAC or segment thereof further comprises an inducible origin of replication.", "132.The bacteriophage of claim 131, wherein the inducible origin of replication is oriV.", "133.The bacteriophage of claims 125-126, comprising a bacterial artificial chromosome (pBAC) or a segment thereof comprising an inducible origin of replication.", "134.The bacteriophage of claim 125, comprising at least two recombination sites selected from the following: (a) two recombination sites, wherein either site does not recombine with the other; (b) two lox recombination sites, wherein either site is capable of recombining with each other; (c) two homing endonuclease asymmetric recognition site sequences; (d) two restriction asymmetric endonuclease cleavage site sequences, wherein either site sequence does ligate with the other, recognizable by class IIS restriction enzymes.", "135.The bacteriophage of claim 134, wherein the two recombination sites (a) are selected from the group consisting of attB, attP, attL, attR and derivatives thereof.", "136.The bacteriophage of claim 134, wherein the two recombination sites (a) are lox recombination sites derivative, which do not recombine with each other.", "137.The bacteriophage of claim 134, wherein the two recombination sites (b) are loxp sites.", "138.The bacteriophage of claim 134, wherein the two homing endonuclease site sequences (c) are selected from the group consisting of: I-CeuI, PI-SceI, PI-PspI, and I-SceI.", "139.The bacteriophage of claim 125, further comprising at least a background-reducing sequence.", "140.The bacteriophage of claims 139, wherein the at least background-reducing sequence is selected from: a) the ccdB gene; b) the lacZ gene; c) a lox sequence.", "141.A method for cloning a nucleic acid of interest or for preparing a bulk nucleic acid library of interest comprising the steps of: (a) preparing a bacteriophage cloning vector according to claim 125; (b) inserting a nucleic acid of interest into the bacteriophage cloning vector; (c) allowing the in vivo or in vitro excision of the BAC plasmid comprising the nucleic acid insert of interest; (d) recovering the BAC plasmid carrying the nucleic acid insert of interest or a library of these BAC plasmids." ], [ "<SOH> BACKGROUND ART <EOH>Efficient genomic and cDNA cloning vectors are important tools in molecular genetic research, because high quality, representative libraries are rich sources for the analysis of many genes.", "Full-length cDNAs are the starting material for the construction of the full-length libraries (for example, the RIKEN mouse cDNA encyclopedia, RIKEN and Fantom Consortium, “Functional annotation of a full-length mouse cDNA collection”, Nature, Feb. 8, 2001, Vol.409:685-690).", "In contrast to standard cloning techniques, full-length cDNA cloning has the inherent risk of under representation or absence of long clones from the libraries, and cDNAs deriving from very long mRNAs are not cloned if the capacity of the vector is not sufficient.", "Available plasmid cloning vectors show bias for short cDNAs: shorter fragments are cloned more efficiently than longer ones when competing during ligation and library amplification steps.", "Although plasmid electroporation does not show relevant size bias, during circularization of plasmid molecules in the ligation step, in a mixed ligation reaction, short cDNAs are ligated more efficiently than longer cDNAs (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press, Molecular Cloning, NY, USA).", "Cloning vectors derived from bacteriophage have been disclosed as particularly useful for cloning, propagation of DNAs and for library construction.", "Ligated mixtures of insert and bacteriophage vector DNAs can be efficiently packaged in vitro and introduced into bacteria by infection.", "Bacteriophage vectors allow cloning of cDNAs sequences, however, the final product for large-scale sequencing should be a plasmid for large-scale colony picking, propagation, DNA preparation and sequencing reactions (Shibata et al., 2000, Genome Res.", "10: 1757-1771).", "Cloning vectors for automatic plasmid excision should have a capacity for wide-range cDNA cloning, that is including cDNAs as short as 0.5 Kb and as long as 15 Kb, which are visible on agarose gel when using trehalose during the first strand cDNA synthesis (Carninci et al., 1998, Proc.", "Natl.", "Sci.", "USA, 95:520-524).", "There are a number of bacteriophage vectors allowing whole library bulk excision, but they are not optimal in terms of cloning size or bulk excision protocol.", "Examples of plasmid excision from bacteriophage vector having a cloned insert were obtained with the λ-Zap II (Short et al., 1988, Nucl.", "Acids Res., 16:7853-7600).", "However, the bulk excision from λ-Zap II shows size bias towards short inserts when using a mixed sample like a cDNA library, which contains both short and long clones.", "Using λ-Zap II, long and rare cDNAs are difficult to obtain.", "Other vectors designed for cDNA cloning and plasmid excision like the λ-Lox derivatives (Palazzolo M. et al., 1990, Gene, 88: 25-36), λ-YES (Elledge et al., 1991, Proc.", "Natl.", "Acad.", "Sci.", "USA., 88: 1731-5) and λ-Triplex™ (CLONTECHniques, January 1996), accept cDNAs that do not exceed 9˜10 Kb.", "Alternatively, vectors for genomic libraries construction and Cre-lox mediated plasmid excision accept inserts longer than 7 Kbp, such as λ PS (Nehls et al., 1994a, Biotechniques, 17: 770-775), λpAn (Holt et al., 1993, Gene, 133: 95-97), λGET (Nehls et al., 1994b, Oncogene, 9: 2169-2175), λ-MGU2 (Maruyama and Brenner, 1992, Gene, 120: 135-141) and a vector based on Tn1721 excision system, λRES (Altenbucher, J, 1993, Gene, 123: 63-68).", "However, these vectors do not allow the preparation of wide range size cDNA libraries.", "Only among the λSK series there were some vectors with calculated capacity between 0.2 to 15.4 Kb (Zabarovski et al., 1993, Gene, 127: 1-14), which would be suitable for wide-range size cDNA cloning purpose.", "Unfortunately, the rudimental excision system of λSK is based on simple restriction digestion, which causes internal cleavage of cDNA clones and probably this is the reason why these vectors are not commonly used for cDNA cloning.", "Japanese patent application having publication number P2000-325080A, discloses a modified λ PS vector.", "The new vector, indicated with the term λ-FLC-1, comprised a 6 kb nucleic acid sequence (stuffer II) in the left arm of the λ PS vector so that the size of the vector, without considering the cDNA of interest, was 38 kb.", "This modified λ PS vector was described as being able to insert broad range size of cDNAs.", "The λ-FLC-1, even if useful for generic (or “standard”) large size cDNA libraries, still shows a bias for short and not full-length cDNAs, so that very long, rare and important full-length cDNAs are difficult to obtain, in particular, in case of strongly normalized and/or subtracted cDNA libraries.", "A further problem in the art refers to the efficiency of bulk excision recombination mechanism.", "Bulk cDNAs (cDNA library), that is a library of cDNA comprising a wide range size of cDNAs, short, medium and long ones, are inserted in cloning vectors.", "These inserts are then transferred in other functional or specialized vectors that have desired characteristics, such as expression vectors.", "This transfer is called subcloning.", "The functional or specialized vectors used for subcloning DNA segments are functionally diverse.", "These include but are not limited to: vectors for expressing genes in various organisms; for regulating gene expression; for providing tags to aid in protein purification or to allow tracking of proteins in cells; for modifying the cloned DNA segment (e.g., generating deletions); for the synthesis of probes (e.g, riboprobes); for the preparation of templates for DNA sequencing; for the identification of protein coding regions; for the fusion of various protein-coding regions; to provide large amounts of the DNA of interest, etc.", "It is common that a particular investigation will involve subcloning the DNA segment of interest into several different specialized vectors.", "Traditional subcloning methods, using restriction enzymes and ligase, are time consuming and relatively unreliable.", "The use of recombinase recognition systems using specific recombinase recognition sequences have been proposed and they are known as Cre-lox (Palazzolo et al., 1990, Gene, 88: 25-36) and Gateway™ (Life Technologies Catalogue; Walhout A. J. M., et al., 2000, Methods in enzymology, Vol.328: 575-592; and U.S. Pat.", "No.", "5,888,732).", "The Cre-recombinase solid-phase in vivo excision requires infection of the amplified cDNA library into a bacterial strain, which constitutively express the Cre-recombinase, for instance BNN132 (Elledge et al., 1991, Proc.", "Natl.", "Acad.", "Sci.", "USA., 88: 1731-5).", "However, this is not recommended because of low plasmid yield (Palazzolo et al., 1990, as above) and plasmid instability (Summers et al., 1984, Cell, 36: 1097-1103): in fact, Cre-recombinase is constitutively expressed causing formation of plasmid dimers/multimers leading to high proportion of plasmid-free cells (Summers et al., 1984, as above), impairing the sequencing efficiency.", "The Gateway excision is an alternative system to the Cre-lox excision.", "According to the general Gateway™ system, an insert donor vector carrying a DNA of interest (insert) and a pair of recombinant sites different from each other, recombines with a donor vector comprising a subcloning vector and a pair of recombinant sites different from each other, but able to recombine with the insert donor vector recombination sites.", "The final product is a subclone product carrying the DNA of interest (insert) and a byproduct.", "The recombinant sites are attB, attP, attL and attR.", "However, the Gateway™ system shows a bias for short cDNA; long cDNAs are obtained with low efficiency (Michael A. Brasch, slide “Gateway cloning of attB-PCR products”, GIBCOBRL® Technical Seminar, “Gateway Cloning Technology”, Life Technologies™, 1999).", "Another further problem in the cloning system consists in the presence of background, which is due to environmental DNA contamination and to subcloning process byproducts, that is a non recombinant plasmids (plasmids without the DNA of interest).", "It is instead highly desirable having a background-cutting cloning system, able to eliminate completely or having a little background.", "Some background-cutting strategies have been proposed in the art.", "Walhout et al.", "(as above), for example, reports that the Gateway™ vectors, attP1-attP and attR1-attR2, also contain between the att sites the ccdB gene (Bernard P. and Couturier M., 1992, J. Mol.", "Biol., 226:735-746), whose protein product interferes with DNA gyrase.", "After recombination, only the plasmids that have lost the ccdB gene (and which are recombinant) can grow in E.coli strains not mutated for gyrA, therefore providing a selective advantage.", "Plasmids carrying the gene ccdB can propagate only in specific E.coli strain, DB3.1, which carries a mutation in gyrA gene conferring resistance to ccdB (Walhout et al., as above).", "Therefore, this kind of recombination is limited to plasmids, since other vectors for instance λ substitution vectors used in cloning systems cannot grow and replicate in cells like DB3.1, which miss the recA protein (the recA product is required for the growth of substitution-type bacteriophage λ: Sambrook et al., 1989).", "In conclusion, there is the need in his field of the art of providing of vectors having the characteristics of: i) being size bias free and allowing the preparation of “size balanced” comprising very long, rare full-length cDNAs; ii) capable of improved recombination mechanism; and iii) able of background cutting.", "The cloning vectors available in the state of the art, fail to satisfy the above characteristics.", "The invention disclosed in the present application is addressed to solve the problems in the art." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present inventors provide a new family of vectors capable of cloning nucleic acids of wide range size and preferably very long ones, with high efficiency of excision and reduced background and contamination.", "Also provided are methods of cloning and for preparing bulk library using such vectors.", "According to a first embodiment, the invention provides a cloning vector comprising a construction vector segment (CS) and a replaceable segment (RS), wherein the size of CS is: 36.5 kb≦CS<38 kb, preferably CS is 37.5 kb.", "The construction vector segment preferably is made or comprise a bacteriophage λ vector fragment.", "The replaceable vector segment (RS) represents the segment, which is replaced by the nucleic acid insert of interest, which one intends to clone.", "It has been surprisingly found that a cloning vector with this size is capable of preferably inserting cDNA of very long sizes, and it is therefore particularly advantageous for cloning very full-length cDNAs.", "This vector overcomes the problem in the art of existing vector λ-FLC having a construction vector segment of 38 kb, which showed a strong bias for short size cDNAs (see Table1).", "The selection of a particular advantageous size of the vector for the preparation of full-length cDNAs libraries can also be applied to bacteriophage other than λ.", "Accordingly, the present invention also relates to a cloning bacteriophage vector comprising a construction segment (CS) and a replaceable segment (RS), wherein the size of CS is: X−1.2 kb≦CS<Xkb; X (expressed in kb) corresponding to the minimum size necessary to the bacteriophage vector for undergoing packaging.", "The size of CS is preferably: X−0.2 kb.", "The present invention also relates to a bacteriophage vector, preferably a λ, comprising a bacterial artificial chromosome (pBAC) or a segment thereof comprising at least an origin of replication (ori).", "This vector can also comprise: a site into which a DNA fragment can be cloned; and a pair of inducible excision-mediating sites defining an excisable fragment that comprises the site into which the DNA fragment can be cloned.", "The pair of excision-mediating sites are preferably FRT sites.", "This vector may further comprise an inducible origin of replication, preferably oriV.", "The cloning vectors according to the invention are capable of carrying out plasmid or nucleic acid insert excision using known recombination systems, for example the Cre-lox and/or Gateway™ system.", "The vectors of the invention can also comprise a background-reducing system, as ccdB gene, a lox sequence or the lacZ gene or asymmetric site sequences recognized by restriction endonuclease.", "The invention also relates to cloning method using the above vectors.", "According to another embodiment, the invention relates to a system for reducing background or contamination by providing a cloning vector comprising a background-reducing sequence like ccdB gene and/or a lox sequence comprised into RS segment of the vector of the invention, or in case of the Gateway™ system into the RS segment of a destination or receiving vector.", "RS of phage or plasmid vectors can also be flanked by two asymmetric site sequences recognized by restriction endonuclease.", "The invention also relates to a method for reducing background or contamination by using these vectors.", "The invention also relates to methods for efficient excision of plasmid or nucleic acid of interest providing improved Cre-recombinase or Gateway™ system using the vectors according to the invention.", "Preferably, the present invention relates to method for the preparation of bulk of long or full-length cDNA libraries, by using the vectors according to the invention.", "The present invention also relates to a kit comprising at least a cloning vector or at least a library of vectors according to the invention.", "The present invention further relates to a method for preparing at least a normalized and/or subtracted library comprising using a plasmid vector obtained with the excision method according to the invention or destination vector according to the invention, preferably reduced at single strand, as normalization and/or subtraction driver." ], [ "FIELD OF THE INVENTION The present invention relates to recombinant DNA technology.", "In particular, it is disclosed a novel cloning vector family and in vitro and in vivo method for cloning of nucleic acids of interest.", "BACKGROUND ART Efficient genomic and cDNA cloning vectors are important tools in molecular genetic research, because high quality, representative libraries are rich sources for the analysis of many genes.", "Full-length cDNAs are the starting material for the construction of the full-length libraries (for example, the RIKEN mouse cDNA encyclopedia, RIKEN and Fantom Consortium, “Functional annotation of a full-length mouse cDNA collection”, Nature, Feb. 8, 2001, Vol.409:685-690).", "In contrast to standard cloning techniques, full-length cDNA cloning has the inherent risk of under representation or absence of long clones from the libraries, and cDNAs deriving from very long mRNAs are not cloned if the capacity of the vector is not sufficient.", "Available plasmid cloning vectors show bias for short cDNAs: shorter fragments are cloned more efficiently than longer ones when competing during ligation and library amplification steps.", "Although plasmid electroporation does not show relevant size bias, during circularization of plasmid molecules in the ligation step, in a mixed ligation reaction, short cDNAs are ligated more efficiently than longer cDNAs (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press, Molecular Cloning, NY, USA).", "Cloning vectors derived from bacteriophage have been disclosed as particularly useful for cloning, propagation of DNAs and for library construction.", "Ligated mixtures of insert and bacteriophage vector DNAs can be efficiently packaged in vitro and introduced into bacteria by infection.", "Bacteriophage vectors allow cloning of cDNAs sequences, however, the final product for large-scale sequencing should be a plasmid for large-scale colony picking, propagation, DNA preparation and sequencing reactions (Shibata et al., 2000, Genome Res.", "10: 1757-1771).", "Cloning vectors for automatic plasmid excision should have a capacity for wide-range cDNA cloning, that is including cDNAs as short as 0.5 Kb and as long as 15 Kb, which are visible on agarose gel when using trehalose during the first strand cDNA synthesis (Carninci et al., 1998, Proc.", "Natl.", "Sci.", "USA, 95:520-524).", "There are a number of bacteriophage vectors allowing whole library bulk excision, but they are not optimal in terms of cloning size or bulk excision protocol.", "Examples of plasmid excision from bacteriophage vector having a cloned insert were obtained with the λ-Zap II (Short et al., 1988, Nucl.", "Acids Res.,16:7853-7600).", "However, the bulk excision from λ-Zap II shows size bias towards short inserts when using a mixed sample like a cDNA library, which contains both short and long clones.", "Using λ-Zap II, long and rare cDNAs are difficult to obtain.", "Other vectors designed for cDNA cloning and plasmid excision like the λ-Lox derivatives (Palazzolo M. et al., 1990, Gene, 88: 25-36), λ-YES (Elledge et al., 1991, Proc.", "Natl.", "Acad.", "Sci.", "USA., 88: 1731-5) and λ-Triplex™ (CLONTECHniques, January 1996), accept cDNAs that do not exceed 9˜10 Kb.", "Alternatively, vectors for genomic libraries construction and Cre-lox mediated plasmid excision accept inserts longer than 7 Kbp, such as λ PS (Nehls et al., 1994a, Biotechniques, 17: 770-775), λpAn (Holt et al., 1993, Gene, 133: 95-97), λGET (Nehls et al., 1994b, Oncogene, 9: 2169-2175), λ-MGU2 (Maruyama and Brenner, 1992, Gene, 120: 135-141) and a vector based on Tn1721 excision system, λRES (Altenbucher, J, 1993, Gene, 123: 63-68).", "However, these vectors do not allow the preparation of wide range size cDNA libraries.", "Only among the λSK series there were some vectors with calculated capacity between 0.2 to 15.4 Kb (Zabarovski et al., 1993, Gene, 127: 1-14), which would be suitable for wide-range size cDNA cloning purpose.", "Unfortunately, the rudimental excision system of λSK is based on simple restriction digestion, which causes internal cleavage of cDNA clones and probably this is the reason why these vectors are not commonly used for cDNA cloning.", "Japanese patent application having publication number P2000-325080A, discloses a modified λ PS vector.", "The new vector, indicated with the term λ-FLC-1, comprised a 6 kb nucleic acid sequence (stuffer II) in the left arm of the λ PS vector so that the size of the vector, without considering the cDNA of interest, was 38 kb.", "This modified λ PS vector was described as being able to insert broad range size of cDNAs.", "The λ-FLC-1, even if useful for generic (or “standard”) large size cDNA libraries, still shows a bias for short and not full-length cDNAs, so that very long, rare and important full-length cDNAs are difficult to obtain, in particular, in case of strongly normalized and/or subtracted cDNA libraries.", "A further problem in the art refers to the efficiency of bulk excision recombination mechanism.", "Bulk cDNAs (cDNA library), that is a library of cDNA comprising a wide range size of cDNAs, short, medium and long ones, are inserted in cloning vectors.", "These inserts are then transferred in other functional or specialized vectors that have desired characteristics, such as expression vectors.", "This transfer is called subcloning.", "The functional or specialized vectors used for subcloning DNA segments are functionally diverse.", "These include but are not limited to: vectors for expressing genes in various organisms; for regulating gene expression; for providing tags to aid in protein purification or to allow tracking of proteins in cells; for modifying the cloned DNA segment (e.g., generating deletions); for the synthesis of probes (e.g, riboprobes); for the preparation of templates for DNA sequencing; for the identification of protein coding regions; for the fusion of various protein-coding regions; to provide large amounts of the DNA of interest, etc.", "It is common that a particular investigation will involve subcloning the DNA segment of interest into several different specialized vectors.", "Traditional subcloning methods, using restriction enzymes and ligase, are time consuming and relatively unreliable.", "The use of recombinase recognition systems using specific recombinase recognition sequences have been proposed and they are known as Cre-lox (Palazzolo et al., 1990, Gene, 88: 25-36) and Gateway™ (Life Technologies Catalogue; Walhout A. J. M., et al., 2000, Methods in enzymology, Vol.328: 575-592; and U.S. Pat.", "No.", "5,888,732).", "The Cre-recombinase solid-phase in vivo excision requires infection of the amplified cDNA library into a bacterial strain, which constitutively express the Cre-recombinase, for instance BNN132 (Elledge et al., 1991, Proc.", "Natl.", "Acad.", "Sci.", "USA., 88: 1731-5).", "However, this is not recommended because of low plasmid yield (Palazzolo et al., 1990, as above) and plasmid instability (Summers et al., 1984, Cell, 36: 1097-1103): in fact, Cre-recombinase is constitutively expressed causing formation of plasmid dimers/multimers leading to high proportion of plasmid-free cells (Summers et al., 1984, as above), impairing the sequencing efficiency.", "The Gateway excision is an alternative system to the Cre-lox excision.", "According to the general Gateway™ system, an insert donor vector carrying a DNA of interest (insert) and a pair of recombinant sites different from each other, recombines with a donor vector comprising a subcloning vector and a pair of recombinant sites different from each other, but able to recombine with the insert donor vector recombination sites.", "The final product is a subclone product carrying the DNA of interest (insert) and a byproduct.", "The recombinant sites are attB, attP, attL and attR.", "However, the Gateway™ system shows a bias for short cDNA; long cDNAs are obtained with low efficiency (Michael A. Brasch, slide “Gateway cloning of attB-PCR products”, GIBCOBRL® Technical Seminar, “Gateway Cloning Technology”, Life Technologies™, 1999).", "Another further problem in the cloning system consists in the presence of background, which is due to environmental DNA contamination and to subcloning process byproducts, that is a non recombinant plasmids (plasmids without the DNA of interest).", "It is instead highly desirable having a background-cutting cloning system, able to eliminate completely or having a little background.", "Some background-cutting strategies have been proposed in the art.", "Walhout et al.", "(as above), for example, reports that the Gateway™ vectors, attP1-attP and attR1-attR2, also contain between the att sites the ccdB gene (Bernard P. and Couturier M., 1992, J. Mol.", "Biol., 226:735-746), whose protein product interferes with DNA gyrase.", "After recombination, only the plasmids that have lost the ccdB gene (and which are recombinant) can grow in E.coli strains not mutated for gyrA, therefore providing a selective advantage.", "Plasmids carrying the gene ccdB can propagate only in specific E.coli strain, DB3.1, which carries a mutation in gyrA gene conferring resistance to ccdB (Walhout et al., as above).", "Therefore, this kind of recombination is limited to plasmids, since other vectors for instance λ substitution vectors used in cloning systems cannot grow and replicate in cells like DB3.1, which miss the recA protein (the recA product is required for the growth of substitution-type bacteriophage λ: Sambrook et al., 1989).", "In conclusion, there is the need in his field of the art of providing of vectors having the characteristics of: i) being size bias free and allowing the preparation of “size balanced” comprising very long, rare full-length cDNAs; ii) capable of improved recombination mechanism; and iii) able of background cutting.", "The cloning vectors available in the state of the art, fail to satisfy the above characteristics.", "The invention disclosed in the present application is addressed to solve the problems in the art.", "SUMMARY OF THE INVENTION The present inventors provide a new family of vectors capable of cloning nucleic acids of wide range size and preferably very long ones, with high efficiency of excision and reduced background and contamination.", "Also provided are methods of cloning and for preparing bulk library using such vectors.", "According to a first embodiment, the invention provides a cloning vector comprising a construction vector segment (CS) and a replaceable segment (RS), wherein the size of CS is: 36.5 kb≦CS<38 kb, preferably CS is 37.5 kb.", "The construction vector segment preferably is made or comprise a bacteriophage λ vector fragment.", "The replaceable vector segment (RS) represents the segment, which is replaced by the nucleic acid insert of interest, which one intends to clone.", "It has been surprisingly found that a cloning vector with this size is capable of preferably inserting cDNA of very long sizes, and it is therefore particularly advantageous for cloning very full-length cDNAs.", "This vector overcomes the problem in the art of existing vector λ-FLC having a construction vector segment of 38 kb, which showed a strong bias for short size cDNAs (see Table1).", "The selection of a particular advantageous size of the vector for the preparation of full-length cDNAs libraries can also be applied to bacteriophage other than λ.", "Accordingly, the present invention also relates to a cloning bacteriophage vector comprising a construction segment (CS) and a replaceable segment (RS), wherein the size of CS is: X−1.2 kb≦CS<Xkb; X (expressed in kb) corresponding to the minimum size necessary to the bacteriophage vector for undergoing packaging.", "The size of CS is preferably: X−0.2 kb.", "The present invention also relates to a bacteriophage vector, preferably a λ, comprising a bacterial artificial chromosome (pBAC) or a segment thereof comprising at least an origin of replication (ori).", "This vector can also comprise: a site into which a DNA fragment can be cloned; and a pair of inducible excision-mediating sites defining an excisable fragment that comprises the site into which the DNA fragment can be cloned.", "The pair of excision-mediating sites are preferably FRT sites.", "This vector may further comprise an inducible origin of replication, preferably oriV.", "The cloning vectors according to the invention are capable of carrying out plasmid or nucleic acid insert excision using known recombination systems, for example the Cre-lox and/or Gateway™ system.", "The vectors of the invention can also comprise a background-reducing system, as ccdB gene, a lox sequence or the lacZ gene or asymmetric site sequences recognized by restriction endonuclease.", "The invention also relates to cloning method using the above vectors.", "According to another embodiment, the invention relates to a system for reducing background or contamination by providing a cloning vector comprising a background-reducing sequence like ccdB gene and/or a lox sequence comprised into RS segment of the vector of the invention, or in case of the Gateway™ system into the RS segment of a destination or receiving vector.", "RS of phage or plasmid vectors can also be flanked by two asymmetric site sequences recognized by restriction endonuclease.", "The invention also relates to a method for reducing background or contamination by using these vectors.", "The invention also relates to methods for efficient excision of plasmid or nucleic acid of interest providing improved Cre-recombinase or Gateway™ system using the vectors according to the invention.", "Preferably, the present invention relates to method for the preparation of bulk of long or full-length cDNA libraries, by using the vectors according to the invention.", "The present invention also relates to a kit comprising at least a cloning vector or at least a library of vectors according to the invention.", "The present invention further relates to a method for preparing at least a normalized and/or subtracted library comprising using a plasmid vector obtained with the excision method according to the invention or destination vector according to the invention, preferably reduced at single strand, as normalization and/or subtraction driver.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a general scheme of the vector family according to the invention.", "The following functional elements (not in scale) are produced in this work.", "In FIG.", "1(a), the functional elements of the vector construction segment (CS) are: the left and right arms; the cloning size regulator (or stuffer II); a plasmid derivative of pBluescript; and the bulk excision elements (recombination sites) loxP; the size of the construction segment (CS) is between 32 and 38.3 kb.", "The replaceable vector segment (indicated as stuffer I or RS) is flanked by the excision Gateway™ elements (attB1 and attB2); this is the segment that will be replaced by the cDNA.", "At the right side of FIG.", "1(a), it is shown the mechanism of plasmid excision according to the cre-lox system or the excision of cDNA inserts into a destination or receiving vector with the Gateway™ system.", "In FIGS.", "1(b)-(f) various constructions and sizes of the stuffer I (RS) are shown: stuffer I of (b) is 10 Kb as from λ-PS vector; (c) is a short version of the stuffer I to simplify the arms purification; (d) is a 10 Kb stuffer with 4 ccdB and two LacZ to cut the background; (e) is a 5 Kb stuffer with 2 ccdB and one Lac Z; (f) is a stuffer for the ccdB and lox P double background cutting.", "In particular, in (g), it is shown a non recombinant plasmid comprising the ccdB gene which inhibits growth, while LacZ (h) allows color selection.", "In (i) it is shown the background-reducing system using a loxP site, which separates the origin of replication and the resistant gene.", "Abbreviations: Sw=SwaI, Sf=SfiI, Sp=SpeI, Fs=FseI, Pa=PacI, Xa=XbaI.", "The PacI, FseI, SfiI, SwaI, and the cloning sites cut only the sites that are shown and do not cut elsewhere in the vectors.", "FIG.", "2.Several constructions for vectors according to the invention, which are for simplicity indicated with the generic name of λ-FLC are shown.", "(a) λ-FLC-I-B and λ-FLC-I-E, having the stuffer I of FIGS.", "1b and 1e, respectively.", "(b) λ-FLC-I-L-B and A-FLC-I-L-D, which lack the stuffer II and have a stuffer I of FIGS.", "1b and 1d, respectively, cloning site as in (a).", "(c) λ-FLC-II-C carrying the Gateway™ attB1 and attB2 sequence for bulk transfer of clones; it has a stuffer I like FIG.", "1c.", "(d) λ-FLC-III-F having the stuffer I like in FIG.", "1f for background reduction.", "(e) λ-FLC-III-L-D which lack the stuffer II and has the stuffer I like in FIG.", "1d.", "(f) λ-FLC-III-S-F, having the stuffer I like in FIG.", "1f but having a longer stuffer II (6.3 Kb).", "Vectors (d-e) have sites for homing endonucleases (I-Ceul and PI-SceI) next to the cloning site for easy transfer of inserts to other vectors; the cloning site is shown in (d) only.", "Vectors (g-j) show polylinker sequences which are placed at left and right side flanking the stuffer I (indicated in FIGS.", "1(b-f)) or cDNAs (which is represented by a sequence of asterisks).", "The underlined sequences into the polylinkers represent primers, recombination sites, restriction sites, and the like.", "These restriction sites do not cut elsewhere in the λ-vectors or in the plasmids at all.", "More specifically, in pFLC-I, the left polylinker (SEQ ID NO:1) comprises: Forward (Fwd) M13 primer site, site for T7 polymerase, recombination site loxP, restriction sites SfiI and SalI site sequences; the right polylinkers (SEQ ID NO:2) comprises: restriction sites BamHI and SfiI, site for T3 polymerase, Reverse (Rev) M13 primer site.", "In pFLC-II, the left polylinker (SEQ ID NO:3) comprises: Fwd M13 primer site, T7, attB1, XhoI and SalI; the right polylinker (SEQ ID NO:4) comprises: BamHI, attB2, loxP, T3, Rev M13 primer site.", "In pFLC-III, the left polylinker (SEQ ID NO:5) comprises: Fwd M13 primer site, T3, I-CeuI, SalI; the right polylinker (SEQ ID NO:6) comprises: BamHI, PI-Sce T7, Rev M13 primer site.", "In pFLC-DEST, the left polylinker (SEQ ID NO:7) comprises: Fwd M13 primer site, T3, attB1, XhoI, SalI; the right polylinker (SEQ ID NO:8) comprises: BamHI, attB2, T7, Rev M13 primer site.", "The general pFLC-II of FIG.", "2h (i.e.", "without mentioning the specific stuffer I or the “insert cDNA”) can be constructed by using a modified pBluescriptII SK.", "A general pFLC-II having this construct is shown in FIG.", "13 and the entire sequence (without stuffer I or “insert cDNA”) is shown in SEQ ID NO:51.FIG.", "3.Excision protocols.", "From left to right, in vivo solid phase Cre-recombinase (state of the art), in vivo liquid phase Cre-recombinase, in vitro Cre recombinase.", "On the right side, the “direct”, “indirect”, and “amplified indirect” protocols, which are mediated by the Gateway™ (GW) sequences and enzymes for in vitro excision.", "FIG.", "4.Average size of obtained cDNA libraries prepared with λ-Zap II or λ-FLC-I-B.", "FIG.", "5.This Figure shows possible vector constructions according to the present invention.", "The vector according to the invention can be circular or linear, comprising a first segment indicated as construction segment (CS) and a second segment indicated as replaceable segment (RS).", "In linear form the construction segment (CS) of the vector is represented comprising a left segment and a right segment.", "RS is the segment which will be replaced by the nucleic acid insert of interest, for example a full-length cDNA.", "The vector according to the invention can be circular or linear.", "In (a) and (b) recombination sites (here generally indicated as att1 and att2), which do not recombine with each other, flanking RS, according to the Gateway™ recombination/excision system (Gateway™ Cloning Technology Manual, GIBCOBRL®, Life Technologies®) are shown.", "In c) and d), recombination sites (lox site in this case), which recombine with each other by the Cre-lox recombination mechanism are present in CS.", "In e) and f) it is shown that the Gateway-like sites flanking a RS and the recombination sites like the lox sites (shown in c) and d)) can be present at the same time.", "In (g), recombination sites flanking RS are two lox sites, which do not recombine with each other.", "They work in the same way as the Gateway sites do.", "In (h), it is shown the presence into RS of the gene ccdB as background-reduction.", "In (i), it is shown the presence of a “third” lox recombination site as background-reducing sequence, capable of recombination with the lox site sequences in CS.", "FIG.", "6.Mechanism of action of a cloning vector comprising two homing endonuclease asymmetric recognition site sequences (a).", "These two sequences not capable of ligating with each other, are placed flanking a RS during the ligation process.", "Each of these sequences recognizes and ligates to one sequence flanking a nucleic acid insert of interest (b).", "Only ligation vector-insert is allowed.", "Ligations insert-insert or vector-vector are in this way avoided.", "FIG.", "7.It is described an example of preparation of λ-FLC-III-F.", "The stuffer If, is the stuffer I of FIG.", "1f.", "FIG.", "8.It is disclosed an example of excision of asymmetric recognition site sequences, in the specific example using homing endonuclease I-CeuI and PI-SceI.", "FIG.", "9.It is described the preparation of a modified pBAC for the preparation of a λ-BAC vector.", "A detailed explanation of the process is disclosed in Example 20.FIG.", "10.It is described the insertion of loxP and XbaI sites into the modified pBAC of FIG.", "7.A detailed explanation of the process is disclosed in Example 20.FIG.", "11.It is described a chart comprising the steps for the preparation of the stuffer II (“component 5”).", "A detailed explanation of the process is disclosed in Example 20.FIG.", "12.It is described a chart comprising the steps for the preparation of the λ-FLC-III-pBAC.", "A detailed explanation of the process is disclosed in Example 20.FIG.", "13.It is reported the full nucleotide sequence of an example of a general pFLC-II as described in FIG.", "2h (that is, without showing the sequence of the stuffer I or the “insert cDNA”).", "The “insert cDNA” or stuffer I (indicated in FIG.", "2h with a line of asterisks) is indicated in FIG.", "13 by a line between the sequences CTCGAG - - - GGATCC.", "This construct of a general pFLC-II is a modified pBluescriptII SK(+).", "The sequence of the plasmid of FIG.", "13 is indicated in SEQ ID NO:51 as a single sequence starting from the sequence GGATCC (above), and terminating with the sequence CTCGAG (above), therefore without indicating the sequence of specific stuffer I or cloning cDNA.", "FIG.", "14.This graph compares cloning vector λ-FCL-I-B of the present invention and conventional ZAP vector in terms of cloning efficiency.", "DETAILED DESCRIPTION OF THE INVENTION Full-length cloning has been hampered by problems related to both the preparation and cloning of long cDNAs.", "A consistent part of the problems has been overcome with the preparation of long cDNAs with thermostabilized and thermoactivated reverse transcriptase (Carninci et al., 1998, Proc.", "Natl.", "Acad.", "Sci.", "USA.", "95: 520-524) and the development of cap-based full-length cDNA selecting techniques (Carninci et al., 1996, Genomics, 37: 327-336; Carninci et al., 1997, DNA Res., 4: 61-66; Carninci et al., 1999, Methods Enzymol., 303: 19-44; Carninci et al., 2000, Genome Res., 10: 1617-1630).", "However, cloning methods and methods for preparing bulk cDNA libraries still showed a bias for short size cDNAs.", "The present inventors provide a new family of vectors capable of cloning nucleic acids with wide range size and preferably very long and full-length cDNAs, high efficiency of excision and reduced background and contamination.", "Also provided are methods of cloning using such vectors.", "According to a first embodiment, the invention provides a cloning vector comprising a construction vector segment (CS) and a replaceable segment (RS) (also indicated as “stuffer I”) (FIG.", "1).", "RS is the segment that will be replaced by the nucleic acid insert of interest, which one intends to clone.", "The bacteriophage or plasmid vector of the invention can be both linear or circular (FIG.", "5,a-i).", "In case of a linear vector, the segment CS can be graphically considered as divided into two arms or segments, one at left side and the other at right side of RS.", "However, for more clarity the terminology of left arm or segment and right arm or segment of CS will be also maintained in case of circular vector.", "The vector available in the state of the art was a modified λ PS vector having a “basic” size of 32 kb plus a 6 kb nucleic acid sequence (stuffer II), so that the size of the vector, without considering the cDNA of interest, was 38 kb (Japanese patent application having publication number P2000-325080A filed by the same applicant of the present invention).", "However, this vector had the disadvantage of bias for short and non full-length cDNAs, the presence of which are inconvenient for the preparation of a full-length cDNA library or encyclopedia.", "The present inventors have surprisingly found that a vector, preferably a bacteriophage, more preferably a λ bacteriophage, having the size of CS of: 36.5 kb≦CS<38 kb, preferably CS is 37.5 kb, allowed the selection of long and full-length cDNA avoiding the problem of the λ phage of 38 kb.", "The preferred size of 37.5 kb of CS according to the vector of the present invention is 0.2 kb shorter than the minimum size necessary for a λ-phage to undergoing packaging, which corresponds to 37.7 kb (Zabarovski et al., 1993, as above).", "The advantages of the vector of CS 37.5 kb according to the invention compared to that of the state of the art of CS 38 kb is showed in Table 1.The system for avoiding the bias for short and for the preferable preparation of full-length cDNAs can also be applied for bacteriophages different from λ.", "Accordingly, the invention also relates to a cloning bacteriophage vector comprising a construction segment (CS) and a replaceable segment (RS), wherein the size of CS is: : X−1.2 kb≦CS<X; X (expressed in kb) corresponding to the minimum size necessary to the bacteriophage vector for undergoing packaging (which nominally is 37.7 kb for λ, as reported in Zabarowski et al., as above).", "The size of CS is preferably: X−0.2 kb.", "The diminution of a short fragment from the size of X renders the CS fragment below the packaging level, however, the presence of the RS (also indicated as “stuffer I”) makes the bacteriophage vector capable of packaging.", "In FIGS.", "1 and 2, the vector according to the invention is constructed inserting a stuffer II of the desired size.", "Preferably, of 5.5 kb, so that the CS corresponds to a size of 37.5 kb.", "However, the stuffer II can be: 4.5≦stuffer II<6.The stuffer II can be of any origin and any nucleic acid.", "It can be a foreign sequence fragment, for example a mouse genomic DNA or can be taken from plasmid.", "The stuffer II can also be already originally present in the vector.", "The CS of the vector according to the invention can preferably be a bacteriophage segment, or comprise a bacteriophage fragment.", "Preferably, the bacteriophage is a λ bacteriophage.", "A list of available bacteriophage and λ bacteriophage has been reported in the state of the art of the present application (see for example those reported in Sambrook et al., 2.16-2.53) or derivatives thereof.", "CS can also be modified by comprising a plasmid segment at least comprising a ori.", "The plasmid comprising ori is preferably selected from the group of: pBluescript (+), pUC, pBR322, and pBAC.", "In FIG.", "1, for example, a fragment of a modified pBluescript(+) comprising ori has been inserted into the left arm of CS.", "An example of use of pBAC or derivative thereof for the preparation of vectors according to the invention is given, for example in FIGS.", "9-12 and Example 20.However, pBAC or its derivative can be efficiently used for the preparation of any vector contruct according to the invention.", "Examples of vectors and linker, adapter, primer sequences and the like that can be used in the construction of the vectors according to the invention are reported in the NCBI VecScreen, UNIVEC Build #3.2 Database (National Centre for Biotechnology Information, National Library of Medicine, National Institute of Health, US).", "Specific information about these vectors can also be found in the Catalog of Amersham Pharmacia Biotech, Inc., US; Clontech Laboratories, Inc, US; Invitrogen Corporation, US; Life Technologies, Inc., US; New England Biolabs, Inc., US; Promega Corporation, US; and Stratagene, US.", "The cloning vector according to the invention can also comprise a selectable marker.", "Accordingly, CS comprises at least a selectable marker selected from the group consisting of: a DNA segment that encodes a product that provides resistance against otherwise toxic compounds (e.g.", "antibiotic resistant gene); a DNA segment that encodes a product that suppresses the activity of a gene product; a DNA segment that encodes a product that is identifiable (e.g.", "phenotypic markers such as beta-galactosidase, green fluorescent protein,(GFP), and cell surface proteins); a DNA segment that encodes a product that inhibits a cell function; a DNA segment that provides for the isolation of a desired molecule (e.g.", "specific protein binding sites); and a DNA segment that encodes a specific nucleotide recognition sequence which is recognized by an enzyme.", "The selectable marker is more specifically at least a marker selected from the group consisting of an antibiotic resistance gene, an auxotrophic marker, a toxic gene, a phenotypic marker, an antisense oligonucleotide; an enzyme cleavage site, a protein binding site; and a sequence complementary to a PCR primer sequence.", "Amp as an example of selectable marker is showed in FIGS.", "1 and 2.The RS of the vectors of the invention can be flanked by two recombination sites (as showed in FIGS.", "1, 5) wherein these two recombination sites do not recombine with each other.", "More in particular, these recombination sites are selected from the group consisting of attB, attP, attL, and attR or their derivatives for carrying out the recombination excision according to the Gateway™ methodology (Walhout et al., 2000, as above; Life Technologies catalogue; Gateway Cloning Technologies, Instruction Manual, GibcoBRL, Life Technologies; and U.S. Pat.", "No.", "5,888,732).", "The complete list of Gateway recombination sites and derivatives is disclosed in the above Life Technologies references.", "The Gateway™ system has been proposed in the art for exchange of components between plasmids and for transferring a nucleic acid insert of interest into a specific functional plasmid.", "However, the Gateway system showed a bias for short cDNA; long cDNAs are obtained with low efficiency (Michael A. Brasch, slide “Gateway cloning of attB-PCR products”, GIBCOBRL® Technical Seminar, “Gateway Cloning Technology”, Life Technologies™, 1999).", "The present inventors have instead surprisingly found that when Gateway recombination sites are transferred into a bacteriophage vector according to the present invention and positioned flanking the RS (as shown in FIGS.", "1, 2 and 5,a,b,e,f) the cloned cDNA library did not show bias for short cDNAs.", "The present invention therefore, provides a bacteriophage vector, preferably having a CS size of: 32 kb≦CS<45 kb, in particular 36.5 kb≦CS<38 kb, more preferably CS is 37.5 kb comprising two recombination sites, which do not recombine with each other, flanking RS (FIG.", "5,a-g).", "The bacteriophage is preferably a λ bacteriophage.", "The bacteriophage vector according to the present invention, however, is not limited to λ bacteriophage but other bacteriophage known in he art can be used (for example those described in Zabarovski et al., 1993, as above).", "In the vector according to the present invention, in alternative to the Gateway attB, P, L or R or their derivatives, two lox recombination sites flanking RS (for example, two generic lox1 and lox2 sites are shown in FIG.", "5,g) can be used.", "These lox recombination sites can be any mutated or derived lox sites, for example a mutated or derived loxP site (for example loxP511) as described in Hoess et al., NucleicAcids Res., 1986, 14(5):2287.The vector according to the invention can also comprise two lox recombinant sites each of them placed in each arm (or segment portion) of CS (FIGS.", "1, 2, and 5,c-f,i), that is, one lox site placed in the CS, at the left side of the RS (or of the nucleic acid of interest) and the other lox site in the CS, at the right side of the RS (or of the nucleic acid insert of interest); these lox recombination sites being capable to recombine with each other.", "These sites can be two lox recombination sites modified, mutated or derived lox site (Hoess et al., 1986, as above), preferably a loxP or a modification or derivative thereof For example, the lox sites can be loxP 511 (Hoess et al, 1986, as above).", "A loxP 511 recombines with another loxP 511 site, but not with a loxP site.", "All the above variation, mutation, modification or derivation of lox site, will be generally indicate as “lox site and derivative thereof”, for the purpose of the present application.", "In this case, after the RS is substituted by the nucleic acid insert of interest, the recombination is carried out by a Cre-lox recombinase.", "The Cre-lox recombination system is described in several prior art references, for example, Palazzuolo et al., 1990, as above; Elledge et al., 1991, as above; and Summers et al., 1984, as above.", "In alternative, to the Cre-lox recombinase system, other recombination systems can be used for the purpose of the present invention.", "Among them, Kw recombinase (Ringrose L., et al., 1997, FEBS, Eur.", "J Biochem., 248:903-912), hybrid site-specific recombination system with elements from Tn3 res/resolvase (Kilbride E., et al., 1999, J. Mol.", "Biol., 289:1219-1230), β recombinase system (Canosa I., et al., 1998, Journal Biological Chemistry, Vol.273, No.22, May 29:13886-13891); FLP recombinase system (Huffman K. E., and Levene S. D., 1999, J. Mol.", "Biol.,:286:1-13; and Waite L. L., and Cox M. M., 1995, Journal Biological Chemistry, Vol.270, N.40:23409-23414).", "Modification, mutation or derivative of these recombination sites can also be used and they will be generally indicated as “derivative thereof”.", "The result of this recombination process, mediated by Cre-recombinase or other recombinases, is the excision of a plasmid comprising the nucleic acid of interest.", "According to an embodiment of the invention, the presence of both the recombination sites flanking RS for the recombination Gateway-like system and the recombination sites in the two arms of CS for Cre-lox, Kw, Tn3 res/resolvase, β recombinase, and FLP recombination, into a vector, renders said vector particularly suitable for cloning, transfer of nucleic acid material of interest, and preparation of libraries.", "In fact, according to the particular case, the most convenient excision system can be chosen without changing or modifying the vector.", "According to a further aspect, the cloning vector according to the invention can also be used for cloning or for preparing libraries with low or no background.", "Accordingly, the present invention provides a cloning vector comprising a construction segment (CS) and a replaceable segment (RS), wherein said CS is a bacteriophage vector segment and said RS comprises at least the ccdB gene as background-reducing system.", "The bacteriophage or plasmid cloning vector according to the invention, can also comprises a construction segment (CS) and a replaceable segment (RS), wherein said CS is a bacteriophage or a plasmid vector segment and i) said RS comprises at least a recombination site (capable of recombination with the two recombination sites present in the left and right arms of CS) as background-reducing system, or ii) RS is flanked by two endonuclease asymmetric recognition site sequences which do not ligate with each other and are recognized by restriction endonucleases.", "The recombination site comprised into RS must be able to recombine with the recombination sites present into the left and right arms of CS, therefore, we can address to this RS recombination site as the “third” recombination site.", "The “third” recombination site can be a lox recombination site or a derivative thereof, preferably a loxP site or derivative thereof.", "The two endonucleases asymmetric site sequence background-reducing systems can be for example: i) homing endonuclease asymmetric recognition site sequences, or ii) asymmetric restriction endonuclease cleavage site sequences recognizable by class IIS restriction enzymes.", "The background-reducing bacteriophage vector has preferably the size of CS: 32 kb≦CS≦45 kb, advantageously CS is: 36.5 kb≦CS<38 kb, more preferably CS is 37.5 kb.", "The bacteriophage is preferably a λ bacteriophage.", "The bacteriophage CS or the vector can comprise a plasmid segment at least comprising an ori.", "The plasmid segment comprising an ori is preferably, but not limited to, selected from the group consisting of: pBluescript(+), pUC, pBR322 and pBAC, or any plasmid as included into the NCBI Database, as above.", "In case of the background-reducing plasmid, this can be any kind of plasmid known in the art, for example any of the plasmid above indicated or disclosed in the NCBI Database.", "This vector preferably comprises at least a selectable marker selected from the group as above disclosed.", "In particular, the at least selectable marker can be selected from the group consisting of an antibiotic resistance gene, an auxotrophic marker, a toxic gene, a phenotypic marker, an enzyme cleavage site, a protein binding site; and a sequence complementary to a PCR primer sequence.", "The background-reducing cloning bacteriophage or plasmid vector can also comprise at least one of the recombination system as above described, that is i) two recombination sites which do not recombine with each other flanking RS (Gateway sites or lox modified sites) and/or ii) at least two recombination sites which recombine with each other placed into the two arms of CS, recognized by a recombinase.", "These recombination sites capable of recombining with each other, are preferably selected from the group consisting of: lox sites, Kw, Tn3 res/resolvase, β recombinase sites, and FLP sites, as described above.", "With reference to the background-reducing ccdB system, it has been disclosed into plamids by Bernard P. and Couturier M. (1992, J. Mol.", "Biol., 226:735-746) and also Walhout et al.", "(as above) for the Gateway™ vectors.", "The product of the ccdB gene interferes with DNA gyrase.", "After recombination, only the plasmids that have lost the ccdB gene (and which are recombinant) can grow in E.coli strains not mutated for gyrA, therefore providing a selective advantage (see Life Technologies references).", "Plasmids carrying the gene ccdB can propagate only in specific E.coli strains.", "For example in DB3.1, which carries a mutation in gyrA gene conferring resistance to ccdB (Walhout et al., as above).", "Therefore, this kind of recombination is limited to plasmids, because bacteriophage vectors, for instance λ substitution vectors, used in cloning systems cannot grow and replicate in cells like DB3.1, which lack the recA protein (the recA product is required for the growth of substitution-type bacteriophage λ: Sambrook et al., 1989).", "The present inventors have instead surprisingly found that a bacteriophage, preferably a λ bacteriophage, comprising at least a ccdB gene into the RS, according to the invention can propagate and multiply on a culture of C600cells.", "On the contrary, plasmids comprising the ccdB gene cannot propagate in C600 cells.", "The mechanism of the background-reducing ccdB system in the vector of the invention is shown in FIG.", "1,g.", "During the replacement of the RS with the nucleic acid insert of interest, it may happen that no replacement occurs or an imperfect ligation or replacement is realized.", "In this case, bacteriophage or plasmid vectors without complete nucleic acid insert of interest are present in the culture creating background.", "With the presence of ccdB, the “suicide gene”, the background or byproduct can be reduced about or very closed to zero.", "A problem of background contamination can also occur during the purification, when the removal of stuffer I (RS) is realized on gel (for example agarose gel) and fragment of stuffer I nucleic acid is collected with CS and can therefore be reinserted into the vectors.", "Another background-reducing system is the “third” recombination site, which is placed into RS and is capable to recombine with the recombination sites present into the left and right arms of CS of the bacteriophage or plasmid vector of the invention (FIG.", "1,i; FIG.", "5,i).", "This “third” recombination site can be in presence or in absence of the ccdB gene.", "Preferably, this background-reducing “third” recombination site is a lox site or a derivative thereof, more preferable a loxP site or a derivative, modification or mutation thereof, as above described.", "However, the background recombination site present into RS, must be capable of recombination with the two recombination sites present in the two arms of CS.", "Therefore, in case of recombination mediated by Cre-recombinase, all the three sites have to be lox-recombination or derivatives thereof, capable of recombining with each other.", "For example, in FIGS.", "1,a and 1,f, the two recombination sites present in the left and right arms of CS (of a bacteriophage or a plasmid vector) and the background-reducing “third” recombination site into RS (stuffer I) are all loxP sites.", "In FIG.", "1.i), it is explained the mechanism of action of the “third” recombination site.", "In case of imperfect ligation of the nucleic acid insert of interest, one of the loxP site in arms of CS preferably recombine with the “third” loxP forming, during the excision step, an excised plasmid, which in one case lack the ori and cannot replicate, and in the other case lack the selectable marker (Amp in the Figure) and cannot grow up.", "Accordingly, the present invention also relates to a method for cloning or preparing bulk library with low or no background using a bacteriophage or plasmid vector comprising at least the “third” recombination site as described.", "The background-reducing “third” recombination site can be any recombination site other than lox, for example the recombination sites used for the recombination as above described.", "The background-reducing bacteriophage or plasmid cloning vector according to the invention, can also comprises the lacZ gene into RS even in presence of the ccdB gene or the “third” recombination site or the like, or in presence.", "The bacteriophage or plasmid cloning vector according to the invention, in alternative or in presence of the background-reducing sequences above described, can also comprise two asymmetric sites recognized by restriction endonucleases.", "These two asymmetric site sequences flank the RS of the vector (FIG.", "6).", "Asymmetric site sequences useful for the purpose of the present invention are: i) two homing endonuclease asymmetric recognition site sequences or ii) restriction endonuclease asymmetric cleavage sites sequences recognizable by class IIS restriction enzymes.", "Homing endonucleases are sold and described by New England Biolabs, Inc. A; a description of the asymmetric site sequences is also available in the New England Biolabs Catalog.", "These homing endonuclease asymmetric recognition site sequences are from 18 to 39 bp.", "However, in the present invention the recognition site sequences are not limited to those sequences nor to these sizes.", "The New England Biolabs Catalog reports that after 5-fold overdigestion with I-Ceu-I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.", "Preferably, the restriction homing endonucleases capable of cutting the asymmetric site sequences are selected from the group consisting of: I-CeuI, PI-SceI, PI-PspI and I-SceI.", "FIG.", "6,a) shows a vector being removed of its RS, bringing two homing endonoclease recognition site sequences, which do not ligate with each other, at the extremities of the CS arms; the RS being removed by using the homing endonucleases specific for those site sequences.", "In FIG.", "6,b) a nucleic acid insert of interest having a pair of homing endonuclease site sequences placed flanking said insert of interest (these sequences being the same of those of the vector) is provided for the ligation to a vector having RS removed.", "In FIG.", "6,c) one homing endonuclease site sequence of the vector recognizes and hybridizes to a complementary homing endonuclease site sequence of the insert.", "In FIG.", "6,d), the second homing endonuclease site sequence of the vector, after a certain time, preferably overnight, recognizes and hybridizes the complementary homing endonuclease site sequence placed on the other extremity of the insert of interest.", "In conclusion, using this system, after a certain time, all the complementary site sequences of the inserts recognizes and hybridize with their complementary site sequences of the vectors.", "As consequence, insert-vector ligation is carried out.", "Both insert-insert and vector-vector ligations are not realized since they extremities are not complementary reducing by-products.", "With this system, also nucleic acid contamination entering the vector is reduced.", "The homing endonuclease recognition site sequences can also be placed into a destination vector, preferably a plasmid, and the subcloning process can be advantageously carried out.", "This vector ligates with the nucleic acid insert of interest, which brings two endonuclease recognition site sequences, which are the same of the destination vector, placed flanking this nucleic acid insert of interest.", "The same process can be realized when asymmetric site sequences recognized by class IIS endonuclease enzymes are used instead of the homing endonuclease site sequences.", "Examples of class IIS restriction enzymes include, AlwI, AlwXI, Alw26I, BbsI, BbvI, BbvII, BcsfI, BccI, BcgI, BciVI, BinI, BmrI, BpmI, BsaI, BseRI, BsgI, BsmAI, BsmBI, BspMI, BsrDI, BstF5I, EarI, Eco31I, Eco57I, Esp3I, FauI, FokI, GsuI, HgaI, HinGUII, HphI, Ksp632I, MboII, MmeI, MnlI, NgoVIII, PleI, RlaAI, SapI, SfaNI, TaqII, TthlllII, BsnIs, BsrIs, BsmFI, BseMII, and the like (see Szybalski W., et al., 1991, Gene, 100, 13-26; and Catalog of New England Biolabs, Inc.).", "Examples of recognition sites and cleavage sites of several restriction enzymes are (into parenthesis are the recognition site and the cleavage site): BbvI (GCAGC 8/12), HgaI (GACGC 5/10), BsmFI (GGGAC 10/14) SfaNI (GCATC 5/9), and Bsp I (ACCTGC 4/8).", "The endonuclease asymmetric recognition site sequences as described above can be placed into the bacteriophage or plasmid cloning vector according to the invention also in presence of, the ccdB gene, the lacZ gene, and/or the “third” background-reducing recombination site (for example lox) into RS.", "The vector ligated with the endonuclease asymmetric system as described above can then be excised by any of the recombination system present in CS, as above described, for example cre-lox recombinase, preferably loxP, Kw, FLP, Tn3 res/resolvase, β recombinase, etc.", "The vector comprising the endonuclease asymmetric according to the invention, therefore, also comprises at least a pair of recombination sites into the CS.", "The RS (or stuffer I) of the cloning vector according to the invention is removed by the vector and it is replaced by the nucleic acid insert of interest with the ligation process.", "The nucleic acid insert of interest which is used in all of the embodiments of the present application is selected from the group consisting of DNA, cDNA, RNA/DNA hybrid.", "Advantageously, long cDNA and preferably full-length cDNA.", "The full-length cDNA is preferably a normalized and/or subtracted full-length cDNA.", "Any of the vectors according to the invention has proven to be particularly useful for cloning nucleic acids of interest and for the preparation of library, in particular full-length cDNA library/libraries.", "Accordingly, the present invention relates to a method for cloning at least a nucleic acid insert of interest or for preparing at least a bulk nucleic acid library of interest, comprising the steps of: a) preparing at least a cloning vector according to the invention; b) replacing RS with a nucleic acid insert of interest into the cloning vector obtaining a vector comprising the nucleic acid insert of interest; c) allowing the in vivo or in vitro excision of the nucleic acid insert of interest or of the plasmid comprising the nucleic acid insert of interest; d) recovering the (recombinant) plasmid carrying the nucleic acid insert of interest or the library of (recombinant) plasmids carrying the nucleic acid inserts of interest.", "Optionally, between step b) and c), a step of amplification of cloning vector can be carried out.", "The method according to the invention can also be used for cloning nucleic acid insert of interest or for preparing a bulk nucleic acid library of interest with reduced or no background.", "Accordingly, the present invention provides a method for cloning a nucleic acid insert of interest or for preparing a bulk nucleic acid library of interest, with low or no background, comprising the steps of: (a) preparing at least a cloning vector according to the invention comprising a background-reducing system as above described; (b) replacing RS of vector of step (a) with a nucleic acid insert of interest; (c) allowing the in vivo or in vitro excision of the nucleic acid insert of interest or of the plasmid comprising the nucleic acid insert of interest; (d) recovering the (recombinant) plasmid carrying the nucleic acid insert of interest and lacking of the background-reducing sequence or a library of said plasmids.", "Optionally, an amplification step is carried out between the steps b) and c).", "The background-reducing system according to the invention can be the gene ccdB or a “third” recombination site sequence (capable of recombination with the two lox recombination sites present into the left and right arm of CS), which is placed into the RS of the bacteriophage or plasmid vector according to the invention.", "The “third” recombination site is preferable a lox site or derivatives thereof, more preferably a loxP site or derivatives thereof.", "In case of a Gateway-like method, the gene ccdB is instead placed into the RS of a destination vector.", "The bacteriophage or plasmid vector or the destination vector can also comprise the lacZ gene.", "In Alternative, in the background-reducing method according to the invention, the bacteriophage or plasmid vector can comprise two endonuclease asymmetric recognition site sequences flanking RS.", "Accordingly, the present invention also relates to a method for cloning a nucleic acid insert of interest or for preparing a bulk nucleic acid library of interest, comprising the steps of: (a) preparing at least a bacteriophage or plasmid vector comprising two endonuclease asymmetric recognition site sequences placed flanking RS of said vector; (b) replacing RS with a nucleic acid insert of interest comprising two endonuclease asymmetric recognition site sequences flanking said insert of interest, said sequences being capable of ligating with the two sequences placed into the vector of step a), and obtaining a vector comprising the nucleic acid insert of interest; (c) allowing the in vivo or in vitro excision of the nucleic acid insert(s) of interest or of at least a plasmid comprising the nucleic acid insert of interest; (d) recovering the (recombinant) excised plasmid or destination plasmid carrying the nucleic acid of interest or a library of said plasmid(s) with low or no background.", "Further, the present invention relates to in vivo and in vitro Cre-lox recombination system, using the vector according to the invention.", "As discussed in the state of the art section, the Cre-recombinase solid-phase in vivo excision (see also FIG.", "3 of the present application) known in the art (Palazzolo et al., 1990, Gene, 88:25-36) shows drawbacks as low plasmid yield (Palazzolo et al., 1990, as above) and plasmid instability; in fact Cre-recombinase is constitutively expressed causing formation of plasmid dimmers/multimers leading to high proportion of plasmid-free cells, impairing the sequencing efficiency (Summers et al., 1984, Cell, 36:1097-1103).", "A Cre-recombinase liquid-phase in vivo excision, however, has not been successufully used in the state of the art because in liquid culture, cells comprising short plasmids replicate faster than cells comprising very long plasmids creating a bias for short plasmids (that is short nucleic acid insert of interest), and serious difficulty in obtaining long or full-length nucleic acid inserts.", "The present inventors have surprisingly found that the drawbacks of the state of the art could be avoided essentially by allowing an excision of plasmids in liquid-phase under condition of very low or no growth (replication) and amplification, extraction of nucleic acid inserts of interest, preparation of different plasmids capable to growth in cells do not expressing Cre-recombinase, and further growth (amplification) in solid phase (on plate).", "Accordingly, the present invention provides a method for cloning at least a nucleic acid insert of interest or preparing at least a bulk nucleic acids library of interest comprising the steps of: a) preparing at least a cloning vector, comprising a construction segment (CS) and a replaceable segment (RS), wherein said CS is a bacteriophage vector comprising at least two lox recombination sites or derivatives thereof positioned in the left and right arm of CS.", "; a) replacing RS with a nucleic acid insert of interest into the cloning vector; b) packaging of the vector; c) in vivo in liquid-phase infection of at least a cell expressing cre-recombinase; d) allowing the in vivo in liquid-phase excision of a plasmid comprising the nucleic acid insert of interest under condition of short-time growth or no growth of the excised plasmid; e) carrying out the cellular lysis and recovering the plasmid carrying out the insert or of a library of these plasmids.", "This method, optionally comprises the steps of: f) electroporating or transforming at least a cell, not expressing Cre-recombinase, making the plasmid(s) of step f) penetrating into said cell(s); g) plating of cell(s) infected as at step g) and recovering the plasmid carrying the nucleic acid insert of interest or a library of said plasmids.", "The electroporation is carried out according to the well-known mwthodology in the art.", "The transformation is preferally carried out by chemical treatment, for example, according to Sambrook et al., 1.71-1.84.The bacteriophage vector according to this method is preferable a λ bacteriophage.", "The lox recombination sites, which recombine with each other, can be any mutated, modified or derived lox site as above described, preferable a loxP, which can be mutated, modified or derived (therefore, generally indicated as loxP or derivatives thereof”).", "The step e) of this method is preferably carried out in 0-3 hours at a temperature of 20-4° C. The temperature is preferably from room temperature to 37° C. The present inventors have also developed a new and inventive in vitro Cre-lox recombination method.", "In this in vitro method, a bacteriophage vector comprising the nucleic acid insert of interest is packaged in vitro in presence of (bacterial) packaging extract as known in the state of the art (for example, Gigapack® or Gigapack Gold® or the like, Stratagene, US).", "The nucleases present in the extract cut the short nucleic acids which have not been packaged and the nucleic acid contamination in general.", "The result is that the nucleic acid of the vector which has been packaged result purified.", "In a preferred case, when a vector comprising the stuffer II of 5.5 kb (or a bacteriophage vector having the size of CS of 37.5 kb) is used, the short and not full-length cDNA having sizes below 0.5 kb are not packaged and are removed by the esonuclease.", "The result is a library with low or without bias for short cDNA.", "This library results to be very useful for the preparation of very long and full-length cDNAs.", "Accordingly, the present invention provides a method for cloning at least a nucleic acid insert of interest or at least a bulk nucleic acid library of interest comprising the step of: (a) preparing at least a cloning vector, comprising a construction segment (CS) and a replaceable segment (RS), wherein said CS is a bacteriophage vector segment comprising two lox recombination sites or derivatives thereof positioned in the left and right arm of CS; (b) replacing RS with a nucleic acid insert of interest into the at least a cloning vector; (c) in vitro packaging of the bacteriophage cloning vector of step b) in presence of packaging extract; (d) extraction of bacteriophage cloning vector(s) from the capside; (e) in vitro excision of the plasmid(s) comprising the nucleic acid insert(s) of interest from the vector in presence of Cre-recombinase; (f) recovery of said plasmid or library of plasmids.", "This method may further comprise the steps of: (g) electroporating or transforming at lest a cell, not expressing Cre-recombinase, making said plasmid(s) entering into said cell(s); (h) plating the cell(s) of step g) and recovering plasmid carrying the nucleic acid insert of interest or a library of said plasmids.", "Optionally, between the steps c) and d) an amplification step on plate of the bacteriophage can be carried out.", "The lox recombination sites can be lox sites mutated, modified or derivative thereof, preferably loxP or derivatives thereof.", "The bacteriophage used in this in vitro Cre-lox method is preferably a λ bacteriophage.", "Further, the present inventors have developed a method based on the Gateway mechanism from transferring nucleic acid insert of interest from the vector according to the invention into at least a destination functional vector.", "This functional vector can be utilized for different uses, for example for sequencing, for expressing a protein in bacteria or eukaryotic cells, making a protein fusion product, and so on.", "The Gateway method as already said above is related only to plasmids and shows a strong bias for short cDNAs.", "In the Gateway method, cDNAs are amplified by PCR and inserted into the plasmid destination vector.", "However, the reaction times of PCR or full-length cDNAs are very long and generally the reaction is carried out overnight, which means low efficiency and size bias.", "Fragments with short insert recombine faster than fragment with long inserts.", "Therefore, when mixed, there is always size bias, the shortest competes with longer and the short is more efficiently cloned causing size bias.", "The present inventors have solved this bias problem of the Gateway method.", "The method according to the present invention comprises a step of ligating nucleic acids of interest (of different size) into the bacteriophage vector.", "The bacteriophage vector according to the invention has bigger size (for example 37.5 kb plus the nucleic acid insert) than the donor vector of the Gateway method.", "A vector having the CS size according to the invention does not discriminate between short and long insert and vectors comprising both kid of inserts can be amplified and/or excised with a similar efficiency, so that there is no bias for short nucleic acid inserts.", "Accordingly, the present invention provides a “Gateway-like” method for cloning at least a nucleic acid insert of interest or for preparing at least a bulk nucleic acid library of interest, comprising the steps of: (a) preparing at least a cloning vector comprising a construction segment (CS) and a replaceable segment (RS), wherein said CS is a bacteriophage vector segment and RS is flanked by two recombination sites, wherein these recombinant sites do not recombine with each other; (b) replacing said RS with a nucleic acid insert according to the invention; (c) in vitro packaging the at least one bacteriophage cloning vector of step b); (d) allowing the in vitro excision of the nucleic acid insert of interest by providing to the cloning vector of step c) at least a destination vector comprising a destination replaceable segment (RS) flanked by two recombination sites, which are capable of recombining with the recombination site of cloning vector(s) of step (a); (e) recovering a recombinant plasmid carrying the nucleic acid insert of interest or a library of said plasmids.", "Preferably, the bacteriophage is a λ bacteriophage.", "The two recombination sites which do not recombine with each other flanking the RS of the bacteriophage cloning vector or of the destination vector, can be i) recombination sites selected from the group consisting of attB, attP, attL, and attR or derivatives thereof, or ii) lox recombination site or derivatives thereof, preferably loxP or derivative thereof (for example loxP and loxP511).", "After the nucleic acid of interest has been transferred into the destination vector using the Gateway technology, said acid nucleic of interest can be transferred in a further destination or receiving vector according to the following procedures named as: i) GW direct; ii) GW indirect; and iii) GW amplification method, according to FIG.", "3 and to the examples.", "The excised plasmid or destination plasmid bringing the nucleic acid insert of interest according to the invention can be used as driver in a normalization and/or subtraction method.", "A method for normalization and/or subtraction of a cDNA library, preferably a full-length cDNA library, has been disclosed by Carninci et al., 2000, Genome Res.,10:1617-1630.Accordingly the present invention relates to a method for preparing at least a normalized and/or subtracted library comprising the steps of: (a) providing at least a plasmid excised or a destination plasmid prepared according to the method of the present invention; (b) providing the plasmid of step b) to a pool of nucleic acid targets; (c) removing the plasmid/target hybrids; (d) collecting the normalized and/or subtracted nucleic acid targets, which did not hybridize to the plasmid of the invention.", "According to an embodiment, the plasmid of step a) is rendered as single strand.", "For example, it is treated by making at least a nick into one strand of the double stranded plasmid.", "Then, the strand which has been nicked is removed, finally steps (c)-(d) are applied.", "Preferably, the nick is introduced by using the protein GeneII (Gene-trapper Kit, Gibco, Life Technologies, US) and the strand which has been nicked is removed by an exonuclease.", "The exonuclease is preferably ExoIII.", "According to a further embodiment, the present invention relates to a method for preparing at least a normalized and/or subtracted library comprising the steps of: (a) providing at least a vector according to the invention comprises a construction segment (CS) and a replaceable segment (RS), wherein CS comprises a F1 ori; (b) replacing RS with a nucleic acid insert of interest according to the invention; (c) adding an helper phage and producing a number of a single strand DNA (ssDNA) vector copies, secreted from the cells; (d) providing the copies of step c) to a pool of nucleic acids targets; (e) removing the plasmid/target hybrids; (f) collected the normalized and/or subtracted nucleic acid targets, which did not hybridize with the target(s).", "Helper phage is preferably obtainable from Stratagene.", "A more detailed description of a method for preparing ssDNA vector, consisting in infecting the bacterial cells with a helper phage (Stratagene catalog), then recovering the single strand plasmid secreted from the cell, extracting the DNA, and finally recovering the DNA from single strand plasmid can be found in the Stratagene User Manual of pBluescript.", "A method using the helper phage for reducing the vector at single strand is also described in (Bonaldo et al, 1996, Genome Res., 6:791-806).", "When using the f1(+) origin of replication, an helper phages such as R408 can be used (Short et al., 1988, as above).", "The bacteriophage vectors according to the invention can be prepared using any kind of plasmid or plasmid fragment known in the art, for instance pBluescript(+), pUC, pBR322, bacterial artificial chromosome plasmid (pBAC), pBeloBAC11 (Kim et al., 1996, Genomics, 34:213-218, a modified or derivative pBeloBAC11 according to U.S. Pat.", "No.", "5,874,259 (herein incorporated by reference), or any other plasmid as listed public database or available from Company's Catalogues as above indicated.", "Acording to one embodiment, the invention provides a bacteriophage vector comprising a bacterial artificial chromosome (pBAC) or pBAC derivative or a segment thereof comprising at least an origin of replication (ori).", "The bacteriophage is preferably a λ bacteriophage.", "The ori can preferably be an ori capable of maintaining the plasmid at single copy.", "The pBAC or segment thereof, comprised into the bacteriophage, may further comprise: a site into which an DNA fragment can be cloned; at least one pair of inducible excision-mediating sites flanking the site into which the DNA fragment can be cloned, the excision-mediating sites being provided in parallel orientation relative to one another and defining an excisable fragment that comprises the site into which the DNA fragment can be cloned.", "The pair of inducible excision-mediating sites can be, for example, sites provided in parallel orientation relative to one another (see U.S. Pat No.", "5,874,259).", "The pair of excision-mediating sites are preferably FRT sites.", "The bacteriophage may further comprises into pair of excision-mediating sites a sequence as shown in SEQ ID NO:45 (according to U.S. Pat.", "No.", "5,874,259).", "The pBAC or segment thereof, comprised into the bacteriophage, may further comprise an inducible origin of replication, preferably oriV.", "Thus oriV may be induced to produce multiple copies of the BAC plasmid (the pBAC is usually present at single copy).", "This bacteriophage can comprise one or more of the recombination sites described in the present application.", "For example, this bacteriophage may comprise at least two recombination sites selected from the following: (a) two recombination sites, wherein either site does not recombine with the other; (b) two lox recombination sites, wherein either site is capable of recombining with each other; (c) two homing endonuclease asymmetric recognition site sequences; (d) two restriction asymmetric endonuclease cleavage site sequences, wherein either site sequence does ligate with the other, recognizable by class IIS restriction enzymes.", "The two recombination sites (a) may be selected from the group consisting of attB, attP, attL, attR and derivatives thereof.", "The two recombination sites (a) may also be lox recombination sites derivative, which do not recombine with each other.", "The two recombination sites (b) are preferably loxP sites.", "The two homing endonuclease site sequences (c) are preferably selected from the group consisting of: I-CeuI, PI-SceI, PI-PspI, and I-SceI.", "The excision used can be any excision system, included those described in FIG.", "3.The bacteriophage may further comprise at least a background-reducing sequence, for example: a) the ccdB gene; b) the lacZ gene; c) a lox sequence.", "It is also provided a method for cloning a nucleic acid of interest or for preparing a bulk nucleic acid library of interest comprising the steps of: (a) preparing a bacteriophage cloning vector comprising a pBAC (or a pBAC derivative) or a fragment thereof: (b) inserting a nucleic acid of interest into the bacteriophage cloning vector; (c) allowing the in vivo or in vitro excision of the plasmid (pBAC or derivative thereof) comprising the nucleic acid insert of interest; and (d) recovering the BAC plasmid carrying the nucleic acid insert of interest or a library of these BAC plasmids.", "The present invention also relates to a kit comprising at least a cloning vector or at least a library of vectors according to the invention.", "The present invention will be further explained more in detail with reference to the following examples.", "EXAMPLES Bacterial Strains The following not limitative list of bacterial strains were used in the following examples: C600, F− thi-1 thr-1 leuB6 lacYl tonA21 supE44-λ−; XL1-Blue-MRA(P2), Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 gyrA96 relA1 Jac (P2 lysogen); DB3.1, F− gyrA462 endA Δ(srl-recA) mcrb mrr hdsS20(rB−, mB−) supE44 ara-14 gaIK2 lacYl proA2 rpsL20 xyl-5 λ leu mt/1; BNN132, e14(McrA) Δ (lac-proAB) thi-1 gyrA96 endal hsdR17 rela1 supE44 [F traD36 proAB lacZ Δ M15] constitutively expressing Cre-recombinase (Elledge et al., 1991, Proc.", "Natl.", "Sci.", "USA, 88:1731-1735); and DH10B, F− mcrA A(mrr-hsdRMS-mcrBC) Φ80 lacZΔM15 ΔlacX74 deoR recA1 endA1 araDl39 (ara-leu)7697 galU galKλ− rpsL nupG (these bacterial strains are all commercially available).", "Structure and Nomenclature of λ-FLC Vectors The basic name of the constructed vectors used in the present description derives from full-length cDNA; the roman numerals indicate: I, general use; II, presence of Gateway sequence (Life Technology); and III, presence of homing endonuclease sites.", "L and S indicate whether the cloning capacity of the vector better accommodates long (size-selected) or short cDNAs.", "B, C, D, E, and F indicate the type of stuffer I, as described in FIGS.", "1b-f.", "Basic Components of λ-FLC Vectors We constructed a series of λ-based cloning vectors for broad-size directional cloning of full-length cDNAs.", "These λ-FLC vectors can nominally package inserts of approximately 0.2 to 15.4 kb.", "Another benefit of our λ-FLC vectors is that they accommodate cloning and bulk-excision of short and long cDNAs at similar efficiencies within the same library.", "Then, we adapted these vectors for additional purposes, for example, for selecting very long or full-length cDNAs by using the stuffer II of 5.5 kb (that is a complete size of the construction segment CS of 37.5 kb).", "The components used to construct the vectors were assembled to produce several constructs shown in FIGS.", "1 and 2.FIG.", "1a illustrates the general scheme for the assembly of the λ-FLC vectors and excision into a plasmid library by using Cre-recombinase or Gateway recombination system.", "The basic structure of the λ-based vectors according to the present invention, consists of the left and right λ-arms, which are functionally the same as those of λ-2001 (Karn et al., 1984, Gene, 32:217-224).", "Between the left and right arms, we inserted a stuffer (stuffer I) and a modified pBluescript or pBAC, flanked on both sides, by two lox P sites for the bulk excision of the plasmid cDNA library, analogous to the structure of λ-PS (Nehls et al., 1994a, as above).", "An example of pBluescript construct is shown in FIG.", "13 and SEQ ID NO:51.The calculated size of the λ arms plus the plasmid, but excluding stuffer I (which is substituted with the cDNA in a library) and stuffer II, is about 32 kb.", "Stuffer II is the “cloning size regulator” and determines the size of the insert, given that the nominal lambda packaging capacity (Zabarovsky et-al., 1993, Gene, 127:1-14).", "When stuffer II is 5.5 kb long, as in several constructs presented here, the size of the vector, excluding stuffer I, (that is the size of the construction segment CS) is calculated to be 37.5 kb.", "As reported in Table1, the vector having a stuffer II of 5.5 kb (CS size of 37.5 kb) is particularly useful in selecting long and full-length cDNAs compared to the use of the same vector having a stuffer II of 6 kb (CS size of 38 kb).", "Alternative stuffer II elements of 0 and 6.3 kb or even more, were also used to shift the cloning size and collect wide range size of cDNAs.", "Type I stuffers (FIGS.", "1d-f) can contain the background indicator LacZ and a background-reducing element, such as the ccdB-toxic element or an additional lox P site, which separates the antibiotic resistance gene and the origin of replication during excision (FIG.", "1i).", "All of the excised plasmids contain conventional forward (Fwd) and reverse (Rev) primer sequences and T7/T3 RNA polymerase promoters, to allow transcriptional sequencing (Sasaki et al., 1998, Proc.", "Natl.", "Acad.", "Sci.", "USA, 95:3455-3460) and transcription (FIGS.", "2g-j, underlined sequences).", "In addition, all plasmids can be used to produce single-stranded DNA (ssDNA), and all of them carry the f1(+) origin (Short et al., 1988, as above).", "When using the f1(+) origin of replication with helper phages such as R408 (Short et al., 1988, as above) to rescue ssDNA, the strand that is rescued is the opposite of the strand represented in FIGS.", "2g-j.", "In some constructs, we have also introduced cloning or recombination sites such as Gateway sequences flanking RS or the cDNA of interest or placing site sequences for homing endonucleases (New England Biolabs, Inc. also indicated as NEB) for bulk or individual excision of the cloned insert.", "Example 1 Construction of Vectors Any vector according to the invention was generated by following standard molecular biology techniques (Sambrook et al., 1989) and using the components shown in Figures.", "The λ arms (that is the portions at left and right side of Stuffer I) in vectors according to the invention were derived from λ-PS (Nehls et al., 1994a, as above) and were originally described for λ-2001 (Karn et al., 1984, Gene, 32:217-224).", "Into the XbaI site in the left arm of λ-PS, we inserted a 5.5-kb genomic fragment obtained by PCR amplification of mouse genomic DNA that was cleaved with XbaI and to which was ligated a linker/primer adapter containing an AscI restriction site for later removal or modification of the insert: the linker/primer upper oligonucleotide is: 5″-CTAGGCGCGCCGAGAGATCTAGAGAGAGAG (SEQ ID NO:9); the lower oligonucleotide is: 5′-CTCTCTCTCTAGATCTCTCGGCGC-3′ (SEQ ID NO:10).", "The upper is also used for PCR amplification.", "Before PCR amplification, the genomic DNA also was cleaved with XhoI, SalI, and SfiI to eliminate these sites from the amplified fragment.", "The amplification and agarose gel-purification steps (Boom et al., 1990, J. Clin.", "Microbiol., 28:495-503) were repeated 3 times.", "The 5.5-kb fragment size was chosen as the size regulator (stuffer II) for the λ-FLC-I-B vector, and its derivatives were created by cloning similarly obtained fragments of approximately 4.5 to 5.5 kb and we verified that inserts as short as 0.5 kb were clonable.", "In addition, the sequences of the polylinkers (sequences as appears in the excised plasmids of FIG.", "2) and stuffer I (FIG.", "1) were changed to accommodate directional cloning (according to Standard molecular biology techniques, for example Sambrook et al.", "), basically, restriction digestion, followed by re-ligation (T4 DNA ligase) with linker having the desired sequences which are inserted between the previous fragments of the phage.", "The 10-kb stuffer I (FIG.", "1b) was obtained from λ-PS (Nehls et al., 1994a, as above).", "The 3-kb shorter fragment of the stuffer (FIG.", "1c) was obtained by digesting the 10-kb stuffer I with XhoI and SalI.", "Subsequently, we amplified this 3-kb with the primers 5′-GAGAGACTCGAGGTCGACGAGAGAGGCCCGGGCGGCCGCGATCGCGGCCGGCCAGTCTTTAATTAACT-3′ (SEQ ID NO:11) and 5′-GAGAGAGGATCCGAGAGAGGCCAGAGAGGCCATTTAAATGCCCGGGCTGCAGGAATTCGATAT-3′ (SEQ ID NO:12) to add several restriction sites to the 3-kb stuffer (FIG.", "1c).", "To this modified stuffer (FIG.", "1c), we inserted the blunt-ended LacZ cassette into the SwaI site.", "Then, we restricted the modified stuffer with SfiI and inserted the ccdB gene as a triple ligation to obtain the stuffer I in FIG.", "1e.", "The ccdB gene was obtained by PCR amplification of the template pDEST-C, which can be propagated in E. coli DB3.1 (Life Technologies); the primer pairs were 5′-GAGAGAGCGGCCGCCCGGGCCATTTAAATCCGGCTTACTAAAAGCCAGA-3′ (SEQ ID NO:13) and the reverse primer 5′-AGCGGATAACAATTTCACACAGGA-3′ (SEQ ID NO:14)(as in pBluescript, Stratagene), and 5′-GAGAGAGGCCTCTCTGGCCACTAGTCTGCAGACTGGCTGTGTATA-3′ (SEQ ID NO:15) and the forward primer 5′-TGTAAAACGACGGCCAGT-3′ (SEQ ID NO:16).", "The LacZ cassette was obtained by digesting a pUC18 with NaeI and AflII and then blunting the appropriate fragment by using the Klenow fragment of DNA polymerase before cloning.", "Lox P, attB, and the modified polylinker sequences were prepared by annealing complementary oligonucleotides.", "The stuffer I of FIG.", "1e, after blunting the SalI and BamHI restriction sites, was dimerized by ligation with DNA ligase (New England Biolabs) to obtain the stuffer in FIG.", "1d.", "The stuffer in FIG.", "1f was obtained by PCR amplifying the stuffer in FIG.", "1c with a primer containing the Lox P site, 5′-GAGAGAGGATCCAGAGAGATAACTTCGTATAATGTATGCTATACGAAGTTATGAGAGAGGCCAGAGAGGCCATTTAA-3′ (SEQ ID NO: 17)(on the BamHI side), and the primer 5′-GAGAGACTCGAGGTCGACGAGAGAGGCCCGGGCGGCCGCGATCGCGGCCGGCCAGTCTTTAATTAACT-3′ (SEQ ID NO: 18)(on the SalI side).", "After purification (according to Boom et al., 1990, as above) and restriction digestion, this fragment was ligated with DNA ligase (according to Sambrook et al., 1989) to the ccdB fragment to yield the stuffer in FIG.", "1f.", "The plasmids obtained after excision (described later) are derivatives of pBluescript+ (Stratagene) or pBAC.", "The pDEST-C vector (Life Technologies) is the acceptor plasmid of the LxR reaction (Gateway System, Life Technologies) and, after excision, produces pFLC-DEST (FIG.", "2.j).", "pDEST is prepared from pBluescript II SK+ (Stratagene) by removal of the polylinker by digesting the pBluescript II SK+ with the restriction enzymes SacI and KpnI.", "Then, blunting the cleaved extremities with T4 DNA polymerase (according to Sambrook et al., 1989).", "The rfB II cassette (purchased by Life Technologies) comprising the ccdB gene was then inserted and ligated into the cleaved plasmid following the instruction of Gateway Cloning System Manual, Version 18.4, Life Technologies.", "The ligated plasmid vector was then cleaved with BssHI restriction enzyme and the cleaved fragment inverted (that is rotated of 180 degrees) and re-entered into the vector (according to known methodologies, Sambrook et al, 1989).", "The pDEST-C vector was used in the same way as is pDEST12.2 (Catalog and Instruction Manual, Gateway™ Cloning Technology, GIBCOBRL®, Life Technologies®).", "The λ-FLC-I-B vector was in general used as starting point for the construction of the other vectors according to the invention.", "λ-FLC-I-E was obtained by substituting the stuffer in FIG.", "1e for that of λ-FLC-I-B.", "λ-FLC-I-L-B was obtained by removing stuffer II from λ-FLC-I-B, and λ-FLC-I-L-D was created by substituting the stuffer shown in FIG.", "1e for that of λ-FLC-I-B.", "λ-FLC-II-C was obtained by joining a modified pBluescript II KS+ (purchased from Stratagene) with a stuffer like that in FIG.", "1c; the rest of the vector was as in λ-FLC-I-B.", "λ-FLC-III-F was created by inserting a construct containing the plasmid sequence and stuffer I of FIG.", "1f (the construct is shown FIG.", "2d) into λ-FLC-I-B-derived phage arms (including the 5.5-kb stuffer II) in the same way as described in the example “preparation of λ-FLC-III-C (but introducing the stuffer 1f instead of the stuffer 1c).", "The vector λ-FLC-III-F was also prepared as shown in FIG.", "7.λ-FLC-III-L-D was obtained from λ-FLC-III-F by first substituting the stuffer I of FIG.", "1f with the one of FIG.", "1d, followed by deletion of stuffer II.", "λ-FLC-III-S-F was obtained by ligating (using DNA ligase, as described in Sambrook et al., 1989) the concatenated arms from λ-FLC-I-B (devoid of stuffer II) with a 6.3 Kb long stuffer II and the “plasmid+stuffer I” derived from λ-FLC-III-F. Vector λ-FLC-III-E was prepared in the same ways as described for λ-FLC-III-F (and λ-FLC-III-C) introducing the stuffer 1e instead of the stuffer 1c or 1f; with “stuffer 1e” it is intended the stuffer I of FIG.", "1e, and the like for the other stuffers).", "Vectors comprising a pBAC or pBAC derivative can be prepared as shown in Example 20 and according to FIGS.", "9-12.Example 2 Preparation of λ-Arms for Cloning The final λ-DNA constructs were prepared by using standard methods (Sambrook et al., 1989) or the Lambda Maxi Prep Kit (#12562, Qiagen).", "The cohesive termini (cos ends) of 10 μg of λ-DNA were annealed by incubating for 2 h at 42° C. in 180 μl 10 mM Tris.Cl (pH 7.5)/10 mM MgCl2.We then added 20 μL 10× ligation buffer and 400 U T4 ligase (New England Biolabs) and incubated the mixture for 5 h at room temperature.", "The ligase was inactivated by incubating for 15 min at 65° C. At this point, the λ-DNA was digested with the required restriction enzymes (as described below; all purchased from New England Biolabs) in 3 steps because of the different concentrations of NaCl needed.", "For the first step, restriction was done in 50 mM NaCl by the addition of 2 μL 5 M NaCl, 6 U FseI, and 8 U PacI for each vector.", "The sample (the vector) was incubated for 4 h or overnight at 37° C. The second step was done in 100 mM NaCl by adding 2 μL 5 M NaCl, 30 μL 10×NEB 3 buffer, 270 μL H2O, and 20 U SwaI to the previous reaction and incubating for 2 h at room temperature.", "After this step, the reaction tube was heated for 15 min at 65° C. Finally, the third step was done in 150 mM NaCl by adding 5 μL 5 M NaCl, 40 U XhoI (in the cases of the μ-FLC-I and -III vectors, to reduce the background by reducing the size of the E. coli genomic DNA fragments; and for the μ-FLC-II vectors, to create the cloning site), 40 U SalI, and 40 U BamHI to the heat-inactivated reaction and incubating for 4 h at 37° C. For λ-FLC-II vectors, the SalI may be omitted or may be used to generate an alternative to the XhoI cloning site.", "The FseI, PacI and SwaI step are omitted for the λ-FLC-I-B, which does not carry these sequences.", "After restriction, the DNA was purified by proteinase λ treatment in the presence of 0.1% SDS and 20 mM EDTA, extracted with 1:1 phenol/chloroform and chloroform, and precipitated with ethanol (Sambrook et al., 1989).", "To avoid problems during resuspension, the DNA concentration did not exceed 20 μg/mL.", "After careful resuspension for at least 30 min, the digested DNA was separated in a 0.66% low-melting point agarose gel (Seaplaque®, FMC) according to the followings steps.", "The wells were in the middle of the gel.", "After electrophoresis for 1.5 h at 8 V/cm, the DNA fragments of the StyI-digested λ-DNA that were shorter than 19 kb were cut from the gel and discarded (step 1).", "Then, the electrophoresis buffer 1×TBE (electrophoresis buffer Tris-Borate-EDTA; see Sambrook et al., 1989) was replaced with fresh buffer, and the DNA remaining in the gel was electrophoresed in the opposite direction at 8 V/cm for 2.5 h. Then the DNA shorter than 19 kb again was discarded (step 2).", "The buffer was changed again.", "To condense the region containing the λ-arm DNA to decrease reaction volumes, the DNA remaining in the gel was electrophoresed at 8 V/cm for 30 min in the same direction as for step 1.Finally, the portion of the gel containing the λ-arm DNA was removed (step 3), the gel was equilibrated with TE buffer (Sambrook et al., 1989), and the λ-arms were purified and checked as described (Carninci and Hayashizaki, 1999, Methods Enzymology, 303:19-44) by using β-agarase (New England Biolabs).", "We typically recovered 30% to 50% of the starting λ-DNA.", "The purified λ-arms were stored indefinitely in single-use aliquots at −80° C. or at +4° C. for up to 1 week.", "A typical cloning efficiency was 1-2×107 pfu/μg λ-FLC-I-B vector with a test insert of 6 kb and less than 1% background of non-recombinant clones.", "Example 3 Preparation of λ-FLC-I-B λ-PS vector has been cleaved using BamHI restriction enzymes and stuffer I inserted using a left linker adapter comprising two complementary oligonucleotides: upper oligonucleotide 5′-GATCAGGCCAAATCGGCCGAGCTCGAATTCG-3′ (SEQ ID NO: 19) and lower oligonucleotide 5′-TCGAGAATTCGAGCTCGGCCATTTGGCCT-3′ (SEQ ID NO:20), and a right linker adapter comprising two complementary oligonucleotides: (SEQ ID NO:21) upper oligonucleotide 5′-GATCAGGCCCTTATGGCCGGATCCACTAGTGCGGCCGCA-3′ and (SEQ ID NO:22) lower oligonucleotide 5′-TCGATGCGGCCGCCTAGTGGATCCGGCCATAAGGGCCT-3′.", "Each one of two oligonucleotides of the left adapter, that is SEQ ID NO:19 and SEQ ID NO:20 was treated with Kinase with cold ATP for 20 min at 37° C. as follows: 1 μg of each oligonucleotide, 1 μl of ATP 5mM, 2 μl of PNK buffer (New England Biolabs), 0.5 μl of PNK (Polynucleotide Kinase; New England Biolabs), and water up to 20 μl.", "The obtained products were the two complementary oligonucleotides 5′-phosphorilated.", "The two oligo (SEQ ID NOS:19 and 20) solutions were mixed together and NaCl added to a final concentration of 100 mM.", "The mixer was incubated 15 min at 65° C. and then for 10 min at 45° C. to carry out the annealing.", "The annealed oligos were diluted at the concentration 0.5 ng/μl suitable for cloning.", "The same procedure was carried out for the oligo pair (SEQ ID NOS: 21 and 22) which were also annealed forming the right adapter.", "200 ng of λ-PS vector above cleaved with BamHI (that is the left and the right arms) were mixed with 0.4 ng of the left adapter and 0.4 ng of the right adapter, and 60 ng of the stuffer I, in a final volume of 5 μl.", "The ligation was carried out overnight (alternatively the ligation can also be carried out for 2 hours and 16° C.).", "The ligated vector/adapters/stuffer I was packaged according to the methodologies known in the art Sambrook et al., 1989).", "A stuffer II of 5.5-kb genomic fragment obtained by PCR amplification of mouse genomic DNA that was cleaved with XbaI was ligated at both extremities with a linker/primer adapter containing an AscI restriction site for later removal or modification of the insert.", "The linker/primer upper oligonucleotide is: 5″-CTAGGCGCGCCGAGAGATCTAGAGAGAGAG (SEQ ID NO:9); the lower oligonucleotide is: 5′-CTCTCTCTCTAGATCTCTCGGCGC-3′.", "(SEQ ID NO:10) The stuffer II with the adapter was introduced into the XbaI site in the left arm of λ vector above prepared, obtaining the vector λ-FCL-I-B.", "From this vector after the excision with in vitro Cre-lox recombinase (as described later), the plasmid pFLC-I-b (the plasmid of FIG.", "2g comprising the stuffer I of FIG.", "1b) was obtained.", "Example 4 Preparation of λFLC-III-C Plasmid pFLC-I-b, obtained from excision of λ-FLC-I-B as described above, was used as template and amplified by PCR.", "The primers used were: T7 Rev (56 mer) 5′-GTGTGATATCGCCCTATAGTGAGTCGTATTACATAGCTGTTTCCTGTGT GAAATTG-3′ (SEQ ID NO:23) and T3 Fwd (70 mer) 5′-GAGAGATATCTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCAATTCA CTGGCCGTCGTTTTACAACGTC-3′ (SEQ ID NO:24) obtaining the linear “product 1”.", "Plasmid pFLC-IIc was used as a template and amplified by PCR.", "The primers used were: FLCIIX2 (68 mer) 5′-GAGAGACTCGAGGTCGACGAGAGAGGCCCGGGCGGCCGCGATCGCG GCCGGCCAGTCTTTAATTAACT-3′ (SEQ ID NO:25) and primer FLCIIB2 (63 mer) 5′-GAGAGAGGATCCGAGAGAGGCCAGAGAGGCCATTTAAATGCCCGGGC TGCAGGAATTCGATAT-3′ (SEQ ID NO:26).", "The product of this PCR was cleaved with XhoI and BamHI restriction enzyme obtaining a linear fragment of 3 bk.", "This fragment was used as template for PCR amplification with the primers: 5′ I-CeuI-SalI (59 mer) 5′-GTGTAACTATAACGGTCCTAAGGTAGCGAGTCGACGAGAGAGGCCCG GGCGGCCGCGAT-3′ (SEQ ID NO:27) and 3′PI-SceI-BamHI (67 mer) 5′-GCATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGCAGGATCCGA GAGAGGCCAGAGAGGCCA-3′ (SEQ ID NO:28), obtaining the linear “product 2”.", "The “product 2” was then phosphorilated with PNK-polynucleotide kinase and gamma-ATP according to Sambrook et al., 1989.Then, the “product 1” was cleaved with the EcoRV restriction enzyme and the fragment obtained was ligated (according to the standard methodology, Sambrook e al., 1989) with the “product 2” prepared as above.", "A (circular) plasmid indicated as “product 3” was obtained.", "The plasmid “product 3” was used as template and amplified by PCR using the primers: XbaI-LoxP Tag primer 3F (69 mer) 5′-GAGAGTCTAGATAACTTCGTATAGCATACATTATACGAAGTTATAAATC AATCTAAAGTATATATGAGT-3′ (SEQ ID NO:29) and XbaI-LoxP Tag primer 3R (69 mer) 5′-GAGAGTCTAGATAACTTCGTATAATGTATGCTATACGAAGTTATAAAAC TTCATTTTTAATTTAAAAGG -3′ (SEQ ID NO:30) obtaining a linear product, which was then cleaved with XbaI restriction enzyme, obtaining the linear “product 4”.", "A λ-FLC-I-B was cleaved with XbaI restriction enzyme, then purified with electrophoresis according to the standard methodology (Sambrook, et al., 1989) and the resulting λ left arm, λ right arm, and stuffer II were recovered from the purification by electrophoresis.", "200 ng of λ left arm, 90 ng of λ right arm, 55 ng of Stuffer II, and 60 ng of the “product 4” were ligated overnight according to the standard methodology (Sambrook et al., 1989).", "The obtained vector λ-FLC-III-C was packaged according to the methodologies known in the art (Sambrook et al., 1989).", "By treatment with Cre-recombinase, the in vitro cre-lox recombinase excision was carried out and the plasmid pFLC-III-c (plasmid of FIG.", "2i comprising the stuffer I of FIG.", "1c)) obtained.", "Other λ-FLC vectors can be prepared starting from λ-FLC-III-C vector.", "For example, vector λ-FLC-III-F or λ-FLC-III-E can be prepared by substituting the stuffer Ic of λ-FLC-III-C with the stuffer If or Ie, respectively.", "Example 5 Preparation of λ-FLC-II-C pBluescript II SK+ (purchased from Stratagene) was digested with Kpn I and Not I.", "The large fragment was separated by agarose gel electrophoresis and purified.", "λ-FLC-I-B was digested with XhoI and SalI and blunted by T4 DNA polymerase, according to standard methodology (Sambrook et al., 1989).", "A 3 kb fragment was separated by agarose gel and purified.", "Then three double stranded linkers (AttB1, AttB2 and LoxP) were synthesized as follows.", "AttB1 linker: upper oligonucleotide is 5′-CGGGCCACAAGTTTGTACAAAAAAGCAGGCTCTCGAGGTCGACGAGA GGCCAGAGAGGCCGGCCGAGATTAATTAA-3′ (SEQ ID NO:31), lower oligonucleotide is 5′-TTAATTAATCTCGGCCGGCCTCTCTGGCCTCTCGTCGACCTCGAGAGC CTGCTTTTTTGTACAAACTTGTGGCCCGGTAC-3′ (SEQ ID NO:32).", "AttB2 linker: upper oligonucleotide is 5′-GGCCATGACGGCCGAGAGATTTAAATGAGAGAGGATCCACCCAGCTT TCTTGTACAAAGTGGTCTAGACCTCTCTTGG-3′ (SEQ ID NO:33), lower oligonucleotide is 5′-GAGGTCTAGACCACTTTGTACAAGAAAGCTGGGTGGATCCTCTCTCAT TTAAATCTCTCGGCCGTCATGGCC-3′ (SEQ ID NO:34).", "LoxP linker: upper oligonucleotide is 5′-CCGCATAACTTCGTATAGCATACATTATACGAAGTTATGC-3′ (SEQ ID NO:35), lower oligonucleotide is 5′-GGCCGCATAACTTCGTATAATGTATGCTATACGAAGTTATGCGGCCAA GA-3′ (SEQ ID NO:36).", "The lower strand of attB2 linker and the upper strand of LoxP linker were phospohorylated by using polynucleotide kinase PNK; New England Biolabs) according to how described above in the preparation of λ-FLC-I-B.", "The two oligos (SEQ ID NO:31 and 32) solutions were mixed together and NaCl added to a final concentration of 100 mM.", "The mixer was incubated 15 min at 65° C. and then for 10 min at 45° C. to carry out the annealing.", "The annealed oligos were diluted at the concentration 0.5 ng/μl suitable for cloning.", "The same procedure was carried out for the oligo pairs (SEQ ID NO: 33 and 34; and for SEQ ID NO:35 and 36) which were annealed respectively.", "AttB2 linker (0.5 ng) and LoxP linker (0.5 ng) were mixed and ligated in the volume of 5 μl.", "The tube was incubated at 16° C. After 20 min, attB1 linker (0.5 ng), pBluescript cleaved with KpnI and NotI (25 ng) and the 3 kb fragment from λ-FLC-I-B (25 ng) were added in the tube in the volume of 10 μl.", "Then, it was incubated overnight at 16° C. obtaining a ligation solution comprising a plasmid comprising the ligated fragment.", "The ligation solution comprising a plasmid was then introduced by electrophoresis into DH10B cells and plated on a medium.", "Plasmids was prepared from the recombinant cells.", "The cells were lysed and the plasmids cleaved with XbaI and a plasmid fragment was obtained “fragment 1”.", "A junction linker was prepared, having an upper oligonucleotide: 5′-GGCCATGAGAT-3′ (SEQ ID NO:37), and a lower oligonucleotide is: 5′-CTAGATCTCAT-3′ (SEQ ID NO:38).", "These two oligonucleotide were annealed and the “fragment 2” obtained.", "λ-FLC-I-B was cut with NotI and a 26 kb fragment was separated with agarose gel and purified “fragment 3”.", "A 9 kb fragment was also prepared by cleavage with XbaI of λ-FLC-I-B “fragment4”.", "These “fragments 1-4” (26 kb left arm, the junction linker, stuffer-plasmid, 9 kb right arm) were ligated in the volume of 5 μA.", "The ligation solution was packaged and amplified obtaining the vector λ-FLC-II-C.", "These steps were carried out according to standard procedures (Sambrook et al., 1989).", "From the vector λ-FLC-II-C after in vitro excision with Cre-recombinase (see later), the plasmid pFLC-II-c (the plasmid of FIG.", "2j comprising the stuffer I of FIG.", "1c) was obtained.", "Example 6 Preparation of λ-FLC-III-F A λ-FLC-III-F vector can be prepared as described at the end of Example 4, however, other methods of preparation are also possible.", "One alternative way of preparation of λFLC-III-F, which will be described in the present example is represented in FIG.", "7.To obtain lambda arms and stuffer II (5.5 kb), the cohesive termini of 10 μg of λ-FLC-I-B were annealed by incubating for 2 h at 42° C. in 180 μl 10 mM Tris.Cl (pH 7.5)/10 mM MgCl2.We then added 20 μL 10× ligation buffer and 400 U T4 DNA ligase (New England Biolabs) and incubated the mixture for 5 h at room temperature.", "The ligase was inactivated by incubating for 15 min at 65° C. The concatemerized λ-FLC-I-B was digested with 30 units of Xba I (NEB) in 1× manufactures recommendation buffer.", "The tube was incubated for 2 h at 37° C. After restriction, λ-FLC-I-B/XbaI DNA was purified by proteinase K (Qiagen) treatment in the presence of 0.1% SDS and 20 mM EDTA, extracted with 1:1 phenol/chloroform and chloroform, and precipitated with ethanol (Sambrook et al., 1989).", "To avoid problems during resuspension, the DNA concentration did not exceed 20 μg/mL.", "After careful resuspension for at least 30 min, the digested DNA was separated in a 0.6% low-melting point agarose gel (Seaplaque®, FMC) for 1.5 h at 8 V/cm.", "The portion of the gel containing the 29 kb λ DNA (ligation product between L-arm and R-arm) and 5.5 kb stuffer II were cut out and equilibrated with TE buffer (Sambrook et al., 1989).", "The DNAs were purified and checked as described (Carninci and Hayashizaki, 1999, Methods Enzymology, 303:19-44) by using β-agarase (New England Biolabs).", "3 μg of pBS II SK+ (Stratagene) was digested with 9 unit of Bss HII (NEB) at 37° C. for 2 h and dephosphorylated by CIP (Takara, Japan) (Sambrook et al., 1989, standard technique).", "To introduce homing nuclease sites (I-CeuI and PI-SceI) into pBS II SK+, double strand, an I-CeuI/PI-SceI adaptor oligonucleotide comprising an oligonucleotide up adaptor strand: 5′-pCGCGCTAACTATAACGGTCCTAAGGTAGCGAGTCGACGAGAGAGAG AGGATCCATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGCAG-3′ (SEQ ID NO:39) and an oligonucleotide down adaptor strand: 5′-pCGCGCTGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATGGATC CGAGAGAGAGAGTCGACTCGCTACCTTAGGACCGTTATAGTTAG-3′) (SEQ ID NO:40) was prepared (according to standard technique), and ligated with pBS II SK+/BssHII (NEB)/CIP (Takara, Japan).", "pBS II SK+/BssHII/CIP and I-CeuI/PI-SceI adaptor were ligated, by mixing 100 ng of pBS II SK+/BssHII/CIP, 2 ng of I-CeuI/PI-SceI adaptor, 400 unit T4 DNA ligase, 1× ligation buffer in a total volume of 5 μl.", "The tube was incubated overnight at 16° C. The ligation products were introduced into DH10B and cultured.", "The clones containing the proper plasmid were selected by preparing plasmid and restriction using I-CeuI (Sambrook et al., 1989, standard technique).", "Then the I-CeuI/PI-SceI adaptor was substituted with Stuffer If (the stuffer I of FIG.", "10 described as following.", "3 μg of plasmids comprising I-CeuI/PI-SceI adaptor were digested with 9 units of Sal I and 9 units of Bam HI in 30 μl.", "To remove the SalI-BamHI short fragment, the plasmid/SalI and BamHI were separated in a 0.6% low-melting point agarose gel (Seaplaque®, FMC) for 1.5 h at 8 V/cm.", "The 3 kb DNA was cut out and equilibrated with TE buffer (Sambrook et al., 1989).", "The 3 kb DNA were purified and checked as described (Carninci and Hayashizaki, 1999, Methods Enzymology, 303:19-44) by using β-agarase (New England Biolabs).", "We typically recovered 30% to 50% of the starting DNA.", "100 ng of the plasmid DNA and 140 ng of stuffer If were ligated with 400 unit T4 DNA ligase, 0.5 μl of 10× ligation buffer in a total volume of 5 μl.", "The tube was incubated overnight at 16° C. The ligation products were introduced into DH10B and cultured.", "The clones containing the proper plasmid were selected by preparing plasmid and restriction using BamHI and SalI (Sambrook et al., 1989, standard technique).", "In the next step loxP sites were introduced into the vector between ampr gene and ori.", "LoxP was introduced by PCR using XbaI-LoxP Tag primer 3F (69 mer) having the sequence: 5′-GAG-AGT-CTA-GAT-AAC-TTC-GTA-TAG-CAT-ACA-TTA-TAC-GAA-GTT-ATA-AAT-CAA-TCT-AAA-GTA-TAT-ATG-AGT-3′ (SEQ ID NO:41) and XbaI-LoxP Tag primer 3R (69 mer) having the sequence: 5′-GAG-AGT-CTA-GAT-AAC-TTC-GTA-TAA-TGT-ATG-CTA-TAC-GAA-GTT-ATA-AAA-CTT-CAT-TTT-TAA-TTT-AAA-AGG-3′ (SEQ ID NO:42) (according to standard technique).", "Using 3 μg of the resulting PCR product (7.2 kb), the PCR product was digested with 9 units of XbaI at 37° C. for 1 h (Sambrook et al.,).", "To remove short DNA fragment resulting from PCR product/XbaI, the digested product was separated in a 0.6% low-melting point agarose gel (Seaplaque®, FMC) for 1.5 h at 8 V/cm.", "The 7.2 kb DNA was cut out and equilibrated with TE buffer (Sambrook et al., 1989).", "The 7.2 kb DNA were purified and checked as described (Carninci and Hayashizaki, 1999, Methods Enzymology, 303:19-44) by using β-agarase (New England Biolabs).", "The 7.2 kb PCR product, the purified arms and stuffer II (5.5 k) were ligated in the ratio of 25 ng: 100 ng: 19 ng with 400 units of T4 DNA ligase (Sambrook et al., 1989).", "The ligation solution was packaged and amplified obtaining the vector λ-FLC-III-F.", "These steps were carried out according to standard procedures (Sambrook et al., 1989).", "Example 7 Preparation of λ-FLC-III-E The λ-FLC-III-E vector can be prepared by substituting the stuffer I of other FLC-III vectors with the stuffer Ie.", "In the present example, λ-FLC-III-E was obtained by substituting the stuffer If of the λ-FLC-III-F vector prepared in Example 6 with the stuffer Ie (i.e.", "the stuffer I of FIG.", "1e) according to the following steps.", "The cohesive termini of 10 μg of λ-FLC-III-F were annealed by incubating for 2 h at 42° C. in 180 μl 10 mM Tris.Cl (pH 7.5)/10 mM MgCl2.We then added 20 μL 10× ligation buffer and 400 U T4 DNA ligase (New England Biolabs) and incubated the mixture for 5 h at room temperature.", "The ligase was inactivated by incubating for 15 min at 65° C. At this point, the concatemerized λ-FLC-III-F was digested with the required restriction enzymes, by adding 30 units of BamHI, 30 units of SalI and 40 μl 10× BamHI buffer (all purchased from New England Biolabs) in a total volume of 400 μl.", "The tube was incubated for 2 h at 37° C. After restriction, the DNA was purified by proteinase K (Qiagen) treatment in the presence of 0.1% SDS and 20 mM EDTA, extracted with 1:1 phenol/chloroform and chloroform, and precipitated with ethanol (Sambrook et al., 1989).", "To avoid problems during resuspension, the DNA concentration did not exceed 20 μg/mL.", "After careful resuspension for at least 30 min, the digested DNA was separated in a 0.6% low-melting point agarose gel (Seaplaque®, FMC) for 1.5 h at 8 V/cm.", "The portion of the gel containing the λ DNA was cut out and equilibrated with TE buffer (Sambrook et al., 1989).", "The λDNA were purified and checked as described (Carninci and Hayashizaki, 1999, Methods Enzymology, 303:19-44) by using β-agarase (New England Biolabs).", "We typically recovered 30% to 50% of the starting λ-DNA.", "To obtain stuffer Ie (FIG.", "1e), 10 μg of λ-FLC-I-E were digested with 30 units of BamHI, 30 units of SalI in 200 μl 1×BamHI buffer.", "The tube was incubated for 2 h at 37° C. After restriction, the 5 kb DNA fragment was separated in a 0.6% low-melting point agarose gel (Seaplaque®, FMC) for 1.5 h at 8 V/cm.", "The 5 kb DNA (stuffer Ie) was cut out and equilibrated with TE buffer (Sambrook et al., 1989).", "The 5 kb DNA were purified and checked as described (Carninci and Hayashizaki, 1999, Methods Enzymology, 303:19-44) by using β-agarase (New England Biolabs).", "We typically recovered 30% to 50% of the starting DNA.", "The λ-FLC-III-F having the stuffer If removed, and stuffer Ie (prepared as above) were ligated (the ratio was 210 ng to 30 ng) by mixing with 400 units T4 DNA ligase in 10 ul of 1× ligation buffer (NEB).", "The tube was incubated overnight at 16° C. The ligation solution was packaged and amplified obtaining the vector λ-FLC-III-E.", "These steps were carried out according to standard procedures (Sambrook et al., 1989).", "Example 8 Preparation of pDEST-C pBluescript II SK+ (purchased from Stratagene) was cleaved with SacI and KpnI restriction enzymes followed by blunting with T4 DNA polymerase (Sambrook et al., 1989) and two fragments were obtained.", "The short fragment was removed by agarose gel electrophoresis and the long fragment purified and recovered.", "The purified long fragment was ligated with RfB cassette overnight at 16° C. according to standard methodology (Sambrook et al.", "1989) and introduced into DH10B cells by electroporation (Sambrook et al.", "1989).", "Recombinant clone was amplified and plasmid extracted (pDEST-A) In order to invert the BssHII fragment in pDEST-A, pDEST-A was cut with BssHII restriction enzyme and then extracted by using phenol/chloroform and precipitated by ethanol (Sambrook et al., 1989) and two fragments were obtained.", "These two fragments, digestion products of pDEST-A, were ligated overnight at 16° C. by inverting the RfB cassette of 180 degrees (Sambrook et al., 1989) and the obtained plasmid introduced into DH10B cells by electroporation.", "The clone having the fragment inverted was selected (pDEST-C) by restriction mapping (Sambrook et al.", "1989).", "Example 9 Preparation of pFLC-DEST λ-FLC-II-C and pDONR201 (Life Technologies) were recombined by BP clonase (Life Technologies).", "Then the recombination vector was mixed with pDEST-C and recombined by LR clonase.", "The reaction solution was introduced into DH10B cells by electroporation and the recombinant clone selected on LB plate containing ampicillin.", "Recombinant cells were amplified and the plasmid (pFLC-DEST) was prepared.", "Example 10 Preparation of Purified pFLC-III-f 100 ng of λ-FLC-III-F were treated with 1 U Cre-recombinase (in vitro cre-lox mediated recombinase) at 37° C. for 1 hour in 300 μl, and the FLC-III-f plasmid was excised.", "The plasmid was then extracted with phenol/chloroform, and chloroform, and precipitated with ethanol (according to Sambrook et al., 1989).", "The recovered plasmids were electroporated into DH10B (Life Technologies) at 2.5 kb/cm.", "The cells were spread on LB agar containing ampicillin, X-gal (Sambrook et al., 1989) and cultured overnight at 37° C. Blue colony from LB plate containing ampicillin were picked up and plasmids prepared using QIAGEN kit.", "The plasmids were digested-with restriction enzymes (I-CeuI, PI-Sce I) according to the following steps.", "First restriction step: a solution of 20 μl of 10×I-Ceu I buffer, 20 μl of 10×BSA and 3U of I-Ceu I (total volume 200 μl) was prepared in a tube and incubated for 5 hour at 37° C. Second step of restriction: 22.5 μl of 10×PI-Sce I buffer and 3 U PI-Sce I were added and the obtained solution incubated for 5 hour at 37° C. After this step, the tube was heated for 15 min at 65° C. Then, the digested DNA was purified by proteinase K treatment (Sambrook et al., 1989), extracted with phenol/chrolofolm, chroloform,and prepicipated with ethanol (as described in Sambrook et al., 1989).", "After careful resuspension, the digested DNA was separated in 0.8% low melting agarose gel as follows.", "After electrophoresis for 1.5 hours at 50V, the DNA fragments (2.9 kb) were cut off from gel and recovered.", "They were purified with QIAGEN QIAquick Gel Extraction kit and then used for the ligation.", "Example 11 Preparation of cDNA and Cloning Full-length cDNAs were prepared as described (Carninci and Hayashizaki, 1999, as above; Carninci et al., 1997, DNA Res., 4:61-66) and normalized and/or subtracted (Carninci et al., 2000, Genome Res., 10:1617-1630) before cloning.", "After digestion with 25 U BamHI (New England Biolabs)/μg cDNA (to cleave the 3′ end) and 25 U XhoI (Fermentas Vilnius, Lithuania)/μg cDNA (to cleave the 5′ end), the cDNA was treated with 1.3 U thermosensitive shrimp alkaline phosphatase (SAP; Amersham Pharmacia Biotech)/μg cDNA to avoid concatenation and chimerism of cDNAs, which are concerns when working with large-capacity cloning vectors.", "Then the cDNA was treated with proteinase K, extracted with phenol/chloroform, and applied to a CL-4B spin column (Amersham Pharmacia Biotech).", "The purified cDNA was ethanol-precipitated (Carninci and Hayashizaki, 1999, as above) or size-fractionated.", "Normalization/subtraction was not used for cDNA that was size-fractionated by using an agarose gel.", "This process was similar to that used in the isolation of the λ arms of the vectors: the direction of electrophoresis was inverted after short fragments were run out of the gel (we changed the buffer before resuming the electrophoresis).", "cDNA was isolated from the gel either by using β-agarase (New England Biolabs) as described or by binding in the presence of 7 M guanidine-Cl to double-acid-washed and size-fractionated diatomaceous earth (Sigma) essentially as described (Boom et al., 1990, J. Clin.", "Microbiol., 28:495-503).", "cDNA and vectors were always ligated(according to Carninci and Hayashizaki, 1999, Methods Enzymology, 303:19-44) at an equimolar ratio in a 5-μL reaction containing T4 DNA ligase (New England Biolabs).", "The quantity of cDNA was estimated by the radioactivity incorporated during synthesis of the first and second strands (Carninci and Hayashizaki, 1999, as above).", "The cloning sites on the vectors were the SalI (cohesive ends with XhoI) and BamHI sites, except that XhoI and BamHI sites were used for the λ-FLC-II-C vector.", "cDNA sequencing was performed as described (Shibata K., et al., 2000, Genome Res., 10:1757-1771), and sequence analysis and clustering were performed as described (Konno et al., 2001, Genome Res., 11:281-289).", "Example 12 Bulk Excision of cDNA Libraries I) In Vivo, Solid-Phase Excision (State of the Art) cDNA libraries were amplified in E. coli C600 cells.", "Approximately 1-5×104 pfu were plated on 150-mm dishes of LB-agar, topped with LB-agar containing 10 mM MgSO4, and grown overnight to confluence (Sambrook et al., 1989, as above).", "Subsequently, phage particles were eluted with SM-buffer and titered.", "Then, BNN132 cells were grown overnight in LB-broth plus 10 mM MgSO4.Cells were pelleted, resuspended in 10 mM MgSO4, and immediately infected with the phage library, which was converted in vivo to a plasmid DNA library and plated on LB-ampicillin plates.", "II) In Vivo, Liquid-Phase Excision Up to 5×1010 phage particles prepared as above were used to infect 10 mL of overnight-grown BNN132 cells (OD600=˜0.5) after pelleting and resuspending in 10 mM MgSO4, which were then cultured in 90 LB medium supplemented with 100 μg/ml of ampicillin.", "After 1, 2 or 3 h at either 30° C. or 37° C., the cultures were stopped, and we extracted the plasmid by using the Wizard Plus Midiprep DNA Purification System (Promega).", "The plasmid library was electroporated into DH10B cells (Life Technologies) at 2.0 Kv/cm, which are suitable for sequencing operations as described (Shibata K., et al., 2000, as above).", "III) In Vitro Cre-Lox-Mediated Excision Phage cDNA libraries were amplified in C600 cells as described.", "We isolated the library phage DNA from the amplified phage solution by using the Wizard Lambda Preps DNA Purification System (Promega).", "We converted one fourth of the obtained phage DNA to plasmid by treating with 1 U Cre-recombinase at 37° C. for 1 h in 300 μL as recommended (Novagen), and then purified (proteinase K treatment, phenol/chloroform extraction and ethanol precipitation, according to Sambrook et al., 1989).", "The bulk-excised plasmid libraries were electroporated into DH10B cells (Life Technologies) at 2.0 kV/cm.", "IV) Gateway-Mediated Bulk-Excision (“Indirect”) Protocol We mixed 16 ng library phage DNA, 300 ng pDONR201(Instruction Manual, Gateway Cloning Technology, GibcoBRL, Life Technologies), 4 μL BP buffer, and BP Clonase enzyme mix (Life Technologies) in 20 μL.", "Overnight incubation at 25° C. was followed by proteinase K treatment in the presence of 0.2% SDS and 10 mM EDTA at 45° C. for 15 min.", "We added 1 μg glycogen and extracted the reaction by using phenol/chloroform and chloroform; the sample was precipitated by using isopropanol.", "The precipitate was mixed with 300 ng pDEST12.2 (Life Technologies), 4 μL LR buffer, and 4 μL LR Clonase enzyme mix in a volume of 20 μL.", "The sample was further purified with proteinase K/phenol chloroform extraction followed by ethanol precipitation.", "V) “Amplified Indirect” Protocol The sample was treated as in the previous protocol (Gateway mediated bulk excision-“indirect”) until the BP Clonase reaction.", "We electroporated 1 μL of the 20-μL reaction into DH10B cells.", "The cells were spread on LB containing kanamycin, and the resulting colonies underwent plasmid extraction (Sambrook et al., 1989).", "The prepared plasmids were each reacted with LR Clonase and purified and then electroporated as before.", "VI) “One-Tube” (“Direct”) Protocol The procedure was the same as that for the indirect protocol until the BP Clonase reaction (Life Technologies).", "Then, we added 450 ng pDEST12.2, 6 μL LR Clonase enzyme mix, and 1 μL 0.75 M NaCl to the tube (total volume, 30 μL).", "The sample was treated with LR Clonase and purified as described.", "The BP/LR-reacted samples were dissolved in sterile water and electroporated into DH10B cells.", "The transformed cells were spread on LB plates containing either ampicillin or kanamycin and cultured overnight at 37° C. To assess the conversion frequency of each excision method, we prepared the plasmids from 60 random colonies from LB plates.", "The plasmids were cut with PvuII, and the sizes of the inserts were analyzed by using 0.8% agarose gels.", "We also could assess the conversion efficiency by counting the colonies that grew on ampicillin- or kanamycin-containing plates.", "Example 13 Homing Endonuclease System: A Vector For Ligation-Mediated Transfer of Inserts: λ-FLC-III-F 1) Insert cDNA Preparation cDNA libraries were prepared by cloning the cDNA (prepared as in Carninci et al., 2000, Genome Research, 10:1617-1630) into the λ-FLC-III-F vector (Example 6), which carries the homing endonucleases I-CeuI and PI-SceI (New England Biolabs) at either side of the cloning sites (SalI and BamHI).", "These homing endonucleases, which recognize and cleave sequences of 26 and 39 bp respectively, do not cleave mouse genome (in fact, these homing endonucleases statistically cut once every 1.8×1018 base pairs and once every 1.2×1024, respectively and therefore are very unlikely to cut even once high complex genomes such as Human and Mouse, whose total size is about 3×109 base pairs).", "Therefore, they are optimal for subcloning cDNAs without internal cleavage of any of the tens of thousand clones in a library.", "A phage cDNA library was prepared according to one variant of the cap-trapper technology (Carninci et al., 2000, Genome Research, 10:1617-1630) and cloned into λ FLC-III-F and amplified in C600 cells (Sambrook et al., 1989).", "We isolated the library phage DNA from 1 ml of the amplified phage solution by using the Wizard Lambda Preps DNA Purification System (Promega).", "Purified library phage DNA was digested with restriction enzymes (I-CeuI, PI-Sce I).", "First restriction step: a solution of 5 μl of 10×I-Ceu I buffer, 5 μl of 10×BSA and 2.5 U of I-Ceu I (total volume 50 μl) was prepared in a tube and incubated for 4 hour at 37° C. After this step, the restriction tube was heated for 15 min at 65° C. The digested DNA was purified by proteinase K treatment (Sambrook et al., 1989), extracted with phenol/chloroform, and chloroform, and precipitated with isopropanol, and very carefully resuspended.", "The second step restriction was carried out as follows: redissolve the DNA in 40 μl of water, add 5 μl of 10×PI-Sce I buffer and, 4 U PI-Sce I (New England Biolabs, total volume 50 μl),and incubate for 4 h at 37° C. After this step, the restriction tube was heated for 15 min at 65° C. The digested DNA was purified by proteinase K treatment, extracted with phenol/chloroform, and chloroform, and precipitated with isopropanol, and very careful resuspension.", "(as in Sambrook et al., 1989).", "2) pFLCM-f Preparation λ-FLCIII-F vector (Example 6) was excised with in vitro cre-lox mediated recombinase.", "At first, 100 ng of λ-FLCIII-F were treated with 1 U cre-recombinase at 37° C. for 1 hour in 300 μl final volume.", "Then, extracted with phenol/chloroform, and chloroform, and precipitated with isopropanol (Sambrook et al., 1989).", "The plasmids were electroporatetd into E. coli DH10B (Life Technologies) at 2.5 kv/cm following the instruction of the manufacturer.", "Cells were spread on LB-agar (Sambrook et al., 1989) containing 50 μg/ml of ampicillin.", "To the surface of the agarose in the 9 cm petri dish, we added also 40 microliters of 2% X-gal and 7 microliters of 200 mM IPTG for colorimetric detection of the plasmid carrying the LacZ stuffer I to facilitate later identification of the background (for a theoretical consideration: Sambrook et al., 1989).", "The plate was cultured overnight at 37° C. and the day later several dozens colonies appear.", "We picked one blue colony from the above LB, inoculated in 50 ml of LB-broth/50 microgram/ml ampicillin and let grow overnight with 300 rpm shaking (Sambrook et al., 1989).", "Next day we prepared plasmid DNA by QIAprep spin mini prep kit (QIAGEN).", "3) Plasmid Vector Preparation (Removal of the Stuffer I) (See Also FIG.", "8) This step is to prepare a plasmid (in this case pFLC-III-f) devoid of the stuffer I (in this case stuffer of FIG.", "1f) to maximize the recombination.", "Three μg of plasmids cDNA were digested with restriction enzymes (I-Ceu I, PI-Sce I ).", "In the first step restriction was done in total volume 50 μl in presence of 5 μl of 10×I-Ceu I buffer, (New England Biolabs), 5 μl of 10×BSA (bovine serum albumine supplied by New England Biolabs with the enzyme) and 4 U of I-Ceu I (New England Biolabs, and incubation for 4 hour at 37° C. After this step, the restriction tube was heated for 15 min at 65° C. Digested DNA was purified by proteinase K treatment, extracted with phenol/chloroform, and chloroform, and precipitated with isopropanol, and very carefully resuspended (Sambrook et al., 1989).", "The second restriction step was done in a total volume of 50 μl supplemented with.", "5 μl of 10×PI-Sce I buffer (New England Biolabs), 4 U PI-Sce I (New England Biolabs,), and incubated for 4 hour at 37° C. After this step, the restriction tube was heated for 15 min at 65° C. Digested DNA was purified by proteinase K treatment, extracted with phenol/chloroform, and chloroform, and precipitated with isopropanol (Sambrook et al., 1989).", "After very careful resuspension, the digested DNA was separated in 0.8% low melting agarose gel (seaplaque agarose FMC) buffered with TAE (Tris-acetate-EDTA; see Sambrook et al., 1989).", "In the following step: after electrophoresis for 1.5 h at 50V, the DNA fragment corresponding to the empty plasmid vector (2.9 kb) was cut off from gel and purified by QIAGEN QIAquick Gel Extraction kit (QIAGEN).", "4) Ligation of Cleveaged Plasmid pFLC-III-f and cDNA Insert (See Also FIG.", "8) 7.5 ng of prepared insert and 100 ng of pFLCIII-f plasmid vector, prepared in the above step 3), were mixed in a final volume of 100 μl, containing also 10×T4 DNA ligase buffer (New England Biolabs) and DNA 200 U of T4 ligase (New England Biolabs) and incubated at 16° C. overnight.", "Ligated palasmids were electroporated into DH10B at 2.5 Kv(Kilovolt)/cm (Invitrogen) following the manufacturer's instruction.", "Cell were spread on LB containing ampicillin (as above), and cultured overnight at 37° C. We picked then randomly 12 colonies and prepared plasmids (inoculation in 3 ml LB-broth/50 microgram/ml ampicillin and let grow overnight with 300 rpm shaking (Sambrook et al., 1989).", "Plasmid DNA was prepared with a Quiagen plasmid DNA extraction kit.", "The plasmids were cut with PvuII (New England Biolabs) in presence of 1×Pvu II buffer) and their insert size was analyzed using 0.8% TBE agarose gel stained with Ethidiumbromide (Sambrook et al., 1989).", "5) Result Titer: pFLCIII-f+ insert (cDNA):2.1×104 pfu/ml Insert size check (average size) Excision protocol here presented: 3.07 kb In vitro Cre-lox mediated recombinase (control experiment): 3.1 kb.", "The control experiment consisted in the same library excised with the Cre-lox following protocol as the example 12, (number III, in vitro Cre-lox mediated excision).", "It has been known in the art that the use of restriction enzymes give high size bias.", "In fact, usually plasmid libraries prepared by ligation show half the size of lambda-excised cDNA libraries (in Table 2 the cerebellum library is 1.4 Kb in pBluescript while 3.36 Kb with λ-FLC-I-B: the size is only 41.6%, and therefore not very efficient).", "In the current example, instead, the size with the homing nucleases is 3.07 kb versus 3.0 kb, the 99%, which is almost not relevant size bias (a 1% size bias enters in the statistical variability).", "In conclusion, we proved that the excision system using homing endonucleases restriction enzymes is an efficient excision system.", "Example 14 Vectors For Size Selection and Background-Reducing Systems The λ-FLC-I-B and other vectors shown in the FIGS.", "1 and 2 has been used to successfully prepare libraries of full-length mouse cDNA, and showed to having a cloning capacity of ˜0.2 to 15.4 kb cDNAs.", "When we tried to clone strongly subtracted cap-trapped cDNAs (according to the method described in Carninci et al., 2000, Genome Res., 10: 1617-1630), we found that because of the paucity of cDNA (less than 10 ng), using λ-FLC-I-B led to a certain background.", "When this background exceeded 20% to 30%, it affected the cost-performance of subsequent large-scale sequencing operations.", "To develop a vector associated with less background, we prepared a new, very effective method to decrease the background of λ-phage libraries that are excised into plasmids.", "We substituted the stuffer I in λ-FLC-I-B with that in FIG.", "1e to produce the λ-FLC-I-E.", "The stuffer of this vector carries 2 copies of the “suicide gene” ccdB (Bernard and Couturier, 1992, J. Mol.", "Biol., 226: 735-745) and a functional LacZ for blue-white selection (FIG.", "1f).", "Notice that the LacZ present in the pBluescript-derived fragment is nonfunctional because it is disrupted by either stuffer I or the cloned cDNA.", "Interestingly, λ phages carrying the cob gene can replicate in E. coli C600; this suggests that during the lytic cycle of the λ phage, DNA gyrase, the target of the ccdB gene product, is dispensable.", "After the excision procedure, we plated the equivalent of up to 300 pg of the excised vector (without insert) but did not obtain any colonies.", "On the contrary, in a control experiment, we obtained more than 1175 colonies (equivalent to the background) when we plated the equivalent of ˜3.5 pg of a similar construct containing a 3.6-kb insert but without ccdB instead of the stuffer.", "This difference constitutes an impressive background reduction of at least 105-fold, similar to that of λ-FLC-III-F (described later).", "Example 15 DNA Contamination Background All of the tested background-reducing stuffers like those in FIGS.", "1d-f yielded undetectable background derived from nonrecombinant vectors and therefore can be considered interchangeable.", "With the vectors λ-FLC-I-E, λ-FLC-III-F, λ-FLC-III-D, λ-FLC-III-S-F, and λ-FLC-I-L-D, the background depend on the environmental DNA contamination.", "In a test experiment, we did not ligate any cDNA to λ-FLC-I-E. Because there was no background to reduce at the λ-plating stage, we obtained 8.4×104 pfu/μg vector, which included the contribution of non recombinant vector, compared with typical values of >107 pfu/μg for positive controls.", "We amplified the background plaques, excised the plasmids, analysed 12 clones, and sequenced representative samples showing different electrophoretic patterns.", "The background clones that remained after the selection were derived only from the E. coli genome, which was probably a residual from the dead E. coli cells during the vector DNA preparation, whereas no vector sequence was found in any insert.", "Therefore, if a goal is the complete absence of background, all contaminating genomic DNA must be eliminated from the λDNA preparations and, perhaps more importantly, cDNAs must have intact ends so that they are easily clonable.", "Example 16 Background-Reduction loxP System The background reduction associated with stuffer I differs from that of the stuffer in λ-FLC-I-E, because we independently tested a double strategy using a single copy of ccdB and an additional lox P site inserted into the stuffer I (FIG.", "1f).", "During the excision process, the third lox P site favours the separation of the origin of replication from bla (the gene for β-lactamase, for conferring resistance to ampicillin), as shown in FIG.", "1i.", "To eliminate this problem, we manipulated the order of the plasmid sequence and lox P elements in the λ-vector so that the lox P on stuffer I was between bla and the origin of replication.", "Neither of the defective excised plasmids can replicate or confer antibiotic resistance (FIG.", "1i).", "In a preliminary experiment, we constructed a λ-FLC-III-type vector that contained as a stuffer only the background-reducing sequence of FIG.", "1i but without the ccdB gene.", "We obtained 43 colonies from ˜3.5 pg of the excised plasmid compared with 771 from ˜3.5 pg of a control excised plasmid of the same size that lacks both the lox P background reducing sequence and the ccdB gene.", "Therefore, the lox P background-reducing sequence eliminated 94.4% of the background.", "When ccdB was added to the lox P-containing stuffer, the resulting vector did not yield any colonies even when we electroporated up to 350 pg of excised plasmid, which had a background-reducing element like that in FIG.", "1f.", "This result corresponds to a background reduction of at least 7.7×104-fold, a factor similar to that obtained with the background-reducing element of the λ-FLC-I-E vector.", "The background-reducing systems of both the λ-FLC-III-F and λ-FLC-I-E vectors were considered sufficient for our full-length cDNA cloning purpose.", "Example 17 Bulk Excision of cDNA Libraries Before bulk excision, cDNA libraries are optionally amplified on a solid-phase medium according to the standard procedure (Sambrook et al., 1989).", "This process does not decrease the size of the cDNA library, but because of the preferential packaging of long phages, decreases (but does not eliminate) the frequency of the phages that carry cDNA inserts of approximately ≦0.5 kb.", "Amplification in C600 cells eliminates hemimethylation, which is used to clone the cDNA (Carninci and Hayashizaki, 1999, as above).", "Hemimethylated cDNA of a primary cDNA library would be cleaved during the in vivo excision in BNN132 (described later).", "I) Cre-Lox-Based Excision—In Vivo Solid-Phase Excision The in vivo solid-phase excision process (representing the state of the art) seems straightforward (FIG.", "3), simply requiring infection of the amplified cDNA library into the BNN132 bacterial strain, which constitutively expresses Cre-recombinase (Elledge et al., 1991, Proc.", "Natl.", "Acad.", "Sci.", "USA, 88:1731-5).", "However, this practice is not recommended, because of plasmid instability (Summers et al., 1984, as above) and low plasmid yield (Palazzolo et al., 1990, as above).", "In fact, Cre-recombinase is expressed constitutively, causing formation of plasmid dimers and multimers and leading to a high proportion of plasmid-free cells (Summers et al., 1984, as above), thereby impairing the sequencing efficiency.", "We confirmed that low plasmid yield and plasmid loss after prolonged culture are the rule when using BNN132 as a host strain for cDNA libraries.", "II) Cre-Lox-Based Excision—In Vivo Liquid-Phase Excision The in vivo liquid-phase excision process overcomes this problem of plasmid loss and poor yield after prolonged culture: we extracted the excised plasmid cDNA library after a brief culture at 30° C. or 37° C. and electroporate into any convenient E. coli strain, such as DH10B.", "Similar results in terms of size of the excised library were obtained after culture/excision for 1, 2, or 3 h at either 30° C., which is supposed to preserve the size of the library unbiased by keeping the plasmid at a low copy number (Lin-Chao et al., 1992, Mol.", "Microbiol., 6:3385-3393), or 37° C., at which plasmids are expressed at increased copy number.", "The copy number is also inversely proportional to the size of the cDNA inserts.", "When we excised a cDNA library cloned in λ-FLC-I-B, the final titer after the excision was 2.4×108 cfu/μg after culture for 1 h at 30° C., 9.1×108 cfu/μg after 2 h at 30° C., and 1.4×109 cfu/μg after 3 h at 30° C. The titers after growth at 37° C. were 1.5×109 cfu/μg after incubation for 1 h, 9.8×108 cfu/μg after 2 h, and 2.8>109 cfu/μg after 3 h. The average insert size was 4.1, 3.9, and 3.3 kb for 1, 2, and 3 h at 30° C., and 2.9, 3.6, and 3.8 kb for 1, 2, and 3 h at 37° C., respectively.", "These results suggested that there were no noteworthy excision-associated problems related to the length of inserts or to the temperature and duration of the BNN132 E. coli culture.", "To better quantify the size bias associated with the Cre-lox excision system, we mixed an equal number of non-recombinant λ-FLC-I-B vectors carrying the 10-kb stuffer with phages from the amplified cDNA library, then infected the cells.", "The ratio of clones containing the 10-kb insert was close 50% at all of the described conditions.", "This result confirms the robustness against size bias of the Cre-lox excision system.", "Among the advantages of this in vivo liquid-phase excision method is the high DNA yield, which facilitates downstream operations, such as the production of consistent quantities of single-stranded plasmid DNA by using GeneII-ExoIII, which can be used for further normalization/subtraction of existing cDNA libraries (Bonaldo et al., 1996, Genome Res., 6:791-806) while avoiding plasmid amplification steps that could decrease the size of the amplified library.", "III) Cre-Lox-Based Excision—In Vitro Excision Although it does not show size bias, the in vivo liquid-phase excision procedure still involves a brief round of library amplification, which might cause sequence-specific representational bias.", "Therefore, we developed the in vitro excision method, which is based on Cre-mediated recombination.", "This excision system uses purified λDNA from the amplified cDNA library, followed by electroporation.", "For this application, we tested the electroporation conditions described for long BAC inserts (Sheng et al., 1995, Nucl.", "Acids Res., 23:1990-1996).", "In light of our results from sizing 60 plasmids after restriction with PvuII, we did not find significant differences in the final size of the plasmid cDNA library when we used pulses between 1.7 and 2.5 kV/cm.", "We regard the Cre-lox in vitro excision protocol as the most suitable of those we tested, because it does not require even a brief amplification step of cDNA libraries in BNN132, is robust in terms of size bias, and can be used with all of the vectors described here.", "IV) Gateway™-System-Mediate Excision For λ-FLC-II-C, in addition to the Cre-lox excision protocol for excising a pFLC-II plasmid (FIG.", "2h), we have developed protocols for bulk excision which are based on the Gateway system.", "Inserts are at first transferred into an entry vector, the pDONR201 (Life Technologies), followed by transferring to a destination vectors, the pDEST12.2 (Life Technologies, structure not shown).", "λ-FLC-II-C vector that we prepared carries the Gateway attB1 and attB2 sequences for transferring individual clones (Walhout et al., 2000, as above) or bulk libraries into different functional vectors (FIG.", "2c) or into pFLC-DEST (FIG.", "2j) for sequencing.", "The three Gateway excision protocols (the “indirect”, “amplified indirect”, and “direct” protocols) are outlined in FIG.", "3 and described above in the experimental part.", "Any of the Gateway-mediated bulk-excision protocols was a valid alternative to the Cre-lox bulk excision procedure.", "In fact, the average size of 60 clones from the excised cDNA sublibraries was 2.3 kb for the control Cre-lox reaction (in vitro Cre-recombinase protocol), 2.4 kb with the “indirect” protocol, 2.5 kb with the “amplified indirect” protocol, and 3.3 kb with the “direct” protocol.", "The average size of this cDNA before excision was 3.7 Kb.", "Considering the final size close to the average size of mRNAs on gel, we considered the excision systems satisfactory.", "The Gateway-mediated excision system is anyway very attractive when sufficient cDNA is available for cloning into λ-FLC-II-C, which accommodates the use of the Gateway excision protocols.", "In light of the requirements of our sequencing operation, we used pFLC-DEST (FIG.", "2j) as our destination vector.", "Example 18 Comparative Example Between 6.0 kb and 5.5 kb Stuffer II Vectors 1) Vectors Construction λ-FLC-I with 5.5 Kb stufferII was constructed as described before in the examples above.", "To compare the cloning size, λ-FLC-I with 6.0 Kb stufferII was constructed.", "We added a 0.5 Kb fragment in the HindIII site on the 5.5 Kb stufferII.", "0.5 Kb fragment was obtained by restriction digestion with HindIII of mouse genomic DNA.", "Mouse genomic DNA was digested with HindIII and 0.5 Kb fragment was separated by gel electrophoresis.", "The fragment was subcloned into the pBluescript+ (stratagene) and cleaved by HindIII and inserted into HindIII site on the 5.5 Kb stufferII fragment subcloned into the pBluescript.", "The 6.0 Kb stufferII was recovered by the restriction digestion of AscI and ligated into λ left arm and right arm with 10 Kb stufferI and pBluescript.", "2) Preparation of Arms For Cloning λ-DNA was prepared by QIAGEN lambda Midi kit (#12543).", "The cohesive termini of 10 μg of the lambda DNA were annealed by incubation for 2 hours at 42° C. in 180 μl of 10 mM Tris-Cl pH 7.5, 10 mM MgCl2, and we added 20 μl of 10×Ligation buffer and 400 unit of T4 Ligase (both of NEB Kit), and incubated for 7 hours at room temperature, followed by ligase inactivated for 15 min at 65° C. The above λ-DNA was digested with restriction enzymes (all purchased from New England Biolabs, Inc.) in 3 steps by addition of 50 mM, 100 mM and then 150 mM NaCl (final concentration at each of the three steps).", "The first step restriction was done in 50 mM NaCl by addition of 2 μl of 5M NaCl, 10 μl of NEB 2 buffer, 73 μl of H2O, 40 units of XhoI, 20 units of SpeI and 32 units of PacI for both vectors and then the sample was incubation for 2 hours at 37° C. The second step was done in 100 mM NaCl by addition of 2 μl of 5M NaCl, 20 μl of 10×NEB 3 buffer, 180 μd of H2O and 20 units of SwaI and incubation for 2 hours at room temperature.", "After this step the reaction tube was heated for 15 min at 65° C. Finally, the third step was done in 150 mM NaCl by addition of 5 μl of 5M NaCl, 60 units of SalI and 60 units of BamHI, and incubation for 4 hours at 37° C. After restriction the DNA was purified by Proteinase K treatment in presence of 0.1% SDS and 20 mM EDTA, extracted with phenol/chloroform and chloroform, and precipitated with ethanol (Sambrook, et al., 1989).", "DNA concentration should not exceed 20 μg/ml to avoid resuspension problems.", "After very careful resuspension for at least 30 min the digested DNA was separated in 0.7% low-melting agarose gel (Seaplaque, FMC) in the followings steps.", "After electrophoresis for 1.5 hours at 8 V/cm the DNA fragments which was shorter than 19 Kb of the StyI-digested λ DNA were cut off from the gel (step 1).", "Then, the electrophoresis buffer (1×TBE) was changed for fresh one and the remained DNA in the gel were electrophoresed to the opposite orientation at 8 V/cm for 2.5 hours.", "At this point the shorter DNA than 19 kb were cut off again (step2).", "The buffer was changed again.", "The remainder of DNA in the gel were electrophoresed to the same orientation of the step 1 at 8 V/cm for 30 min in order to compact the region containing the λ arms DNA for shorter reaction volumes.", "Finally the λ arms DNA were cut off (step 3), and purified and checked as previously described (Carninci and Hayashizaki, 1999, as above) with β-agarase (NEB) after equilibration of the gel with TE buffer (Sambrook et al., 1989).", "3) Construction of the Test Insert 250 bp Test Insert λ-DNA was digested with PstI and electrophoresed in the 2% low melting agarose gel.", "200-300 bp bands were cut off and purified by QIAquick Gel Extraction Kit (Qiagen).", "200-300 bp PstI fragments were subcloned into the pBluescript and digested with BamHI and SalI.", "250 bp BamHI-SalI fragmet was separated in 2.0% low-melting agarose gel and cut off and purified by Qiagen Kit.", "2 kb Test Insert The plasmid containing 2.0 Kb mouse cDNA was used as PCR template.", "2 Kb insert was amplified with the 1stBS primer and 2ndXprimer and purified by Proteinase K treatment in presence of 0.1% SDS and 20 mM EDTA, extracted with phenol/chloroform and chloroform and precipitated with ethanol (Sanbrook, et al., 1989, as above).", "PCR products were digested with BamHI and XhoI (cohesive ends with SalI) and purified as described above.", "6 Kb Test Insert 6 Kb test insert was prepared as described above for the previous inserts.", "10 Kb Test Insert p-FLC-I with 10 Kb stufferi was digested with BamHI and SalI and purified by proteinase K as described above.", "The 10 Kb BamHI-SalI fragment was separated with 0.7% low-melting agarose gel electrophoresis and isolated from gel with β-agarase (NEB) after equilibration of the gel with TE buffer (Sambrook et al., 1989) 4) Insert Size Check 4 kinds of test insert was ligated into μ-FLC-I with 5.5 Kb stufferIl and μ-FLC-I with 6.0 Kb stufferll.", "200 bp, 2 Kb, 6 Kb and 10 Kb test inserts were ligated at ratio 1:1:1:1 or 3:1:1:1 to the both vectors, respectively.", "Subsequently, the packaging reaction was performed using MaxPlax Lambda Packaging Extract (Epicentre Technologies).", "The phage solutions were amplified in C600 cells.", "1×104 pfu were plated on 90 mm dishes of LB-agar and topped with LB-agar containing 10 mM MgSO4 and let grow overnight to confluence (Sambrook et al., 1989).", "The phages particles were eluted with SM-buffer and titered.", "The phage DNA was extracted and converted to plasmid with 1 U Cre-recombinase at 37° C. for 1 hour in 300 uL as recommended (Novagen, Madison, Wis., USA), and the purified by S400 spun column (Pharmacia).", "The excised plasmids were electroporated into DH10B cells (Life Technologies) at 2.5 KV/cm and plated on the LB-agar plate containing 100 ug/ml ampicillin.", "Each 96 colonies were picked up and the plasmid preparation was performed by the plasmid extraction automatic instrument, solutions and protocols obtained by KURABO (however, any other method of purification of plasmid, for instance according to Sambrook et al., 1989, can be used).", "The plasmids were digested with PvuII and insert size was checked by agarose gel electrophoresis.", "Results are shown in Table 1.TABLE 1 5.5 kb stuffer II 6.0 kb stuffer II 10.0 kb insert 5 3 6.0 kb insert 43 27 2.0 kb insert 42 50 0.25 kb insert 3 2 Vectors stuffer II of 5.5 kb were able in 43 cases to accept inserts of 6 kb and in 5 cases inserts of 10 kb.", "The inserts of 6 and 10 kb corresponding to long and full-length cDNAs.", "The result demonstrated that vectors comprising a stuffer II of 5.5 kb, allowed the insertion of cDNA inserts of long sizes (6.0 and 10.0 kb) more efficiently than vectors comprising a stuffer II of 6.0 kb.", "A vector having CS of 31.5 kb (that is stuffer II of 5.5 kb) is advantageous for preparing full-length cDNAs libraries than a vector having the CS size of 30 kb (that is stuffer II of 6 kb).", "Example 19 The Gene Discovery is Correlated With the Average Insert Size of the cDNA Library I) A Vector for Cloning Size-Selected cDNA With Ligation-Mediated Clone Transfer.", "λ-FLC-III-L-D (FIG.", "2e) Similar to λ-FLC-I-L-B and λ-FLC-I-L-D, λ-FLC-III-L-D lacks stuffer II and therefore is used for cDNA libraries with large inserts.", "This vector carries the same background-reducing element as λ-FLC-I-L-D, but λ-FLC-III-L-D differs from λ-FLC-I-L-D in that excision of λ-FLC-III-L-D yields a pFLCIII-d plasmid (the plasmid of FIG.", "2i comprising the stuffer I of FIG.", "1d), which is suitable for subcloning without internal cleavage of cDNAs.", "II) A Vector for Short cDNAs and Ligation-Mediated Transfer of Inserts: λ-FLC-III-S-F (FIG.", "2f) The mRNA of many organisms that are evolutionarily far from vertebrates, such as Arabidopsis thaliana and Oryza sativa (rice), is shorter (typically 1 to 1.5 kb on an agarose gel) than that of vertebrates.", "When working with invertebrates, size selection like that used in all of the previously described examples may bias for long inserts, which may not be representative of the starting mRNA.", "Even though gene discovery from 3 rice libraries has been excellent even when we use λ-FLC-I-B, we prepared λ-FLC-III-S-F to address this concern.", "λ-FLC-III-S-F is the same as the previously described λ-FLC-III-F but has a longer stuffer 11 (6.3 kb).", "With the 6.3-kb stuffer II, the nominal cloning size is 0 to 14.9 kb, which facilitates cloning relatively short cDNAs.", "The background-reducing element of λ-FLC-III-S-F is that in FIG.", "1f, and this vector produces, after excision, a pFLCIII-f plasmid (the plasmid of FIG.", "2i comprising the stuffer I of FIG.", "1f).", "III) Full-Length cDNAs The full-length cDNA we used was prepared as described (Carninci and Hayashizaki, 1999, as above) and was normalized/subtracted (Carninci et al., 2000, Genome Res., 10:1617-1630).", "cDNA prepared with any other technique can be directionally cloned into the λ-FLC vectors, provided that the restriction sites are compatible or that the vector is properly modified.", "The average insert size of cDNA cloned into λ-FLC-I-B was always longer than that for the same cDNA cloned into other vectors (Table 2; average size of cDNA libraries using various vectors).", "TABLE 2 Tissue Vector titer size (Kbp) Placenta λ-ZAP II 4.6 × 105 1.3 Placenta λ-FLC-I-B 1.8 × 105 2.34 Cerebellum pBluescript 8.6 × 104 1.4 Cerebellum λ-FLC-I-B 3.7 × 105 3.36 The average insert size of the λ-FLC-I-B library was 1.8 times larger than that of the λ-ZapII library and 2.4 times larger than that of the plasmid cDNA library.", "We correlated the average insert size of each cDNA library in Table 3 and FIG.", "4 with the complexity of the library.", "In fact, these libraries were sequenced for the gene discovery program during the construction of the full-length cDNA encyclopedia (RIKEN mouse cDNA encyclopedia, RIKEN and Fantom Consortium, Nature, Vol.", "409: 685-690.The redundancy obtained by sequencing randomly picked clones and clustering clones with the same ends (Konno et al., 2001, as above) was compared by using 7 cDNA libraries cloned in λ-Zap II (conventional vector) and 9 cDNA libraries cloned in λ-FLC-I-B (Table 3).", "To facilitate comparing differences in the complexity of these libraries, we show not only the clustering data after completion of sequencing of a given library but also the number of clusters after the available number of runs closest to 5000 sequencing passes.", "The conventional vector did not accommodate the preparation of complex, low-redundancy cDNA libraries from any tissue.", "In contrast, all of the normalized/subtracted cDNA libraries cloned into λ-FCL-I-B showed higher complexity (average, 3392 clusters/4826 reactions; redundancy, 1.42) than did normalized/subtracted libraries with the conventional vector (average, 2089 clusters/4773 reactions; redundancy, 2.28).", "Even if we cannot expect to know a priori the variety (or complexity) of gene expression in a given organ, the complexity was supposed to be very high for the pooled total “embryo 10+11” library (Table 3).", "However, the “embryo 13 forelimb” library, which is cloned in λ-FCL-I-B and which covers a relatively restricted biological phenomenon, showed higher complexity than did the “embryo 10+11” library, which surely contains an increased variety of genes because it includes many developing organs and neuronal tissues.", "A more direct comparison comes from the libraries made from embryonic stem cells (ES cells); these libraries were all prepared from the same starting RNA.", "The number of clusters after 5104 sequencing reactions (total number of sequenced samples) is 3068 for the λ-FCL-I-B-cloned cDNA but just 2362 after 5160 sequencing reactions for the library in the conventional vector.", "That is, 31% more clusters were discovered by using λ-FCL-I-B.", "The difference is even more striking after additional sequencing reactions : 4971 clusters were categorized after 10514 sequencing reactions for the λ-FCL-I-B-based library and only 3795 clusters after 10492 sequencing reactions of the conventional ZAP vector library (see FIG.", "14); then, 15 520 sequencing passes of the conventional ZAP vector library (48% more) led to only 4566 clusters (9% fewer)) FIG.", "14).", "Notice also that although both the ES cell libraries were normalized and mildly subtracted with the same drivers, the C3 library (which was in λ-FCL-I-B) was also subtracted with genes that were already categorized.", "Although we expected that a strongly subtracted library would contain a lower variety of genes, this was not the case.", "These data support the notion that the capacity to clone long cDNAs accelerates new gene discovery when full-length approaches are used.", "In addition, the introduction of the λ-FCL vectors during the course of the preparation of the mouse cDNA encyclopedia restored a high rate of gene discovery (Table 3).", "Noteworthy also is the increased rate of new genes identified by using 5λ-end readings of λ-FLC-based libraries, which suggested that previously available cloning protocols and vectors have biased the gene discovery for short cDNAs.", "The λ-FLC vector family according to the invention demonstrated to be a powerful tool for high-efficiency cloning of full-length cDNA, gene discovery, and bulk transfer of selected cDNA clones into vectors for functional analysis, such as expression vectors.", "Example 20 λ-BAC vector construction 1) Preparation of “Component 1” (FIG.", "9) 10 μg of plasmid named pFLC-III-e were digested with 10 units of restriction enzyme BssHII (New England Biolabs also indicated as NEB) in 20 μl of 1× supplied buffer (NEB) at 37° C. for 1 hour.", "The pFLC-III-e/BssHII was separated with TAE (Tris-acetate-EDTA buffer, Sambrook et al., 1989) 0.8% low-melting agarose gel (SeaPlaque, FMC) at 50 V for 1 hour (see Sambrooket al, 1989).", "The plasmid band was cut out from the gel and digested with β-agarase (New England Biolabs) as suggested by the manufacturer (alternatively, also the standard technique described in Sambrook et al., 1989 can be used).", "The 5 kb of stuffer I was cut out from the gel and sliced.", "The gel was mixed with 1 ml of 1× β-agarase buffer (NEB).", "The tube containing the gel was put on ice for 30 min to equilibrate with 1× β-agarase buffer.", "The buffer was removed from the tube by pipetting and put a new 1× β-agarase buffer.", "The tube was put on ice for 30 min.", "This buffer exchange cycle was repeated once more.", "The buffer was removed and the tube was incubated at 65° C. for 5 min to melt the gel.", "10 unit of β-agarase (NEB) were added to the tube and incubated for 5 hours.", "Phenol/chloroform extraction was done and precipitated with ethanol according to standard techniques (Sambrook et al., 1989).", "The precipitated 5 kb fragment was dissolved with 5 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and indicated as “component 1”.", "2) Preparation of “component 2” (FIG.", "9) A pBeloBAC11 derivative prepared according to FIG.", "1 of U.S. Pat.", "No.", "5,874,259 (herein incorporated by reference) was used in the following “preparation of component 2” experiment.", "According to the description of U.S. Pat.", "No.", "5,874,259, the basic pBeloBAC11 (Kim et al., 1996, Genomics, 34:213-218) was modified by as following: ligating together the oriV element (SEQ ID NO:43) and the FRT element (SEQ ID NO:44) and the resulting fragment was made blunt and ended and then ligated into the XhoI site which had been made blunt end.", "The orientation of the two joined fragments is such that when the fragment is cloned into the XhoI site, the ori is physically located between the nearby FRT site and the insert cloning site.", "3 μg of this pBeloBAC11 derivative (FIG.", "9) was cleaved with 10 U of the restriction enzyme SalI (NEB) in 30 μl as recommend by the manufacturer (37° C. in the supplied buffer) and then dephosphorylated by adding 1 unit of CIP (Calf Intestinal Phosphatase)(Takara, Japan) at 37° C. for 30 min (a general use of dephosphorylation to reduce the cloning background is disclosed in Sambrook et al., 1989) followed by separation using TAE 0.8% low-melting point agarose (SeaPlaque, FMC) at 50 V for 1 hour (standard technique, Sambrook et al., 1989).", "The agarose gel region containing the plasmid fragment of 6.7 kb indicated in FIG.", "9 as “component 2” was cut out of the gel (approximately 200 microliters) and digested with 10 units of β-agarase (NEB) for 5 hours, extract with phoenol/chloroform and then followed by ethanol precipitation same as shown in component 1.3) Preparation of “Component 3” (FIG.", "9) A double strand oligonucleotide “adaptor” (FIG.", "9) comprising the upper strand: 5′-pTCGAAGCTTCCG-3′ (SEQ ID NO:45) phosphorylated at the 5′ end and the lower strand: 5′-CGCGCGGAAGCT-3′ (SEQ ID NO:46) was prepared using oligosynthesized using an automated synthesizer (EXPEDITE 8909 using the standard protocol and reagents).", "4) Ligation of “Components 1, 2 and 3” (FIG.", "9) “Component 1” (pFLC-III-e/BssHII fragment), “component 2” and “component 3” were mixed together in the ratio of 50 ng: 37 ng: 0.1 ng in the presence of 1÷ buffer (prepared by dilution to 1/10 from a stock of 10× supplied by the manufacturer NEB), 400 units of T4 DNA ligase (NEB) in final 5 μl of final volume reaction (buffer 1× dilution, DNA, adaptor, DNA ligase).", "The mixture was incubated at 16° C. overnight to complete the ligation reaction.", "After the addition of NaCl at 0.2 M final concentration into the ligation reaction, the ligation products were precipitated with 2 volumes of 96% ethanol and 1 μg of Glycogen (Roche)—according to the standard techniques (Sambrook et al., 1989) and the ligated products were recovered by ethanol precipitation according to standard protocol (Sambrook et al., 1989).", "The ligation products were dissolved in 10 μl of H2O.", "1 μl of the recovered ligation products were electropotrated into 20 μl of DH10B electrocomponent cells (Invitrogen) at 2.5 KV.cm (according to Invitrogen) instructions followed by plating the elctroporeted plasmid cells on LB-agar-supplemented with ampicillin at 50 μg/ml.", "To select positive clone which has modified pBAC, having the construct with the desired insert (“component 1”), randomly picked clones were cultured and plasmids checked (see Sambrook et al for general strategy of selecting and analyzying recombinants plasmids).", "A plasmid (modified pBAC of FIG.", "9) having the stuffer I as indicated in FIG.", "1e as insert is then selected for the next step 5) Introduction of loxP and XbaI Sites (FIG.", "10) In order to introduce loxP and XbaI sites into the modified pBAC prepared as above, 1 μg of the modified pBAC was mixed with 0.5 μM of “primer 1” (5′-AGAGAGAGAGATCTAGAATAACTTCGTATAATGTATGCTATACGAAGTTA TCTGTCAAACATGAGAATTG-3′)(SEQ ID NO:47), 0.5 μM of “primer 2”: (5′-GAGAGAGAGATCTAGATAACTTCGTATAGCATACATTATACGAAGTTATC GAATTTCTGCCATTCAT-3′)(SEQ ID NO:48), 125 μM dNTP mix, 1× “GC buffer 1” (Takara, Japan), 5 units of LA-Taq (Takara, Japan) in a volume of 50 μL.", "Then, the following PCR amplification cycle was repeated for 25 times; step 1: 94° C. for 5 sec; step 2: 50° C. for 5 sec, 72° C. for 12 min.", "After amplification, 1 μl of 0.5M EDTA, 1 μl of 10% SDS and 1 μl of proteinaseK, (10 mg/ml stock) (Sigma) were added to the PCR products obtained, incubated at 45° C. for 15 min and followed by phenol/chloroform treatment, chloroform extraction and then ethanol precipitation (Sambrook et al., 1989).", "After ethanol precipitation, the pellet was dissolved with water and cut with 15 units of restriction enzyme XbaI (NEB) in the buffer supplied by the manufacturer (NEB).", "PCR product was purified after electrophoretic separation with TAE 0.8% low-melting agarose gel (SeaPlaque, FMC) at 50 V for 1 hour (Sambrook et al., 1989).", "The PCR product was cut and digested with 10 units of beta-agarase (NEB) as suggested by the manufacturer (alternatively, also the standard technology disclosed in Sambrook et al., 1989 can be used).", "The 11.7 kb of PCR product was cut out from the gel and sliced.", "The gel was mixed with 1 ml of 1× β-agarase buffer (NEB).", "The tube containing the gel was put on ice for 30 min to equibrate with 1× β-agarase buffer.", "The buffer was removed from the tube and put a new 1× β-agarase buffer.", "The tube was put on ice for 30 min.", "This buffer exchange cycle was repeated once more.", "The buffer was removed and the tube was incubated at 65° C. for 5 min to melt the gel.", "10 unit of β-agarase (NEB) were added to the tube and incubated for 5 hours.", "Phenol/chloroform extraction was done and precipitated with ethanol following standard techniques (Sambrook et al., 1989).", "The precipitated 11.7 kb fragment was dissolved with 5 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and indicated as “component 4” (FIG.", "10).", "6) Preparation of Stuffer II (“Component 5”)(FIG.", "11) To prepare the 1.8 kb stuffer as a size balancer (also indicated as “stuffer II”), 3 μg of mouse genomic DNA was digested with 20 units of Sau3AI and 1× supplied buffer (Nippon Gene, Japan) for 2 hours at 37° C. in a volume of 20 μl.", "The digested DNA was separated with 1.2% low-melting agarose gel at 50 V for 2 hours with lambda/Styl molecular marker (Nippon Gene, Japan).", "DNA fragments that migrated showing a size of about 1 1.8 kb were cut out of the gel and sliced.", "The gel was mixed with 1 ml of 1× β-agarase buffer (NEB).", "The tube containing the gel was put on ice for 30 min to equibrate with 1× β-agarase buffer.", "The buffer was removed from the tube and put a new 1× β-agarase buffer.", "The tube was put on ice for 30 min.", "This buffer exchange cycle was repeated once more.", "The buffer was removed and the tube was incubated at 65° C. for 5 min to melt the gel.", "10 unit of β-agarase (NEB) were added to the tube and incubated for 5 hours.", "Phenol/chloroform extraction was done and precipitated with ethanol following standard techniques (Sambrook et al., 1989).", "The precipitated 1.8 kb stuffer II DNA was dissolved with 10 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).", "The purified 1.8 kb DNAs (100 ng) was ligated with 10 ng Sau3AI/XbaI adaptor comprising the upper strand: 5′- GAGAGAGAGATCTAGAAAGCTCCA-3′ (SEQ ID NO:49), and the lower strand: 5′- GATCTGGAGCTT-3′ (SEQ ID NO:50) for 16 hours at 16° C. in the presence of 1× ligation buffer (diluted stock as above described) and 400 units of T4 DNA ligase (NEB) in a final volume of 5 μl.", "After inactivation of the ligase at 65° C. for 5 min, the ligation products were separated by TAE 1.2% low-melting agarose gel (SeaPlaque, FMC) at 50 V for 1 hour (Sambrook et al., 1989).", "again and 1.8 kb DNA was cut and digested with beta-agarase (NEB) as suggested by the manufacturer (alternatively, the technique described in Sambrook et al., 1989 can be used).", "The 1.8 kb of PCR product was cut out from the gel and sliced.", "The gel was mixed with 1 ml of 1× β-agarase buffer (NEB).", "The tube containing the gel was put on ice for 30 min to equibrate with 1× β-agarase buffer.", "The buffer was removed from the tube and put a new 1× β-agarase buffer.", "The tube was put on ice for 30 min.", "This buffer exchange cycle was repeated once more.", "The buffer was removed and the tube was incubated at 65° C. for 5 min to melt the gel.", "10 unit of β-agarase (NEB) was added to the tube and incubated for 5 hours.", "Phenol/chloroform extraction was done and precipitated with ethanol following standard techniques (Sambrook et al., 1989).", "The precipitated 1.8 kb fragment was dissolved with 5 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).", "The 1.8 kb of the purified DNA was amplified using 0.5 μM XbaI primer (5′-GAGAGAGAGATCTAGAAAGCTCCA-3′)(SEQ ID NO:49), 125 μM dNTPs mix, 1× GC buffer I (Takara, Japan), 5 units of LA-Taq (Takara)in a final volume of 50 μl.", "For the PCR amplification of DNA, the following cycle was repeated 25 times: step 1: 94° C. for 5 sec; step2: 68° C. for 1.5 min.", "After amplification, 1 μl of 0.5M EDTA, 1 μl of 10% SDS and 1 μl of proteinaseK, (10 mg/ml stock) (Qiagen) were added to the PCR products obtained, incubated at 45° C. for 15 min and followed by phenol/chloroform treatment, chloroform extraction and then ethanol precipitation (Sambrook et al, 1989).", "After ethanol precipitation, the pellet was dissolved with water and cut with 15 units of restriction enzyme XbaI (NEB) in the buffer supplied by the manufacturer (NEB).", "PCR products/Xbalwere separated with TAE 0.8% low melting point gel at 50V for 1 hour and cut out a 1.8 kb DNA fragment.", "This DNA fragment was digested with beta-agarase (NEB) as suggested by the manufacturer.", "The 1.8 kb of PCR product was cut out the gel and sliced.", "The gel was mixed with 1 ml of 1× β-agarase buffer (NEB).", "The tube containing the gel was put on ice for 30 min to equibrate with 1× β-agarase buffer.", "The buffer was removed from the tube and put a new 1× β-agarase buffer.", "The tube was put on ice for 30 min.", "This buffer exchange cycle was repeated once more.", "The buffer was removed and the tube was incubated at 65° C. for 5 min to melt the gel.", "10 unit of β-agarase (NEB) were added to the tube and incubated for 5 hours.", "Phenol/chloroform-extraction was done and precipitated with ethanol following standard techniques (Sambrook et al., 1989).", "The precipitated 1.8 kb fragment was dissolved with 5 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).", "The purified PCR products/XbaI were named “component 5” (see FIG.", "11).", "7) Preparation of “Component 6” (FIG.", "12) The cohesive termini (cos ends) of 10 μg of the (linear) λ-FLC-I-E (FIG.", "2a) annealed (the two complementary cos ends and the ends anneal to each other after this treatment; this increase ligation efficiency in later steps and simplify further procedures) by incubation for 2 hours at 42° C. in 180 μl of 10 mM Tris-Cl (pH 7.5), 10 mM MgCl2, and 20 μl of 10× ligation buffer provided by NEB.", "400 units of T4 DNA ligase (NEB) were added to the solution, and the sample was incubated for 5 hours at room temperature, followed by ligase inactivation for 15 min at 65° C. The k DNA with the cos-ends ligated in the previous step was digested with 5 units of XbaI (Nippon Gene, Japan), 1× manufacturers supplied buffer for 2 hours at 37° C. in a volume of 50 μl.", "After digestion, 1 μl of 0.5M EDTA, 1 μl of 10% SDS and 1 μl of proteinaseK, (10 mg/ml stock) (Qiagen) were added to the DNA obtained, incubated at 45° C. for 15 min and followed by phenol/chloroform treatment, chloroform extraction and then ethanol precipitation (Sambrook et al., 1989).", "After ethanol precipitation, the pellet was dissolved with water for 30 min while the tube was kept on ice, the digested DNA was separated in TAE 0.6% low-melting agarose gel at 50 V for 5 hours.", "Cos-ligated fragment (29 kbp) was cut out the gel and sliced.", "The gel was mixed with 1 ml of 1× β-agarase buffer (NEB).", "The tube containing the gel was put on ice for 30 min to equibrate with 1× β-agarase buffer.", "The buffer was removed from the tube and put a new 1× β-agarase buffer.", "The tube was put on ice for 30 min.", "This buffer exchange cycle was repeated once more.", "The buffer was removed and the tube was incubated at 65° C. for 5 min to melt the gel.", "10 unit of β-agarase (NEB) were added to the tube and incubated for 5 hours.", "Phenol/chloroform extraction was done and precipitated with ethanol following standard techniques (Sambrook et al.", "1989).", "The precipitated 29 kb cos-ligated fragment was dissolved with 5 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), named “component 6” (FIG.", "12).", "8) Ligation of “Components 4, 5 and 6” (FIG.", "12) The “component 4” (modified pBAC), “component 5” (stuffer) and “component 6” (arms) were mixed in the following ratio: 120 ng: 19 ng: 300 ng, in presence of 1× ligation buffer (NEB ligation buffer) and 400 units of T4 DNA ligase NEB in 5 μl for 16 hours at 16° C. After in vitro packaging (“MaxPlax™ Lambda Packaging Extract”, EPICENTRE TECHNOLOGIES,.", "Madison Wis., US) and plating the recombinant λ-phage (as described in Sambrook et al., 1989), a few hundreds plaques of λ phages were obtained.", "5 clones (phage plaques) were randomly selected according to the method described in Sambrook et al., 1989.The picked phage plaques were put in SM Buffer (Sambrook et al., 1989) and left at room temperature for 1 hour.", "Then, the eluted phage solution was used to infect C600 cells and were amplified according to the standard protocol (Sambrook et al., 1989).", "In 3 out 5 clones we obtained the desired inserts (corresponding to “component 1”) by analysis with restriction enzymes (XbaI+BamHI+SalI, XbaI+BamHI, XbaI+SalI) (Sambrook et al., 1989).", "One of this clone, named λ-FLC-III-pBAC (FIG.", "12) shown the same cloning range of other described λ-vectors (for example, λ-FLC-I-B, λ-FLC-II-C, λ-FLC-III-F) which was 0.2-15.4 kb.", "TABLE 3 Lambda-Flcl allows preparing longer cDNA libraries, which is correlated to higher complexity and higher gene discovery rate Clusters Final 5′ at fixed sequence (1) extent of sequencing (2) Coding novelty Code Tissue Titer Size (Kbp) sequences clusters redundancy sequences clusters redundancy (3) % (4) % Conventional vectors (5) 6-100 kidney 3 × 10exp5 1.21 4680 1439 3.25 99.1 6.5 22-100 stomach 3.5 × 10exp5 1.33 4447 1987 2.24 82.1 12.4 22-104 stomach 2.0 × 10exp5 1.08 4068 1960 2.08 82.1 6.38 23-100 tongue 4.1 × 10exp4 1.81 5016 2514 2 10295 4021 2.56 76.8 9.8 24-100 ES cells 1.3 × 10exp5 1.69 5160 2362 2.18 15520 4566 3.4 88.6 7 25-100 embryo 13, liver 8.5 × 10exp4 1.63 5005 1502 3.33 5864 1679 3.49 92.2 5.85 28-104 total embryo 10 + 11 8.8 × 10exp5 1.8 5040 2859 1.76 9450 4470 2.11 93.9 5.69 Average 2.8 × 10exp5 1.51 4773 2089 2.28 10,282 3681 2.79 87.8 7.66 Lambda Flc-l (6) 49-304 testis 2.6 × 10exp6 2.36 5000 3520 1.42 9015 5502 1.64 93.1 46.57 49-305 testis 8.9 × 10exp5 2.52 5120 3606 1.42 11564 6605 1.75 93.1 36.57 53-304 pituitary gland 2.1 × 10exp6 2.93 5073 3242 1.56 8059 4662 1.73 100 17.41 58-304 thymus 1.7 × 10exp6 3.81 5085 3742 1.36 10259 6445 1.59 80 21.6 59-304 embryo 13, forelimb 3.9 × 10exp6 3.19 3908 2865 1.36 60 16.05 63-304 medulla oblungata 6.0 × 10exp5 2.89 4001 2998 1.33 75 21.7 63-305 medulla oblungata 4.8 × 10exp5 2.97 5060 3654 1.38 8339 5358 1.56 75 29.7 64-305 olfactory brain 5.7 × 10exp5 3.01 5085 3835 1.33 10179 6394 1.59 80 23.9 C3-300 ES cells 1.5 × 10exp5 2.45 5104 3068 1.66 10,514 4971 2.12 78.8 19 Average 1.4 × 10exp6 2.9 4826 3392 1.42 9704 5705 1.71 81.6 25.8 (1) calculated by using a number of plates that give the value closest to 5000, for easy comparison of library complexity (2) Some libraries were further sequenced (3) Presence of the first ATG of annotated mouse genes (4) Novelty of 5′ end ESTs versus databases (5) Lambda ZAP II.", "cDNA size is shown after bulk excision of to plasmid library (6) After in-vitro excision and electroporation into DH10B cells" ] ]	Patent_10469508
[ [ "Optical cross-connect system", "An optical cross-connect switch comprises a base (216), a flap (211) and one or more electrically conductive landing pads (222) connected to the flap (211).", "The flap (211) has a bottom portion that is movably coupled to the base (216) such that the flap (211) is movable with respect to a plane of the base (216) from a first orientation to a second orientation.", "The one or more landing pads (222) are electrically isolated from the flap (211) and electrically coupled to be equipotential with a landing surface." ], [ "1.An optical cross-connect switch, comprising: a base; a flap having a bottom portion movably coupled to the base such that the flap is movable with respect to a plane of the base from a first orientation to a second orientation; and one or more electrically conductive landing pads connected to the flap, wherein the one or more landing pads are electrically isolated from the flap and electrically coupled to be equipotential with a landing surface.", "2.The switch of claim 1, wherein at least one landing pad is substantially parallel to the flap.", "3.The switch of claim 1 or 2, wherein the base further includes at least one cantilevered anti-stiction bar.", "4.The switch of claim 1, 2 or 3, wherein a magnetic field is used to create a first actuation force to manipulate the flap.", "5.The switch of claim 1,2 or 3, wherein an acoustic pulse is used to create a first actuation force to manipulate the flap.", "6.The switch of claim 1,2 or 3, wherein a pneumatic pressure is used to create a first actuation force to manipulate the flap.", "7.The switch of claim 1, 2, 3, 4, 5 or 6 further including a stop configured to contact a portion of the flap in a contact area sized so that, upon application of an electrostatic bias between the flap and the stop, a sufficient second force holds the flap against the stop.", "8.The switch of claim 7 wherein stop is comprised of two vertical walls.", "9.The switch of claim 7 or 8 wherein the stop includes a sidewall made of silicon and having crystalline orientation of <111>.", "10.The switch of claim 1,2,3,4,5,6, 7, 8 or 9 further including a microcontroller electrically coupled to the flap to enable the flap to be grounded or held at a voltage potential.", "11.The switch of claim 10, wherein the microcontroller also controls the first actuation force.", "12.The switch of claim 10, further comprising a magnet assembly electrically coupled to the microcontroller and sized less than 0.5″ in width, said magnet assembly positioned to surround the switch and provide magnetic force along both a Z and an X axis, wherein the microcontroller also controls the magnet assembly and wherein the first actuation force is magnetic.", "13.The switch of claim 10, 11 or 12 wherein the microcontroller stores the state of the flap and capacitive sensing is used to confirm that the flap is in the correct state.", "14.The switch of claim 10, 11, 12 further including a magnetoresistive element coupled to the flap, wherein the microcontroller stores the state of the flap and magnetoresistive sensing is used to confirm that the flap is in the correct state.", "15.The switch of claim 14 further including a magnetoresistive element coupled to the base, wherein the microcontroller stores the state of the flap and magnetoresistive sensing is used to confirm that the flap is in the correct state.", "16.The switch of claim.", "14 or 15 further including a magnetoresistive element coupled to the stop, wherein the microcontroller stores the state of the flap and magnetoresistive sensing is used to confirm that the flap is in the correct state.", "17.The switch of claim 7, 8 or 9, further including a charge-storing circuit electrically coupled between the stop and an electrical ground, an isolator element electrically coupled between the clamping surface and a source of clamping voltage, wherein the isolator element is configured to electrically isolate the source of clamping voltage from the clamping surface in the event of a power failure.", "18.The switch of claim 17 wherein the isolator element is an opto-isolator or a low leakage diode.", "19.The switch of claim 17 or 18, wherein the charge-storing circuit includes a capacitor.", "20.The switch of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, further including an enclosure for sealing the flap and having one or more sidewalls, an optical element coupled to at least one of the sidewalls, wherein at least one of the one or more sidewalls or the optical element includes a surface that is angled with respect to an optical plane.", "21.The switch of claim 19, wherein the enclosure is evacuated.", "22.The switch of claim 19, wherein the enclosure is filled with a gas.", "23.The switch of claim 10, 11, 12, 13, 14, 15 or 16 wherein the microcontroller also monitors voltage levels and engage a signal transmission in the event of a power failure.", "24.The switch of claim 17, 18, 19, 20 or 21 further including a microcontroller to monitor voltage levels, whereby the microcontroller may engage a signal transmission in the event of a power failure.", "25.The switch of claim 10, 11, 12, 13, 14, 15 or 16, further including a stop configured to contact a portion of the cantilever in a contact area sized so that, upon application of an electrostatic bias between the flap and the stop, a sufficient second force holds the flap against the stop; and a charge-storing circuit electrically coupled between the stop and an electrical ground, an isolator element electrically coupled between the clamping surface and a source of clamping voltage, wherein the isolator element is configured to electrically isolate the source of clamping voltage from the clamping surface in the event of a power failure." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Microelectromechanical systems (MEMS) are miniature mechanical devices manufactured using the techniques developed by the semiconductor industry for integrated circuit fabrication.", "Such techniques generally involve depositing layers of material that form the device, selectively etching features in the layer to shape the device and removing certain layers (known as sacrificial layers, to release the device.", "Such techniques have been used, for example, to fabricate miniature electric motors as described in U.S. Pat.", "No.", "5,043,043.Recently, MEMS devices have been developed for optical switching.", "Such systems typically include an array of mechanically actuatable mirrors that deflect light from on optical fiber to another.", "The mirrors are configured to translate and move into the path of the light from the fiber.", "Mirrors that move into the light path generally use torsion flexures to translate mirror position vertically while and changing its angular from a horizontal to a vertical orientation.", "MEMS mirrors of this type are usually actuated by magnetic interaction, electrostatic interaction, thermal, pneumatic actuation or some combination of these.", "The design, fabrication, and operation of magnetically actuated micromirrors with electrostatic clamping in dual positions for fiber-optic switching applications are described, e.g., by B. Behin, K. Lau, R. Muller in “Magnetically Actuated Micromirrors for Fiber-Optic Switching,” Solid-State and Actuator Workshop, Hilton Head Island, S.C., Jun.", "8-11, 1998 (p. 273-276).", "When the mirror is in the horizontal position, it rests against a substrate that forms a base.", "Often, the mirror is subject to electromechanical forces, sometimes referred to as “stiction” that cause the mirror to stick to the substrate and prevent the mirror from moving.", "The same stiction forces can also prevent the mirror from being properly released from the substrate during manufacture.", "To overcome stiction problems, landing pads (also called dimples or bumps have been used in MEMS devices to minimize or otherwise control the contact area between the device and the underlying substrate.", "In the prior art, such landing pads are formed prior to deposition of a device layer either by etching pits in an underlying sacrificial layer or by depositing pads of another material prior to the deposition of the layer forming the device.", "The problem of stiction with respect to an example of a MEMs mirror device 100 is shown in FIG.", "1 .", "The device 100 includes a mirror 111 formed from the device layer 112 of a substrate 110 .", "The mirror 111 may be movably attached to the device layer by a flexure 114 , actuated by an off-chip electromagnet, and individually addressed by electrostatic clamping either to a surface of the substrate 110 or to a vertical sidewall 114 of a top mounted chip 106 .", "A first actuation force may move the mirror 111 between a rest position parallel to the substrate 110 and a position nearly parallel to the vertical sidewall 104 of the top-mounted chip 106 , while the application of a second force (i.e electrostatic field) may clamp the mirror 111 in the horizontal or vertical position.", "The electrostatic field used to hold the mirror 111 in a position regardless of whether the first actuation force is on or off can increase the level of stiction between the mirror 111 and each landing surface.", "When clamped to either the substrate 110 or the vertical side-wall surface 104 , the mirror 111 may rest on a set of landing pads or dimples 122 , 124 , which may lie level with or protrude below or above the mirror surface, respectively.", "These landing pads 122 , 124 may minimize the physical area of contact between the mirror 111 and the clamping surface, thus reducing stiction effects.", "However, since the mirror 111 and clamping surface (either the side wall 104 or the substrate 110 ) may be at different potentials, the landing pads 122 , 124 may be made of an insulating material in order to prevent an electrical short between the mirror 111 and the clamping surface.", "While the insulating landing pad material does, indeed, prevent an electrical short, its inherent properties can lead to other problems.", "Firstly, most insulating materials have the capacity to trap electrical charge and can, in some cases, maintain that charge for long periods of time—sometimes indefinitely.", "As a result, the potential of the landing pads 122 , 124 can drift to an arbitrary value, resulting in either parasitic clamping potential between the mirror 111 and the clamping surface, even when both are externally driven to the same voltage, or a reduced clamping force by shielding the mirror potential.", "Second, since the insulating landing pads 122 , 124 will typically be at a potential close to the mirror potential when not in contact with the clamping surface, a rapid discharge can occur when the landing pads 122 , 124 first come into the contact with the clamping surface that is a kept at a potential different than the mirror 111 .", "This rapid discharge may be exhibited as arcing or short pulses of high current.", "Such surges can lead to physical damage to the landing pads 122 , 124 or the clamping surface, or may produce micro-welding, where the landing pad is welded to the clamping surface—resulting in the mirror 111 being stuck.", "There is a need, therefore, for a MEMS device having stiction resistant landing pads and a method of operating a MEMS device configured in a stiction reduced mode.", "Modern communications systems require a level of robustness that protects the state of the optical switches from being lost in the event of a power failure.", "MEMS optical switches typically include an array of mechanically actuatable mirrors that deflect light from one optical fiber to another.", "A mirror may be retained in a specific ON or OFF state by use of an electrostatic clamping voltage.", "In the event of a power failure, the clamping voltage may be lost and any MEMS mirrors that were clamped in a specific state may revert to the opposing state when under the influence mechanical restoring forces.", "In this manner, the state of the switch may be lost in the event of a power failure.", "Thus, there is a need in the art, for a method of maintaining the state of a MEMS device in the event of a power failure and an apparatus for implementing such a method.", "The increasing complexity of optical switching systems has lead to development of switching fabrics that are larger than say 8×8.When scaling to such larger optical switch fabrics (e.g., 16×16, 32×32), the yield of the optical MEMS die will decrease with the increasing die size.", "This places a feasible upper bound on such scaling.", "One proposed solution to this problem is to develop a new technology with a finer pitch and, therefore, a smaller die.", "Unfortunately this is a lengthy development process.", "Another alternative solution is to use redundant mirrors on the device die.", "Unfortunately, this complicates the overall design of the optical switch.", "It is known to tile two or more smaller dies together to form a larger device.", "For example, Minowa et al.", "uses four 4×4 arrays tiled together in a mosaic fashion to form an 8×8 array.", "However, for 16×16 arrays and larger, the size of the array still presents problems even if smaller devices are tiled together.", "For example, as the array size increases the distance between input and output fibers increases.", "The increased optical path between the fibers can lead to undesirable beam spreading.", "The beam spreading may be overcome by placing collimator lenses between the arrays.", "However, the alignment of the collimator lenses to the switching elements is difficult and even slight misalignment will result in optical loss that degrades switch performance.", "Another problem with tiling two or more dies is that the dies must be very accurately aligned with each other in order to ensure that the mirrors on one die will align with those on the other dies in the mosaic.", "Prior art alignment techniques include self-alignment and active-alignment.", "In self-alignment, metallized bonding pads are placed on two different pieces, e.g.", "a MEMS device die containing rotating mirrors and a corresponding top chip.", "Solder is applied to the bonding pads and the two pieces are brought together such that corresponding bonding pads roughly align with each other.", "When solder is heated through reflow, surface tension forces between the solder and the bonding pads pull the two pieces into alignment.", "In active-alignment, the pieces are placed, within micron tolerances, using a pick and place tool and held in place until the solder freezes.", "Active-alignment allows for the use of epoxies as well as solders for attachment of the top chip to the device die.", "However, even using these techniques, alignment can be particularly problematic with a tiled device having four 8×8 MEMS mirror arrays totaling 256 MEMS mirrors.", "Thus, there is a need in the art, for a self aligned or actively aligned optical MEMS device and a method for making it." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The disadvantages associated with the prior art are overcome by an inventive optical cross-connect switch and methods.", "The optical cross-connect switch comprises a base, a flap and one or more electrically conductive landing pads connected to the flap.", "The flap has a bottom portion that is movably coupled to the base such that the flap is movable with respect to a plane of the base from a first orientation to a second orientation.", "The one or more landing pads are electrically isolated from the flap and electrically coupled to be equipotential with a landing surface." ], [ "FIELD OF THE INVENTION This invention is related to optical communications and more specifically to microelectromechanical systems (MEMS) optical cross-connect switches.", "BACKGROUND OF THE INVENTION Microelectromechanical systems (MEMS) are miniature mechanical devices manufactured using the techniques developed by the semiconductor industry for integrated circuit fabrication.", "Such techniques generally involve depositing layers of material that form the device, selectively etching features in the layer to shape the device and removing certain layers (known as sacrificial layers, to release the device.", "Such techniques have been used, for example, to fabricate miniature electric motors as described in U.S. Pat.", "No.", "5,043,043.Recently, MEMS devices have been developed for optical switching.", "Such systems typically include an array of mechanically actuatable mirrors that deflect light from on optical fiber to another.", "The mirrors are configured to translate and move into the path of the light from the fiber.", "Mirrors that move into the light path generally use torsion flexures to translate mirror position vertically while and changing its angular from a horizontal to a vertical orientation.", "MEMS mirrors of this type are usually actuated by magnetic interaction, electrostatic interaction, thermal, pneumatic actuation or some combination of these.", "The design, fabrication, and operation of magnetically actuated micromirrors with electrostatic clamping in dual positions for fiber-optic switching applications are described, e.g., by B. Behin, K. Lau, R. Muller in “Magnetically Actuated Micromirrors for Fiber-Optic Switching,” Solid-State and Actuator Workshop, Hilton Head Island, S.C., Jun.", "8-11, 1998 (p. 273-276).", "When the mirror is in the horizontal position, it rests against a substrate that forms a base.", "Often, the mirror is subject to electromechanical forces, sometimes referred to as “stiction” that cause the mirror to stick to the substrate and prevent the mirror from moving.", "The same stiction forces can also prevent the mirror from being properly released from the substrate during manufacture.", "To overcome stiction problems, landing pads (also called dimples or bumps have been used in MEMS devices to minimize or otherwise control the contact area between the device and the underlying substrate.", "In the prior art, such landing pads are formed prior to deposition of a device layer either by etching pits in an underlying sacrificial layer or by depositing pads of another material prior to the deposition of the layer forming the device.", "The problem of stiction with respect to an example of a MEMs mirror device 100 is shown in FIG.", "1.The device 100 includes a mirror 111 formed from the device layer 112 of a substrate 110.The mirror 111 may be movably attached to the device layer by a flexure 114, actuated by an off-chip electromagnet, and individually addressed by electrostatic clamping either to a surface of the substrate 110 or to a vertical sidewall 114 of a top mounted chip 106.A first actuation force may move the mirror 111 between a rest position parallel to the substrate 110 and a position nearly parallel to the vertical sidewall 104 of the top-mounted chip 106, while the application of a second force (i.e electrostatic field) may clamp the mirror 111 in the horizontal or vertical position.", "The electrostatic field used to hold the mirror 111 in a position regardless of whether the first actuation force is on or off can increase the level of stiction between the mirror 111 and each landing surface.", "When clamped to either the substrate 110 or the vertical side-wall surface 104, the mirror 111 may rest on a set of landing pads or dimples 122, 124, which may lie level with or protrude below or above the mirror surface, respectively.", "These landing pads 122, 124 may minimize the physical area of contact between the mirror 111 and the clamping surface, thus reducing stiction effects.", "However, since the mirror 111 and clamping surface (either the side wall 104 or the substrate 110) may be at different potentials, the landing pads 122, 124 may be made of an insulating material in order to prevent an electrical short between the mirror 111 and the clamping surface.", "While the insulating landing pad material does, indeed, prevent an electrical short, its inherent properties can lead to other problems.", "Firstly, most insulating materials have the capacity to trap electrical charge and can, in some cases, maintain that charge for long periods of time—sometimes indefinitely.", "As a result, the potential of the landing pads 122, 124 can drift to an arbitrary value, resulting in either parasitic clamping potential between the mirror 111 and the clamping surface, even when both are externally driven to the same voltage, or a reduced clamping force by shielding the mirror potential.", "Second, since the insulating landing pads 122, 124 will typically be at a potential close to the mirror potential when not in contact with the clamping surface, a rapid discharge can occur when the landing pads 122, 124 first come into the contact with the clamping surface that is a kept at a potential different than the mirror 111.This rapid discharge may be exhibited as arcing or short pulses of high current.", "Such surges can lead to physical damage to the landing pads 122, 124 or the clamping surface, or may produce micro-welding, where the landing pad is welded to the clamping surface—resulting in the mirror 111 being stuck.", "There is a need, therefore, for a MEMS device having stiction resistant landing pads and a method of operating a MEMS device configured in a stiction reduced mode.", "Modern communications systems require a level of robustness that protects the state of the optical switches from being lost in the event of a power failure.", "MEMS optical switches typically include an array of mechanically actuatable mirrors that deflect light from one optical fiber to another.", "A mirror may be retained in a specific ON or OFF state by use of an electrostatic clamping voltage.", "In the event of a power failure, the clamping voltage may be lost and any MEMS mirrors that were clamped in a specific state may revert to the opposing state when under the influence mechanical restoring forces.", "In this manner, the state of the switch may be lost in the event of a power failure.", "Thus, there is a need in the art, for a method of maintaining the state of a MEMS device in the event of a power failure and an apparatus for implementing such a method.", "The increasing complexity of optical switching systems has lead to development of switching fabrics that are larger than say 8×8.When scaling to such larger optical switch fabrics (e.g., 16×16, 32×32), the yield of the optical MEMS die will decrease with the increasing die size.", "This places a feasible upper bound on such scaling.", "One proposed solution to this problem is to develop a new technology with a finer pitch and, therefore, a smaller die.", "Unfortunately this is a lengthy development process.", "Another alternative solution is to use redundant mirrors on the device die.", "Unfortunately, this complicates the overall design of the optical switch.", "It is known to tile two or more smaller dies together to form a larger device.", "For example, Minowa et al.", "uses four 4×4 arrays tiled together in a mosaic fashion to form an 8×8 array.", "However, for 16×16 arrays and larger, the size of the array still presents problems even if smaller devices are tiled together.", "For example, as the array size increases the distance between input and output fibers increases.", "The increased optical path between the fibers can lead to undesirable beam spreading.", "The beam spreading may be overcome by placing collimator lenses between the arrays.", "However, the alignment of the collimator lenses to the switching elements is difficult and even slight misalignment will result in optical loss that degrades switch performance.", "Another problem with tiling two or more dies is that the dies must be very accurately aligned with each other in order to ensure that the mirrors on one die will align with those on the other dies in the mosaic.", "Prior art alignment techniques include self-alignment and active-alignment.", "In self-alignment, metallized bonding pads are placed on two different pieces, e.g.", "a MEMS device die containing rotating mirrors and a corresponding top chip.", "Solder is applied to the bonding pads and the two pieces are brought together such that corresponding bonding pads roughly align with each other.", "When solder is heated through reflow, surface tension forces between the solder and the bonding pads pull the two pieces into alignment.", "In active-alignment, the pieces are placed, within micron tolerances, using a pick and place tool and held in place until the solder freezes.", "Active-alignment allows for the use of epoxies as well as solders for attachment of the top chip to the device die.", "However, even using these techniques, alignment can be particularly problematic with a tiled device having four 8×8 MEMS mirror arrays totaling 256 MEMS mirrors.", "Thus, there is a need in the art, for a self aligned or actively aligned optical MEMS device and a method for making it.", "SUMMARY OF THE INVENTION The disadvantages associated with the prior art are overcome by an inventive optical cross-connect switch and methods.", "The optical cross-connect switch comprises a base, a flap and one or more electrically conductive landing pads connected to the flap.", "The flap has a bottom portion that is movably coupled to the base such that the flap is movable with respect to a plane of the base from a first orientation to a second orientation.", "The one or more landing pads are electrically isolated from the flap and electrically coupled to be equipotential with a landing surface.", "BRIEF DESCRIPTION OF THE DRAWINGS The teachings of the present invention can be readily understood by considering the following detailed description in conjunction with the accompanying drawings, in which: FIG.", "1 depicts a cross-sectional schematic diagram of a prior art MEMS device; FIG.", "2A depicts a cross-sectional schematic diagram of a MEMS device according to an embodiment of the present invention; FIG.", "2B depicts a top plan view schematic diagram of the MEMS device of FIG.", "2A; FIGS.", "3A-3F depict simplified cross sectional schematic diagrams depicting the fabrication of a MEMS device according to an embodiment of the invention.", "FIG.", "4 is a flow diagram of a method of maintaining the state of a MEMS device in the event of a power failure; FIG.", "5 is a schematic diagram of an apparatus for maintaining the state of a MEMS device in the event of a power failure; FIG.", "6 is a schematic diagram of a first alternative isolator circuit that may be used in the apparatus of FIG.", "6.FIG.", "7 is a schematic diagram of a second alternative isolator circuit that may be used in the apparatus of FIG.", "6; FIG.", "8 is a schematic diagram of a system for maintaining the state of a MEMS device in the event of a power failure according to an alternative embodiment of the invention; FIG.", "9 is a schematic diagram of an alternative system for maintaining the state of a MEMS device in the event of a power failure according to an alternative embodiment of the invention.", "FIG.", "10 is a diagrammatic perspective view of a movable microstructure apparatus.", "FIG.", "11a is a diagrammatic top view of a movable microstructure apparatus having a different lateral position with respect to an anchor location and a different angular orientation about an anchor location with respect to a first angular orientation.", "FIG.", "11b is a diagrammatic side view of a movable microstructure apparatus having a different lateral position with respect to an anchor location and a different angular orientation about an anchor location with respect to a first angular orientation.", "FIGS.", "12a-12f are diagrammatic perspective views of the movable microstructure apparatus of FIG.", "10 upon application of various combinations of a magnetic field and an electrostatic bias.", "FIG.", "13a is a diagrammatic side view of a movable microstructure apparatus, where a base and a stop have a slight misalignment.", "FIG.", "13b is a diagrammatic side view of the movable microstructure apparatus of FIG.", "13a upon application of a magnetic field.", "FIG.", "13c is a diagrammatic side view of the movable microstructure apparatus of FIG.", "13a upon application of an electrostatic bias between the plate and the stop.", "FIG.", "13d is a diagrammatic front view of the movable microstructure apparatus of FIG.", "13c.", "FIG.", "14a is a diagrammatic side view of a movable microstructure apparatus, where a base and a stop have a slight misalignment.", "FIG.", "14b is a diagrammatic side view of the movable microstructure apparatus of FIG.", "14a upon application of a magnetic field.", "FIG.", "14c is a diagrammatic side view of the movable microstructure apparatus of FIG.", "14a upon application of an electrostatic bias between the plate and the stop.", "FIG.", "15a is a diagrammatic perspective view of an N×M system movable microstructure apparatus used as an optical switch.", "FIG.", "15b is a diagrammatic top view of the N×M system movable microstructure apparatus of FIG.", "15a.", "FIG.", "16a is a diagrammatic side view of the movable microstructure apparatus of FIG.", "10 upon application of a magnetic field, where the magnetic field is not parallel to a sidewall of a stop.", "FIG.", "16b is a diagrammatic front view of an alternate embodiment of a movable microstructure apparatus.", "FIG.", "16c is a diagrammatic side view of the movable microstructure apparatus of FIG.", "16b upon application of a magnetic field, where the magnetic field is not parallel to a sidewall of a stop.", "FIG.", "17A is an exploded isometric diagram of a MEMS device according to an embodiment of the present invention; FIG.", "17B is an isometric assembly diagram of the MEMS device of FIG.", "17A; and FIG.", "18 is a flow diagram of a method of making a MEMS device according an embodiment of the present invention FIGS.", "19A depict an isometric diagram illustrating an apparatus for reducing stiction in a MEMS device according to an embodiment of the present invention; FIGS.", "19B-19D depict schematic diagrams illustrating alternative configurations for anti-stiction members for use with an apparatus of the type depicted in FIG.", "19A; FIGS.", "19E-19G depict a series of isometric diagram illustrating a method for reducing stiction in a MEMS device according to an embodiment of the present invention; FIG.", "19H depicts an isometric diagram illustrating an alternative version of an apparatus for reducing stiction in a MEMS device according to an embodiment of the present invention; FIGS.", "20A-20C depict cross-section schematic diagrams illustrating a MEMS device according to an embodiment of the invention; FIGS.", "21A-21E depict a series of cross-sectional schematic diagrams illustrating the fabrication of a MEMS device according to an embodiment of the present invention; FIGS.", "22A-22B depict alternative versions of MEMS devices according to an embodiment of the present invention; FIG.", "23 depicts an isometric schematic diagram illustrating an optical switch according to an embodiment of the present invention.", "FIG.", "24 is a perspective diagram of a an example of a MEMS moveable element that may be used in the magnet assembly of FIGS.", "27A-27D; FIG.", "25 is a an exploded isometric schematic diagram of a MEMS optical switch according to an embodiment of the present invention; and FIG.", "26 is a partially exploded isometric diagram of an alternative MEMS optical switch according an embodiment of the present invention.", "FIG.", "27A is an elevational schematic view of a magnet assembly according to an embodiment of the present invention; FIG.", "27B is an exploded isometric view of the magnet assembly of FIG.", "27A; FIG.", "27C is an exploded schematic cross sectional view of the magnet assembly of FIG.", "27A; FIG.", "27D is an exploded isometric view of a portion of the magnet assembly FIG.", "27A; FIG.", "28A is an exploded cross-sectional schematic diagram of an x-coil assembly for a magnet assembly of the type depicted in FIGS.", "27A-27D; FIGS.", "28B-28E are plan view of selected layers of the x-coil assembly of FIG.", "28A; FIG.", "29 is a cross-sectional schematic diagram of an alternative x-coil assembly; FIG.", "30 depicts a simplified cross-sectional schematic diagram of a MEMS device with pneumatic actuation and common gas pulse from the backside of a substrate according to an embodiment of the present invention; FIG.", "31 depicts a simplified cross-sectional schematic diagram of a MEMS device with pneumatic actuation using a common gas pulse from above a substrate according to an embodiment of the present invention; FIG.", "32 depicts a simplified cross-sectional schematic diagram of a MEMS device with individual pneumatic actuation of each movable element using multiple electro-pneumatic control valves according to an embodiment of the present invention; FIG.", "33 depicts a simplified cross-sectional schematic diagram of a MEMS device with pneumatic actuation of each movable element using MEMS pneumatic control valves according to an embodiment of the present invention; FIG.", "34 depicts a simplified cross-sectional schematic diagram of a MEMS device with pneumatic actuation of each movable element using Knudsen compressors according to an embodiment of the present invention; FIG.", "35 depicts a simplified cross-sectional schematic diagram of a MEMS device with pneumatic actuation of elements using a micro-pump according to an embodiment of the present invention; FIG.", "36 depicts a simplified cross-sectional schematic diagram of a MEMS device with acoustic pulse actuation from the backside in a gaseous environment according to an embodiment of the present invention; and FIG.", "37 depicts a simplified cross-sectional schematic diagram of a MEMS device with acoustic pulse actuation from the backside in a liquid environment according to an embodiment of the present invention.", "FIG.", "38A-28B depict simplified schematic diagrams of a MEMS device that incorporates capacitive state sensing according to an embodiment of the present invention; FIG.", "39 depicts a block diagram of an MEMS apparatus that incorporates capacitive sensing according to an embodiment of the present invention; FIG.", "40A depicts simplified cross sectional schematics of the apparatus of FIG.", "3 in three different positions; FIGS.", "40B-40C depict capacitance values corresponding to the three positions depicted in FIG.", "40A; FIG.", "41 depicts a simplified timing diagram for operation of a MEMS device according to an embodiment of the present invention; FIG.", "42 depicts a block diagram depicting an optical communications system according to an additional embodiment of the invention FIG.", "43 is a flow diagram of a method for measuring the position of a micro machined optical element using a magnetic sensor according to an embodiment of the present invention FIG.", "44A an isometric schematic diagram of an apparatus according to an alternative embodiment of the present invention; FIG.", "44B is a cross-sectional schematic diagram taken along line 2B-2B of FIG.", "44A: FIG.", "45A is an isometric schematic diagram of an apparatus according to an alternative version of an embodiment of the invention.", "FIG.", "45B is a schematic diagram of a Wheatstone bridge circuit that may be used with the apparatus of FIG.", "45A; FIG.", "46 is an isometric schematic diagram of a MEMS optical switch according to another embodiment of the invention; FIG.", "47A is a plan view schematic diagram of an apparatus according to another alternative version of an embodiment of the invention; FIG.", "47B is a plan view schematic diagram of an apparatus according to another alternative version of an embodiment of the invention; FIG.", "47C is a cross-sectional schematic diagram of an apparatus according to another alternative version of an embodiment of the invention; FIG.", "47D is a plan view schematic diagram of an apparatus according to another alternative version of an embodiment of the invention; FIG.", "47E is a plan view schematic diagram of an apparatus according to another alternative version of an embodiment of the invention; and FIG.", "48 depicts an example schematic diagram of an optical switching system according to another embodiment of the present invention; FIG.", "49 depicts a simplified side cross-sectional schematic diagram of an enclosed MEMS apparatus according to an embodiment of the invention; FIG.", "50 depicts a simplified side cross-sectional schematic diagram of an enclosed MEMS apparatus according to an alternative embodiment of the invention; FIG.", "51 depicts a side cross section of an enclosure in the form of a cap assembly with an optical element attached to the side-wall of the cap according to an embodiment of the present invention; FIG.", "52 depicts a side cross section of a portion of an enclosure having sidewall assembly with a window, attached to a recessed, angled surface according to an embodiment of the present invention; FIG.", "53 depicts a side cross section of a portion of an enclosure having sidewall assembly with a window, attached to a recessed, angled surface according to an embodiment of the present invention; FIG.", "54 depicts a simplified block diagram of a MEMS module according to an alternative embodiment of the invention; FIG.", "55 depicts a simplified side cross-sectional schematic diagram of an enclosed MEMS device according to an alternative embodiment of the invention; FIG.", "56 depicts a graph of pressure versus switching time for a MEMS device; and FIG.", "57 depicts a flow diagram of a high-speed optical switching method according to an embodiment of the invention.", "FIG.", "58A depicts a landing pad structure according to an embodiment of the present invention; FIG.", "58B depicts a landing pad structure according to an embodiment of the present invention; FIG.", "58C depicts a multilayer landing pad structure according to an embodiment of the present invention; FIGS.", "59A-59E depict fabrication of a device according to an embodiment of the present invention; FIGS.", "60A-60E depict fabrication of a device according to an embodiment of the present invention; FIGS.", "61A-61B depicts a microelectromechanical mirror element according to an embodiment of the present invention; FIGS.", "62A-62F depict fabrication of a device according to an embodiment of the present invention; FIGS.", "63A-63B depict fabrication of a device according to an embodiment of the present invention; FIGS.", "64A-64B depict fabrication of a device according to an embodiment of the present invention.", "DESCRIPTION OF THE SPECIFIC EMBODIMENTS Although the following detailed description contains many specific details for the purposes of illustration, anyone of ordinary skill in the art will appreciate that many variations and alterations to the following details are within the scope of the invention.", "Accordingly, the examples of embodiments of the invention described below are set forth without any loss of generality to, and without imposing limitations upon, the claimed invention.", "Like number refer to like elements throughout A. Equipotential Landing Pads FIGS.", "2A and 2B respectively depict cross-sectional and top plan schematic diagrams of a MEMS device 200 having equipotential landing pads according to an embodiment of the present invention.", "The device 200 includes a flap 211 formed from the device layer 212 of a SOI substrate 210 containing the device layer 212 an insulating layer 215 and a base 216.The flap 211 may be movably attached to the device layer 212 by one or more flexure 214.Flexure 214 may be electrically conductive and coupled to one or more topside dimples 224, one or more bottomside dimples 222 or the flap 211.Multiple flexures can provide unique electrical paths to achieve equipotential design, while MEMs springs can also be used to couple connections to a movable flap.", "FIG.", "2B shows a configuration of three flexures 214A, 214B and 214C that provide equipotential to the dimples and clamping voltage to the flap while also providing torsion restoring force thereto.", "The fourth flexure 214D which may be coupled between the flap 211 and the device layer 212 may have an electrical and/or mechanical function.", "While FIG.", "2 illustrates a flap design having top- and bottom-side equipotential dimples, it is understood to be part of the present invention that a flap may be configured with one or more equipotential dimples that, in addition to contacting a vertical sidewall in the ON state, may also contact the substrate in the OFF state.", "This design combines topside and bottomside dimples into a single landing pad structure.", "In this configuration, it is preferred that the dimple is flat, electrically isolated from, and substantially parallel to the mirror.", "A single clamping voltage may be used to secure the flap in the ON and OFF states.", "A flap may be electrostatically held to one or more vertical sidewall structures.", "The flap may include a reflecting surface 213 so that the device 200 acts as a MEMS mirror.", "The flap 211 may be actuated by any first actuation force (i.e.", "an off-chip electromagnet) and be individually addressed by electrostatic clamping either to a surface of a base 216 of the substrate 210 or to one or more vertical sidewall 204.The flap may include a metallic or magnetic material 240, e.g., Nickel.", "An external magnetic field produced by the electromagnet exerts forces on the magnetic material that move the flap 211 between an “off” position parallel to the substrate 210 and an “on” position nearly parallel to the vertical sidewall 204 of the top-mounted chip 206.The sidewall 204 and a surface of the base 216 may serve as landing surfaces for the flap 211.A voltage source 230 may be coupled between the top-mounted chip 206 and the base 216.In the embodiment shown in FIGS.", "2A-2B, the voltage source 230 applies a voltage VCC, e.g., about 40 V, between the top chip 206 and the base 216.By way of example, the voltage source 230 may apply a positive VCC to the top chip 206 while the base 216 is grounded.", "However, the top chip 206 and base 216 may be held at the same potential and the flap grounded at the appropriate time instance in the switching cycle to secure the flap ON or OFF state.", "Similarly, the top chip 206 and base 216 may be grounded and a positive Vcc applied to the flap at the at the appropriate time instance in the switching cycle to secure the flap ON or OFF state.", "Flap 211 may be selectively coupled through switch 232 to VCC or ground to provide electrostatic clamping.", "For example, if the top chip is at VCC, and the flap 211 is in the “on” position, the switch 232 couples the flap 211 to ground, e.g., to the base 216.A voltage difference between the flap 211 and the top chip 206 produces an electric field that clamps the flap 211 in the ON position.", "When the flap is in the “off” position and the base 216 is grounded, the switch 232 couples the flap 211 to VCC.", "A voltage difference between the base 216 and the flap 211 produces an electric field that clamps the flap 211 against a surface of the base 216.The electrostatic fields may hold the flap 211 in position regardless of whether the magnetic field is on or off.", "When clamped to the landing surfaces, e.g., the surface of the base 216 or the vertical side-wall surface 204, the flap 211 may rest on a set of electrically conductive landing pads or dimples 222.These landing pads or dimples may lie level with, protrude below or protrude above the surface of an underside of the flap 211 and may insulated from the flap 211 by an insulating material 223.The landing pads 222 (Shown in phantom in FIG.", "2B) may be electrically coupled to the base 216 through a first flexure 214A or an electrically conductive MEMs spring, to reduce stiction effects.", "The first flexure 214A may be electrically insulated from the flap 211, the device layer 212 and the top chip 206.Thus, when the flap 211 and the base 216 are at different potentials, the landing pads 222 are equipotential to, i.e., at the same potential as, the base 216.This prevents trapping of electrical charge and arcing due to different potentials.", "The insulating materials 223 prevent an electrical short between the flap 211 and the base.", "The flap 211 may optionally include a set of electrically conductive top landing pads 224.The top landing pads may lie level with, protrude above or protrude below a top surface of the flap 211.The top landing pads 224 may be electrically connected to each other and may be electrically insulated from the flap 211 by an insulating material 225.To reduce stiction effects, the top landing pads 224 may be electrically coupled so that it is substantially equipotential to the top chip 206 through a second flexure 214B or an electrically conductive MEMs spring.", "The second flexure 214B may be electrically insulated from the flap 211, the device layer 212 and the base 216.Third flexure 214C or MEMs spring may connect to mirror 213 and through programmable switch 232 to Vcc such that the voltage potential can be programmably coupled through the flap 211.When switch 232 is OFF, Vcc charges mirror 211 with a potential, flap 211 is electrostatically attracted and clamped on the base 216, and the bottomside landing pads 222 make contact with the base 216 at the ground equipotential.", "When switch 232 is ON, Vcc grounds the flap 211, the flap 211 is electrostatically attracted to the top chip 206 and the topside landing pads 224 make contact with top chip 206 at Vcc equipotential.", "It must be stated that in an alternative design the polarity may be reversed so that a negative voltage VCC may be applied to the top chip 206 or the top chip may be grounded and the voltage VCC may be applied to the base 216.Furthermore, it must be stated that the device 200 may optionally include one or more electrically conductive base landing pads 226 disposed on a surface of the base 216 and insulated from the base 216 by an insulating material 227.The device 200 may also optionally include one or more electrically conductive sidewall landing pads 228 that are electrically isolated from the sidewall.", "The base landing pads 226 and sidewall landing pads 228 may be electrically coupleable such that they may be selectively made substantially equipotential to the flap 211, e.g., by a third flexure 214C and the switch 232.The landing pads 222, 224, 226, 228 may be in the form of a plug that protrudes through an opening in the insulating material 223, 225, 227.Alternatively, the landing pads 222, 224, 226 may be in the form of plugs with flanges attached to the flap by a layer of electrically insulating support material similar to that shown in FIGS.", "7A-7B.", "Alternatively, the device 200 may optionally include conductive sidewall landing pads 228 disposed on the sidewall 204 that are electrically isolated from the sidewall 204 and electrically coupled to the flap 211, e.g.", "via an insulated connector on the flexure 214C.", "The conductive landing pads 222, 224, 226, 228 may be made from those materials that exhibit conductive properties, as one skilled in the art would be capable of applying.", "Such materials include, but are not limited to polysilicon, amorphous silicon, single crystal silicon, conductive diamond films, silicon germanium, and metals.", "The insulating materials 223, 225, 227 may be any of those materials that exhibit insulative properties, as one skilled in the art would be capable of applying.", "Such materials include, but are not limited to silicon nitride, silicon oxide, undoped single crystal silicon, undoped polysilicon and undoped silicon germanium.", "FIGS.", "3A-3F depict simplified cross sectional schematic diagrams depicting the fabrication of a MEMS device of the type shown in FIG.", "2.The method starts at FIG.", "3A with a SOI substrate 301 having a device layer 302, a sacrificial insulating layer 304 and a base layer 306.Next, as shown in FIG.", "3B, several openings 308 are made in the device layer 302.The openings 308 are filled with an insulating material 310 as shown in FIG.", "3C.", "The insulating material 310 may also cover the surface of the device layer 302.Next vias 312 are formed through the insulating material 310 that fills the openings 308 as shown in FIG.", "3D.", "The vias may be formed by a dry etch process or an anisotropic wet etch process.", "The vias 312 penetrate partly into the sacrificial insulating layer 304.Next the vias 312 are filled with a conducting material 314 to form conductive plugs 316.The conducting material 314 may also cover the surface of the insulating material 310 that overlies the device layer 302 to provide a common electrical connection between the conductive plugs 316.The sacrificial insulating layer 304 is removed, e.g.", "by isotropic etch, as shown in FIG.", "3F.", "The ends of the conductive plugs 316 project slightly beyond a lower surface of the device layer 302 and insulating material 310 to form landing pads 318 that are electrically isolated from the device layer 302.The landing pads 318 may then be electrically coupled to a landing surface of the base layer 306 by an electrical connection 322 coupled to the conducting material 314.The electrical connection 322 may be formed contemporaneously with the layer of conducting material 314 and electrically insulated from the device layer 302, e.g., by the insulating material 310.B.", "Capacitive Latching A method and/or system for maintaining the state of a MEMS device in the event of a power failure may be incorporated into the MEMS optical cross-connect switch to sustain the state of the switch during a power outage.", "An example capacitive latching method 400 of the present invention is depicted in the flow diagram of FIG.", "4, and FIG.", "5 depicts a schematic diagram of an apparatus 500 that may implement the method of FIG.", "4.The apparatus 500 generally includes a charge-storing circuit 540 and an isolator circuit (ISO) 550 that operates with a MEMS optical switch 501.More specifically, the optical switch 501 has a substrate 502, a moveable element 504 moveably coupled to the substrate 502.By way of example, the moveable element 504 may move between a horizontal “OFF” position (shown in phantom) and a vertical “ON” position.", "In the “ON” position, the moveable element 504 may be retained against a clamping surface 506 whose height is lower than the movable element 504.The moveable element 504 may include one or more equipotential landing pads of the type described above.", "The optical switch 501 may operate in response to signals from a controller 510.Controller 510 may execute code 516 for implementing certain steps of the method 400.The code 516 may conform to any one of a number of different programming languages such as Assembly, C++, JAVA or a number of other languages.", "The switch 501, controller 510, high voltage (HV) driver 520, DC-DC converter 530, charge-storing circuit 540 and isolator element 550 may be subsystems or components of a network element e.g., as shown below with respect to FIG.", "8.Switch 501 may be configured on a removable card and the network element may be part of a network (see FIG.", "6).", "The controller 510 may include network element interface 517 which may be implemented in software e.g.", "in a subroutine in memory 512 or hardware to allow the controller 510 to communicate with the network element.", "Such communication may include, but is not limited to, switching commands issued from the network element to the switch 501 and switch state data transmitted from the switch 501 to the network element.", "In the example depicted in FIG.", "5, the clamping surface 506 may be a “top chip” having one or more openings that receive the moveable element 504 and/or others like it.", "The openings may have sidewalls against which the moveable element 504 may be retained.", "It should also be understood that the term “top chip”, as referenced herein, refers to any platform attached to a substrate containing one or more moveable elements to which a movable element may be clamped.", "One top chip design may be comprised of a single fabricated MEMS structure having an array of 8×8, 16×16 or 32×32 openings that align with each movable element in a corresponding array of moveable elements such as moveable element 504.Another top chip design may be a single or multiple array of high aspect vertical sidewalls; in this case two walls may be associated with each movable element.", "While it should be understood that a top chip may be located at the bottom or side of the movable element as anticipated by the plurality of design abstractions, it should also be stated that the clamping surface 506 may be part of a single-layer device as opposed to a chip layer bonded to a substrate or base such as substrate 502.The clamping surface 506 may be electrically charged to an electrostatic potential Vclamp with respect to 0 volts (ground), as well as isolated from the substrate 502 and all other MEMS elements.", "The moveable element 504 may be selectively coupled to either a source of clamping voltage Vclamp or to a ground potential, e.g.", "0 volts.", "In an 8×8 switching fabric, a high voltage driver 520 may be a 64 channel latch such as the Supertex HV58908 which contains 64 channels of output 525, each of which may couple to movable switch element such as moveable element 504.Commands sent by the microcontroller 510 are received by the high voltage driver via bus 523 to configure each of the 64 outputs to a HIGH or LOW value.", "In the example shown in FIG.", "5, when channel 525A is set HIGH, the clamping voltage Vclamp, e.g., 40 volts, is applied to movable element 504 and no electrostatic clamping is realized since the adjacent clamping surface 506 is also at the clamping voltage Vclamp.", "However, when channel 525A is set LOW, the movable element 504 is grounded, resulting in electrostatic attraction to the adjacent clamping surface 506 which is set at Vclamp.", "The charge-storing circuit 540 is electrically coupled between a clamping surface and ground to hold a clamping charge on the clamping surface to sustain switch state in the event of power failure.", "The example charge-storing circuit 540 has a capacitor 544 and an optional series resistor 542.In the event of a power failure, the capacitor 544 sustains a voltage potential on the clamping surface 506 in the event of a power failure to ensure that the configuration of the optical cross connect switch is maintained.", "In a particular embodiment of the invention, the capacitor 544 has a capacitance that is less than about 20 microfarads (μF).", "Although a capacitive charge-storing circuit 540 is depicted in FIG.", "5, the charge-storing circuit 540 may alternatively include a battery or other circuit element that is capable of storing an electrical charge.", "The series resistor 542 is employed to limit the charge rate of the capacitor at power-up, thus preventing overloading or performance degradation of the clamping voltage source (e.g., DC-DC converter 530).", "In the case where it is desirable to limit the discharge current flowing from the capacitor 544 into the clamping surface 506 (say, for example, to protect the clamping surface 506 and/or MEMS moveable element 504 from further damage should a short suddenly occur), the series resistor 542 could be suitably modified to provide such limiting.", "If, as a result of this modification, the resistance of the series resistor 542 becomes so large as to increase the charge time of the capacitor 544 to an unreasonably long period of time, then a diode 546 in series with an additional resistor 548 may optionally be connected across the series resistor 542 in order to control the charging rate of the capacitor 544 independently of the discharge rate.", "In some cases it may be desirable for the charge-storing circuit 540 to charge up quickly if this will not overload the DC-DC converter 530 with the charging current To facilitate this, the charge-storing circuit 540 may optionally include a “one-way” short circuit around the resistor 542, e.g., in the form of a diode configured to provide a low resistance path for charging the capacitor 544 and a high resistance path (compared to that of resistor 542) for discharging the capacitor 544.The clamping voltage Vclamp from the DC-DC converter 530 is coupled to the clamping surface 506 through the isolator circuit 550.In the event of a power failure, the isolator circuit 550 prevents charge from leaking to ground through the DC-DC converter 530 and/or the HV driver 520.The isolator circuit 550 may be coupled to the microcontroller 510.The isolator circuit 550 may be configured to electrically isolate the DC-DC converter 530 and the HV driver 520 from the clamping surface 506 in the event power is lost to the controller 510.The isolator 550 may optionally include connections to a logic voltage Vcc and/or ground to facilitate isolation when the logic voltage Vcc drops due to a power failure.", "Furthermore, the isolator 550 may include a connection to the microcontroller 510 so that the controller 510 may signal the isolator circuit 550 to isolate the DC-DC converter 530 and the HV driver 520 from the clamping surface 506 if power is lost to the DC-DC converter 530.Preferably, the charge-storing circuit 540 and the isolator element 550 have a total current leakage that is low enough that the clamping surface 506 retains sufficient charge to clamp the moveable element 504 for a sufficient period of time depending upon the requirements of the system of which the switch 501 is a part.", "By way of example this could be as short as a few milliseconds or as long as several days.", "During this period of time, the clamping voltage Vclamp may drop below its initial value.", "The movable element 504 will still be retained against the clamping surface 506 as long as the clamping voltage remains above some minimum value.", "The capacitor 544 preferably has a low current leakage across its leads and is made using a high resistance dielectric.", "Capacitors are generally rated by a capacitance, and a maximum voltage.", "In general, capacitors having a higher maximum voltage rating tend to exhibit a lower leakage current.", "A particular example of a capacitor 544 that is suitable for use with a clamping voltage Vclamp of about 40V is a model ECQE(F) 10-microfarad (μF) 250-volt metallized polyester capacitor made by Panasonic of Osaka, Japan.", "Testing has shown this device to sustain over 168 hours of clamping voltage to 64 movable mirror elements in a prototype 8×8 optical cross-connect switching fabric.", "By way of example, the isolator element 550 may be an opto-isolator.", "FIG.", "6 depicts a schematic diagram of an example of an opto-isolator 650 that may be used as the isolator element 550 of FIG.", "5.The opto-isolator 650 generally includes a phototransistor 652 and a source 654 of light 656.As used herein, the term “phototransistor” refers to a circuit element that is electrically conductive in response to light and electrically isolating in the absence of light.", "By way of example, the phototransistor 652 includes a source 651, a drain 653 and a gate 655.As long as light 656 from the source 654 impinges on the gate 655 of the phototransistor 652 electric current may flow between the source 651 and the drain 653.As used herein the term “light” generally refers to electromagnetic signals that may be transmitted through free space or through a dielectric medium.", "As such, the term “light” includes, but is not limited to, infrared light, visible light, ultraviolet light, and the like.", "The source 654 provides light 656 as long as long as power is on, e.g., there is a voltage difference across the LED.", "Thus current may flow through the phototransistor as long as the power is on.", "By way of example, the source 654 may be a light emitting diode (LED).", "The LED may be coupled between VCC and ground.", "The VCC connection may be provided by one of the I/O functions 514 of the controller 510 or a separate power supply.", "Furthermore the ground connection may be provided through the controller 510 or a separate ground connection.", "A resistor 658 may be coupled in series with the LED to limit an electrical current through the LED.", "For a clamping voltage Vclamp of 40 V, an example of a suitable opto-isolator is a model AQV225N(A) PhotoMOS relay manufactured by Aromat Corporation of San Jose, Calif. Alternatively, the isolator element 550 may be a low leakage diode.", "If the isolator element 550 is a low leakage diode, connection to the controller 510 is not required.", "FIG.", "7 depicts a partial schematic diagram illustrating the how a low leakage diode 750 would be incorporated into the apparatus of FIG.", "5 as the isolator element 550.The diode 750 has an anode 752 and a cathode 754.The diode 750 easily conducts electric charge flowing from the anode to the cathode and is highly isolating for electric charge attempting to flow from the cathode 754 to the anode 752.The anode may be connected to the DC-DC converter 530 and the high voltage driver 520.The cathode 754 may connect to the charge-storing circuit 540 such that the resistor 542 and the capacitor 544 are between the cathode 754 and ground.", "The cathode 754 may be connected to the clamping surface 506.In this configuration, the diode 750 allows electric charge to flow to the clamping surface 506 from the DC-DC converter 530 but inhibits charge from flowing from the clamping surface 506 through the DC-DC converter 530 or through the HV driver 520 to ground.", "For a clamping voltage Vclamp of about 40 V, an example of a suitable low leakage diode is a model BAS116 Low Leakage Diode manufactured by Phillips Corporation of Eindhoven, The Netherlands.", "Alternatively, the diode 750 may be replaced with a high isolation transistor, such as a field effect transistor (FET) or bipolar transistor having sufficiently low leakage.", "Alternatively an Analog Switch or Multiplexer (MUX) may be used to provide an equivalent function to that of the diode 750.Power failure detection may be implemented by real-time monitoring of voltage levels through an A/D pin on controller 510.The code 516 may include software for analyzing voltage over time to calculate slope trends and track the sharp voltage drop that occurs at the instant that power is failing so that the system controller can take action in response to the power failure event.", "Monitoring may be facilitated by an analog to digital (A/D) converter 765, which may be implemented as one or more of the I/O circuits 514 of the controller 510.By way of example, the controller 510 may sense a loss of power by comparing the logic voltage level VCC to a reference voltage level VREF.", "Such a comparison may be implemented, for example, by use of a voltage divider network 760, a resistor 770, and a Zener diode 775.The voltage divider network 760 is coupled between a source of logic voltage VCC and ground.", "The voltage divider network 760 is coupled to the controller 510, e.g., through the A/D converter 765.The voltage divider network 760 provides a voltage that is some known fraction of the actual voltage from the source of logic voltage, e.g., ½VCC.", "Thus, if the source provides a VCC level of 5 volts, the voltage divider network 760 provides 2.5 volts.", "If the VCC level drops to 4.0 volts, the voltage divider network 760 provides 2.0 volts.", "The resistor 770 and Zener diode 775 provide a reference voltage VREF that is substantially fixed, e.g., at 2.5 volts.", "An example of a suitable Zener diode is a model ZRC250F01 from Zetex of Oldham, United Kingdom.", "The reference voltage VREF is also provided from the Zener diode 775 to the controller 510, e.g.", "via the A/D converter 765.The controller 710 may then compare the value of ½VCC to the reference voltage VREF.", "If the value of VCC drops due to a power failure the value of ½VCC also drops, but the Zener diode 775 retains the reference voltage VREF at a sufficiently fixed value so that the controller 510 can sense a power failure by comparing ½VCC to VREF.", "The controller 510 may also monitor the clamping voltage Vclamp provided by the DC-DC converter 530.This is useful, for example, where the controller stabilizes the value of Vclamp.", "However, the clamping voltage Vclamp may be higher than a maximum voltage that can safely be applied to the A/D converter 765.In such a case it is useful to reduce the voltage provided to the controller 510, e.g., through the use of a voltage divider network 760.Although the voltage divider network 760 and A/D converter 765 are shown in the low leakage diode example of FIG.", "7, those skilled in the art will also recognize that a similar voltage divider network may be used with an opto-isolator or other type of isolator 550.The method 400 of the present inventions begins at step 402 by coupling the charge-storing circuit 540 between the clamping surface 406 and ground.", "By way of example, the charge-storing circuit 540 may be hard wired to a circuit board containing the controller 510, high voltage driver 520, DC-DC converter 530, or it may be located externally and coupled there said components via standard I/O ports as one skilled in the art would be capable of applying.", "The isolator element 550 may be hard wired to the circuit board.", "It is desirable to have a high impedance and low leakage current between the components and ground.", "To reduce leakage currents it is often desirable to ensure that the various components of the apparatus 500, and the board or substrate to which they are mounted, are clean.", "At step 406, a clamping voltage is applied to the clamping surface 506 via the isolator 550, which couples the DC-DC converter 530 to the clamping surface 506.In the example shown, if the moveable element 504 is to be retained in the “ON” position, the high voltage driver 520 may electrically couple the moveable element 504 to ground.", "The clamping voltage Vclamp produces an electric force that retains the moveable element 504 in the “ON” position when the moveable element is connected to ground e.g., through the high voltage driver 520.Although the charge-storing circuit 540 is designed to hold the charge on the clamping surface 506 in the event of a power failure, charge may leak to ground through the DC-DC converter 530 and/or the high voltage driver 520.Therefore, at step 408 it is important to electrically isolate the source of clamping voltage from the clamping surface 506 in the event of a power failure.", "Furthermore, it is important to keep the MEMS element 504 at a suitably low voltage if the MEMS element 504 is to be clamped to the clamping surface.", "By way of example, the high voltage driver 520 may simply operate in a “fail safe” mode, in which it couples the MEMS element 504 to ground automatically in the event of a power failure.", "Additional actions may be taken in association with step 408, e.g., where the MEMS optical switch 501 is part of a network.", "Examples of such steps may be understood by simultaneously referring to FIG.", "4 and FIG.", "8, which illustrates a system 790 according to an alternative embodiment of the present invention.", "The system 790 generally includes a network element 799 which may be coupled to one or more other network elements 870, 880 via a network 890.The network elements 799, 870, 880 may operate in response to instructions from a network management software 892 coupled to the network 890.The network element 799 includes a switch fabric 800 and other network element components 860.The switch fabric 800 includes an optical switch 801, a controller 810, a high voltage driver 820, a DC-DC converter 830, a charge-storing circuit 840 and an isolator 850.These components may have features in common with the corresponding components described above with respect to FIG.", "5 and/or FIG.", "7 and may be configured in a similar fashion.", "The optical switch 801 may include an array of moveable elements 804 that are moveably coupled to a substrate 802.The moveable elements 804 may be selectively clamped to a clamping surface 806, e.g.", "at top chip, as described above with respect to FIG.", "5.For example, the high voltage driver 820 may have a set of outputs 825 that are coupled to the moveable elements 804.The optical switch 801 may operate in response to signals from a controller 810 having features in common with the controller 510 described above.", "By way of example, the controller 810 may be configured to include a CPU 811, memory 812 input/output (I/O) functions 514, and an analog to digital (A/D) I/O function 819, all of which may communicate with each other via a system bus 815.The A/D I/O function 819 may be coupled to the HV driver 820 and or DC-DC converter 830 to facilitate power failure monitoring.", "The memory 812 may contain instructions, e.g., in the form of the program code 816.The code 816 may include instructions for implementing certain steps of the method 400.The program code 816 may include network element interface 817 which may be implemented in software e.g.", "in a subroutine in memory 812 or hardware to allow the controller 810 to communicate with the network element 799 and/or the network 890.Such communication may include, but is not limited to, switching commands issued from the network element 799 to the switch fabric 800 and switch state data transmitted from the switch fabric 800 to the network element 799.The other network element components 860 may include, but are not limited to multiplexers, demultiplexers, photo detectors, variable optical attenuators, optical amplifiers, packet routers, optical-electronic-optical (OEO) components, such as non-optical routers, port cards, and the like.", "An alternative system 790′ is depicted in FIG.", "9.The system 790′ has features in common with the system 790 of FIG.", "8.Specifically, the system 790′ has a network element 799′ and switch fabric 800′ with a charge storage circuit 840′.", "These elements are configured in a substantially similar fashion to that shown and described with respect to FIG.", "8.However, the charge storage circuit 840′ is separate from the switch fabric 800′ and is coupled to it through a port 845.Thus, the charge storage circuit may be provided, e.g., sold, separately from the switch fabric 800′ and the other components in the system 790′.", "Referring back to FIG.", "4, at optional step 410 the controller 810 may implement a controlled shutdown feature during power failure.", "The controlled shutdown may include, but is not limited to, saving the state of the switch 801 in the memory 812 and communicating to the host Network Element 799 the fact that the switch 801 lost power at a particular time, e.g., mm:dd:yy at hh:mm:ss.", "This is useful because it allows the Network Element 799 or higher-level switch that controls the switch fabric 800, of which the switch 801 may be a part, to trigger maintenance alarms 895 with respect to the discharge and time remaining in the latching period.", "The alarms 895 may be communicated to the network management software 892 or to the network element 799.Flags triggered by the alarms 895 can also be stored in the memory 812, e.g.", "FLASH memory, with the state of the switch to enable certain actions to be taken on power up after the switch has recovered power.", "These actions may include informing the Network Element that power has been recovered.", "The method 400 may also optionally include related features such as a controlled shutdown and boot-up.", "During power failure detect, the controlled shutdown feature may save the current state of the switch 801.In the event of a power failure, there is often a time lag, e.g.", "al milliseconds of clock cycle time remaining before the value of VCC drops below a level at which the controller ceases to function.", "During this interval, the controller program code 816 may execute instructions for the controller 810 to signal the host Network Element 799 with the event, date and time the switch fabric lost power.", "By signaling the Network Element 799 during shutdown, the Network Element 799 can prioritize maintenance alarms with respect to discharge period and track the time remaining in the latching period, i.e., the time remaining before charge leaks from the clamping surface 806 to the point that there is no longer sufficient force to clamp the moveable element in the “ON” position.", "With reference to FIG.", "8, the signal alarm 895 transmitted to the Host Network Element 799 may be relayed to the network 890 so that network management control software 892 that manages control of the network elements 799,870, 880 can manage network resources in a contingency plan.", "The network management software 892 generally keeps track of and controls the inventory of network element assets.", "When a signal alarm 892 is transmitted from the network element 799, it may contain a node I.D.", "enabling network management software 892 to reference in a database those features associated with the network element node.", "As so the network management software 892 may derive from the signal alarm the amount of latching time associated with the network element 799 which can then be used to trigger and prioritize maintenance schedules and redirect traffic in response to the downed system.", "It should be understood that the signal alarm 895 itself may include data encoding the latching duration associated with downed system.", "In addition to notification of power failure, the program code in the system of FIG.", "3 may also direct the controller 810 to flag the power failure event in a non-volatile memory, e.g., a FLASH memory, to enable a smart boot-up of the fabric, so that the fabric can handle special circumstances and signal the network element on power recovery, such as signaling an alarm to the host network element so that itself and/or the network management software can be configured in response thereto.", "Referring to both FIG.", "5 and FIG.", "8, when the power returns after a power failure, the DC-DC converter 530, 830 may require some finite amount of time to ramp up to the clamping voltage Vclamp.", "To restore the state of the switch 501, 801 it is often desirable to include in the method 400 an optional step 412 of doing a proper power-up sequence for the HV driver 520, 820 and restoring the states of the HV driver outputs 525, 825.Furthermore, when the power returns after a power failure, the DC-DC converter 530, 830 may require some finite amount of time to ramp up to the clamping voltage Vclamp.", "The method 400 may include a step 414 of waiting for a voltage provided by the source of clamping voltage Vclamp (e.g., the DC-DC converter 530) to ramp up to the clamping voltage level Vclamp in the event power returns after the power failure.", "This step is useful if an opto-isolator, photoMOS relay, or other such bi-directional current switching device is used to isolate the clamping surface from the DC-DC converter.", "If the output of the DC-DC converter 530, 830 were applied to the top chip 506, 806 in this manner before the output voltage has reached the minimum clamping voltage Vclamp, the top chip 506, 806 will experience a sudden dropout in clamping voltage and the moveable elements 504, 804 being held up may drop.", "By way of example, the controller 510, 810 may be programmed to read the voltage produced by the DC-DC converter 530, 830 to ensure that the desired clamping voltage level has been attained.", "Alternatively, the controller 510, 810 may be programmed to wait for a predetermined amount of time that is sufficient to allow the DC-DC converter to ramp up to the clamping voltage level.", "In either case, the source of clamping voltage may subsequently be reconnected to the clamping surface 506, 806 in step 416.Some systems may require all of steps 402-416 to ensure that the moveable elements 504, 804 do not fall in the event of a power failure.", "In the diode switching implementation of FIG.", "7, however, clamping voltage dropout is less likely due to the unidirectional current regulation characteristic of the diode 750 and thus step 416 is unnecessary for this case.", "It is possible to use various alternatives, modifications and equivalents.", "It should be understood that the clamping voltages may take on various values and that the polarity of clamping components may be reversed; for example the clamping surface 506, 806 may be held at ground, the substrate 502, 802 may be held at 30 volts, and 30 volts may be applied to the movable element(s) 504, 804 to clamp it in the ON state.", "It should be understood that the clamping surface 506, 806 may exert an electric force on the movable element such that the moveable element(s) 504, 804 need not make physical contact with the respective clamping surface 506, 806.It should also be understood that, though specific example applications are shown that relate to a specific sub-field of optical communications, the present invention may be applied to maintain the state of a MEMS device in a variety of other applications within optical communications as well as other applications utilizing MEMS moveable elements.", "Such applications may include or relate to, but are not limited to, waveguides, relays, mixers, pumps, accelerometers, RFMEMS, bioMEMS etc.", "C. Cantilevered Microstructures Equipotential landing pads of the type described above with respect to FIGS.", "2A-2B may be incorporated into a cantilevered microstructure.", "Cantilevered microstructures are particularly useful for optical cross-connect (OXC) applications such as crossbar switches.", "An optical crossbar switch can provide interchange of data paths between different fibers, at multi-gigabit data rates, without having to first convert them into the electronic domain as is being done in existing networks.", "An N×N optical crossbar switch consists of N input and N output optical fiber ports, with the capability of selectively directing light from any input port to any output port in a “non-blocking” fashion.", "Currently, switches deployed in the communication infrastructure operate by converting the input optical signals to electronic signals, directing the electronic signals to the proper output channels, and converting them back into optical signals.", "In an all-optical OXC, the light is directly deflected from an input fiber port into an output fiber port without any electrical conversion.", "Each of the optical beams can be expanded and collimated by inserting a microlens at the tip of each input and output fiber port.", "By propagating an array of optical beams in free space and selectively actuating reflectors in an array of movable reflectors, any one of the N input optical beams can be directed to any one of the N output fibers ports.", "The core of each input and output fiber port is the region in which most of the optical beam travels.", "Due to the small diameter of the core, the optical crossbar switch requires the reflectors to be maintained at a precise position in order to direct each optical beam from one fiber port to another.", "An example of a cantilevered microstructure apparatus is depicted in FIGS.", "10-11b.", "FIG.", "10 shows an apparatus 1000 having a stop 1002, a base 1004 and a plate 1006.The plate 1006 is coupled to the base 1004 and is movable between a first angular orientation and a second angular orientation.", "The stop 1002 has at least one substantially planar sidewall 1008 that is configured to contact the plate 1006 in a contact area when the plate 1006 is in the second angular orientation.", "In one implementation, a substantially planar sidewall 1008 is constructed to lie in a plane which is orthogonal to the top surface of the base 1004.The apparatus 1000 is fabricated by a MEMS process.", "The base 1004 may be composed of an insulating layer disposed over a semiconductor substrate; for example, silicon nitride, silicon oxide, or a combination of both, may be disposed over a silicon substrate.", "The plate 1006 may be a rectangular beam formed from a conductive material or a semiconductive material such as polycrystalline silicon.", "A layer of magnetic material may be plated onto the plate 1006.More than one region of the plate 1006 may be so plated.", "The magnetic material may be one of various combinations of nickel, iron, or other elements, and is usually ferromagnetic characterized by a high saturation magnetization.", "The plate 1006 may be coupled through flexures 1010 and 1012 to the base 1004 at anchor locations.", "In one implementation, insulative anchors 1014 and 1016 are used to attach the flexures 1010 and 1012 to the base 1004.The flexures 1010 and 1012 may be formed from a flexible and resilient conductive or semiconductive material (e.g., polycrystalline silicon).", "The flexible material provides the flexures 1010 and 1012 with a degree of elasticity.", "The flexures 1010 and 1012 allow the plate 1006 to change its angular orientation about the anchors 1014 and 1016 with respect to the first angular orientation and its lateral position with respect to the anchors 1014 and 1016, as shown in FIGS.", "11a and 11b.", "In one implementation, the stop 1002 is coupled to a voltage source 1018 and the base 1004 is electrically grounded.", "An electrostatic clamping circuit can be formed from a switch 120, a contact 1022 for forming a connection between the switch 1020 and the flexure 1010, the plate 1006, the flexure 1012, and the anchor 1016, and is switchable between a voltage source 1024 and electrical ground 1026.The voltage sources 1018 and 1024 may be external sources such as power supplies or batteries, or internal sources on the apparatus 1000.An electrostatic bias can be created between the plate 1006 and one of the clamping surfaces (base 1004 and stop 1002) depending on the position of the switch 1020.Referring to FIG.", "12a, in the absence of any applied force, the plate 1006 lies in the first angular orientation substantially parallel to the base 1004.The voltage source 1024 may be coupled to the electrostatic clamping circuit to create an electrostatic bias between the plate 1006 and the base 1004 upon application of a voltage V1.If a sufficient voltage V1 is applied, the plate 1006 is “clamped” to the base 1004 and restrains the plate 1006 from rotating in the presence of an applied force, for example, a magnetic field 1026 as shown in FIG.", "12b.", "If the plate 1006 is not clamped to the base 1004, application of the magnetic field 1026 would cause the plate 1006 to be rotated about the anchors 1014 and 1016 between the first angular orientation and the second angular orientation until there is an equilibrium between the resultant torque from the torsional stretching of the flexures 1010 and 1012 and the force on the plate 1006 caused by the magnetic field 126.The angular orientation of the plate 1006 at the equilibrium point defines a static equilibrium position.", "In one implementation, the static equilibrium position is the second angular orientation, as shown in FIG.", "12c.", "In another implementation, the static equilibrium position is between the first angular orientation and the second angular orientation, as shown in FIG.", "12d.", "In this implementation, the force on the plate 1006 resulting from the application of the magnetic field 1026 can be time-varying, such that the plate 1006 is provided with a momentum that rotates the plate 1006 beyond the static equilibrium position to the second angular orientation.", "The time-varying force on the plate 1006 may have a step profile, a ramp profile, a sinusoidal profile or a pulse profile.", "Once the plate 1006 is in the second angular orientation, an electrostatic bias may be created between the plate 1006 at electrical ground and the stop 1002 having a voltage V2, as shown in FIG.", "12e.", "The plate 1006 clamps to the sidewall 1008 in a contact area characterized by a height b and a width w provided the following condition is satisfied: torque about axis defined through anchors torque resulting from torsional 1014 and 1016 resulting from electrostatic bias≧stretching of flexures 1010 and created between plate 1006 and stop 102 1012 ɛ ⁢ wV 2 2 ⁢ g 2 × b 2 + 2 ⁢ ab 2 ≥ k θ ⁢ θ ( 1 ) where ε is a constant representing the permittivity of a material separating the electrically conductive portion of the plate 1006 and the electrically conductive portion of the stop 1002 when plate 1006 is in contact with the stop 1002, V is a voltage applied to create an electrostatic bias between the plate 1006 and the stop 1002, g is a distance separating the electrically conductive portion of the plate 1006 and the electrically conductive portion of the stop 1002 when the plate 1006 is in contact with the stop 1002, kθ is a torsional spring constant of the flexures 1010 and 1012, θ is the angular orientation of the plate 1006 about the anchors 1014 and 1016 with respect to the first angular orientation, and a is a distance separating the stop 1002 and the base 1004.Once the plate 1006 is clamped to the sidewall 1008, removing the magnetic field 1026 has no effect on the angular orientation of the plate 1006, as shown in FIG.", "12f.", "FIG.", "13a shows the stop 1002 coupled to the base 1004 with a slight misalignment In this example, the anchors 1014 and 1016 are offset from a plane 1302 defined through the contact area of stop 1002.The plate 1006 is movable through an obtuse angle θ about the anchors 1014 and 1016 with respect to the first angular orientation.", "In the absence of an applied force, the plate 1006 lies in the first angular orientation substantially parallel to the base 1002.If the plate 1006 is not clamped to the base 1004, application of the magnetic field 1026 may rotate the plate 1006 about the anchors 1014 and 1016 until the plate 1006 contacts a top edge 1028 of the stop 1002 in the second angular orientation, as shown in FIG.", "13b.", "The plate 1006 clamps to the sidewall 1008 in a contact area characterized by a height b and a width w as shown in FIGS.", "4c and 4d provided two conditions are satisfied: (i) condition 1 defined above; and (ii) the following condition: ti torque about axis defined through top torque resulting from the lateral edge 1028 resulting from electrostatic bias≧stretching of flexures 1010 and 1012 created between plate 1006 and stop 1002 ɛ ⁢ wV 2 2 ⁢ g 2 × b 2 2 ≥ kd ⁡ ( a + b ) ( 2 ) where k is a lateral spring constant of the flexures 1010 and 1012, and d is a distance separating the location of the anchors 1014 and 1016 and a plane defined by the contact or overlap area 1304 of the stop 1002.FIG.", "14a shows the stop 1002 coupled to the base 1004 with an alternative misalignment.", "In this example, the anchors 1014 and 1016 are offset from a plane 1402 defined through the contact area of the stop 1002.The plate 1006 is movable through an acute angle θ about the anchors 1014 and 1016 with respect to the first angular orientation.", "In the absence of an applied force, the plate 1006 lies in the first angular orientation substantially parallel to the base 102.If the plate 1006 is not clamped to the base 1004, application of the magnetic field 1026 may rotate the plate 1006 about the anchors 1014 and 1016 until the plate 1006 contacts a bottom edge 1030 of the stop 1002 in the second angular orientation, as shown in FIG.", "14b.", "The plate 1006 clamps to the sidewall 1008 in a contact area characterized by a height b and a width w as shown in FIG.", "14c provided two conditions are satisfied: (i) condition 1 defined above; and (ii) the following condition: torque about axis defined through bottom torque resulting from the lateral edge 1030 resulting from electrostatic bias≧stretching of flexures 1010 and created between plate 1006 and stop 1002 1012 ɛ ⁢ wV 2 2 ⁢ g 2 × b 2 2 ≥ kda ( 3 ) In the three cases described above and shown in FIGS.", "12f, 13c and 14c, in the absence of an applied force, the plate 1006 returns to the first angular orientation substantially parallel to the base 1004 as the torsional and lateral stretching of the flexures 1010 and 1012 are relaxed.", "FIGS.", "15a and 15b show an apparatus 1500 having a stop assembly 1502 coupled to a base assembly 1504.The base assembly 1504 has an array of plates 1506.Each plate may be coupled to the base assembly 1504 by at least one flexure which permits each plate to change its angular orientation and lateral position.", "The stop assembly 1502 may have an array of substantially planar surfaces.", "Each substantially planar surface may be configured to contact a respective plate in a contact area sized so that, upon application of a force to the plate substantially normal to the substantially planar surface of the stop assembly 1502, a sufficient force holds the plate against the stop assembly 1502 in a plane substantially parallel to the substantially planar surface of the stop assembly 1502.In one implementation, the stop assembly 1502 defines an array of apertures 1508, as shown in FIG.", "15a.", "Each aperture has at least one substantially planar surface 1510 that contacts a respective plate in a contact area.", "Each substantially planar surface 1510 is constructed to lie in a plane normal to the base assembly 1504.The array of plates 1506 may be coupled to an electrostatic clamping circuit such that each plate may be individually selected to be clamped to its respective surface 1510 or to the base assembly 1504.In an alternative implementation (not shown), the stop assembly may define an array of cavities, each cavity having at least one substantially planar surface that contacts a respective plate in a contact area.", "One application of the apparatus 1500 is an optical switch.", "In one implementation, the array of plates 1506 act as reflectors.", "A suitable reflector coating may be deposited on the portion of each plate above the plane of a top surface 1512 of the stop assembly 1502 to enhance reflectivity if desired.", "In an alternative implementation, the array of plates 1506 act as beam splitters.", "Each plate may be constructed from a material that transmits and reflects different parts of an optical beam.", "Each plate may be similarly sized and constructed such that each sidewall 1510 contacts a bottom portion of its respective plate.", "FIG.", "15a shows the apparatus 1500 having three optical inputs 1514, 1516, and 1518, and three optical outputs 1520, 1522, and 1524.The N inputs (1514, 1516, and 1518) are along one side of the apparatus 1500 and the M outputs (1520, 1522, and 1524) are along an adjacent side.", "The switching elements are the array of plates 1506.Each plate is oriented at a similar angle, for example, 45 degrees to an incoming optical beam.", "If the mth plate along one of the N input beams is clamped to its respective sidewall, that beam is reflected into the mth of the M outputs.", "All but one plate in a given input line may be held down by the electrostatic bias applied between the plate and the base assembly 1504.The plate that is clamped to its respective sidewall selects the output for that input line.", "The materials from which the apparatus 1500 is fabricated, the voltage sources, the applied electrostatic bias, and the applied magnetic fields may be chosen by a user to adjust the sensitivity of the apparatus 1500 for any particular purpose or application.", "The apparatus 1500 may be fabricated using techniques including “lithographic, galvanoformung and abformung” (LIGA), traditional machining, deep anisotropic plasma etching and laser machining.", "The stop assembly 1502 may be fabricated by anisotropic etching of (110)-oriented silicon which ensures the angular uniformity of all the sidewalls 1510 on the stop assembly 1502.The array of plates 1506, sidewalls 1510 and the base assembly 1504 may be fabricated to have textured surfaces on one or more surfaces to reduce sticking when a plate is clamped to its respective sidewall or to the base assembly 1504.The textured surface may include dimples, bumps, and ridges such that the contact area may include the overlap area between the plate and the stop at a distance gap generally equal to the effective height of the texture.", "The number of plates defining the N-by-M array of plates 1506 may be adjusted based on the application of the apparatus 1500.The apparatus 1500 may be fabricated in a single batch-process and consist of a single stop-base module.", "Alternatively, the apparatus 1500 may be fabricated in a two-part process, one process for fabricating the stop assembly 1502 and the other process for fabricating the base assembly 1504.The stop assembly 1502 may be aligned with the base assembly 1504 in a separate alignment step.", "The applied electrostatic bias may be an attractive force applied by the electrostatic clamping circuit described above or by other means, where the attractive force is defined as any force that pushes or pulls a plate towards a stop.", "The magnetic fields may be applied using coils located internal or external to the apparatus 1500, or a permanent magnet located internal or external to the apparatus 1500.Current-carrying coils, hard magnetic materials, soft magnetic materials, or a combination of the three formed on each of the array of plates 1506 may apply a force to the plate in the presence of magnetic fields.", "Rotational magnetic fields may be used to apply torque to the flap.", "An applied magnetic field 1602 that is not perfectly parallel to the sidewall 1604 may induce a slight torque and resultant bending in the portion of the plate 1606 containing a reflective surface when the plate 1606 is clamped to the sidewall 1604, as shown in FIG.", "16a.", "The resultant bending may cause a misalignment of a reflected beam.", "FIG.", "16b shows an alternative implementation to the plate 1606 that reduces the bending effects on optical performance.", "The magnetic portion 1608 of the plate 1610 is connected to the rest of the plate 1610 by support arms 1612.The support arms 1612 isolate the portion of the plate 1610 containing a reflective surface 1614 from the magnetic field 1602 that is applied on the magnetic portion 1608 of the plate 1610.In this implementation, when the plate 1610 is clamped to the sidewall 1616, application of the magnetic field 1602 that is not perfectly parallel to the sidewall 1616 results in minimal bending in the portion of the plate 1610 containing the reflective surface 1614 as shown in FIG.", "16c.", "Other embodiments are within the scope of the present invention.", "For example, the steps of the method can be performed in a different order and still achieve desirable results.", "D. Mosaic Tiling Problems associated with large scale MEMS arrays may be overcome by tiling two or more MEMS device dies together to form a tiled MEMS device.", "Such a tiled device generally includes a substrate with two or more device dies attached to the substrate.", "Each device die includes one or more microelectromechanical (MEMS) optical elements.", "A common clamping die is attached to the device dies such that each MEMS optical element aligns with a corresponding clamping surface on the common clamping die.", "Such tiling techniques may be applied to MEMS devices that include equipotential landing pads of the type described above.", "FIG.", "17A depicts an exploded isometric diagram of a MEMS device 1700 according to a first embodiment of the present invention.", "An assembly diagram of the device 1700 is depicted in FIG.", "17B.", "The device 1700 may be used, for example, as an optical switch, as shown in FIG.", "17B.", "The device 1700 may selectively couple optical signals 1701 between a first set of optical input/output (I/O) ports 1752 and a second set of I/O ports 1754 as shown in FIG.", "17B.", "The I/O ports 1752, 1754 may be collimator lenses, such as graded refractive index (GRIN) lenses that couple the optical signals 1701 to and from optical fibers (not shown).", "To facilitate optical coupling to the I/O ports 1752, 1754, the device 1700 may include collimator arrays 1756 that are disposed along the perimeter of the device 1700.The device 1700 generally includes a substrate 1710, MEMS device dies 1720A, 1720B, 1720C, 1720D, and a common clamping die 1730.Each MEMS device die 1720A, 1720B, 1720C, 1720D may have an array of MEMS optical elements 1722.By way of example, each optical element 1722 may be in the form of a flap attached to the rest of the device die by one or more flexures 1724.The flap may include a reflective surface so that it may act as a MEMS mirror.", "The optical element 1722 may move between an “OFF” position and an “ON” position under the influence of an actuating force, such as a magnetic force.", "By way of example the optical elements 1722 may be oriented substantially parallel to the substrate 1710 in the “OFF” position and substantially perpendicular to the substrate in the “ON” position.", "In the “ON” position, the optical elements 1722 deflect the optical signals 1701.By way of example each of the four device dies 1720A, 1720B, 1720C, 1720D includes a 3×3 array of MEMS optical elements 1722.When assembled the four device dies are arranged in a 2×2 tiled configuration to provide a device 1700 with a 6×6 array of MEMS optical elements.", "The tiling concept may be extended to encompass any number of device dies greater than one.", "The device dies 1720A, 1720B, 1720C, 1720D may be of almost identical appearance and construction, with the possible exception of the routing of electrical circuits to the MEMS optical elements 1722 and or the placement of bond pads (not shown).", "The device dies 1720A, 1720B, 1720C, 1720D are attached to the substrate 1710.The device dies 1720A, 1720B, 1720C, 1720D may be accurately self-aligned to the substrate 1710, e.g., to within a few microns using known solder attachment processes.", "Alternatively, the device dies may be accurately aligned by active-alignment and bonded using a solder or epoxy.", "Because each device die contains a relatively small number of MEMS optical elements 1722, each of the device dies may be manufactured by a process having a higher yield than a process for producing a larger single device die having the same total number of MEMS optical elements as the device 1700.Thus, the overall yield for the tiled MEMS device 1700 is greater than for MEMS device made with a single large device die covering the same area and having the same number of MEMS elements.", "To facilitate attachment to the substrate 1710, each device die may include on a backside one or more metallized bonding pads 1726 (shown in phantom).", "The bonding pads 1726 may align with corresponding metallized bonding pads 1716 on the substrate 1710.The common clamping die 1730 may be in the form of a “top chip” having openings 1732 that may receive all of the optical elements 1722 of the device dies 1720A, 1720B, 1720C, 1720D.", "The openings 1732 may include clamping surfaces 1734 in the form of sidewalls.", "The clamping surfaces 1734 provide reference stopping-planes for the MEMS optical elements 1722.The clamping die 1730 may include clamping surfaces 1734 in the form of a single vertical wall or two vertical walls with a hole therebetween to allow light to pass.", "Such a vertical wall or walls may be higher than the MEMS optical elements 1722.Clamping die 1730 may also contain a magnetic pole piece and/or be bonded to substrate 1710 at perimeter referential locations to enable clamping surface 1734 positioning structures that extend downward second substrate clamping die 1730.Finally, positioning structure clamping surface walls 1734 may form an air gap between the substrate 1710 and the optical elements (not shown).", "A voltage may be applied between individual optical elements 1722 and the common clamping die 1730 to electrostatically clamp the optical elements 1722 in the “ON” position.", "The voltage may be applied through an elongated oval shaped hairpin tether device or any flexure 1724 to optical elements 1722.Optical elements 1722 may contain restriction tabs that contact the positioning structure walls or clamping surfaces 1734 when in the “ON” position.", "The optical elements 1722 may be electrically insulated from the clamping surfaces 1734 by an insulating gap, such as an air gap.", "The larger common clamping die 1730 may be manufactured by a simple semiconductor process, and thus does not present the same yield problems as the more complicated MEMS device dies 1720A, 1720B, 1720C, 1720D.", "Furthermore, since the clamping surfaces 1734 are all formed on the same clamping die 1734 the common clamping die 1730 very accurately registers the individual MEMS optical elements 1722 on the different device dies 1720A, 1720B, 1720C, 1720D.", "This facilitates accurate alignment of the MEMS optical elements 1722 to the I/O ports 1752, 1754.Even if one or more of the device dies is slightly misaligned with respect to the others, the optical elements 1722 will still register to the clamping die 1730 as long as there is sufficient play in the flexures 1724 to accommodate the misalignment.", "The collimators 1756 may be disposed along the sides of the device 1700.The collimators 1756 may couple light into and out of fiber associated with each switching channel.", "Device 1700 may be configured with two, three or four sides of switching channels and collimators 1756 may be in the form of collimator arrays or individual collimator lenses, e.g., ball lenses, microlenses, and the like.", "The collimators 1756 may fit into slots 1758 in the clamping die 1730 to align them with the optical elements 1722.Collimators 1756 may also be actively aligned whereby each lens manipulated actively via control signals to optimize coupling into the fiber input/output channels.", "Depending upon the size of the device dies 1720A, 1720B, 1720C, 1720D it may be desirable to counteract beam spreading over long optical path lengths through the device 1700.To facilitate this collimators 1740 may optionally be disposed between adjacent device dies.", "Each collimator includes lenses 1742.The lenses 1742 may be any suitable type of lens such as GRIN lenses, ball lenses, microlenses, and the like.", "The collimators 1740 may be in the form of pre-assembled collimator arrays or individual lenses.", "The collimators 1740 may be aligned with the optical elements 1722 using through-slots 1736 in the clamping die 1730.The through-slots 1736 allow the collimators 1740 to be inserted after the clamping die 1730 has been attached to the device dies 1720A, 1720B, 1720C, 1720D.", "The substrate 1710 may include corresponding slots 1712 to allow some vertical adjustment in the positioning of the collimators.", "Depending upon the configuration of the optical switch, it may be desirable to accommodate signal regeneration, attenuation, power monitoring, wavelength switching and wavelength detection feature functions.", "To facilitate these and other feature functions, collimators 1740 and/or collimators 1756 may be replaced with a feature module that includes the suitable type of detectors, filters, actuators and sensors to support the accommodated function.", "Feature module may perform collimation in addition to its feature function and be aligned with the optical elements 1722 using through-slots 1736 in the clamping die 1730.The through-slots 1736 allow the feature modules to be inserted after the clamping die 1730 has been attached to the device dies 1720A, 1720B, 1720C, 1720D.", "The substrate 1710 may include corresponding slots 1712 to allow some vertical adjustment in the positioning of the feature module.", "MEMS devices of the type shown in FIGS.", "17A-17B may be manufactured according to an inventive method 1800 according to a second embodiment of the invention.", "The steps of the method 1800 are illustrated in the flow diagram of FIG.", "18.For the purposes of example, the steps of the method 1800 are described below with respect to the device 1700 of FIGS.", "17A-17B.", "The method begins with the fabrication of MEMS device dies 1720A, 1720B, 1720C, 1720D by standard semiconductor processes.", "At an optional step 1802, the device dies may be tested prior to further assembly to ensure proper operation.", "At step 1804, the device dies are attached to the substrate 1710.By way of example, the device dies 1720A, 1720B, 1720C, 1720D may be attached with a solder attachment process.", "For example, the metallized bonding pads 1716, 1726 react with a solder to form strong self-aligned bonds.", "The bonding pads 1716 on the substrate 1710 are preferably defined with the highest resolution processes available (e.g., thin film technology on ceramic substrate).", "In addition, the metallized bonding pads 1726 on the backside of the MEMS device die 1720 may be formed by patterning and etching with semiconductor photolithographic processes.", "The type of metal used in the bonding pads may depend on the type of solder.", "For example, if a Pb/Sn eutectic is used as the solder, a Cr/Ni/Au layer could be used as the patterned metal thin film on the device dies 1720A, 1720B, 1720C, 1720D and substrate 1710.Once the metallized bonding pads 1716, 1726 are defined, the solder may be applied, e.g., in paste form, to the substrate 1710 or device dies 1720A, 1720B, 1720C, 1720D.", "The device dies are then placed on the substrate 1710, such that the bonding pads 1716 on the substrate 1710 align with the bonding pads 1726 on substrate 1720.This may be accomplished, e.g., with a standard “pick and place” tool.", "The solder is heated through reflow, e.g., with a belt furnace.", "This allows the solder to react to the metal, of the bonding pads, pulling the device die into a preferred alignment, accurate, e.g., to a few microns.", "Preferably, the bonding pad pattern on the substrate and die will maximize the surface to volume ratio of solder after reflow.", "A large number of small solder bumps (and thus small metal pads) are preferred.", "This tends to enhance the solder surface tension effects for a given amount of solder.", "Furthermore, to improve angular alignment of a given device die, it is desirable to place the solder acting to align the device die as far from the centroid of the die as possible.", "In addition, a sufficient amount of solder must be used to obtain sufficient alignment.", "Alternatively, the device dies 1720A, 1720B, 1720C, 1720D may be attached to the substrate 1710 using an active-alignment process.", "For example, each of the device dies may be accurately placed, e.g., to within a few microns, on the substrate 1710 using a pick and place tool.", "An example of a suitable pick and place tool is a KS model number FC150 manufactured by Karl Suss of Germany.", "The device dies 1720A, 1720B, 1720C, 1720D may be held in place by surface tension with respect to the substrate 1710.A solder, placed e.g., on the bonding pads 1716, 1726, is heated through reflow.", "The device dies are held in place until the solder cools and freezes.", "Alternatively, an epoxy may be used to bond the device dies to the substrate.", "If an epoxy is used, the device dies may be aligned without the use of metallized bonding pads.", "After the device dies 1720A, 1720B, 1726C, 1720D are attached to the substrate 1710, the common clamping die 1730 may be attached to the device dies in step 206.In order to improve the inter-die alignment of the MEMS optical elements 1722, when in the “ON” state, a single monolithic clamping die 1730, e.g., a “top chip”, can be used.", "The semiconductor processes typically used to fabricate a top chip are simple, and so yields for large clamping die are less of a concern than for device dies.", "The clamping die 1730 may be bonded to the MEMS device dies, again using a technique that provides very accurate alignment.", "In a preferred embodiment, the clamping die 1730 is attached using an active alignment process in which a pick and place tool places the clamping die 1703 over the device dies and holds it there as solder is heated and cooled.", "The attachment method for the clamping die 1730 must be at a lower temperature than the temperature used for attaching the optical MEMS die to the substrate e.g., to keep the solder holding the device dies 1720A, 1720B, 1720C, 1720D to the substrate 1710 below the solder reflow temperature.", "Provided there is enough “play” in the mechanical flexures 1724 of the MEMS optical elements 1722, the optical elements 1722 will be able to register to the clamping die (clamping is most likely achieved electrostatically).", "As the clamping die 1730 is a monolithic structure, the inter-die alignment of the MEMS optical elements 1722 will be superior.", "Subsequent assembly of the device 1700 may proceed according to standard processes.", "The order of steps 1804 and 1806 is not critical and may be reversed as indicated by the dashed arrows 1805.Specifically, the common clamping die 1730 may be attached to the device dies (or vice versa) before attaching the device dies to the substrate 1710.Of course, if step 1806 takes place before step 1804 it is important that the device dies are attached to the substrate by a process that takes place at a lower temperature than the process for attaching the clamping die 1730 to the device dies.", "The device dies 1720A, 1720B, 1720C, 1720D may be self aligned to the common clamping die 1730 using solder and metallized bonding pads on the backside of the clamping die and the front sides of the device dies.", "It is possible to use various alternatives, modifications and equivalents on the embodiments described above.", "For example, in the embodiment depicted in FIGS.", "17A-17B each of four device dies 1720A, 1720B, 1720C, 1720D contains a 3×3 array of optical elements 1722.The device 1700 is shown this way for the sake of clarity.", "The invention is not limited to four device dies having 3×3 arrays of MEMS optical elements.", "For example four device dies each having an NXN array of MEMS optical elements may be arranged in a 2×2 tiled configuration to form a MEMS device having a 2N×2N array, where N is an integer greater than or equal to 1.Furthermore, two or more device dies having any number of optical elements may be used without departing from the scope of the present invention.", "E. Anti-Stiction Bars Although the equipotential landing pads described above with respect to FIGS.", "2A-2B can reduce stiction in MEMS devices, other stiction-reducing techniques may be used in conjunction.", "Such techniques may include interposing an anti-stiction member between the moveable element and the substrate.", "Such a technique is particularly useful, e.g., to prevent the mirror from being properly released from the substrate during manufacture.", "FIG.", "19A depicts an example of an apparatus 1999 for reducing stiction according to an embodiment of the present invention.", "The apparatus 1999 generally includes a MEMS device 1900 having a moveable element 1906 moveably coupled to a substrate 1901.In the example depicted in FIG.", "19A the MEMS device 1900 is formed from a silicon-on-insulator (SOI) substrate 1901.The SOI substrate 1901 includes an insulator layer 1902 disposed between a support layer 1903 and a device layer 1904.The apparatus 1999 includes one or more an anti-stiction members 1910 that are interposable between the moveable element 1906 and the support layer 1903.The moveable element 1906 may be formed from a portion of the device layer 1904.The moveable element 1906 may include a light-deflecting component 1907 so that the apparatus 1900 may operate as part of a MEMS optical switch.", "By way of example, the light-deflecting component 1907 may be a simple plane reflecting (or partially reflecting) surface, curved reflecting (or partially reflecting) surface, prismatic reflector, refractive element, prism, lens, diffractive element, e.g.", "grating or fresnel lens, a dichroic coated surface for wavelength specific and bandpass selectivity, a waveguide or some combination of these.", "A hinge 1908 moveably attaches the moveable element 1906 to the rest of the device layer 1904.The hinge 1908 is attached to the device layer and the moveable element The hinge.", "1908 may be made of a flexible material that flexes when a force or torque is exerted on the moveable element 1906.In the embodiment shown, the hinge 1908 allows the moveable element 1906 to rotate with respect to the substrate 1901.The hinge 1908 may provide a torque that counters rotation of the movable element 1906 with respect to the plane of the substrate 1901.The hinge may be any suitable structure such as one or more torsion hinges, cantilever flexures, serpentine flexures, or pin-and-staple hinges combined with one or more springs.", "The hinge 1908 may also be a flexible member that allows vertical movement of the movable element with respect to the plane of the substrate.", "The anti-stiction members significantly decrease the area of contact between the moveable element 1906 and the substrate 1901.Many designs are possible for the anti-stiction members 1910.In the example depicted in FIG.", "19A, the anti-stiction members 1910 are in the form of cantilevered bars that are attached to the device layer 1902 but not to the moveable element 1906.The anti-stiction members 1910 substantially overhang the moveable element 1906.The anti-stiction members 1910 may be made from a flexible material such as polysilicon or metals commonly used in the semiconductor industry, e.g., Nickel, Tungsten, and the like.", "Alternatively, the anti-stiction members 1910 may be made from a suitable polymer material.", "Furthermore, if the moveable element 1906 is formed using a lithography and etch process, it is often desirable that the anti-stiction members 1910 are made from a material that is resistant to the final release etch process that forms the moveable element 1906.For example, polysilicon is resistant to hydrofluoric acid (HF).", "Although bar-shaped anti-stiction members are depicted in FIG.", "19A, the invention is not limited to this particular configuration.", "Anti-stiction members having other shapes, such as serpentine, U-shaped, or L-shaped may also be used.", "As used herein, the term flexible means that the anti-stiction members 1910 have at least one portion that is capable of flexing.", "Although, flexibility may often be imparted by choice of material, the shape of the anti-stiction member may also impart some degree of flexibility.", "By way of example, and without loss of generality, FIGS.", "19B-19D depict possible alternative shapes for the anti-stiction member 1910.In FIG.", "19B an anti-stiction member 1910B has a serpentine portion 1913B disposed between an anchor 1911B and a stand-off 1912B.", "The serpentine portion may impart flexibility to the anti-stiction member 1910B.", "The anti-stiction member 1910B may be attached to a substrate at the anchor 1911B.", "The stand-off 1912B at a free end of the anti-stiction member 1910B reduces the contact area between the anti-stiction member and the underside of a MEMS device.", "A serpentine shape such as that depicted in FIG.", "19B may have an undesirable tendency to twist.", "To overcome this an anti-stiction member 1910C may have double-serpentine hinge portion 1913C located between a fixed end 1911C and a free end 1912C, as shown in FIG.", "19C.", "The double-serpentine hinge portion 1913C may be formed by making a hole in a widened portion of the anti-stiction member 1910C.", "The double-serpentine hinge 1913C is less susceptible to undesired twisting that the serpentine portion 1913B depicted in FIG.", "19B.", "Additional flexibility may be imparted by using two double-serpentine hinges 1913D as shown in FIG.", "19D.", "The double-serpentine hinges 1913D are disposed between a fixed end 1911D and a free end 1912D of an anti-stiction member 1910D.", "The operation of the anti-stiction bars is best understood by reference to FIGS.", "19E-19G, which depict an example of a method of reducing stiction in a MEMS device according to an embodiment of the invention.", "The method begins at FIG.", "19E by providing the substrate 1901 with one or more anti-stiction members 1910.The anti-stiction members 1910 are then interposed between the moveable element 1906 and the substrate 1901 as illustrated in FIGS.", "19F-19G.", "By way of example, the anti-stiction members 1910 may be interposed between the moveable element 1906 and the substrate 1901 as follows.", "First, the moveable element 1906 is actuated such that it engages the anti-stiction members 1910, thereby causing them to flex.", "Any suitable mechanism may be used to actuate the moveable element 1906.For example, a magnetic force or an electrostatic force may actuate the moveable element 1906.The actuating force may cause the moveable element to rotate as shown in FIG.", "19F.", "The more the moveable element 1906 rotates, the more the anti-stiction members 1910 flex.", "At some point the moveable element 1906 will move so far that the anti-stiction members 1910 flex past the moveable element 1906 and snap into place between the moveable element 1906 and the substrate 1901.More specifically, the anti-stiction members 1910 flex into position between the moveable element 1906 and the support layer 1903 as shown in FIG.", "19G.", "In this position, the anti-stiction members 1910 support the moveable element 1906 and inhibit direct contact between the moveable element 1906 and the underlying portion of the substrate 1901, e.g.", "either the support layer 1903 or the oxide layer 1902.Although the anti-stiction members 1910 may bias the moveable element 1906 in a position that is slightly out of the plane and/or out of parallel with respect to the device layer 1904 this is not a serious drawback.", "In MEMS applications, this position may correspond to an “OFF” state where the alignment of the moveable element is not critical.", "The out-of-parallel orientation may be corrected by using many pairs of anti-stiction members 1910 to bias the moveable element 1906 in a position that is substantially parallel to the device layer 1904.In a particular version of the method, the moveable element 1906 may be actuated while it is immersed in a liquid.", "The surface tension forces that tend to cause stiction between the moveable element 1906 and the substrate 1901 may be eliminated when both are immersed in a liquid.", "Such actuation may be motivated, e.g., by a magnetic field provided by a magnet located outside the liquid.", "Post release stiction problems may be avoided by actuating the moveable element 1906 in liquid and interposing the anti-stiction members 1910 between the movable element 1906 and the substrate 1901 before removing the moveable element 1906 and substrate 1901 from the liquid.", "Such a procedure is useful, for example, after a wet etching process that releases the moveable element 1906.It is often desirable to electrically isolate the moveable element 1906 from the substrate 1901.The moveable anti-stiction member 1910 must not create an undesirable short circuit between the moveable element 1906 and the substrate 1901.For example, if the moveable element 1906 is to be electrostatically clamped to the substrate 1901 a short circuit between them will undesirably cause a current to flow.", "The moveable element 1906 may be electrically isolated, e.g., by an insulating material disposed between the anti-stiction member 1910 and the device layer 1904.Alternatively, a portion of the oxide layer 1902 may electrically isolate the moveable element 1906 from the support layer 1903.An alternative scheme for electrically insulating a moveable element from anti-stiction members is depicted in FIG.", "19H, which shows an apparatus 1950 that has features in common with the apparatus 1900 of FIG.", "19A.", "In the apparatus 1950 a moveable element 1956 is formed from a device layer 1954 of a substrate 1951, which may also include an insulating layer 1952 and a support layer 1953.A hinge 1958 moveably connects the moveable element to the device layer 1954.Anti-stiction members 1960 are interposeable between the moveable element 1956 and the rest of the substrate 1951.The moveable element 1956 includes insulating portions 1957 that contact anti-stiction members 1960.The insulating portions 1960 electrically isolate the anti-stiction members 1960 from an electrically conductive portion of the moveable element 1956 thereby electrically isolating the anti-stiction members 1960 from the device layer 1954.The insulating portions 1957 may be formed by etching out sections of the moveable element 1956 and filling in the etched out sections with insulating material.", "Similar insulating portions may be used to isolate the hinge 1958 from the device layer 1954.The present invention also includes embodiments directed to MEMS devices.", "An example of such a MEMS device 2000 is depicted in the cross-sections shown in FIGS.", "20A-20C.", "The MEMS device 2000 generally includes a moveable element 2006, a substrate 2001 and one or more an anti-stiction members 2010 that are interposable between the moveable element 2006 and the substrate 2001.A hinge 2008 moveably attaches the moveable element 2006 to the rest of the device layer 2004.The hinge 2008 is attached to the device layer and the moveable element 2006.The hinge 2008 may be made of a flexible material that flexes when a torque is exerted on the moveable element 2006.In the example depicted in FIGS.", "20A-20C the MEMS device 2000 is formed from a silicon on insulator (SOI) substrate 2001 having an insulator layer 2002 disposed between a support layer 2003 and a device layer 2004.The moveable element 2006 is formed from a portion of the device layer 2004.The moveable element 2006 may include a light-deflecting component 2007 of any of the types described above with respect to FIGS.", "19A-19D.", "A magnetic material 2009 such as nickel may be deposited on the moveable element 2006 for magnetic actuation.", "The moveable element 2006 may optionally include one or more standoffs 2013 formed on an underside of the moveable element.", "The anti-stiction member 2010 significantly decreases the area of contact between the moveable element 2006 and the underlying portion of the substrate 2001, e.g.", "insulating layer 2004 and/or support layer 2003.In the example depicted in FIGS.", "20A-20C, the anti-stiction member 2010 is in the form of a cantilevered bar that is attached to the device layer 2004 but not to the moveable element 2006.The anti-stiction member 2010 substantially overhangs the moveable element 2006.The overlap between the anti-stiction member 2010 and the moveable element is preferably smaller than the overlap between the anti-stiction member and the device layer 2004.The anti-stiction member may include a standoff 2012 that minimizes the contact area between the anti-stiction member and the moveable element 2006.The standoff 2012 may be made from an insulating material to help electrically isolate the moveable element 2006 from the substrate 2001.The anti-stiction members 2010 may be made from a flexible material and may have any suitable shape as described above.", "Although bar-shaped anti-stiction members are depicted in FIGS.", "20A-20C, the invention is not limited to this particular configuration.", "The operation of the anti-stiction members 2010, as illustrated in FIGS.", "20B-20C, proceeds substantially as described above with respect to FIGS.", "19B-19D.", "Specifically , the anti-stiction members 2010 may be interposed between the moveable element 2006 and the substrate 2001 by actuating the moveable element 2006 such that it engages the anti-stiction members 2010, thereby causing them to flex as shown in FIG.", "20B.", "For example, a magnetic field B may exert a force on the magnetic material 2009 to actuate the moveable element 2006.At some point the moveable element 2006 will move so far that the anti-stiction members 2010 flex past the moveable element 2006 and snap into place between the moveable element 2006 and the substrate 2001.In this position, the anti-stiction members 2010 inhibit direct contact between the moveable element 2006 and the underlying portion of the substrate 2001, e.g.", "the oxide layer 2002.There are many ways of making a MEMS apparatus or device with anti-stiction members for reducing stiction as described above.", "FIGS.", "21A-21E depict a series of cross-sections that illustrate an example of a method of fabricating of a MEMS device according to another embodiment of the invention.", "The method begins as shown in FIG.", "21A with an SOI substrate 2101 having an oxide layer 2102 disposed between a support layer 2103 and a device layer 2104.One or more trenches 2105 are etched in the device layer to define a moveable element 2106 from the device layer 2104 as shown in FIG.", "21B.", "A light-deflecting component (not shown) may be formed on the moveable element either before or after forming the trenches 2105.The trenches 2105 are formed all the way through the device layer 2104 to the oxide layer 2102.Next a sacrificial layer 2107 is formed over the device layer 2104 as shown in FIG.", "21C.", "The sacrificial layer may be, e.g., an oxide layer such as SiO2.The sacrificial layer 2107 is patterned with vias 2109A, 2109B, and 2109C.", "One or more patterns of flexible material are then deposited over the sacrificial layer 2107 and into the vias 2109A, 2109B, and 2109C as shown in FIG.", "21D.", "By way of example, the flexible material may be polysilicon deposited by low pressure chemical vapor deposition (LPCVD).", "Alternatively, the flexible material may be a metal, such as Nickel or Tungsten that may be deposited by evaporation, sputtering, plating and the like.", "The flexible material provides a hinge 2108 and an anti-stiction member 2110.The anti-stiction member 2110 substantially overhangs the moveable element 2106 but is not attached to it.", "Via 2109A provides a point of attachment between the anti-stiction member 2110 and the device layer 2104.Via 2109B provides a point of attachment between the hinge 2108 and the moveable element 2106.Via 2109C provides a point of attachment between the hinge 2108 and the device layer 2104.The anti-stiction member 2110 and the hinge 2108 may be formed from the same flexible material and they may be formed at the same time.", "Alternatively, the hinge 2108 and the anti-stiction member 2110 may be formed of different materials at different times.", "A standoff 2112 may be formed at a free end 2111 of the anti-stiction member 2110, e.g.", "by patterned deposition of an insulating material.", "Once the anti-stiction member 2110 and hinge 2108 have been formed, the moveable element 2106 may be released by etching away the sacrificial layer 2107 as show in FIG.", "21E.", "Such an etch process may be an isotropic etch in HF.", "The process that etches the sacrificial layer 2107 may also remove a portion of the oxide layer 2102.The moveable element 2106 remains attached to the device layer 2104 by the hinge 2108.The anti-stiction member 2110 is attached to the device layer 2104 but not the moveable element 2106.The free end 2111 of the anti-stiction member overhangs the moveable element 2106 and may be interposed between the moveable element 2106 and the support layer 2103 in a manner similar to that shown and described above with respect to FIGS.", "19B-19D and 20A-20C.", "The MEMS devices described above may be varied in many ways without departing from the scope of the invention.", "For example, anti-stiction members may be employed in beam steering MEMS elements.", "FIG.", "22A depicts an isometric schematic diagram of such a MEMS device 2200.The device 2200 generally comprises a substrate 2201 having, e.g., an insulator layer 2202 disposed between a support layer 2203 and a device layer 2204.A moveable element 2206 is formed from the device layer 2204 and is attached to the rest of the device layer 2204 by torsion hinges 2208A, 2208B.", "The moveable element 2206 may include a light-deflecting element 2207.The moveable element 2206 may rotate about an axis through the torsion hinges 2208A, 2208B, e.g.", "under the influence of an actuating force, e.g., an electrostatic or magnetic force.", "Alternatively, the moveable element may move by translation, e.g., in a direction substantially perpendicular to the plane of the device layer 2204.Anti-stiction members 2210A, 2210B may be interposed between the moveable element 2206 and the support layer 2203 as described above.", "Specifically, the moveable element 2206 may rotate in one direction to interpose anti-stiction member 2210A and then in an opposite direction to interpose anti-stiction member 2210B.", "The anti-stiction members 2210A, 2210B may also provide mechanical biases to the moveable element 2206.Although, moveable elements that rotate are described herein, the present invention is in no way limited to in rotating devices.", "An example of a MEMS device 2250 that uses anti-stiction members with a translating moveable element is depicted in FIG.", "22B.", "The device 2250 generally comprises a substrate 2251 and a moveable element 2256.Flexible anti-stiction members 2260 are interposable between the moveable element 2206 and the substrate 2251.In the device 2250, the moveable element 2256 is configured to translate in direction substantially perpendicular to the substrate 2251 as shown by the double-ended arrow.", "By way of example, the moveable element is retained between the substrate 2251 and a cap 2255.The moveable element may move under the influence of a pneumatic force, e.g.", "provided by gas that enters the space between the substrate and the cap through a passage 2253.Alternatively, the moveable element 2256 may move under the influence of an electrostatic or magnetic force.", "The anti-stiction members 2260 may be interposed between the substrate 2251 and the movable element 2256 by exerting an actuating force on the moveable element 2256 causing it to move away from the substrate.", "Once the moveable element moves far enough, the anti-stiction members 2260 flex past the moveable element 2256 and into position between the moveable element 2256 and the substrate 2251.The present invention also includes embodiments directed to systems that incorporate two or more MEMS apparatus, e.g.", "arranged in an array.", "An example of such an array is an optical switch 2300 depicted in FIG.", "23.The switch 2300 generally comprises a substrate 2301 having an array of moveable elements 2302.Each moveable element is associated with one or more anti-stiction members 2304.The anti-stiction members 2304 are interposable between the associated moveable element 2302 and the substrate 2301.Each moveable element includes a light-deflecting component 2303, e.g.", "of any of the types described above.", "By way of example, and without loss of generality, the light deflecting component 2303 one each moveable element 2302 may be a mirror.", "The light deflecting components 2303 on the moveable elements 2302 selectively couple optical signals 2305 between one or more input fibers 2306 and one or more output fibers 2308.While the above includes a description of a preferred embodiment of the present invention, it is possible to use various alternatives, modifications and equivalents.", "For example, the anti-stiction bar may be made of a thermal bimorph that may be actuated to lift the flap a few degrees to increase switching time.", "It should be understood that, though specific example applications are shown that relate to optical communications, the present invention may be applied to reduce stiction effects in a plurality of applications utilizing a moveable element.", "Such applications may include, but not be limited to, relays, mixers, pumps, accelerometers, RFMEMS, bioMEMS etc.", "F. Actuation 1.Magnetic Actuation In magnetic actuation schemes a magnetic field actuates one or more magnetically actuatable MEMS optical elements.", "FIG.", "24 depicts one possible example of a magnetically actuatable MEMS 2400.The optical element 2400 generally comprises a base 2406 and a flap 2411 coupled to the base 2406, e.g.", "by one or more flexures 2414, so that the flap 2411 is movable out of the plane of the base 2406 from a first angular orientation to a second angular orientation.", "By way of example, the first position may be substantially horizontal, i.e., substantially parallel to a plane of the base, and the second position may be substantially vertical, i.e., substantially perpendicular to the plane of the base.", "The flap 2411 may include a light-deflecting element 2413 to deflect optical signals.", "By way of example, the light-deflecting element 2413 may be a mirror, e.g., a simple plane reflecting (or partially reflecting) surface, curved reflecting (or partially reflecting) surface, prismatic reflector, refractive element, prism, lens, diffractive element, e.g.", "fresnel lens, a dichroic coated surface for wavelength specific and bandpass selectivity, or some combination of these.", "The flap 2411 and the base 2406 may be formed from a portion of a starting material 2401 in order to avoid alignment problems associated with post-process bonding associated with a two wafer approach.", "For example, the starting material 2401 may be formed from a silicon-on-insulator (SOI) wafer having a device layer 2402, an insulator layer 2404 and a substrate layer as the base 2406.The starting material 2401 may include an opening or cavity 2415 having sidewalls 2417 that are vertical, i.e., substantially perpendicular to a plane of the base 2406.One or more of the sidewalls 2417 may contain an electrode 2416 that may be electrically isolated from the base 2406.The flap 2411, flexures 2414, and sidewalls 2417 may be positioned so that a bottom portion of the flap 2411 contacts one of the sidewalls 2417 when the flap 2411 is in the second angular orientation such that the flap 2411 may assume an orientation substantially parallel to that of the sidewall 2417.A voltage applied between the electrode and the flap may attract the flap to the sidewall to secure the flap in place.", "Preferably, the flap 2411 is attracted to the electrode 2416 such that such that the flap 2411 may assume the angular orientation of the sidewall 2417.In an alternative configuration, clamping surface, such as a top chip, may be bonded to the base to provide a reference stopping plane and electrode for retaining the flap 2411.The flap 2411 contains a magnetically active element 2440 to facilitate movement of the flap by interaction with an externally applied magnetic field.", "The magnetically active element 2440 may be a magnetically active material having, e.g.", "a fixed magnetic moment, i.e., it may be a permanent magnet.", "Magnetically active materials may include Nickel, Nickel-Iron, Iron-Cobalt, Aluminum-Nickel-Cobalt, Neodymium-Iron-Boron, etc., and, may be deposited in a uniform or stepped pattern.", "The magnetically active element 2440 may optionally include one or more coils 2420.The coils 2420 may interact with an externally applied magnetic field B produced by a magnet assembly 2450 of the type described above with respect to FIGS.", "1A-3.The magnetic field B has a z-component Bz and an x-component Bx that is directed substantially perpendicular to the z-component Bz.", "The magnetic field B interacts with the magnetic material 2440 and/or coils 2420 in a way that causes a flap 2411 to move from one angular position to another with respect to a base 2406.In a particular configuration, the coil 2420 may interact with a magnetic material deposited in close proximity to the flap 2411′.", "The magnetic material e.g., Nickel, may be applied through suitable techniques such as sputtering or electroplating.", "In configurations where the flap 2411 includes a coil 2420, the polarity of current that runs through the coil 2420 may be reversed to apply an opposite force to the flap 2411.Two or more optical elements of the type depicted in FIG.", "24 may be arranged in an N×N array to form an N×N 2-dimensional optical switch.", "According to an embodiment of the invention, an optical switch that may utilize a magnet assembly, e.g., of the type described below with respect to FIGS.", "27A-29.A schematic diagram of such an apparatus 2500 is shown in FIG.", "25.The apparatus 2500 generally includes an optical switch 2501 and a magnet assembly 2509.The optical switch 2501 includes an N×N array of magnetically actuatable MEMS optical elements 2504 that are moveably coupled to a substrate 2502.The moveable elements 2504 may have features in common with the optical element 400 described above with respect to FIG.", "4.The optical elements 2504 may be actuated by a magnetic field B having an x-component Bx and a z-component Bz.", "The optical elements 2504 selectively couple optical signals between a first set of optical fibers 2506 and a second set of optical fibers 2508.The magnet assembly 2509 produces the magnetic field B.", "The magnet assembly 2509 generally includes first and second z-coil assemblies 2510, 2530, and first and second x-coil assemblies 2520, 2540.The first and second x-coil assemblies may have features in common with the x-coil assemblies described below with respect to FIGS.", "28A-28F and 29.The first and second z-coil assemblies 2510, 2530 may have features in common with the z-coil assemblies described below with respect to FIGS.", "27A-27D.", "In particular, the z-coil assemblies 2510, 2530 may respectively include flat spiral z-coils 2512, 2532 and magnetically permeable yoke plates 2514, 2534.A magnetically permeable rib 2550 may be connected between the yoke plates 2514, 2534 such that the rib 2550 and yoke plates 2514, 2534 form a cantilevered “C”-shape.", "Spacers 2555, 2557 may be coupled at the open end of the “C”-shape to provide structural rigidity.", "The spacers 2555, 2557 may be made of a non-magnetically permeable material, e.g.", "stainless steel or aluminum.", "The spacers 2555, 2557 may be sized and positioned such that the end and sides of the “C”-shape remain substantially open.", "The open end and sides of the “C”-shape provide access for the optical fibers 2506, 2508.Additional access at the back of the “C”-shape may be provided by replacing the rib 2550 with two magnetically permeable spacers with a gap in between them when assembled.", "In the apparatus 2500 the optical elements 2504 that are oriented at approximately 45° with respect to an edge of 2505 the substrate 2501.The fibers 2506, 2508 are oriented a approximately 45° to the moveable optical elements 2504, e.g.", "substantially perpendicular to the edge of edges of the substrate 2501.Other configurations of the fibers and movable element are possible without departing from the scope of the present invention.", "For example, an apparatus 2600, which is a variation on the apparatus 2500, is depicted in FIG.", "26.The apparatus 2600 generally includes an optical switch 2601 and a magnet assembly 2609.The optical switch 2601 includes an N×N array of magnetically actuatable MEMS optical elements 2604 that are moveably coupled to a substrate 2602.The optical elements 2604 may have features in common with the optical element 400 described above with respect to FIG.", "4.The optical elements 2604 are oriented approximately parallel to an edge of the substrate 2602.The fibers 2606, 2608 are oriented a approximately 45° to the moveable optical elements 2604, e.g.", "at substantially 45° with respect to edges 2605A, 2605B of the substrate 2602.The optical elements 2604 may be actuated by a magnetic field B provided by the magnet assembly 2609.The magnetic field B may have an x-component Bx and a z-component Bz.", "The magnet assembly 2609 may generally include first and second z-coil assemblies 2610, 2630, and first and second x-coil assemblies 2620, 2640.The first and second x-coil assemblies may have features in common with the x-coil assemblies described below with respect to FIGS.", "28A-28F and 29.The first and second z-coil assemblies 2610, 2630 may have features in common with the z-coil assemblies described below with respect to FIGS.", "27A-27D.", "In particular, the z-coil assemblies 2610, 2630 may respectively include flat spiral z-coils 2612, 2632 and magnetically permeable yoke plates 2614, 2634.The optical switch 2601 may be disposed within a gap 2603 between the first and second x-coil assemblies 2620, 2640.A magnetically permeable rib in the form of a post 2650 may be connected between the yoke plates 2614, 2634 such that the post 2650 is disposed approximately midway along an edge of the yoke plates 2614, 2634.A non-magnetically permeable spacer, e.g.", "in the form of a post 2655, may be coupled between the two yoke plates 2614, 2634.to provide structural rigidity.", "This particular construction leaves the corners of the magnet assembly 2609 substantially free of obstruction in order to accommodate the optical fibers 2606, 2608.a.", "Magnet Designs A novel magnet assembly may be utilized with magnetically actuated MEMS devices that incorporate equipotential landing pads.", "The novel magnet assembly typically includes at least one z-coil assembly and at least one x-coil assembly attached to the at least one z-coil assembly.", "The at least one z-coil assembly includes a flat spiral z-coil and a magnetically permeable yoke plate.", "A combined thickness of the at least one z-coil assembly and the at least one x-coil assembly is approximately 0.1″ or less.", "The at least one z-coil assembly is configured to produce a magnetic field of about 100 Gauss or greater when driven by a voltage of about 5 volts or less.", "The magnet assembly may be incorporated into an optical switching apparatus having an array of magnetically actuatable optical switching elements is disposed proximate the magnet assembly.", "An example of a magnet assembly 2700 that may be used with embodiments of the present invention is depicted in FIGS.", "27A-27D.", "The magnet assembly 2700 generally includes top and bottom z-coil assemblies 2710, 2730, top and bottom x-coil assemblies 2720, 2740, and a magnetically permeable rib 2750.The rib 2750 spaces apart the upper and lower z-coil assemblies 2710, 2730 and provides a path for magnetic flux between the two z-coil assemblies 2710, 2730.A MEMS optical switch 2790 may be disposed in the gap 2701.A magnetic field B is present in a gap 2701 between the upper and lower x-coil assemblies 2720, 2740 when current flows through the x-coil assemblies 2720, 2740, z-coil assemblies 2710,2730 or both.", "The magnetic field may actuate one or more moveable optical elements 2794 of the optical switch 2790.The magnetic field B may have both an x-component and a z-component.", "For the purposes of the present discussion, the x-component may be regarded as being directed substantially parallel to a plane containing either x-coil assembly 2720, 2740 or either z-coil assembly 2710, 2730.The z-component may be regarded as being directed substantially perpendicular to a plane containing either x-coil assembly 2720, 2740 or either z-coil assembly 2710, 2730.The upper z-coil assembly 2710 includes a first z-coil 2712 and a yoke plate 2714.The yoke plate 2714 may be attached to the rib 2750 by any suitable means, such as one or more screws 2752.To facilitate manufacturability, the upper and lower z-coil assemblies 2710, 2730 may have similar construction.", "More specifically, the lower z-coil assembly includes a z-coil 2732 and a yoke plate 2734.The yoke plate 2734 may be attached to the rib 2750 by any suitable means, such as one or more screws 2752.Together, the z-coils 2712, 2732, yoke plates 2714, 2734 and the rib 2750 form a magnetic circuit that concentrates magnetic flux within a gap between the upper and lower coil assemblies.", "The magnetic flux provides a first magnetic field component Bz that is directed substantially perpendicular to the plane of the z-coils 2712, 2732.The first and second z-coils 2712, 2732 may be flat pancake type coils having multiple windings disposed substantially within the same plane.", "By way of example, the first and second z-coils 2712, 2732 may be aligned such that they are substantially coaxial.", "Flat coils can have a low inductance, which is often desirable when the current through them is AC.", "A low inductance means a low inductive reactance, which means that for a sufficiently low AC frequency, the power dissipated by the coil is mainly dependent on the coil resistance.", "The z-coils 2712, 2732 may be made from a wound flat ribbon.", "In a particular example the z-coils 2712, 2732 may be made from 80 turns of wire approximately 0.03″ in width and 0.003″ in thickness.", "The overall wire thickness may include an insulating material, e.g.", "polyimide approximately 0.00075″ thick.", "Furthermore the turns of the z-coils 2712, 2732 may be held together with an adhesive, to keep them from unraveling after they have been wound.", "The z-coils 2712, 2732 may respectively include inner and outer leads 2711A, 2711B, 2731A, 2731B that provide electrical connections to the ends of the wire at the inside and outside of the z-coils 2712, 2732.The inner lead 2711A of the lower z-coil 2732 may be connected in series to the outer lead 2731B of the upper z-coil 2712 so that the same current flows through both coils.", "The z-coils 2712, 2732 are preferably oriented such that the magnetic fluxes from upper and lower coils tend to reinforce each other in the gap between the two z-coil assemblies 2710, 2730.Alternatively, the upper and lower z-coils 2712, 2732 may be connected in parallel so that a lower voltage may be applied to them.", "The inner and outer leads 2711A, 2711B, 2731A, 2731B may be respectively insulated from the z-coils 2712, 2732, e.g., by insulating patches 2719, 2739.The yoke plates 2714, 2734 and the rib 2750 are made of magnetically permeable material, e.g., a Nickel-Iron alloy such as μ-metal or Hipernom Alloy.", "Hipernom is a registered trademark of Carpenter Technology Corporation of Reading, Pa.", "The zcoils 2712, 2732 may be respectively insulated from the yoke plates 2714, 2734, e.g.", "by a polymer sheets 2713, 2733 attached by adhesives 2716A, 2736A.", "The z-coil assemblies 2710, 2730 may be attached to the x-coil assemblies 2720, 2740 by adhesive layers 2716B, 2736B.", "FIG.", "27D provides a detailed isometric view of the lower z-coil assembly 2730.The yoke plate 2734 is made from a magnetically permeable material.", "The yoke plate 2734 includes a core 2735 to guide magnetic flux from the z-coil 2732.The z-coil 2732 is disposed about the core 2735, e.g.", "in a groove 2737 machined into the yoke plate 2734.A sheet adhesive 2736 may retain the z-coil in the groove 2737.An inside radius of the z-coil 2732 closely matches an outside radius of the core 2735 so that the z-coil 2732 closely fits about the core 2735.By closely fitting the z-coil 2732 about the core 2735 the z-coil 2732 may be aligned with respect to the yoke plate 2734.The upper z-coil assembly 2710 may be similarly manufactured so that the upper and lower z-coils 2712, 2732 are aligned with sufficient precision to optimize the magnetic field strength and magnetic field uniformity in the region between the two z-coil assemblies.", "For example, a set of registration holes 2717A, 2717B in the upper yoke plate 2714 may align with corresponding sets of registration holes in the lower yoke plate 2734 as well as registration holes in the two x-coil assemblies 2720, 2740, e.g.", "registration holes 2741A, 2741B shown in FIG.", "27A and 27C and registration hole 2738B shown in FIG.", "27C.", "A magnet assembly of the type depicted in FIGS.", "27A-27D may produce a magnetic field having a z-component Bz of approximately 200 gauss when approximately 1 ampere flows through each of the z-coils 2712, 2732.Such a current may be supplied e.g., by connecting a voltage source of approximately 5 volts to the leads 2711A, 2711B, 2731A, 2731B of each of the z-coils 2712, 2732.The z-component Bz may be uniform to with about ±3% of a nominal value.", "In this particular example, the gap 2701 had a thickness of about 0.35″, the z-coils 2712, 2732 each had an inner radius of about 0.43″.", "Furthermore each z-coil was made of about 80 turns of copper ribbon wire about 0.030″ wide by 0.003″ thick (including an insulation thickness of 0.00075″).", "The upper and lower x-coil assemblies 2720, 2740 provide a second magnetic field component Bx that is substantially orthogonal to the magnetic field component Bz produced by the z-coil assemblies 2710, 2730.In the example illustrated in FIGS.", "27A-27D, the x-coil assemblies 2720, 2740 may be fabricated as flex circuits having a layered construction.", "By using flex circuit construction, the x-coil assemblies may be made very thin.", "FIG.", "2A schematically illustrates an example of layered construction in the upper x-coil assembly 2720.The lower x-coil assembly 2740 may be similarly constructed.", "In the example depicted in FIG.", "28A, the x-coil assembly 2720 includes a conductive base layer 2820, two conductive coil layers 2840, 2860, a top coverlay 2810, first and second innerlays 2830, 2850 and a bottom coverlay 2870.The conductive base layer 2820 includes cross connections and blind vias for connecting the coil layers 2840, 2860.An example of a plan view of the base layer 2820 is depicted in FIG.", "28B.", "The coverlays 2810, 2870 and innerlays 2830, 2850 may include registration holes that align with each other and with the registration holes 2717A, 2717B, 2738B in the yoke plates 2714, 2734 and/or with the registration holes 2741A, 2741B in the lower x-coil assembly.", "Examples of such registration holes include registration holes 2721A, 2721B depicted in FIG.", "28B and registration holes 2725A, 2725B depicted in FIG.", "28F.", "The first and second coil layers 2840, 2860 each include first and second flat spiral coil sublayers 2842, 2846, 2862, 2866 respectively as shown in FIGS.", "28C-28F.", "Insulating layers 2844, 2864 are respectively disposed between spiral coil sublayers 2842, 2846 and 2862, 2866.The insulating layers 2844, 2864 may be made of a polymer such as polyimide.", "The insulating layers 2844, 2864 may be thin, e.g., about 1 mil (0.001″) or less in thickness.", "The spiral coil sublayers may be stacked together in any suitable fashion.", "By way of example, the base layer 2820 may be disposed between the top coverlay 2810 and the first innerlay 2830.The first spiral coil sublayer 2840 may be disposed between the first and second innerlays 2830, 2850.The second spiral coil sublayer 2860 may be disposed between the second innerlay and the bottom coverlay 2870.The innerlays 2830, 2850 may respectively include insulating layers 2834, 2854 that are each disposed between corresponding adhesive layers 2832, 2836, 2852, 2856.The coverlays 2810, 2870 may each respectively include an insulating layer 2814, 2874 and an adhesive layer 2812, 2872.The insulating layers 2814, 2834, 2854, 2874 may be made from a polymer material, e.g., polyimide.", "The insulating layers may be about 1 mil (0.001″) or less in thickness.", "The adhesive layers 2812, 2832, 2836, 2852, 2856, 2872 facilitate attachment of the layers of the coil assembly 28720.The spiral coil; sublayers 2842, 2846, 2862, 2866 each respectively include first and second oppositely wound flat spiral x-coils 2843A, 2843B, 2845A, 2845B, 2863A, 2863B, 2865A, 2865B.", "The first and second spiral coils in each sublayer are formed e.g., by etching a pattern in a metallic layer, e.g.", "copper foil.", "The first and second spiral coils are electrically connected to each other in series such that the same electric current may flow through both coils.", "The coil sublayers may be connected to each other in series or in parallel through vias 2821-2828 in the base layer.", "By way of example, vias 2821, 2822 in the base layer 2820 are connected to each other.", "Via 2821 connects to spiral coil 2843A at a via 2841A in sublayer 2842.Via 2822 connects to spiral coil 2863A at a via 2861A in sublayer 2862.Spiral coil 2843B connects in series to spiral coil 2845A through via 2841B in sublayer 2842, vias 2823 and 2824 in the base layer 2820 and via 2847 in sublayer 2846.In a like fashion, spiral coil 2863B connects in series to spiral coil 2865A through via 2861B in sublayer 2862, vias 2825 and 2826 in the base layer 2820, and via 2867 in sublayer 2866.Via 2849 in sublayer 2846 and via 2869 in sublayer 2866 are respectively connected to vias 2827 and 2828 in base layer 2820.Vias 2827 and 2828 may be connected together so that sublayers 2842, 2846 are connected in parallel with sublayers 2862, 2866.The foregoing connections are set forth for the purpose of example and are not intended to limit the scope of the invention.", "Other connections between the sublayers 2842, 2846, 2862, 2866, e.g.", "all four sublayers connected in series or all four sublayers connected in parallel, may be implemented without departing from the scope of the present invention.", "The spiral x-coils are all disposed substantially parallel to the same plane.", "The opposite windings of the spiral coils in each layer produces a magnetic flux that is partially directed parallel to the plane of the coils.", "Although part of the flux generated by the upper and lower x-coil assemblies 2720, 2740 may be directed through the yoke plates 2714, 2734 the flux that passes between the two yoke plates 2714, 2734 may be used to produce the x-component Bx of the magnetic field.", "An alternative configuration of an x-coil assembly 2900 that may be used with apparatus of FIGS.", "27A-27D is depicted in FIG.", "29.In the alternative configuration, an upper x-coil assembly 2901 plurality of windings 2910 of conductive wire are wrapped around a thin rectangular yoke plate 2920.The yoke plate may be made from a magnetically permeable material to guide the magnetic flux produced by a current flowing through the windings.", "The windings 2910 may be wound in multiple layers to increase the number of ampere turns surrounding the yoke plate 2920.The x-coil assembly 2900 may include a similarly constructed lower x-coil assembly 2902 having windings 2930 and a yoke plate 2940.A current flowing in the lower set of windings 2930 is directed such that the flux through the lower yoke plate 2940 is oppositely directed to the flux through the upper yoke plate 2920.The flux in a gap 2904 between the upper and lower x-coil assemblies produces a magnetic field BX that is directed substantially parallel to the yoke plates 2920, 2940.The magnet assembly described above may be used in conjunction with a magnetically actuatable microelectromechanical systems (MEMS) device.", "In a particular example, a magnet assembly of the type described above may be used with a MEMS optical switch.", "2.Pneumatic Actuation Although magnetic actuation is quite common in MEMS applications alternative actuation schemes may also be used in conjunction with the equipotential landing pads described above with respect to FIGS.", "2A-2B.", "One possible alternative schemes involves pneumatic actuation.", "There are many possible ways of implementing pneumatic actuation.", "For example, FIG.", "30 depicts an embodiment of a MEMS device 3000 with pneumatic actuation and common gas pulse from the backside of a substrate.", "The device generally comprises a substrate 3004 with one or more movable elements 3002, such as mirrors, mounted for rotation with respect to the substrate 3004.Alternatively, the movable elements 3002 may translate, e.g.", "vertically or horizontally.", "Gas (preferably nitrogen, although other inert gases will also work) can be supplied from a source 3006 to a device package 3007 and directed to a chamber 3008 under the movable elements 3002 through holes 3010 in the backside of the substrate 3004.In this embodiment a micro valve 3012 and gas regulator 3014 connected between the gas source 3006 and the chamber 3008 control gas pulse duration and flow.", "A filter may optionally be included to remove particles from the gas.", "Different layouts of the movable elements may require a different direction of gas flow.", "For example FIG.", "31 depicts an embodiment of a MEMS device 3100 in which a gas source 3106, microvalve 3112 and regulator 3114 feed a gas pulse to a package 3107 located above a substrate 3104 containing moveable elements 3102.The gas may then be exhausted into an exhaust chamber 3108 through holes 3110 located proximate each movable element 3102.In the embodiments depicted in FIGS.", "30-31 the gas pulse rotates all the mirrors at the same time.", "Individual mirrors may be held in place in an “on” position using conventional electrostatic clamping.", "The movable elements may further include torsional flexures that rotate the elements back to an “off” position in the absence of an actuating force, such as the gas pulse.", "In this fashion individual movable elements may be switched from the “on” position to the “off” position using a combination pneumatic actuation and electrostatic clamping.", "In many MEMS applications it is desirable to actuate only selected movable elements in an array without actuating others.", "Several embodiments of the present invention may be implemented to achieve this.", "For example, FIG.", "32 depicts a MEMS device 3200 that uses multiple electro-pneumatic control valves 3212 to allow separate actuation of each of several movable elements 3202 (or rows or columns of such elements) moveably coupled to a substrate 3204.Thus each element 3202 may be moved only when it needs to be switched between two fixed positions.", "This reduces number of actuations for each movable element 3202 and leads to longer lifetime of the device 3200.The control valves 3212 may be coupled to a manifold 3213 that communicates with a gas source 3206.A device package 3207 may be attached to the substrate to enclose the moveable elements 3202.Each control valve 3212 may be coupled to a corresponding hole 3210 in the substrate 3204 through a dedicated channel 3215.Alternatively, as shown in FIG.", "33, a MEMS device 3300 may include individual arrayable MEMS pneumatic control valves 3314 may be used to feed gas from a source 3306 to each of several movable elements 3302 moveably coupled to a substrate 3304.A device package 3307 may be attached to the substrate to enclose the moveable elements 3302.Arrays of such valves are described in detail, for example, in “Batch Fabrication of Pneumatic Valve Arrays by Combining MEMS with Printed Circuit Board Technology,” Patrick Cheung, Andrew Berlin, David Biegelsen, Warren B. Jackson, DSC-Vol 62/HTD-Vol 354, Microelectromechanical Systems (MEMS) ASME 1997.Such valves may operate in a 1-20 ms range.", "Alternatively, as shown in FIG.", "34, a MEMS device 3400 may include one or more movable elements 3402, moveably coupled to a substrate 3404 that may be actuated by an array of Knudsen Compressor devices 3416.A device package 3407 may be attached to the substrate to enclose the moveable elements 3402.The operation of Knudsen compressors is based on thermal transpiration.", "In a typical Knudsen compressor two volumes of gas are separated by a thin membrane having many holes.", "Each of the holes is characterized by dimensions that are small compared to the mean free paths of the gas.", "If the two volumes are maintained at temperatures T1 and T2, but are otherwise undisturbed, the equilibrium pressures p1 and p2 of the two volumes are related by p1/p2=(T1/T2)1/2.Each Knudsen compressor 3416 in the array can be aligned and attached to the backside of the substrate 3404 proximate a corresponding movable element 3402 to provide a gas pulse on demand to actuate each movable element 3402 individually.", "A device package 3407 may be attached to the substrate to enclose the moveable elements 3402.MEMS type Knudsen compressors are described in detail, for example, in “The Knudsen Compressor as a Micro and Macroscale Vacuum Pump Without Moving Parts or Fluids,” S. E. Vargo, E. P. Muntz and G. R. Shiflett, W. C. Tang.", "Instead of cylinder gas supply one can alternatively use a micro pump (compressor), which generates a positive pressure.", "FIG.", "35 depicts an example of a MEMS device 3500 employing a micropump 3518 coupled to a chamber beneath a substrate 3504.Moveable elements 3502 are moveably coupled to the substrate 3504.Holes 3510 disposed proximate the moveable elements 3502.A device package 3507 may be attached to the substrate to enclose the moveable elements 3502.The micropump 3518 actuates the moveable elements 3502, e.g., by providing a gas pulse to the holes 3510 via a chamber 3508 disposed below the substrate 3504.A microvalve 3514 may be coupled between chamber and the micropump 3518 to control the flow of gas.", "Examples of suitable micro pumps include “AAA” series micro-air pump of Sensidyne, Inc., of Clearwater, Fla. (6 psi, 98% air filtration), or the NMP05 rnicro-diaphragm pump and compressor of KNF Neuberger, Inc of Trenton, N.J. (6 psi, 20 gr.", "Weight, 30×20×17 mm3 volume).", "Examples of suitable micro valves 3514, include control valves of the Lee Company of Westbrook Conn., (2.5 ms response time, 12 mm dia×30 mm, power consumption—780 mW).", "Any of the embodiments of pneumatic actuation means depicted in FIGS.", "30-35 may be incorporated into a MEMS optical switch, such as an NXN crossbar switch of the type shown above.", "Such a switch typically includes a substrate and a plurality of rotatable mirrors, mounted for rotation with respect to the substrate.", "Advantages of such a MEMS optical switch with pneumatic actuation over similar switches with magnetic actuation are as follows: 1.The moveable elements (mirrors) do not require a magnetic pad for actuation.", "The manufacturing is therefore simpler due to elimination of the electroplating process used to deposit the magnetic pads.", "2.The overall weight of the switch is reduced due to elimination of outside electromagnet.", "3.The overall power consumption of the switch is reduced due to elimination of electromagnets normally used for magnetic actuation.", "4.The size of the mirror elements may be made smaller and the scalability of the switch is enhanced since more elements may be incorporated onto the same footprint of the MEMS device due to elimination of the magnet pads.", "5.Eliminating the heavy magnetic pads enhances the reliability of the switch due to reduced overall weight of the movable parts suspended on the hinges.", "6.The absence of magnetic materials on a mirror makes the optical switch insensitive to external electromagnetic fields.", "7.Using nitrogen gas feed for mirror actuation improves reliability of the switch by eliminating external moisture penetration into the package, which can lead to stiction problems.", "3.Acoustic Pulse Actuation Another class of possible alternative schemes that may be used with equipotential landing pads involves acoustic pulse actuation.", "In such application schemes energy in the form of an acoustic pulse actuates a moveable portion of a MEMS device.", "There are many possible ways of implementing acoustic pulse actuation.", "For example FIG.", "36 depicts an embodiment of a MEMS device 3600 with acoustic pulse actuation and from a backside of a substrate.", "The device generally comprises a substrate 3602 with one or more moveable elements 3604, such as mirrors, mounted for rotation with respect to the substrate 3602 between a horizontal position and a vertical position.", "The device 3600 may include clamping mechanisms, such as electrostatic clamping electrodes, to selectively retain each moveable element 3604 in the vertical or horizontal position.", "Each moveable element 3604 may be mounted to the substrate 3602 via one or more flexures that provide a torsional force that biases the moveable element 3604 to return to the horizontal position in the absence of an actuating force or clamping force.", "Alternatively, the movable elements 3604 may translate, e.g.", "vertically or horizontally.", "A package 3606 that covers the movable elements 3604 contains a gas (preferably nitrogen, although other inert gases will also work).", "Gas also fills a chamber 3608 under the movable elements 3604.Of course, the relative positions of the chamber 3608 and package 3606 may be reversed.", "The package 3606 and chamber 3608 are connected through holes 3610 in the backside of the substrate 3602 proximate the movable elements.", "An electromagnetic 3612 is coupled to the chamber 3608 to provide acoustic pulse actuation.", "In this embodiment the chamber 3608 includes a membrane 3609 that divides the chamber into two part 3611, 3613.A first part 3611 communicates with the package via the holes 3610.A second part 3613 is proximate to the electromagnet 3612.Each part of the chamber 3608 may be filled with the same medium, e.g.", "the same gas or liquid.", "Alternatively, the two parts 3611, 3613 may be filled with different media, e.g.", "different gases, different liquids, gas in one part liquid in the other part, or the first part 3611 may be filled with gas or liquid and the second part 3613 may be evacuated.", "A pulse generator 3614 coupled to the electromagnet 3612 provides an electromagnetic pulse.", "Preferably, the membrane 3609 is made of magnetic material in order to be able to interact with electromagnetic force produced by the pulsed magnetic field.", "The pulse of a magnetic field deforms the membrane 3609, which creates acoustic pulse (medium pressure gradient) in the first part 3611 of the chamber 3608.This acoustic pulse propagates through the gas or liquid and actuates the movable elements 3604, e.g., by turning one or more of the moveable elements 3604 90 degrees around a hinge axis.", "The magnitude of a given moveable element's angular movement depends on the maximal deformation of membrane 3609, which controls local gas or liquid pressure gradient.", "The required amount of deformation can be obtained by properly choosing the elastic properties of the material of the membrane 3609, the membrane's geometry and size, and the strength of the electromagnetic pulse.", "The magnitude of angular movement depends also on the moveable element's hinge stiffness and mass as well as the viscosity of the media in the chamber 3608.The pulse of magnetic field may be otherwise inductively coupled to the membrane 3609, which delivers an acoustic pulse to the first part 3611 of the chamber 3608.In such case the membrane 3609 may be dielectric, but could contain a coil, with electric current flowing through it, for interaction with the electromagnetic induction force.", "Such a coil can be deposited and patterned using photolithographic techniques.", "Since to the membrane 3609 need not oscillate, but just create a single deformation from the rest state, a short DC pulse (no frequency requirements).", "It is desirable to make the length of the pulse as short as possible to achieve the desired power or a given amount of membrane deflection.", "The acoustic pulse is transmitted to the movable elements 3604 though the holes 3610 and drives one or more of the movable elements 3604, e.g.", "causing it to rotate from a horizontal position towards a vertical position.", "Selected ones of the movable elements 3604 may then be clamped in the vertical position by electrostatic clamping.", "In a similar fashion, specific movable elements 3604 may be prevented from rotating, e.g.", "by electrostatically clamping them, e.g., against the substrate 3602, in the horizontal position.", "Other means for acoustic pulse actuation may be used in alternative embodiments of the present invention.", "For example, a piezoelectric transducer may be used place of the electromagnet and membrane of FIG.", "36.Furthermore, a miniature piezoelectric transducer may be located proximate each of the holes to provide individual acoustic pulse actuation of each of the movable elements.", "In an alternative embodiment, depicted in FIG.", "37, the sound pulse may be delivered to the movable elements 3604 through a liquid medium 3701.Such a liquid medium is preferably transparent to sound waves in the wavelength range suitable for actuation of the movable elements.", "Since embodiments of the device of the present invention operate with the single pulse of pressure (acoustic pulse), rather than a continuous acoustic wave, the acoustic transparency of the medium is immaterial, as long as the medium will transfer the energy.", "Other parameters, such as the speed of pulse propagation through medium and decay of energy, will differ from one material to another.", "From this point of view, liquids are better than gases.", "Liquid mediums will typically give shorter response time for the switch than gases.", "For optical switch applications, it is desirable that the medium in the package 3606, whether liquid or gas, be optically transparent to the wavelength of light for the optical switch operation, for example 1.3-1.5 micron.", "Furthermore, it is desirable for the liquid medium 3701 to have a low viscosity.", "The viscosity of the liquid medium 3701 should be as low as possible.", "Suitable liquids include water and low viscosity oils will work if the electromagnet pulse is strong enough.", "Any of the embodiments of pneumatic actuation means depicted in FIGS.", "36-37 may be incorporated into a MEMS optical switch, such as an NXN crossbar switch of the type shown in FIG.", "1.Such a switch typically includes a substrate and a plurality of rotatable mirrors, mounted for rotation with respect to the substrate.", "Advantages of such a MEMS optical switch with pneumatic actuation over similar switches with magnetic actuation are as follows: 1.The elements (mirrors) do not require a magnetic pad for actuation.", "The manufacturing is therefore simpler due to elimination of the electroplating process used to deposit the magnetic pads.", "2.The size of the mirror elements may be made smaller and the scalability of the switch is enhanced since more elements may be incorporated onto the same footprint of the MEMS device due to elimination of the magnet pads.", "3.Eliminating the heavy magnetic pads enhances the reliability of the switch due to reduced overall weight of the movable parts suspended on the hinges.", "4.Absence of magnetic materials on a mirror makes optical switch insensitive to external electromagnetic fields.", "5.Using acoustic pulse actuation in an inert gas environment improves reliability of the switch by eliminating external moisture penetration into the package, which can lead to stiction problems.", "6.Using liquid environment eliminates stiction problems and improves the reliability of the switch.", "In accordance with the foregoing, low-cost, high yield scalable MEMS devices and switches may be provided without the disadvantages attendant to magnetic actuation.", "G. State Sensing It is often desirable to allow for detection of whether a rotatable MEMS element is in a first or second position, , e.g., horizontal or vertical, and whether it is properly clamped in either of these two positions.", "This sensing capability is useful, for example, in fault detection.", "By sensing the mirror position, mirror failure can be immediately detected, and traffic through the switch can be appropriately re-routed.", "Such state sensing may be incorporated into MEMS devices that utilize equipotential landing pads.", "1.Capacitive State Sensing One of many possible schemes for state sensing involves sensing a capacitance, e.g., between a MEMS moveable element and a corresponding substrate, or a change in such a capacitance.", "FIGS.", "38A-38B depict one example of an apparatus 3800 the implements capacitive state sensing according to an embodiment of the invention.", "Such an apparatus may be used in conjunction with MEMS devices that are equipped with equipotential landing pads.", "The apparatus generally comprises a rotatable element 3802, and first and second electrodes 3804, 3806.The first electrode 3804 is typically located adjacent to the element 3802 when element 3802 is in its vertical position.", "The second electrode 3806 is typically located adjacent to the element 3802 when element 3802 is in its horizontal position.", "For the purpose of example, and without loss of generality, the rotatable element 3802 may be a MEMS mirror that rotates about a substantially horizontal axis 3801 relative to a static part 3803.The rotatable element may include a separate electrode for clamping or capacitance sensing.", "Alternatively, if the rotatable element 3802 is electrically conductive, the element 3802 itself may be regarded as an electrode.", "In the embodiment depicted in FIGS.", "38A-38B the rotatable element 3802 rotates between two positions that are substantially 90° apart.", "In particular, the rotatable element rotates between a vertical position, as shown in FIG.", "38A, and a horizontal position, as shown in FIG.", "38B.", "The vertical position defines a first or “on” control state.", "The horizontal position defines a second or “off” control state.", "In the embodiments of the present invention the capacitance between the rotatable element 3802 and the electrodes 3804,3806 depends on whether the rotatable element is in the first or second position.", "The first electrode 3804 can be placed so that it is disposed close to and substantially parallel with the rotatable element 3802 in the vertical position.", "The capacitance between the rotatable element 3802 and the first electrode 3806 can be monitored to determine the control state of the rotatable element 3802.For example when the rotatable element 3802 is flipped to the vertical position from the horizontal position, the capacitance between the element 3802 and the first electrode 3804 changes from a low value to a much higher value.", "At the same time, the capacitance between the rotatable element 3802 and the second electrode 3806 changes from a high value to a lower value.", "In a similar fashion, the capacitance between the second electrode 3806 and the rotatable element 3802 can be used to detect the control state of the when it is in the horizontal position.", "The magnitude of the “on”-state capacitance is known, and if the element 3802 is somehow improperly positioned in the “on” state, the capacitance may not reach the known value, and a fault may be indicated.", "In the device 3800, the electrodes 3804, 3806 may also serve as clamping electrodes as well as for capacitive control state sensing.", "Alternatively, the device 3800 may include separate electrodes for sensing and clamping.", "In the case of an array of rotatable elements, e.g., MEMS mirrors, the electrodes for the “off”, or horizontal, state detection may be electrically shorted to each other.", "In such a case, the static part 3803 may comprise a substrate to which the mirrors are mounted.", "Similarly, the “on”, or vertical, state electrodes may comprise a single component with features that define a vertical electrode for each mirror.", "FIG.", "39 depicts a simplified cross-sectional schematic diagram of an apparatus 3900 according to an embodiment of the present invention.", "The apparatus generally comprises a MEMS device 3910, and a device controller 3920.The device 3910 typically includes a substrate 3911 and a rotatable element 3912, such as a mirror.", "The substrate 3911 includes a vertical stop 3915 and a horizontal stop 3917.The rotatable element 3912 rotates about an axis oriented substantially parallel to a plane of the substrate 3911.The rotatable element 3912 may be attached to the substrate 3911 by a torsional flexure 3913.The rotatable element 3912 rotates, e.g.", "under magnetic actuation, between a vertical position proximate the vertical stop 3915 and a horizontal position proximate the horizontal stop 3917.The substrate 3911 further includes vertical and horizontal electrodes 3914, 3916 proximate the vertical and horizontal stops 3915, 3917.The electrodes 3914, 3916 are typically electrically isolated from each other and from the rotatable element 3912.The controller 3920 typically includes a processor 3921, a fault detector 3922, a state selector 3923, vertical and horizontal capacitance sensors 3924, 3926 and vertical and horizontal power voltage sources 3927, 3929.The state selector 3923 and fault detector 3922 are coupled to the processor 3921.The capacitance sensors 3924, 3926, are coupled to the electrodes 3914, 3916 respectively and to the fault detector 3922.Conditioning electronics 3925V, 3925H, such as amplifiers or analog to digital (A/D) converters, may optionally be coupled between the capacitance sensors 3924, 3926 and the fault detector 3922.In the embodiment shown in FIG.", "39, the voltage sources 3927, 3929 are coupled to the electrodes 3914, 3916 respectively.", "The voltage sources 3927, 3929 supply clamping voltages to the electrodes 3914, 3916 to clamp the rotatable element to the vertical stop 3915 or the horizontal stop 3917.Alternatively, the device 3910 may include separate clamping electrodes coupled to the voltage sources 3927, 3929.For example, it is often the case that the capacitance sensors are coupled to conditioning electronics that interpret the signals from the capacitance sensors.", "Such conditioning electronics may include amplifiers, analog-to-digital converters, and the like.", "It is often desirable to ensure that the conditioning electronics receive signals from the sensors having an acceptable level of noise.", "The acceptable value of the noise level depends on the circuit and the required precision in the specific application.", "For very small capacitance signals, e.g., of order 10−15 farads, this may affect the design of the apparatus.", "For example, to reduce the signal to noise ratio, it may be important that the conditioning electronics be located in close proximity to the capacitive sensors.", "A short distance between the sensors and the electronics reduces the amount of wiring between them, thereby reducing noise.", "Close proximity between the sensor and the electronics may be ensured by placing the conditioning electronics in the same packaging as the sensor, e.g., on a die adjacent to a die containing a MEMS device with the sensors.", "The sensors and conditioning electronics may be connected by wire bonding across the die.", "Alternatively, the conditioning electronics may be integrated into the same die as the MEMS die itself.", "Although only a single device 3910 with a rotatable element 3912 is shown in FIG.", "39, those of skill in the art will recognize that the device 3910 may include an array containing any number of such devices.", "Furthermore, the inventive concepts described herein may also be applied to micromirror architectures such as those described in H. Toshiyoshi and H. Fujita, “Electrostatic micro torsion mirrors for an optical switch matrix,” J. Microelectromech.", "Syst., vol.", "5, no.", "4, 231-7, Dec. 1996 and E. L. Goldstein, and R. W. Tkach, “Free-space micromachined optical switches with sub-millisecond switching time for large-scale optical crossconnects,” OFC'98 and IEEE Photonics Technol.", "Lett., April 1998, both of which are incorporated herein by reference.", "The relationship between the position of the rotatable element 3912 and the capacitance values measured by the sensors 3914, 3916 is illustrated in FIGS.", "40A-40C.", "When the rotatable element is in the vertical state, as shown on the left in FIG.", "40A, a large capacitance is detected between the rotatable element 3912 and the vertical electrode 3914, and a small capacitance is detected between the rotatable element 3912 and the horizontal electrode 3916 as shown on the left of FIGS.", "40B and 40C.", "This combination of capacitances indicates that the rotatable element 3912 is in an “up” digital control state.", "When the rotatable element 3912 is switching and is in between the vertical position and the horizontal position (or vice versa), as shown in the middle in FIG.", "40A, a small capacitance is detected between the rotatable element 3912 and both the horizontal electrode 3916 and the vertical electrode 3914 as shown in the middle of FIGS.", "40B and 40C.", "When the rotatable element 3912 is in the horizontal position, as shown on the right in FIG.", "40A, a large capacitance is detected between the rotatable element 3912 and the horizontal electrode 3916, and a small capacitance is detected between the rotatable element 3912 and the vertical electrode 3914 as shown on the right of FIGS.", "40B and 40C.", "This combination of capacitances indicates that the rotatable element 3912 is in a “down” digital control state.", "The capacitance can be measured across the same electrical connections that are used to supply the electrostatic clamping voltages from the voltage sources 3927, 3927 and the electrodes 3914, 3916.The processor 3921 determines the appropriate control state for the rotatable element 3912 and supplies a control signal to the state selector 3923 and the fault detector 3922.The state selector 3923 determines which voltage source 3927, 3929 applies a clamping voltage based on a control signal from the processor.", "Those of skill in the art will recognize that the state selector 3923 may be inplemented in either hardware, software or a combination of both.", "Although two voltage sources 3927, 3929, are depicted in FIG.", "39, the control state selector may alternatively be connected to a single voltage source, which is selectively coupled to the electrodes 3914, 3916 by a switch.", "The fault detector 3922 compares the control signal from the processor to a measured control state determined by measurements from the capacitance sensors 3924, 3926.In either the horizontal or vertical position, the sensors 3924, 3926 can detect exact magnitude of the capacitance to indicate improper clamping of the rotatable element 3912.For example, if a particle (e.g.", "a piece of dust) lands on one of the clamping surfaces and causes the mirror to clamp at an improper angle to the vertical sidewall, the capacitance detected between the rotatable element 3912 and the vertical electrode 3914 will be different than that normally detected in the vertical control state.", "In such a situation the fault detector 3922 would signal a fault to the processor 3921.Those of skill in the art will recognize that the fault detector 3922 may be implemented in either hardware, software or a combination of both.", "The apparatus 3900 may operate according to a method according to an embodiment of the present invention.", "The method 4100 is set forth in the flow diagram of FIG.", "41.In the method 4100 begins at step 4102 with the provision of an apparatus with a rotatable element and such as the apparatus 3900.Electrodes, such as the vertical and horizontal electrodes 3914, 3916 are provided at step 4104.At step 4106 a capacitance between the rotatable element and one or more of the electrodes is measured, e.g.", "with sensors such as the sensors 3924, 3926.In the apparatus 3900, the capacitance sensors 3924, 3926 measure the capacitance between the rotatable element 3912 and the electrodes 3914, 3916 to monitor the control state of the rotatable element 3912.Various methods exist for detecting the capacitance between the rotatable element 3912 and the electrodes 3914, 3916.For example, in step 4106, a small AC signal may be superimposed on top of a DC signal that is supplied by one or more of the voltage sources 3927, 3929 for electrostatic clamping.", "The sensors 3914, 3916 can monitor a current arising from this small AC signal to indicate the capacitance.", "An alternative method employs time-division multiplexing of actuation and sense signals.", "In this scheme, the DC actuation signal is periodically turned off and replaced by a small AC or DC sense signal.", "The sensors 3914, 3916 measure the sense signal to monitor the capacitance.", "Preferably, the time-multiplexing is done at a rate much faster than the natural frequency of the device .", "The capacitance signals from the sensors 3914, 3916 can be used to properly time the electrostatic clamping signals used for clamping the rotatable element 3912 in its two positions.", "For example, when the rotatable element 3912 is actuated up to a position near the vertical stop 3915, the processor signals the state selector to apply a voltage to the vertical electrode 3914 to pull the rotatable element 3912 in to the vertical stop 3915 and clamp it there electrostatically.", "After the rotatable element 3912 is pulled in, the voltage can be reduced to a lower value, since a lower voltage is needed to hold the rotatable element 3912 next to the electrode 3914 than that needed to pull it in.", "Monitoring of the capacitance signal can allow proper timing of these signals.", "That is, the clamp voltage would be lowered only when the capacitance value from the vertical capacitance sensor 3924 indicates that the rotatable element 3912 has reached the vertical position.", "In the descriptions above, it is assumed that the electrodes used for clamping are also used for sensing.", "It is also possible to divide the electrode structures into several isolated regions, in which case one set of electrodes can be used for electrostatic clamping or actuation, and another set for capacitive sensing.", "FIG.", "42 depicts a block diagram depicting an optical communications system 4200 according to an additional embodiment of the invention.", "In the system 4200, a method having features in common with step 4106 of method 4100 of FIG.", "41 is implemented as a computer program code 4205 running on a processor of a computer controlled apparatus having features in common with the apparatus 3900 described above with respect to FIG.", "39.In the embodiment shown, the program code 4205 controls the operation of one or more MEMS mirrors M in a crossbar optical switch S. The switch S may have features in common with the type of switch 100 shown in FIG.", "1.One or more input fibers IF and output fibers OF are coupled to the switch S. Each mirror M is rotatably coupled to a substrate and actuated by electrostatic or magnetic actuators A.", "The mirrors M are clamped in the vertical or horizontal position by voltages applied to clamping electrodes CE.", "The system 4200 includes a controller 4201.The controller 4201 includes a programmable central processing unit (CPU) 4202 that is operable with a memory 4204 (e.g., RAM, DRAM, ROM, and the like) an optional mass storage device, 4206 (e.g., CD-ROM hard disk and/or removable storage), and well-known support circuits 4210 such as power supplies 4212, clocks 4214, cache 4216, input/output (I/O) circuits 4218 and the like.", "All of the above elements may be coupled to a control system bus 4208.The memory 4204 contains instructions that the processor unit 4202 executes to facilitate the performance of the apparatus 4200.The instructions in the memory 4204 are in the form of the program code 4205.The program code may conform to any one of a number of different programming languages.", "For example, the program code can be written in C+, C++, BASIC, Pascal, JAVA or a number of other languages.", "The mass storage device 4206 stores data and instructions and retrieves data and program code instructions from a processor readable storage medium, such as a magnetic disk or magnetic tape.", "For example, the mass storage device 4206 can be a hard disk drive, floppy disk drive, tape drive, or optical disk drive.", "The mass storage device 4206 stores and retrieves the instructions in response to directions that it receives from the processor unit 4202.The processor unit 4202 operates the apparatus 4200 using data and program code instructions that are stored and retrieved by the memory 4204 and/or the mass storage device 4206.The data and program code instructions may be first retrieved by the mass storage device 4206 from a medium and then transferred to the memory 4204 for use by the processor unit 4202.The apparatus 4200 may optionally include a user interface 4220, such as a keyboard, mouse, or light pen, coupled to the processor unit 4202 to provide for the receipt of inputs from an operator (not shown).", "The apparatus 4200 may also optionally include a display unit 4222 to provide information to the operator in the form of graphical displays and/or alphanumeric characters under control of the processor unit 4202.The control system bus 4208 provides for the transfer of data and control signals between all of the devices that are coupled to the control system bus 4208.Although the control system bus 4208 is displayed as a single bus that directly connects the devices in the processor unit 4202, the control system bus 4208 can also be a collection of busses.", "For example, the display unit 4222, user interface 4220 and mass storage device 4206 can be coupled to an input-output peripheral bus 4208, while the processor unit 4202 and memory 4204 are coupled to a local processor bus.", "The local processor bus and input-output peripheral bus are coupled together to form the control system bus 4208.The system controller 4201 is coupled to the elements of the apparatus 4200, for turning off a source of optical power in accordance with embodiments of the present invention via the system bus 4208 and the I/O circuits 4218.These elements include the following: one or more clamping voltage sources CV and capacitance sensors CS coupled to clamping electrodes CE in the switch S, and one or more actuator drivers AD coupled to the actuators A.", "For the sake of clarity, connection is shown to only one of the clamping electrodes CE and one of the actuators A.", "In practice, all the clamping electrodes CE and actuators A could be coupled to the I/O circuits 4218.The system controller 4201 provides signals to the above elements to switch optical signals between the input fibers IF and the output fibers OF.", "The steps of the method of the method described above with respect to FIG.", "41 could be implemented by a suitable computer program running on the CPU 4202 of the controller 4201.The CPU 4202 forms a general purpose computer that becomes a specific purpose computer when executing programs such as the program 4105 of the embodiment of the method of the present invention depicted in the flow diagram of FIG.", "38.Although the invention is described herein as being implemented in software and executed upon a general purpose computer, those skilled in the art will realize that the invention could be implemented using hardware such as an application specific integrated circuit (ASIC), microcontroller or other hardware circuitry.", "As such, it should be understood that the invention can be implemented, in whole or in part, in software, hardware or both.", "Those skilled in the art would be readily able to devise a computer program 4205 to implement step 4106 described above with respect to FIG.", "41.The program 4205 is suitable for monitoring and controlling the switch S in accordance with embodiments of the present invention.", "Although the program 4205 is described herein with respect to a MEMS optical switch, those skilled in the art will recognize that programs embodying the method of the present invention can be applied to any MEMS device.", "2.Magnetic State Sensing An alternative state sensing scheme that may be used in conjunction with equipotential landing pads involves Magnetic state sensing.", "Magnetic sensors may detect changes in a magnetic field by sensing a change in an electrical, mechanical and/or optical property of the sensor that result from changes in the magnetic field.", "The change in the electrical, mechanical and/or optical property may depend upon the strength of the magnetic field or the relative position of the field with respect to the sensor.", "Magnetic sensors include, but are not limited to magnetoresistive sensors, magnetostrictive sensors, Hall-effect sensors, flux sensing coils, magnetostriction sensors and magneto optic sensors.", "Magnetoresistive sensors utilize materials having an electrical resistance that changes in response to a change in a magnetic field.", "Magnetoresistivity in ferromagnetic materials was discovered in 1856 by Lord Kelvin, and has since been used in a variety of magnetic sensors to detect magnetic field strength and direction.", "The change in resistivity is dependent upon the strength of the magnetic field and the relative orientation of the field with respect to a conduction path through the magnetoresistive material.", "The change is usually a minimum when the field is perpendicular to the conduction path and is usually a maximum when the field is parallel to the conduction path.", "As the conduction path of a magnetoresistive sensor changes with respect to an external magnetic field (or vice-versa) the electrical resistance changes.", "The Hall effect is based on the deflection of moving electric charges by a magnetic field.", "In a Hall effect sensor, the electrical property may be a voltage, sometimes referred to as a Hall voltage.", "The Hall voltage is related to the strength of an electric field, referred to herein as the Hall electric field, that results from the interaction of an electric current with a magnetic field.", "The Hall electric field is generally directed perpendicular to both the magnetic field and the direction of flow of the electric current through the Hall effect sensor.", "As the direction of flow of the electric current through the Hall effect sensor changes with respect to an external magnetic field (or vice-versa) the Hall voltage changes.", "Flux sensing coils operate on the principle of electromagnetic induction.", "As the AC magnetic flux through the coil changes a voltage may be induced on the coil.", "The magnetic flux may change due to a change in intensity of an external magnetic field.", "Alternatively, the flux may change due to a change in the relative position of the coil with respect to the magnetic field.", "Flux sensing coils may be characterized by a property known as electrical inductance, which relates the voltage across the coil to the rate of change of electric current through the coil.", "The inductance of a coil may change, e.g., due to a change in proximity of magnetic material with respect to the coil.", "The term magnetostriction refers to the change in the physical dimensions caused by magnetization.", "Magnetostriction sensors utilize this effect to measure field strength.", "Magneto optic sensors utilize materials characterized by optical properties that depend on strength and/or orientation of an applied magnetic field.", "Such optical properties include, but are not limited to, polarizing direction, reflectivity etc.", "For example, in a Kerr or Faraday rotation, the polarization of optical signals is rotated by an amount that depends on the surface magnetization, which in turn depends on the strength and direction of the applied magnetic field.", "Thus, the amount of polarization rotation may be used as an indicator of magnetic field strength and/or orientation.", "FIG.", "43 depicts a flow diagram illustrating an example of a method 4300 for measuring the position of a micro machined (MEMS) optical element according to an embodiment of the invention.", "At step 4302 a magnetic sensor is disposed on a micro machined optical element.", "At step 4304 the magnetic sensor is exposed to a magnetic field.", "At step 4306 a change in an electrical, mechanical and/or optical property of the magnetic sensor is measured as an orientation of the MEMS optical element changes with respect to the magnetic field.", "As used herein, “position” may refer to relative spatial position, relative angular orientation, or some combination of both.", "Furthermore, the position of the MEMS optical element may change with respect to the magnetic field if the magnitude or direction of the magnetic field changes with respect to the MEMS optical element.", "The ON/OFF state of a 2D MEMS optical switch may be determined by comparing the value of the magnetic sensor property measured in step 4306 with one or more predetermined values of the sensor property when the MEMS optical element is known to be in an ON and/or OFF position.", "FIGS.", "44A-44B depicts schematic diagrams of an apparatus 4400 according to another embodiment of the invention.", "The apparatus 4400 includes a micro machined optical element 4410 and a magnetic sensor 4420 disposed on the micro machined optical element 4410.The magnetic sensor 4420 may be coupled to a position detector 4430, e.g.", "by leads 4431, 4432.By way of example, the micro machined optical element 4410 may include a fixed portion, such as a base 4412, and a movable portion, such as a flap 4414.As used herein, the term “moveable” means capable of movement by translation or rotation or some combination of both.", "Translation includes translation with respect to one or more axes.", "Rotation includes rotation with respect to one or more axes.", "By way of example, the flap 4414 may rotate about an axis 4415.The axis 4415 may be oriented substantially parallel to a plane of the flap 4414.Alternatively, the axis 4415 may be substantially perpendicular to the plane of the flap such that the flap is oriented substantially perpendicular to a plane of the base 4412.The flap 4414 may be coupled to the base e.g.", "by one or more flexures, so that the flap 4414 is movable out of the plane of the base 4412.The flexures may apply a torsional, or restoring force that returns the flap 4414 to a rest position when an actuating force is removed.", "Other restoring forces may be applied to flap 4414 to return the flap to the rest position.", "Such forces may be exerted on flap 4414 by biasing mechanisms that operate via pneumatic, thermal, or magnetic principals, including coils that interact with an external magnetic field, electrostatic elements, such as gap closing electrodes, piezoelectric actuators and thermal actuators.", "Multiple restoring forces may also be used together, and the forces may operate along the same or opposing directions.", "A light-deflecting element 4416 may be disposed on the flap 4414 to deflect one or more optical signals.", "By way of example, the light-deflecting element 4416 may be a simple plane reflecting (or partially reflecting) surface, curved reflecting (or partially reflecting) surface, prismatic reflector, refractive element, prism, lens, diffractive element, e.g.", "fresnel lens, a dichroic coated surface for wavelength specific and bandpass selectivity, or some combination of these.", "Any conventional means may be used to provide an actuating force to move the flap 4414.For example, the flap 4414 may contain a magnetically active element 4425 to facilitate movement of the flap by interaction with an externally applied magnetic field.", "The magnetically active element may be a magnetically active material having, e.g.", "a fixed magnetic moment, i.e., it may be a permanent magnet.", "Magnetically active materials may include Nickel, Nickel-Iron, Iron-Cobalt, Aluminum-Nickel-Cobalt, Neodymium-Iron-Boron, etc., and, may be deposited in a uniform or stepped pattern.", "Alternatively, e.g.", "one or more vertical combdrive actuators may be used to tilt the flap 4414 through a continuous range of angles in a controlled fashion.", "The magnetic sensor 4420 may be used to sense the state or position of a flap such as the flap 4414.The magnetic sensor 4420 may operate by sensing a change in an electrical property such as a resistance, reactance, or impedance of the sensor under the influence of a magnetic field B.", "The magnetic field B may be an external field that actuates movement of the flap by interaction with a magnetic material 4425 on the flap 4414.Alternatively, the magnetic field may be a separate sense magnetic field, e.g.", "a magnetic field that is produced by the magnetic material 4425.The magnetic sensor 4420 may include, but is not limited to, magnetoresistive sensors including giant magnetoresistance (GMR) sensors, such as spin valves, colossal magnetoresistance (CMR) sensors, anisotropic magnetoresistance (AMR) sensors, magnetic tunnel junction (MTJ) devices, and Hall effect sensors, flux sensing coils, magnetostriction sensors and magneto optic sensors.", "By way of example and without loss of generality, the magnetic sensor 4420 may be a magnetoresistive sensor that includes a magnetoresistive material.", "Examples of magnetoresistive materials Include Cu, Ni, Fe, Co and their alloys, oxides and structures having multiple layers containing one or more of these.", "A magnetic sensor 4420 in the form of a magneto resistive sensor may be formed by depositing magnetoresistive material and leads on the micro machined optical element 4410.Evaporation and annealing processes may be used for a multiple layer or GMR film.", "The magnetoresistive material may be deposited by suitable techniques including, but not limited to, sputter deposition, evaporation and electroplating FIG.", "44B shows a cross-sectional schematic diagram of the apparatus 4400 taken along line 44B-44B.", "The flap 4414 may make an angle θ with respect to the magnetic field B.", "A sense current I flows through the MR sensor 4420.The MR sensor 4420 may have a thickness that is very small compared to its length and width to constrain the sense current I to flow in a path substantially within a plane.", "The sense current I is directed at an angle θ with respect to the magnetic field B.", "The sensor may be disposed on the flap 4414 as shown in FIG.", "44B, so that the angle θ changes as the flap 4414 rotates with respect to the magnetic field B.", "Since the electrical property of the MR sensor 4420 depends on both B and θ, changes in the angular orientation of the flap produce corresponding changes in the electrical property of the MR sensor 4420.Alternatively, the flap 4414 may translate with respect to the magnetic field B.", "If the magnetic field B is non-uniform in either magnitude or direction, changes in the spatial position of the flap 4414 may produce changes in the electrical property of the magnetic sensor 4420.The position detector 4430 may measure changes in the electrical property of the magnetic sensor 4420 that varies with changes in a magnetic flux through the magnetic sensor 4420.Where, for example, the relevant electrical property of the magnetic sensor is an electrical resistance, the position detector 4430 may include a resistance measuring circuit.", "Such a circuit may supply a fixed sense current I to the magnetic sensor 4420 and measure changes in the voltage across the magnetic sensor 4420.If the relevant electrical property of the MR sensor 4420 is a Hall voltage, the position detector may supply a fixed current to the opposite ends of the magnetic sensor 4420 and detect the Hall voltage that develops across the width of the detector.", "The position detector 4430 may be implemented in hardware, software, firmware, or some combination of these.", "By way of example, the position detector 4430 may be implemented as one or more application specific integrated circuits (ASIC's).", "More than one magnetic sensor may be disposed on the micro machined optical element.", "Furthermore, the magnetic sensor may be disposed on the fixed portion of the micro machined optical element.", "By way of example, FIG.", "45 depicts an isometric schematic diagram of an apparatus 4500 according to an alternative version of the above-described embodiment of the invention.", "Apparatus 4500 may include a micro machined optical element 4510 and first, second, third, and fourth magnetic sensors 4520A, 4520B, 4520C, 4520D disposed on the micro machined optical element 4510.The magnetic sensor 4520 may be coupled to a bridge circuit 4530.The optical element 4510 may include a fixed portion 4512 and a moveable portion 4514.The magnetic sensors 4520A, 4520B, 4520C, 4520D may include, but are not limited to, giant magnetoresistance (GMR) sensors, spin valves, colossal magnetoresistance (CMR) sensors, anisotropic magnetoresistance (AMR) sensors, magnetic tunnel junction (MTJ) devices, and Hall effect sensors, flux sensor coils, magnetostriction sensors and magneto optic sensors.", "By way of example, the magnetic sensors 4520A, 4520B, 4520C, 4520D may be magnetoresistive (MR) sensors.", "The magnetoresistive sensors may be formed from a pattern of magnetoresistive material laid out on the micro machined optical element 4510, e.g., by photolithographic techniques.", "As the position of the movable portion 4514 changes with respect to the magnetic field B during the actuation cycle, the orientation of the sensor 4520A with respect to the magnetic field B also changes, e.g., from a from parallel to a perpendicular orientation.", "In the version of the embodiment depicted in FIG.", "45A the first MR sensor 4520A may be disposed on the movable portion 4514 of the micro machined element 4510 and the other three sensors 4520B, 4520C, 4520D disposed on the fixed portion 4512.As the angular orientation of the movable portion 4514 changes with respect to a magnetic field B an electrical property of the first sensor 4520A on the movable portion 4514 changes correspondingly as described above.", "The electrical properties of the other three sensors 4520B, 4520C, 4520D, however, remain substantially fixed as the angular orientation of the movable portion changes with respect to the magnetic field B.", "The properties of all four sensors 4520A, 4520B, 4520C, 4520D change in proportion to changes in the magnetic field B.", "Thus, if all four sensors 4520A, 4520B, 4520C, 4520D are appropriately coupled to the bridge circuit 4530 an output of the bridge circuit may be made sensitive to changes in the angular orientation of the movable portion 4514 of the micro machined optical element 4510, but substantially insensitive to changes in the magnetic field B.", "FIG.", "45B illustrates a schematic diagram of an example of a bridge circuit 4530′ that may be in conjunction with the apparatus 4500.Although the following relates to the use of a bridge circuit with magnetoresistive sensors, bridge circuits may also be used with other magnetic sensors such as Hall effect sensors, flux sensing coils, magnetostriction sensors and magneto optic sensors.", "The four magnetoresistive sensors 4520A, 4520B, 4520C, 4520D may be connected in a Wheatstone bridge fashion with one sensor 4520A being disposed on the movable portion 4514 of the micro machined optical element 4510.By way of example, each of the four magnetoresistive sensors 4520A, 4520B, 4520C, 4520D may be respectively characterized by an electrical resistance RA, RB, RC, RD that changes in response to changes in the magnetic field B.", "The first and third magnetoresistive sensors 4520A, 4520C may be electrically coupled at a first junction 4531.The second and fourth magnetoresistive sensors 4520B, 4520D may be electrically coupled at a second junction 4532.The first and second magnetoresistive sensors 4520A, 4520B may be electrically coupled at a third junction 4533.The third and fourth magnetoresistive sensors 4520C, 4520D may be electrically coupled at a fourth junction 4534.A current source 4540 may be coupled between the first and second junctions 4531, 4532, and null detector (N) 4550 may be electrically coupled between the third and fourth junctions 4533, 4535.The null detector 4550 may be regarded as a sensitive electric current detector.", "By way of example, the resistance of the circuit between the second and third junctions, e.g., RB, may be varied to change the current through the null detector 4550.When the current through the null detector 4550 is zero, it can be shown that the resistance of the magnetoresistive sensor 4520A may be given by: R A = R C ⁢ R B R D Since RA, RC, RC, RD, are dependent magnetic field B changes in the magnetic field B tend to cancel out.", "However, in this example, only RA depends on the angle θ.", "Thus, the bridge circuit 4530′ may capture information regarding the angular position of the movable portion 4514 of the micro machined optical element 4510.Although the foregoing discussion describes measurement of electrical resistance, Wheatstone bridge circuits may be utilized to measure other electrical properties such as Hall voltages.", "Other bridge circuits, such as Mueller bridge circuits may be used with the apparatus 4500 to measure the resistance or other electrical property of one or more magnetic sensors.", "Furthermore, a single magnetic sensor may be coupled to a bridge circuit to sense a change in resistance or other relevant electrical property.", "One or more magnetic sensors can be employed as sense elements in a feedback loop to control the mirror angle, and to incorporate a diagnostic routine to inform a user of switch level malfunctions in the event that the control loop fails to move the mirror to the desired position.", "Embodiments of the present invention can be used to measure the angular position of the scanning MEMS micro mirrors used in fiber-optic switches for optical communication systems.", "FIG.", "46 depicts an isometric schematic diagram of an example of a MEMS optical switch 4600.According to another embodiment of the invention, switch 4600 may generally includes a plurality of micro machined optical elements 4602 and magnetic sensors 4604.The magnetic sensors 4604 may include, but are not limited to the various types of sensors described above, such as giant magnetoresistance sensors, colossal magnetoresistance sensors, anisotropic magnetoresistance sensors, magnetic tunnel junction devices, Hall effect sensors, flux sensing coils, magnetostriction sensors, magneto optic sensors and the like.", "Each micro machined optical element 4602 may include a movable portion 4606.The sensors 4604 may be disposed on the movable portions 4606 as described above.", "By way of example, the movable portion may rotate about an axis 4607 relative to a fixed portion 4608.The fixed portion 4608 may be a base common to all of the micro machined optical elements 4602.The movable portions 4606 may include a light deflecting elements 4616.By way of example, the light-deflecting element 4616 may be a simple plane reflecting (or partially reflecting) surface, curved reflecting (or partially reflecting) surface, prismatic reflector, refractive element, prism, lens, diffractive element, e.g.", "fresnel lens, a dichroic coated surface for wavelength specific and bandpass selectivity, or some combination of these.", "The light deflecting elements 4616 may deflect optical signals to selectively couple the signals from one optical fiber to another.", "It must be stated that movable portion 4606 is shown for example purposes only, that a plurality of movable element designs exist, and the present invention may be used on various MEMS optical mirror designs that utilize a movable optical element.", "The sensors 4604 may be coupled to a switch controller 4612.The switch controller 4612 may be implemented in hardware, software, firmware, or some combination of these.", "By way of example, the switch controller 4612, may be implemented as one or more application specific integrated circuits (ASIC's).", "The switch controller 4612 may receive information on the angular position of the movable portions of the micro machined optical elements 4602 from the sensors 4604.The switch controller may include a feedback loop to control the angle of the movable portions.", "Alternatively, the switch controller 4612 may incorporate a diagnostic routine to inform a user of switch level malfunctions in the event that the control loop fails to move the micro machined optical element 4602 to a desired position.", "In some versions of the above-described embodiments of the invention, the magnetic sensor may be placed on a fixed portion of a micro machined optical element.", "FIGS.", "47A-47E depict several alternative versions of this embodiment.", "In these versions, a magnetic material is characterized by a permanent magnetic moment is disposed on a moveable portion and the magnetic sensor and its associated leads are disposed on a nearby fixed portion.", "The magnetic material may produce a magnetic flux that passes through a magnetoresistive sensor, Hall effect sensor or coil wherein the flux changes as the position of the magnetic material changes with respect to the sensor.", "Changes in flux through the sensor may cause changes an electrical property of the sensor, e.g.", "electrical resistance, Hall voltage or inductance.", "An advantage of this configuration is that an electrical connection to the moveable portion is not required.", "This greatly simplifies the manufacture of the apparatus and improves the robustness of its operation.", "FIG.", "47A depicts a plan view of an apparatus 4700 according to another alternative versions of the above-described embodiment of the invention.", "The apparatus 4700 generally comprises a micro machined optical element having a fixed portion in the form of a substrate 4702 and a moveable portion in the form of a flap 4706.The flap is movable, e.g.", "rotatable with respect to an axis 4703.The flap may include a light-deflecting element 4707 One or more magnetic sensors 4704A, 4704B are disposed on the substrate 4702 proximate the flap 4706.One or more magnetic elements 4708A, 4708B are disposed on the flap 4706 near the sides thereof proximate the sensors 4704A, 4704B.", "The sensors 4704A, 4704B may be connected to detectors 4701A, 4701B through leads 4705A, 4705B, 4705C, 4705D.", "In the embodiment shown in FIG.", "47A the sensors 4704A, 4704B and the magnetic materials 4708A, 4708B are oriented substantially parallel to each other and substantially perpendicular to the rotation axis 4703.The magnetic elements 4708A, 4708B may be magnetically active materials having, e.g.", "a fixed magnetic moment, i.e., they may be permanent magnets.", "Magnetically active materials may include Nickel, Nickel-Iron, Iron-Cobalt, Aluminum-Nickel-Cobalt, Neodymium-Iron-Boron, etc., and, may be deposited in a uniform or stepped pattern.", "The magnetic elements 4708A, 4708B may alternatively include one or more coils that carry electric current to provide a magnetic moment.", "Each magnetic element 4708A, 4708B may be characterized by a magnetic moment having a direction indicated by the arrows 4709A, 4709B.", "In the embodiment depicted in FIG.", "47B the magnetic moments of the magnetic elements 4708A, 4708B are oriented substantially perpendicular to the axis 4703.As the flap 4706 rotates about the axis 4703 the change in the relative position and/or orientation of the magnetic field produced by the magnetic elements 4708A, 4708B with respect to the sensors 4704A, 4704B causes a change in the magnetic flux passing through the sensors 4704A, 4704B.", "The change in flux causes a change in an electrical property of one or more of the sensors 4704A, 4704B.", "In a preferred embodiment, the sensors 4704A, 4704B may have a C-shape that includes a gap.", "The sensors 4704A, 4705B “wrap around” the magnetic elements 4708A, 4708B.", "As the position of the flap 4706 changes with respect to the substrate 4702 the amount of magnetic flux produced by the magnetic elements 4708A, 4708B that is intercepted by the sensors 4704A, 4704B changes.", "Where the sensors 4704A, 4704B are magnetoresistive sensors, the change in intercepted flux produces a change in one or more sense signals detected at the detectors 4701A, 4701B.", "In the particular version of the above-described embodiment shown in FIG.", "47A,the magnetic flux is a maximum when the flap 4706 is substantially parallel to the substrate 4702.In this configuration, the magnetic elements 4708A, 4708B are disposed within the gaps in the sensors 4704 A, 4704B.", "FIG.", "47B depicts a plan view of an apparatus 4710 according to another alternative version of the above-described embodiment of the invention.", "The apparatus 4710 is a variation on the apparatus 4700 of FIG.", "47A.", "The apparatus 4700 generally comprises a micro machined optical element having a fixed portion in the form of a substrate 4712 and a moveable portion in the form of a flap 4716.A light-deflecting element 4717 may be disposed on the flap 4716.The flap 4716 is movable, e.g.", "rotatable with respect to an axis 4713.A magnetic sensor 4714 may be disposed on the substrate 4712 proximate an end of the flap 4716.A magnetic element 4718 may be disposed on the flap 4716 proximate the sensor 4714.The magnetic moment of the magnetic element 4718 may be oriented substantially parallel to the axis 4713, as indicated by the arrow 4719.As in FIG.", "47A the magnetic sensor 4714 may be in the form of a magnetoresistive element having a C-shape with a gap.", "In the particular embodiment shown in FIG.", "47A the magnetic element lies within the gap when the gap when the flap 4716 is substantially parallel to the substrate 4712.The magnetic sensor 4714 may be coupled to a detector 4711, e.g., by leads 4715A, 4715B.", "Some micro machined optical elements may use a top chip design to provide a sidewall for orienting the flap in an up or “on” position.", "FIG.", "47C depicts a cross-sectional view of an apparatus 4720 according to another alternative version of the above-described embodiment of the invention.", "The apparatus 4720 may be assimilated as a variation on those described with respect to FIGS.", "47A-47B.", "The apparatus 4720 may generally comprises a micro machined optical element having fixed portions in the form of a base 4722 and a top chip 4725.The micro machined optical element has a moveable portion in the form of a flap 4726.In some applications such a two-chip approach is used to align the optical element in an “up” or “on” position with the flap 4726 oriented substantially perpendicular to a plane of the base 4722.The flap 4726 may be formed from one or more layers of the substrate 4722.In an “off” or down-position (shown in phantom), the flap 4726 is substantially parallel to the base 4722.The flap 4726 may be attached for movement with respect to the substrate 4722 by one or more flexures 4733.By way of example, the base 4722 may be a silicon-on-insulator (SOI) substrate.", "The top-chip 4725 has an opening 4723 with perpendicular sidewalls 4727.The term “sidewall” as used herein refers generally to any surface that provides a reference stopping plane for the flap 4726.Although a sidewall 4727 that is part of the substrate is shown in FIG.", "47C the sidewall may alternatively be part of the substrate 4722 or part of a separate structure formed on of the substrate 4722 or on the top chip 4725.The top chip 4725 is aligned with the substrate 4722 such that flap aligns with the opening 4723 and the substrate 4722 and top-chip 4725 are bonded together.", "The opening 4723 receives the flap 4726 when the flap is in an “on” state, i.e., substantially perpendicular to a plane of the substrate 4722.The flap 4726 may be clamped against a sidewall 4727 of the top chip 4725 when the flap is in the “on” state as shown in FIG.", "47C.", "When the top-chip 4725 is properly aligned and bonded to the substrate 4722 the sidewalls 4727 of the openings 4723 can serve as reference stopping planes to fix the up-position of the flap.", "In addition, the sidewalls 4727 may also serve as electrodes to hold the mirrors in the up-position by electrostatic attraction.", "A “top chip” having openings with almost perfectly perpendicular sidewalls may be formed, e.g., by etching a <110> silicon wafer with an anisotropic etchant.", "One or more magnetic sensors 4724 may be disposed on the top chip 4725 proximate the flap 4726.Although FIG.", "47C shows the sensor 4724 disposed on a surface of the top chip 4725, a sensor 4724′ may alternatively be disposed on the sidewall 4727.The sensors 4724, 4724′ may be coupled to a detector 4721, e.g., via leads 4729A, 4729B.", "A magnetic element 4728, such as a magnetic material, may be disposed on the flap 4726 to provide a sense magnetic field that is detected by the sensors 4724, 4724′.", "Alternatively one or more of the sensors 4724, 4724′ may be disposed on the flap 4726 and the magnetic material may be disposed on the substrate 4722, the top chip 4725 or the sidewalls 4727.It need be stated that the top chip associated with each micro machined optical element may also be comprised of two high-aspect-ratio deep vertical walls separated by an air gap.", "Several orientations of the sensors and magnetic elements are possible.", "Two particular configurations are depicted in FIG.", "47D and FIG.", "47E.", "FIG.", "47D depicts a plan view of an apparatus 4730 according to another alternative versions of the above-described embodiment of the invention.", "The apparatus 4730 generally comprises a micro machined optical element having fixed portions in the form of a substrate 4732 and a top chip 4735.The micro machined optical element includes a moveable portion in the form of a flap 4736.One or more magnetic sensors 4734A, 4734B are disposed on the top chip 4735 proximate the flap 4736.The sensors 4734A, 4734B may be coupled to a detector 4731, e.g., via leads 4739A, 4739B.", "The sensors 4734A, 4734B may be in the form of serpentine coils of magnetic material.", "The serpentine shape allows a greater length for the sensors, which increases their sensitivity to changes in magnetic flux.", "One or more magnetic elements 4738A, 4738B are disposed on the flap 4736 near the sides thereof.", "The magnetic elements 4738A, 4738B may be positioned such that they are proximate the sensors 4734A, 4734B when the flap 4736 is clamped against the top chip 4735.In this position, the magnetic flux though the sensors 4734A, 4734B from the magnetic elements 4738A, 4738B may be maximized.", "FIG.", "47E depicts a plan view of an apparatus 4740 according to another alternative version of the above-described embodiment of the invention.", "The apparatus 4740 generally comprises a micro machined optical element having fixed portions in the form of a substrate 4742 and top chip 4745.The micro machined optical element may include a moveable portion in the form of a flap 4746.A magnetic sensor 4744 may be disposed on the top chip 4745 proximate the flap 4746.The magnetic sensor 4744 may be coupled to a detector 4741, e.g.", "through leads 4747A, 4747B.", "The magnetic sensor 4744 may be in the form of a serpentine pattern of magnetoresistive material having features in common with the serpentine patter described with respect to FIG.", "47D.", "One or more magnetic elements 4748 may be disposed on the flap 4716 proximate an end thereof.", "The magnetic element 4748 may be positioned on the flap 4746 such that it is proximate the magnetic sensor 4744 when the flap is in an “on” position.", "Other variations are possible on the above embodiments.", "For example, the magnetic sensor element may include an inductive coil disposed on either a fixed or moveable portion of a micro machined optical element.", "Changes in the position of the moveable portion may lead to changes in an inductance of the coil.", "The change in inductance may be correlated to the change in position.", "Changes in inductance may be less susceptible to noise than changes in capacitance.", "FIG.", "48 depicts a block diagram depicting an optical communications system 4800 according to another embodiment of the invention.", "In the system 4800, a method having features in common with the method 4300 of FIG.", "43 may be implemented as a computer program code 4805 running on a processor of a computer controlled apparatus having features in common with the MEMS optical switch 400 described above with respect to FIG.", "4.In the embodiment shown, the program code 4805 controls the operation of one or more MEMS optical elements 4832A, 4832B, 4832C, 4832D in an optical switch 4830.Although the program 4805 is described herein with respect to a MEMS optical switch, those skilled in the art will recognize that programs embodying the method of the present invention may be applied to any MEMS device.", "The optical elements 4832A, 4832B, 4832C, 4832D may have features in common with the optical elements described above.", "The optical switch 4830 may have features in common with the type of switch 4600 shown in FIG.", "46.By way of example, the switch 4830 may be a 2D MEMS optical switch.", "Each optical element 4832A, 4832B, 4832C, 4832D may include a moveable portion that is moveably coupled to a substrate and actuated by, for example, electrostatic, pneumatic thermal, acoustic or magnetic actuators 4834A, 4834B, 4834C, 4834D.", "The optical elements 4832A, 4832B, 4832C, 4832D may be clamped in vertical or horizontal position by voltages applied to clamping electrodes (not shown).", "One or more magnetic sensors 4836A, 4836B, 4836C, 4836D may be respectively coupled to moveable and/or fixed portions of the optical elements 4832A, 4832B, 4832C, 4832D.", "The magnetic sensors 4836A, 4836B, 4836C, 4836D may be of any of the types described above.", "The magnetic sensors 4836A, 4836B, 4836C, 4836D sense changes in the position or state of the optical elements 4832A, 632B, 4832C, 4832D with respect to a magnetic field B′ provided, e.g., by a magnet 4838.If the actuators 4834A, 4834B, 4834C, 4834D are magnetic actuators, the magnetic field B′ may be the same magnetic field that drives the actuators.", "Alternatively, the magnetic field B′ may be a separate sense magnetic field.", "In some embodiments, a single magnet 4838 may be used to actuate all the optical elements 4832A, 4832B, 4832C, 4832D.", "In such a situation, the actuators 4834A, 4834B, 4834C, 4834D may include electrodes for clamping moveable portions of the optical elements 4832A, 4832B, 4832C, 4832D in their respective “ON” or “OFF” states.", "The switch 4830 may optionally include a temperature sensor 4820 disposed in proximity to switch 4830 or positioned in thermal contact with a portion of the switch, e.g.", "one or more of the optical elements 4832A, 4832B, 4832C, 4832D.", "The temperature sensor may produce a signal that is proportional to a temperature of the switch 4830.By way of example, the temperature sensor 4820 may be a thermocouple, thermistor, infrared (IR) temperature sensor, etc.", "One or more input fibers 4807A, 4807B and output fibers 4807C, 4807D may be optically coupled to the optical switch 4830.Optical sources (OS) 4803A, 4803B may provide optical signals to the input fibers 4807A, 4807B while optical detectors (OD) 4809A, 4809D may be optically coupled to the output fibers 4807C, 4807D to establish, for example, that the micro machined optical elements in the switch are in a known state.", "Alternatively, the optical sources and detectors may be replaced with optical transceivers to allow two-way signal traffic through the switch 4830.A switching sub-system 4800 may typically include a switch 4830 combined with a controller 4801.The controller 4801 may be a self contained microcontroller such as the PICK Microchip, or controller 4801 may be configured to include a CPU 4802, memory 4804 (e.g., RAM, DRAM, ROM, and the like), clock 4814 and well-known support circuits 4810 such as power supplies 4812, input/output (I/O) functions 4818 coupled to a control system bus 4808.The memory 4804 may contain instructions that the processor unit 4802 executes to facilitate the performance of the apparatus 4800.The instructions in the memory 4804 may be in the form of the program code 4805.The code 4805 may conform to any one of a number of different programming languages such as Assembly, C++, JAVA or a number of other languages.", "The controller 4801 typically operates the apparatus 4800 through I/O functions 4818 in response to data and program code instructions stored and retrieved by the memory 4804.The CPU 4802 may be coupled to the elements of the system 4800 via the system bus 4808 and the I/O functions 4818.The elements of system 4800 may include the following: one or more detector circuits (DC) 4835 coupled to one or more of the magnetic sensors 4836A, 4836B, 4836C, 4836D, and one or more actuator drivers (AD) 4833 coupled to one or more of the actuators 4834A, 4834B, 4834C, 4834D.", "If the magnet 4838 is an electromagnet, a magnet driver (MD) 4837 may be coupled to the magnet.", "For the sake of clarity, connection is shown to only one of the magnetic sensors 4836D and one of the actuators 4834D.", "In practice, all the magnetic sensors 4836A, 4836B, 4836C, 4836D and actuators 4834A, 4834B, 4834C, 4834D may be coupled to the I/O functions 4818.One or more clamping voltage sources may be optionally coupled between clamping electrodes in the switch 4801 and the I/O functions 4818.The optical sources 4803A, 4803B and the optical detectors 4809A, 4809B may also be coupled to the I/O functions 4818 and system controller 4801 may provide control to switch optical signals between the input fibers 4807A, 4807B and the output fibers 4807C, 4807D.", "The support circuits 4810 may also include a temperature detector (TD) 4839 coupled to the temperature sensor 4820 and the I/O functions 4818.It should be stated that depending on the configuration or selection of controller 4801 and system 4800, the conditioning circuits, including actuator driver 4833, temperature detector 4839, magnetic driver 4837 and/or detector circuit 4835 may be implemented in software form,, e.g., within code 4805, such that I/O functions 4818 may directly connect to each respective switch component.", "The system 4800 may be a subsystem or component of a network element (not shown).", "The network element may be part of a network (not shown).", "The microcontroller 4801 may include network element interface 4806 which may be implemented in software e.g.", "in a subroutine in memory 4804 or hardware to allow the system 4800 to communicate with the network element.", "Such communication may include, but is not limited to, switching commands issued from the network element to the system 4800 and switch state data from the system 4800 to the network element.", "Certain steps of the method described above with respect to FIG.", "43 may be implemented by a suitable computer program code 4805 running on the CPU 4802 of the controller 4801.The CPU 4802 may form a general-purpose computer that becomes a specific purpose computer when executing programs such as the program 4805.Although the invention is described herein as being implemented in software and executed upon a general purpose computer, those skilled in the art will realize that the invention could be implemented using hardware such as an application specific integrated circuit (ASIC), microcontroller or other hardware circuitry.", "As such, it should be understood that the invention can be implemented, in whole or in part, in software, hardware or both.", "A computer program 4805 may be devised to implement steps 4304 and 4306 described above with respect to FIG.", "43.The program 4805 is suitable for monitoring and controlling the position or state of the optical elements 4803A, 4803B, 4803C, 4803D of the optical switch 4801 in accordance with embodiments of the present invention.", "By way of example, the program 4805 may implement fault detection in the system 4800.For example, suppose that only when the optical element 4832B is in an “ON” state, optical element 4832B deflects optical signals from input fiber 4807B to output fiber 4807C.", "The state of optical element 4832B may be determined by sending an optical signal towards optical element 4832B from the source 4803B to input fiber 4807B and monitoring the optical signal at output fiber 4807C with optical detector 4809A.", "If the optical signal from the optical source 4803B is detected by the optical detector 4809A optical element 4832B is presumably in the “ON” state.", "While the optical element 4832B is known to be in the “ON” state, the property of the magnetic sensor associated with thereto may be recorded through I/O function 4818 and stored in a look-up table in memory 4804.This step may occur when the magnet 4838 is turned on to provide a sense field for the magnetic sensors 4836A, 4836B, 4836C, 4836D or when the magnet 4838 is turned on to perform a switching event.", "Alternatively, a signal from the magnetic sensor 4836B disposed proximate the optical element 4832B may be measured when the movable element associated with the magnetic sensor 4836B is in a known state.", "Signals from sensors 4836 may be measured in batch or selectively addressed in response to code 4805 and through I/O functions 4818 when they are in a known state.", "The position of optical element 4832B changes when it moves from the “ON” state to the “OFF” state.", "Consequently, the magnetic sensor 4836B may produce a different signal when the optical element 4832B is in the OFF state.", "The other magnetic sensors 4836A, 4836C, 4836D may also produce different signals.", "In a manner similar to that described above, a set of signals from the sensors 4836A, 4836B, 4836C, 4836D may be correlated to the “OFF” state of the optical element 4832B.", "In a similar fashion, the known “ON” and “OFF” states of the other optical elements 4832A, 4832C, 4832D may be correlated to measured signals from the magnetic sensors 4836A, 4836B, 4836C, 4836D.", "These signals from the magnetic sensors 4836A, 4836B, 4836C, 4836D may be organized by the program 4805 as a set of predetermined signals, e.g.", "in a look-up table stored in memory 4804.The program 4805 may index the aforementioned look-up table after reading the value or values associated with the magnetic sensor property to determine that the state of the switch is configured according to the requests of network element interface 4806.The properties of the magnetic sensors 4836A, 4836B, 4836C, 4836D may be temperature dependent.", "Consequently, signals from the magnetic sensors 4836A, 4836B, 4836C, 4836D may drift as the temperature of the switch 4830 changes.", "To compensate for such drift, the program 4805 may include instructions for temperature compensation.", "By way of example, such instructions may include measuring the signal from the magnetic sensors 4836A, 4836B, 4836C, 4836D for the “ON” and off states of the optical elements 4832A, 4832B, 4832C, 4832D at different temperatures measured by the temperature sensor 4820.The program may then determine ranges for the values of the magnetic sensor signals that correspond to the “ON” and “OFF” states.", "If, over a certain temperature range, the two ranges do not overlap the state of an optical element may be determined by measuring that magnetic sensor signal to see whether it falls in the “ON” range or the “OFF” range.", "It must be stated that the look-up table storing the predetermined magnetic sensor property values associated with each micro machined movable element, may be configured to allow a test value to fall within a range of predetermined values for added stability.", "For example, the magnetic sensor property values read into memory 4804 through I/O functions 4818, when the optical element is in a known state to achieve the predetermined value for the lookup table, may be configured in code 4805 such that test values read into memory 4804 through I/O functions 4818 when the optical element is not in a known state may be substantially equal to the lookup values in the table.", "This approach results in added stability and may be used to compensate for temperature variation effects.", "If there is substantial overlap between the “ON” and “OFF” ranges it may be desirable to correct for thermal drift in real time.", "The program 4805 may correct for thermal drift by relating the measured magnetic sensor signals in the “ON” and “OFF” states to temperature measurements made during operation of the switch 4830.The relationship may be stored in the form of a look-up table.", "Alternatively, the relationship may be in the form of a temperature correction equation.", "For example, in the case of a linear relationship between temperature and magnetic sensor signal, the program may calculate a temperature drift coefficient.", "The temperature drift coefficient may be used to adjust the predetermined magnetic sensor signals for changes in temperature.", "Alternatively, the system controller 4801 may be coupled to a temperature regulator (not shown) coupled, e.g.", "through the I/O circuits 4818.The program 4805 may instruct the temperature regulator to maintain the temperature of the switch 4830 within a desired temperature range in response to temperature measurements from the temperature sensor 4820.Preferably, the desired temperature range is sufficiently narrow that any thermal drift of magnetic sensor signal may be neglected.", "Furthermore, the system 4800 may employ some combination of thermal drift correction and temperature regulation to compensate for changes in temperature.", "It should also be stated that magnetic sensors may be connected together e.g.", "through a bridge circuit and the output of the connected sensors may be batch read by the controller 4801 to determine the individual state of each movable portion in the batch of elements.", "This can be accomplished by designing or tuning the sensors to produce a unique value in each known ON and OFF state.", "For example, a magnetoresistive element associated with each micro machined optical element may be designed to produce a unique prime resistance value when turned ON or OFF.", "Magnetic sensors may be connected in series or parallel and grouped according to, but not limited, rows or columns.", "As so, the program code 4805 may engage in a row or column select to pull the combined sensor property value into memory for post processing by the CPU.", "Program code 4805 may then perform data processing on the recorded property value to discern the individual state of all member optical flaps contained in group of sensors.", "Memory 4804 may store the predetermined prime values associated with the plurality of sensors and the program 4805 may engage in an process whereby the recorded value of the combined sensor group is compared against various prime number combinations associated with the group, until a match is found.", "When a match is determined, the micro-machine optical elements associated with the match prime numbers set will share the same ON or OFF state, and the individual states of the batch group can be determined.", "While the above is a complete description of several embodiments of the present invention, it is possible to use various alternatives, modifications and equivalents.", "H. MEMS CAP A critical performance parameter of 2D MEMS free-space optical switches is the switching time.", "The switching time may be regarded as the time it takes for a given MEMS mirror to switch from an “OFF” state to an “ON” state or vice versa.", "For high performance switches, it is desirable to make the switching time as short as possible.", "Certain of the obstacles to improved switching times arise from the design of the MEMS mirrors themselves.", "Stiction is one such obstacle.", "In addition to stiction, a drag force, referred to as “squeeze film damping,” may increase the switching time of the MEMS mirror.", "This type of drag may result from a fluid such as air trapped between the MEMS mirror and the underlying substrate.", "Increased switching times due to stiction and squeeze film damping lead to slower switching speeds and poor switch performance.", "Additional embodiments of the present invention may overcome stiction and squeeze film damping by incorporating an apparatus for hermetically sealing MEMS devices with a cap assembly having optical windows perpendicular (or nearly perpendicular) to the plane of the MEMS substrate.", "MEMS devices that utilize equipotential landing pads may incorporate such an enclosure.", "The MEMS device may be an optical switch having one or more MEMS elements, such as movable mirrors that rotate or translate to deflect light from one or more optical fibers.", "Alternatively, the MEMS device may include elements to modify phase, focus, or direction of the input optical beams.", "The dimensions of the enclosure closely match the dimensions of the MEMS device such that the device itself is enclosed, but fibers or lenses that are optically coupled to the device remain outside the enclosure.", "As shown in FIG.", "49, the MEMS package 4900 generally includes a mount 4902, which may be ceramic, FR4, or otherwise, to which a MEMS device 4910 is attached, e.g., by die bonding, and an enclosure 4906 having one or more optical elements such as a window 4908.Optical signals 4901 may be coupled to the MEMS device 4910 from an optical fiber 4903 via the window 4908 and a collimator 4904, such as a graded refractive index (GRIN) lens.", "The window 4908 may be angled to restrict undesired coupling of reflected light back into the fibers or the MEMS device itself.", "The enclosure 4906 may be bonded to the mount 4902 in such a way as to provide a hermetically sealed environment within the enclosure 4906.The enclosure 4906 may be a cap assembly consisting of a ring-frame with cut-outs for windows, a top-cap hermetically attach to a ring-frame, and optical windows that are hermetically attached to the ring-frame.", "In another option, the enclosure 4906 may be fabricated as a single piece.", "The window 4908 may be attached to the enclosure 4906 using solder, glass-frit, glass-to-metal seal, or another method.", "In another option the enclosure 4902 may include an entire ring-frame fabricated of an optically transparent material where the window 4908 would be inherent in the ring-frame.", "The enclosure 4906 may be evacuated, e.g., through a sealable passage 4920 in the mount 4902, to provide improved switching performance as described below.", "As used herein, the term evacuated describes a situation in which the atmospheric pressure has been reduced below an ambient atmospheric pressure.", "By way of example, and without loss of generality, the ambient pressure of the earth's atmosphere is approximately 760 Torr at mean sea level.", "The MEMS device 4910 generally includes a substrate 4912, and an array of MEMS optical elements 4914 moveably attached to the substrate.", "By way of example each of the MEMS device 4910 may include an N×N or N×M array of MEMS optical elements 4914, where N and M are integers.", "By way of example, each MEMS optical element 4914 may be in the form of a flap attached to the substrate 4912 by one or more flexures (not shown).", "The MEMS optical elements 4914 may include light-deflecting elements such as simple plane reflecting (or partially reflecting) surfaces, curved reflecting (or partially reflecting) surfaces, prismatic reflectors, refractive elements, prisms, lenses, diffractive elements, e.g.", "fresnel lenses, dichroic coated surfaces for wavelength specific and bandpass selectivity, or some combination of these.", "In a particular embodiment, the optical elements 4914 may include reflective surfaces so that may act as MEMS mirrors.", "The MEMS optical elements 4914 may move between an “OFF” position and an “ON” position under the influence of an actuating force, such as a magnetic force.", "By way of example the MEMS optical elements 4914 may be oriented substantially parallel to the substrate 4912 in the “OFF” position and substantially perpendicular to the substrate 4912 in the “ON” position.", "In the “ON” position, the MEMS optical elements 4914 deflect the optical signals 4901.The device 4910 may further include clamping surfaces to orient and retain the MEMS optical elements 4914 in the “ON” position.", "Such clamping surfaces may be provided by a “top chip” 4913 having openings 4915 that may receive the optical elements 4914.The openings 4915 may include sidewalls 4917 that provide the clamping surfaces.", "The sidewalls 4917 provide reference stopping-planes for the MEMS optical elements 4914.Alternatively, the top-chip 4913 may include clamping surfaces in the form of a single vertical wall or two vertical walls with a hole therebetween to allow light to pass.", "Such a vertical wall or walls may be higher than the MEMS optical elements 4914.A voltage may be applied between individual optical elements 4914 and the top chip 4913 to electrostatically clamp the optical elements 4914 in the “ON” position.", "The optical elements 4914 may be electrically insulated from the sidewalls 4917 by an insulating gap, such as an air gap.", "In an alternative embodiment depicted in FIG.", "50, an apparatus 200 may, include an enclosure 206 that is directly bonded to a substrate 212 of a MEMS device 5010 having features in common with the MEMS device 4910 of FIG.", "49.The enclosure 5006 may include an optical element such as a window 5008.The window 5008 may be angled to restrict undesired coupling of reflected light back into one or more optical fibers 5003 or the MEMS device 5010 itself.", "The enclosure 5006 may be bonded to the substrate 5012 in such a way as to provide a hermetically sealed environment within the enclosure 5006.The enclosure 5006 may be a cap assembly consisting of a ring-frame with cutouts for the window 5008, a top-cap hermetically attached to a ring-frame, and optical windows that are hermetically attached to the ring-frame.", "In another option, the enclosure 5006 may be fabricated as a single piece.", "The window 5008 may be attached to the enclosure 5006 using solder, glass-frit, glass-to-metal seal, or another method.", "In another option the enclosure 5002 may include an entire ring-frame fabricated of an optically transparent material where the window 5008 would be inherent in the ring-frame.", "The enclosure 5006 may be evacuated e.g., through a sealable passage 5020 in a top of the enclosure 5006, to provide improved switching performance as described below.", "Although FIG.", "49 and FIG.", "50 include enclosures with windows as optical elements, other optical elements may be incorporated into the enclosures.", "For example, FIG.", "51 depicts a side cross-section of an enclosure in the form of a cap assembly 5100 having a ring frame 5102 a top cap 5104 and one or more optical elements 5106.The cap assembly 5100 may be hermetically sealed to a mount as described above with respect to FIG.", "49 and FIG.", "50.The ring frame 5102 has one or more cutouts 5105 on one or more sidewalls that receive the optical elements 5106.Optical signals may travel through the ring frame 5102 via the optical elements 5106 and the cutouts 5105.The top-cap 5104 may be hermetically attached to the top of the ring-frame 5102.The optical elements 5106 may be made of glass, silicon, ceramic, or other optically transmissive materials.", "The optical elements 5106 may be attached to the ring-frame 5104 using solder, glass-frit, glass-to-metal seal, and other methods.", "The optical elements 5106 may be windows, simple refractive surfaces, partially reflective surfaces, curved refracting (or partially reflecting) surfaces, prisms, lenses, diffractive elements, e.g.", "fresnel lenses, dichroic coated surfaces for wavelength specific and bandpass selectivity, or some combination of these.", "If the optical elements 5106 are lenses, they may be fiber lens arrays, graded refractive index (GRIN) lenses, or one or more arrays micro-lenses.", "The optical elements 5106 may be hermetically attached to the sidewalls of the ring-frame 5102 at the cutouts 5105.Such sealing is simpler, higher yielding, less expensive, and more reliable than sealing optical fibers e.g.", "using metallization.", "Although a separate ring frame and top-cap are depicted in FIG.", "51, the ring-frame 5102 and the top-cap 5104 may alternatively be fabricated as a single piece.", "The cap assembly 5100 may be attached to an underlying substrate so as to align the optical elements 5106 to the components inside the cap within the required tolerance.", "The environment within the cap assembly 5100 may be evacuated or partially evacuated to reduce the atmospheric pressure within the space enclosed between the cap assembly and the substrate.", "Optical fibers may be aligned to the optical elements 5106 of the cap assembly 5100 and secured in place.", "As described above, optical elements, such as the windows 4908, 5008 may be tilted with respect to an optical axis to minimize back-reflection and interference effects.", "FIGS.", "52 and 53 depict possible arrangements for the windows that may be used with the apparatus of FIGS.", "49-51.In FIG.", "52, a package sidewall assembly 5200 includes a sidewall 5201 having a front surface 5202 and a back surface 5204.A window 5206 is attached to the front surface 5202.The window 5206 allows optical signals to pass, either selectively by wavelength or over a broad-band of wavelengths.", "The window 5206 can be made from a multitude of glass or ceramic types, Quartz, Sapphire, silicon, and other optically transmissive materials, with or without an anti-reflective coating.", "The window 5206 may be attached by soldering, bonding, epoxy, glass frit, and the like.", "The window 5206 may be partly aligned and supported by an optional ledge 5208 projecting from the front surface 5202.The sidewall 5201 includes an opening 5210 that is aligned with an optical plane 5212 for optical signals that travel through the window 5206.The window 5206 may be angled with respect to the optical plane or axis 5212 along which optical signals travel to reduce undesired back-reflection effects of signals.", "One of the surfaces 5202, 5204 of the sidewall 5201 may be angled to angle the window 5206.The angled surface can be either the innermost or outermost surface of the sidewall 5201.Furthermore, the angled surface can be recessed, to provide support and alignment for the window 5206.The sidewall 5201 does not necessarily have to be part of a package assembly.", "The window 5206 can be pre-attached to a frame if preferred, with the frame being attached to the angled sidewall.", "The ends, sides, or surface of the windows can be used for attachment to the sidewall or frame.", "If preferred, the angled sidewall could be manufactured from glass or other optically transmissive materials, becoming the window.", "In the embodiment shown in FIG.", "52, the front surface 5202 may be tilted with respect to the.", "back surface 5204 by an angle α, e.g., about 3°.", "The front surface 5202 of the sidewall 5201, may be angled, either by machining, molding, or forming, at an angle suitable to minimize the back reflection of coherent light through the attached window 5206, while providing the ledge 5208 as an acceptable surface for window attachment.", "The window 5206 may be a flat window attached to the angled front surface 5202.In the example shown in FIG.", "52 the front surface 5202 is the outside wall of the package assembly 5200.The sidewall 5201 may be a ring-frame, drawn tub, cap, or other package configuration of an enclosure such as those described above with respect to FIG.", "49 and FIG.", "50.The window 5206 may alternatively be attached to an inside wall, recessed or not, hermetically sealed or not, forming an integral enclosure as described above with respect to FIG.", "49 and FIG.", "50.Alternatively, as shown in FIG.", "53, a package assembly 5300 may include wedged window 5306 may be attached to a sidewall 5301 having substantially parallel front and back surfaces 5302, 5304 to provide the desired angle α.", "The wedged window 5306 reduces the back reflection of coherent light through the attached window 5306.Of course, some combination of angled sidewall and wedged window is also possible.", "Enclosed MEMS devices of the types shown in FIG.", "49 and FIG.", "50 with package assemblies of the types shown in FIGS.", "51-53 may be incorporated into an inventive MEMS module 5400 as shown in FIG.", "54.The module 5400 generally includes a mount 5402.The mount 5402 is essentially a board or base to which the elements of the module 5400 are attached.", "The mount 5402 may be made of ceramic, FR4, or another material.", "A MEMS device 5410, such as an optical switch, and control electronics 5420 are attached to the mount.", "The MEMS device 5410 includes an enclosure 5412 having vertical sidewalls with optical elements 5406, 5407 such as windows or lens arrays as described above.", "In the embodiment shown, the MEMS device 5410 is an optical switch having an array of moveable mirrors 5414.The switch may be used to selectively couple optical signals between one or more input fibers 5403 and one or more output fibers 5404.The fibers 5403, 5404 may be attached to the mount by conventional fiber mounts 5405, 5409 such as V-groove arrays and the like.", "The control electronics 5420 may be electrically coupled to the MEMS device 5410, e.g.", "by one or more control lines 5408.An alternative MEMS module 5500, which is a variation on the module 5400, is depicted in FIG.", "55.In this embodiment, a MEMS device 5510 is enclosed by an enclosure 5512 as described above.", "The MEMS device 5510 includes a device driver chip 5511 mounted to a backside of a MEMS substrate 5516.The driver chip 5514 controls the MEMS device 5510, e.g.", "via control lines 5508 or other connectors that pass through the substrate 5516.Such a device provides a completely sealed interchangeable module for use with larger MEMS modules.", "The enclosure 5512 may optionally include an optical element, e.g., in the form of a transparent window 5517 that is parallel to the plane of the substrate 5516 of the MEMS device 5510, e.g.", "on a top side 5513 of the enclosure to facilitate inspection of the device.", "The enclosure 5512 may also include a second optical element 5518 that is attached to a sidewall 5515.By way of example, the second optical element may be a window, lens or lens array as described above.", "The second optical element may facilitate transmission of optical signals 5501 between an externally mounted optical fiber 5503 and the MEMS device 5510.The enclosure 5512 may be evacuated, e.g., through a sealable passage 5520 in the sidewall 5515, to improve switching performance as described below.", "As described above, embodiments of the invention may include an evacuated enclosure that is hermetically sealed.", "The inventors have discovered that the switching time of a MEMS device may be greatly reduced by evacuating, or partially evacuating the environment surrounding the device.", "FIG.", "56 depicts a graph of the rising time versus pressure for a MEMS device having features in common with those described herein.", "As used herein, the rising time is the time that it takes a MEMS optical element to move from an “OFF” position to an “ON” position.", "The particular device used was a magnetically actuated MEMS optical switch.", "It is desirable to reduce this time as much as possible in high speed switching applications.", "FIG.", "56 shows that as the atmospheric pressure decreases in the environment containing the device, the switching time also decreases.", "For example, as the pressure decreased from about 800 Torr to about 100 Torr, the switching time decreased from about 30 ms to about 15 ms, a 50% reduction.", "Further reduction in pressure below about 100 Torr reduced the switching time to a little more than 5 ms. Encouraged by experimental data like that shown in FIG.", "56 the inventors have developed a method for high speed optical switching.", "FIG.", "57 depicts a flow diagram illustrating the steps of the method 5700.At step 5702, the atmospheric pressure proximate a MEMS optical device is reduced to some desired level.", "The MEMS optical device may be one of the types described above.", "In particular, the MEMS optical device may be an optical switch having one or more moveable MEMS optical elements of any of the types described above with respect to FIG.", "49.The amount of pressure reduction depends on the desired switching time as can be seen from FIG.", "56.There are several possible methods of reducing the atmospheric pressure.", "For example, an enclosure may be attached to the device as described above and coupled to an evacuating device, such as a vacuum pump.", "The pump may remove air or other gas from within the enclosure through a passage that may later be sealed after the enclosure has been sufficiently evacuated.", "Alternatively, the enclosure may be hermetically attached to the device in an evacuated environment.", "Furthermore, the device may operate in an evacuated environment.", "At optional step 5704, the MEMS optical device may be hermetically sealed within the enclosure as described above.", "At step 5706 the MEMS optical element moves from a first position to a second position.", "As can be seen from FIG.", "56 this may be accomplished very quickly depending upon how much the pressure has been reduced.", "At step 5708, while in the second position, the MEMS optical element deflects an optical signal from a first optical path to a second optical path.", "The MEMS optical element may return to the first position at optional step 5710.Variations on a device with the inventive equipotential landing pad structure are depicted in FIGS.", "58A-64.In an embodiment of the invention, depicted in FIG.", "58A, a basic device 5800 includes a device layer 5802 and at least one landing pad 5804 protruding from an underside 5806 of device layer 5802.Landing pad 5804 is attached to device layer 5802 by a plug 5808 passing through an opening 5810 in device layer 5802.The landing pad provides a smaller contact area 5812 than an area of underside 5806.The smaller contact area serves to reduce stiction between device 5800 and an underlying substrate 90.Stiction may also be reduced by proper choice of the material comprising landing pad 5804.Device 5800 may be any type of electromechanical device.", "Suitable devices include side-actuated motors, and electromagnetically or thermally actuatable mirrors for optical switches.", "Device layer 5802 is typically a semiconductor material such as silicon, although other possible materials including metals and dielectrics may also be used.", "Depending on the specific application, landing pad 5804 may be made from a dielectric material, such as silicon nitride, or a metal, such as Tungsten, titanium nitride or the like.", "Alternatively the landing pad may be made from polycrystalline silicon or other similar material.", "Two variations on the basic device 5800 are depicted in FIGS.", "58B and 58C.", "FIG.", "58B depicts a second embodiment of the invention.", "The basic structure of the device in this embodiment shares features in common with device 5800 of FIG.", "58A.", "In this embodiment, a device 5820 includes at least one landing pad 5824 having a diameter greater than a plug 5826.This type of device can be fabricated using wet processing, which is a lower cost process than dry processing.", "FIG.", "58C depicts another embodiment, in which a device 5830 includes at least one landing pad 5834 comprised of two or more separate layers 5835 and 5836.Generally, layers 5835 and 5836 are made from different materials.", "For example, layer 5835, which contacts substrate 5812, may be a dielectric layer.", "For example, layer 5836, may be a conductive material, which is part of an electrode structure.", "Layer 5835 insulates layer 5836 from electrical contact with substrate 5812.The devices depicted in FIGS.", "58A-58C may be fabricated by an inventive method according to another embodiment of the invention.", "The basic steps of the method are depicted in FIGS.", "59A-59E.", "FIG.", "59A depicts the basic substrate 5900 from which the device is made.", "Substrate 5900 generally includes a sacrificial layer 5902 disposed between a base layer 5904 and a device layer 5906.The substrate may be formed by a silicon on insulator (SOI) fabrication process.", "When an SOI substrate is used, sacrificial layer 5902 is typically an oxide formed by oxidizing a silicon base layer 5904.Such a structure is sometimes referred to as silicon on oxide (SOI).", "Alternatively, sacrificial layer 5902 may be a nitride layer, in which case the structure is sometimes referred to as silicon on nitride.", "Other possible configurations for substrate 5900 include silicon on polymer, glass on silicon, glass on nitride and other multiple-layer substrates.", "Next one or more vias 5908 are formed through device layer 5906 all the way to sacrificial layer 5902 as shown in FIG.", "59B.", "Vias 5908 may be formed in device layer 5906 by dry etch processes, such as reactive ion etching (RIE) or wet etch processes, e.g., anisotropic etching of Si with KOH.", "In such etch processes, sacrificial layer 5902 often resists attack by etchants used to form vias 5908 and therefore acts as an etch stop.", "Alternatively, vias 5908 may be laser drilled or formed by local oxidation (LOCOS) and oxide etch.", "After vias 5908 have been formed in device layer 5906 sacrificial layer 5902 is partially etched as shown in FIG.", "59C.", "The etching of sacrificial layer 5902 forms one or more depressions 5910 having a depth d at locations corresponding to locations of vias in the device layer.", "A different etch process than that used to form vias 5908 may be used to form depressions 59580.By whatever process they are formed, depressions 5910 do not penetrate all the way through to base layer 5904.In other words the depth d of the depressions is less than the thickness t of sacrificial layer 5902.After forming depressions 5910, vias 5908 and depressions 5910 are filled with a layer landing pad material 5920 as shown in FIG.", "59D.", "Layer 5920 may optionally be planarized down to a top surface 5905 of device layer 5906, e.g.", "by chemical mechanical polishing (CMP).", "Filling depressions 5910 and vias 5908 forms a structure 5922 having one or more landing pads 5924 protruding from an underside 5907 of device layer 5906.Each landing pad 5924 is connected to structure 5920 by a plug 5926 of material that fills via 5908.The depth d of depressions 5910 determines the thickness of landing pads 5924.If depressions 5910 are formed such that they undercut device layer 5906, e.g., by isotropic etching.", "Landing pad 5924 can have a larger diameter than a diameter of plug 5926 resulting in a landing pad structure similar to that shown in FIG.", "58B.", "The landing pad structure shown in FIG.", "58C may be fabricated by partially etching plugs 5926 and filling the resulting void with a layer of material.", "In a particular embodiment landing pad material 5920 is deposited inside vias 5908 to a thickness of at least one-half the diameter of a widest via 5908 to ensure that the landing pad material 5920 plugs the vias.", "Pad material layer 5920 and device layer 5906 generally comprise a device 5930.After landing pad material 5920 has been deposited, sacrificial layer 5902 is removed to release device 5930 as shown in FIG.", "59E.", "Sacrificial layer 5902 may be removed by any suitable method, such as wet etch or other isotropic etch process.", "Devices of the type shown in FIGS.", "58A-58C may alternatively be fabricated by a method according to another embodiment of the invention.", "The basic steps of the method are depicted in FIGS.", "60A-60E.", "FIG.", "60A depicts a basic substrate 6000 from which the device is made.", "Substrate 6000 generally includes a sacrificial layer 6002 disposed on top of a base layer 6004.Substrate 6000 may be formed by a silicon-on-insulator (SOI) fabrication process, e.g.", "by oxidizing a silicon base layer 6004.Alternatively, an oxide or nitride layer may be deposited on top of base layer 6004.Other possible configurations for substrate 6000 include silicon on polymer, glass on silicon, glass on nitride and the like.", "Next sacrificial layer 6002 is partially etched to form one or more depressions 6010 as shown in FIG.", "60B.", "Depressions 6010 having a depth d that is less than the thickness t of sacrificial layer 6002.After depressions 6010 have been formed, a device layer 6006 is bonded to sacrificial layer 6002 as shown in FIG.", "60C.", "Device layer 6006 may be any suitable material depending on the desired application.", "In a specific embodiment, device layer 6006 is a layer of silicon.", "Next one or more vias 6008 are formed through device layer 6006 all the way through to depressions 6010 in sacrificial layer 6002 as shown in FIG.", "60D.", "Vias 6008 may be formed in device layer 6006 by dry etch processes, such as reactive ion etching (RIE) or wet etch processes, e.g., anisotropic etching of Si with KOH as described above.", "Alternatively, vias 6008 may be laser drilled or formed by local oxidation (LOCOS) and oxide etch.", "In the embodiment shown, depressions 6010 have a diameter that is greater than a diameter of vias 6008.Alternatively, the diameter of depressions 6010 may be the same as or smaller than the diameter of vias 6008.After vias 6008 have been formed in device layer 6006, vias 6008 and depressions 6010 are filled with a layer landing pad material 6020 as shown in FIG.", "60E.", "Layer 6020 may optionally be planarized down to a top surface 6005 of device layer 6006, e.g.", "by chemical mechanical polishing (CMP).", "Filling depressions 6010 and vias 6008 forms a structure having one or more landing pads 6024 protruding from an underside 6007 of device layer 6006.Each landing pad 6024 is connected to the structure by a plug 6026 of material that fills via 6008.The depth d of depressions 6010 determines the thickness of landing pads 6024.Because depressions 6010 have larger diameters that vias 6008, landing pads 6024 have a larger diameter than plugs 6026 resulting in a landing pad structure similar to that shown in FIG.", "58B.", "Pad material layer 6020 and device layer 6006 generally comprise a device 6030.After landing pad material 6020 has been deposited, sacrificial layer 6002 is removed to release device 6030 as shown in FIG.", "60F.", "Sacrificial layer 6002 may be removed by any suitable method, such as wet etch or other isotropic etch process.", "Another embodiment of the present invention includes an electromechanically actuatable mirror element of a type used in optical fiber switching arrays.", "Micromechanical elements are described in U.S.", "Provisional Patent application Ser.", "No.", "60/123,496 to Berhang Behin, Kam Lau and Richard Muller, titled “Global Mechanical Stop for Precise Mirror Positioning”” which is incorporated herein by reference.", "An example of an embodiment of such a mirror element 6100 according the present invention is depicted in FIG.", "61A.", "Mirror element 6100 generally is formed from a device layer 6102 as described above with respect to FIGS.", "59A-59E or 60A-60F.", "At least one landing pad 6104 protrudes from an underside 6106 of mirror element 6100.Landing pad 6104 is attached to mirror element 6100 by a plug 6108 passing through an opening 6110 in mirror element 6100.Landing pad 6104 provides a smaller contact area 6112 that serves to reduce stiction between mirror element 6100 and an underlying substrate 6114.Stiction may also be reduced by proper choice of the material comprising landing pad 6104.Mirror element 6100 may be attached to mirror layer 6102 via one or more compliant flexures 6116.Such a mirror element can be actuated between an ‘on’ position, at which it intercepts an optical beam 6111 as shown in FIG.", "61B, and an ‘off’ position, at which it allows optical beam 6111 to pass as shown in FIG.", "61A.", "A second chip 6120 containing vertical sidewall 6122 may be positioned on top of the chip containing mirror 6100, so that when flipped vertically by application of a magnetic field, mirror 6100 can be pulled in to the sidewall by application of an electrostatic force.", "Electrostatic clamping to a precise vertical clamping surface 6124 on a sidewall 6126 defines the mirror position accurately and reproducibly when it is in the ‘on’ position.", "To ensure that all mirrors in an array have the same ‘on’ angle, the clamping surfaces for different mirrors may be constructed at the same angle on a single substrate.", "This provides a global mechanical positioning mechanism for a field of actuated mirrors.", "Mirror element 6100 and the clamping surfaces are typically constructed from electrically conductive material.", "Landing pads 6104 made of an insulating material may prevent electrical contact between the mirrors and the clamping surfaces when the two conductive surfaces are brought together.", "Flexures 6116 allow mirror element 6100 to move out of a plane defined by mirror layer 6102 as shown in FIG.", "61B.", "Mirror actuation may be performed by applying an external magnetic field that interacts with a magnetic material on the mirror element 6100.Lateral compliance of the torsional flexures 6116 reduces the electric field necessary to pull the mirror to sidewall 6122, and allows mirror element 6100 to make contact with sidewall 6122 at three or more points when sufficient electrostatic field is applied.", "After mirror element 6100 is electrostatically clamped to sidewall 6122, the magnetic field can be switched on and off without affecting the mirror position.", "Once the electrostatic field is turned off, torsional flexures 6116 pull mirror element 6100 back to its horizontal position by torsional flexures 6116.A horizontal magnetic field may also be employed to aid in actuating mirror element 6100 back to the horizontal position.", "Mirror 6100, in the horizontal position, can be clamped electrostatically to substrate 6114 to prevent it from responding to an external field.", "Selective electrostatic clamping of mirrors in both the vertical and horizontal positions allows individual addressing of mirrors belonging to an array and subject to the same external magnetic field.", "Mirror 6100 and the top chip 6120 may be fabricated by silicon microfabrication techniques such as polycrystalline-silicon surface-micromachining process.", "The top chip 6120 containing the sidewalls 6122 may be fabricated by anisotropic etching of (110)-oriented Si.", "Such a method ensures angular uniformity of the sidewalls over the area of the top chip.", "Furthermore, mirror 6100 may be fabricated from a silicon on insulator (SOI) substrate by either of the two methods described above.", "Devices of the type shown in FIGS.", "58A-58C and 61 may alternatively be fabricated by a method according to another embodiment of the invention.", "The basic steps of the method are depicted in FIGS.", "62A-62F.", "FIG.", "62A depicts a substrate 6200 from which the device is made.", "Substrate 6200 generally comprises a device layer 6201 and a landing pad material layer 6202.Device layer 6201 may be any suitable material depending on the desired application.", "In a specific embodiment, device layer 6201 is a layer of silicon.", "Landing-pad material layer 6202 may be deposited or formed on a surface of device layer 6201 by any conventional means.", "Next landing-pad material layer 6202 is partially etched to form one or more landing pads 6204 having a height h as shown in FIG.", "62B.", "Landing pads 6204 may be formed by any suitable technique such as reactive ion etching (RIE) or wet etch processes, e.g., anisotropic etching of silicon with KOH as described above.", "Alternatively, landing pads 6204 may be ion milled or formed by local oxidation (LOCOS) and oxide etch.", "Next, a sacrificial layer 6206 is deposited over landing pads and substrate 6200 as shown in FIG.", "62C.", "Typically, sacrificial layer 6206 includes an oxide.", "Alternative sacrificial layers include nitrides, glasses and polymers.", "Sacrificial layer 6206 is typically planarized, e.g.", "by CMP to a thickness t. Preferably, height h of landing pads 6204 is less than thickness t of sacrificial layer 6206 so that landing pads 6204 are not exposed.", "If the landing pads 6204 may have different heights t is preferably greater than the height of the tallest landing pad.", "After sacrificial layer 6206 has been planarized, substrate 6200 may be inverted so that sacrificial layer 6206 faces a base layer 6208 as shown in FIG.", "62D.", "As a result of this step, landing-pads 6204 protrude from an underside 6209 of substrate 6200.Next, substrate 6200 is bonded to base layer 6208 via sacrificial layer 6206 as shown in FIG.", "62E.", "Device layer 6201 and landing pads 6204 generally comprise a device 6220, which may be released by removing sacrificial layer 6206 as shown in FIG.", "62F.", "Sacrificial layer 6206 may be removed by any suitable method, such as wet etch or other isotropic etch process.", "Other variations on the above described devices and fabrication methods are possible.", "For example, in another embodiment of any of the above-described fabrication methods may be used to fabricate a device having landing pads with “air-spaced” standoffs.", "The device 6300, depicted in FIGS.", "63A-63B, includes a device layer 6302 and at least one landing pad 6304 protruding from an underside 6306 of device layer 6302.Landing pad 6304 has a plug 6305 that protrudes through an opening 6310 in device layer 6302.Landing pad 6304 is attached to device layer 6302 by a flange 6308.The landing pad 6304 provides a smaller contact area 6312 than an area of underside 6306.Plug 6305 generally has a diameter that is smaller than a diameter of opening 6310.This configuration produces a gap 6311 between plug 6305 and device layer 6302.The resulting structure provides an air-spaced standoff.", "Flanges 6310 on neighboring landing pads 6304 may be isolated from each other as shown in FIGS.", "63A-63B.", "Alternatively, neighboring landing pads 6304 may protrude from a common layer of landing pad material.", "Device 6300 may be manufactured on a substrate 6301 according to any of the methods described above.", "For example, FIG.", "63A depicts the device 6300 prior to removal of a sacrificial layer 6320.FIG.", "63B depicts the device 6300 after removal of a sacrificial layer 6320.In another embodiment of the invention, a device 6400 may be manufactured with one or more standoffs separated from the rest of the device layer as depicted in FIGS.", "64A-64B.", "The device 6400 is typically manufactured on a substrate 6401 having a sacrificial layer 6420 as shown in FIG.", "64A.", "The device 6400 includes a device layer 6402 and at least one landing pad 6404 protruding from an underside 6406 of device layer 6402.Landing pad 6404 has a plug 6405 that protrudes through an opening 6410 in a standoff region 6403 of device layer 6402.Landing pad 6404 is attached to standoff region 6403 by a flange 6408.One or more trenches 6410 formed in device layer 6402 separate standoff region 6403 from the rest of device layer 6402.During fabrication, sacrificial layer 6420 mechanically supports device layer 6402, standoff region 6403 and landing pad 6404.A layer of support material 6412, formed over trenches 6410 provides a connection between standoff region 6403 and the rest of device layer 6402.Standoff region 6403 and landing pad 6404 form a separated standoff 6414 when sacrificial layer 6420 is removed, as shown in FIG.", "64B.", "Support material 6412 provides a mechanical structural support for standoff 6414 and device layer 6402 after sacrificial layer 6420 is removed.", "Such a configuration is useful, for example, in applications where it is desirable to electrically isolate standoff 6414 from device layer 6402.While the above is a complete description of the preferred embodiments of the present invention, it is possible to use various alternatives, modifications and equivalents.", "Therefore, the scope of the present invention should be determined not with reference to the above description but should, instead, be determined with reference to the appended claims, along with their full scope of equivalents.", "The appended claims are not to be interpreted as including means-plus-function limitations, unless such a limitation is explicitly recited in a given claim using the phrase “means for.”" ] ]	Patent_10469516
[ [ "Data processing apparatus and system and method for controlling memory access", "A data processor comprises a memory having storage elements arranged in columns and a number of column decoders, each having a memory access port.", "The data processor has a plurality of processing elements, and each of the memory ports is coupleable to at least a respective one of the processor elements, such that each processor element is capable of accessing at least one column of storage elements." ], [ "1.A data processor apparatus comprising a plurality of processing elements, a memory having a plurality of storage elements arranged in a plurality of columns, a plurality of column decoders, a plurality of memory ports coupled to said decoders for at least one outputting data from said memory and receiving data for said memory, each of said plurality of memory ports being couplable to at least a respective one of said plurality of processor elements such that each processor element is capable of accessing at least one column of storage elements, a device for accessing said memory, switch means for switchably coupling each one of said plurality of memory ports to a respective processing element and for switchably coupling each one of said plurality of memory ports to said device, and a memory access controller arranged to control said switch means to selectively couple said plurality of memory ports to one of (i) said device and (ii) said plurality of processor elements.", "2.A data processor apparatus as claimed in claim 1, further comprising a data bus for coupling said memory ports to said device and further comprising a bus decoder for coupling selected memory ports to said data bus.", "3.A data processor apparatus as claimed in claim 2, wherein said data bus comprises a plurality of bus lines, and said bus decoder is arranged to selectively couple selected memory ports to selected bus lines.", "4.A data processor apparatus as claimed in claim 3, wherein the number of memory ports is greater than the number of bus lines.", "5.A data processor apparatus as claimed in claim 2, wherein said data bus comprises a plurality of bus lines, and said bus decoder is arranged to selectively couple selected bus lines to said plurality of memory ports.", "6.A data processor apparatus as claimed in claim 5, wherein the number of bus lines is greater than the number of memory ports.", "7.A data processor apparatus as claimed in claim 1, further comprising a data bus for coupling said memory ports to said device, said data bus having a plurality of bus lines wherein the number of bus lines is different to the number of memory ports, and decoding means between said memory ports and said data bus for one of coupling selected ones of said memory ports to said bus lines, if the number of memory ports exceeds the number of bus lines, and coupling selected ones of said bus lines to said memory ports, if the number of bus lines exceeds the number of memory ports.", "8.A data processor apparatus as claimed in claim 1, wherein said processing elements are arranged to perform operations when said plurality of memory ports are coupled to said device.", "9.A data processor apparatus as claimed in claim 8, wherein said processing elements are arranged to process data previously read from said memory when said plurality of memory ports are coupled to said device.", "10.A data processor apparatus as claimed in claim 1, further comprising at least one storage element for storing data received from a memory port before being processed by a respective processor element.", "11.A data processor apparatus as claimed in claim 10, comprising at least one respective storage element coupled to each of said plurality of memory ports for storing data received from said memory ports before being processed by said plurality of processor elements.", "12.A data processor apparatus as claimed in claim 11, comprising two or more storage elements for storing data from each of said plurality of memory ports before being processed by said processor elements.", "13.A data processor apparatus as claimed in claim 1 wherein said device comprises a processor, a direct memory access (DMA) device or an input/output (I/O) device.", "14.A data processor apparatus as claimed in claim 2, further comprising another device coupled to said data bus.", "15.A data processor apparatus as claimed in claim 1, wherein said memory ports each comprise an I/O port.", "16.A data processor apparatus as claimed in claim 1, wherein said memory comprises a random access memory (RAM) and each of said memory ports comprises an input/output (I/O) port of said RAM.", "17.A data processor apparatus as claimed in claim 1, wherein each of said memory ports comprises a one-bit memory port and further comprising a single bit line of a parallel data bus between each memory port and a respective processor element for one of read access and write access.", "18.A data processor apparatus as claimed in claim 2, further comprising an array controller coupled to said data bus for controlling parallel operations of said processing elements.", "19.A data processor apparatus as claimed in claim 1, further comprising a data bus for coupling said memory ports to said device and an array controller coupled to said data bus for controlling parallel operations of said processor elements.", "20.A device comprising a memory having a plurality of memory ports for at least one of outputting data from said memory and receiving data for said memory, a data bus having a plurality of bus lines, wherein the number of bus lines is different to the number of memory ports, and decoding means between said memory ports and said data bus for one of coupling selected ones of said memory ports to said bus lines, if the number of memory ports exceeds the number of bus lines, and coupling selected ones of said bus lines to said memory ports, if the number of bus lines exceeds the number of memory ports.", "21.A device as claimed in claim 20, further comprising a processor coupleable to said plurality of memory ports, independently of said data bus.", "22.A device as claimed in claim 21, wherein said processor comprises a plurality of processing elements, each coupleable to a respective memory port.", "23.A device as claimed in claim 21, further comprising memory access control means for selectively coupling one of said data bus and said processor to said memory ports." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>In a typical computer system having multiple computer processor units (CPUs) which require access to a common memory, the CPUs and memory are connected to a data communication bus for shared memory access.", "An example of a multi-CPU system is shown in FIG.", "1 .", "The system 1 includes a number of microprocessors 3 , 5 and other devices such as a Direct Memory Access (DMA) device 7 and an input/output (I/O) device 9 connected to a data communication bus 11 , which is also connected to a number of shared memory blocks 13 , 15 by respective memory interface units (MIU) 17 , 19 .", "One problem with this implementation is that only one memory can be accessed by only one microprocessor or other device at any one time through the data communication bus, which often leads to a bottle neck or congestion in data transfer.", "For example, if microprocessors 3 , 5 both require access to a memory at the same time, and one of the microprocessors has priority over the other, the microprocessor having lower priority has to wait until memory access by the higher priority microprocessor is complete.", "This problem becomes greater as the number of devices connected to the data communication bus increases, so that, for example, access waiting times for other devices such as the DMA and input/output devices become significantly large.", "Another form of data processor is the single-instruction-multiple-data (SIMD) processor, which has multiple processor units each having its own associated memory space.", "The processor units are simple processors, unable to fetch or interpret instructions, and are controlled by a single control unit, so that the processor units act as slaves to the control unit, performing at its request, arithmatic-logic operations.", "A typical SIMD architecture is depicted in FIG.", "2 .", "The data processor 21 has a number of processing units 23 , 25 each coupled to an associated memory 27 , 29 .", "The data processor has a control unit (not shown) for controlling the processing units in parallel via a data communication bus 33 and other devices such as a DMA 35 and an input/output device 37 , which are also connected to the data communication bus.", "One advantage of this system is that more memory and processor units can be easily added to the computer.", "However, a disadvantage of this system is that when a processor unit requires access to the memory space of another processor unit, the transfer of data is managed by the control unit, which therefore consumes control unit processing time or cycles, and during the time data is being moved around, the processor units remain idle.", "Another example of a SIMD processor is described in U.S. Pat.", "No.", "5,956,274 issued on 21 Sep., 1999 to Duncan G. Elliot, et al, and is shown schematically in FIG.", "3 .", "In this architecture, the processing units 33 are placed within the memory, there being one processor unit per column of storage elements, each processor unit being directly coupled to the sense amplifier of each column, and whose output is coupled to the memory column decoder.", "While this architecture provides a large number of processor units, each tightly coupled to its own memory space, when the microprocessor requires access to memory, the processor elements must remain idle.", "A further disadvantage of this architecture is that the memory must be designed specifically to incorporate the processing elements." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>According to one aspect of the present invention, there is provided a data processor apparatus comprising a memory having a plurality of storage elements arranged in a plurality of columns, a plurality of column decoders, a plurality of memory ports coupled to the decoders for at least one of outputting data from the memory and receiving data for the memory, and a plurality of processing elements, wherein each of the plurality of memory ports is coupleable to at least a respective one of the plurality of processor elements, such that each processor element is capable of accessing at least one column of storage elements.", "In this arrangement, the processor elements are coupleable to the external interface ports of the memory, rather than being embedded in the memory between the sense amplifiers and column decoder.", "Advantageously, this architecture enables a parallel data processor to be realized having a plurality of processing elements each having access to its own portion of memory, but without the requirement for knowledge of the internal memory structure, thereby considerably simplifying design, reducing design time, and offering designers the flexibility of using any suitable memory for the intended application.", "In one embodiment, the data processor apparatus includes switch means between at least one, and preferably each of the memory ports, and at least one, and preferably each of the processor elements, for selectively coupling and decoupling the memory port(s) to and from the processor element(s).", "Advantageously, this arrangement enables the processor elements to be decoupled from the memory, so that the memory can be accessed by another device.", "At the same time, this allows the processor elements to continue to perform operations, for example processing data which was previously read from the memory.", "In one embodiment, at least one storage element is provided for at least one and preferably each processor element for storing data read from the memory before being processed by the processing elements.", "In one embodiment, the storage elements can be decoupled from the memory, again to enable the memory to be accessed by another device while allowing the processor elements to process data stored in the storage element(s).", "According to another aspect of the present invention, there is provided a data processor apparatus comprising a memory having a plurality of memory ports for at least one of outputting data from the memory and receiving data for the memory, a processor coupleable to the memory ports, and a data bus coupleable to the memory ports, and a memory access controller for selectively coupling and decoupling the data bus to and from the memory ports.", "Advantageously, this arrangement allows the data bus to be decoupled from the memory, so that the data bus can be used to transfer data, for example between different devices connected to the data bus, while the memory is being accessed by the processor.", "According to another aspect of the present invention, there is provided a memory device comprising a memory having a plurality of memory ports for at least one of outputting data from the memory and receiving data for the memory, first and second data buses, each being coupleable to the memory ports, and memory access control means for selectively coupling one of the first and second data buses to the memory ports.", "Advantageously, this arrangement enables each of the data buses to be decoupled from the memory so that the decoupled data bus can continue to be used by other devices, while the other data bus is coupled to the memory.", "According to another aspect of the present invention, there is provided a memory device comprising a memory having a plurality of memory ports for at least one of outputting data from the memory and receiving data for the memory, a data bus having a plurality of bus lines, wherein the number of bus lines is different to the number of memory ports, and decoding means between the memory ports and the data bus for one of coupling selected ones of the memory ports to the bus lines, if the number of memory ports exceeds the number of bus lines, and coupling selected ones of the bus lines to the memory ports, if the number of bus lines exceeds the number of memory ports.", "Advantageously, this arrangement provides a decoder coupled between the memory ports and a data bus having a different number of serial bit lines to the number of memory ports, and controls the selection of which memory ports are coupled to which serial bus lines to enable any size of data bus full access to any size of memory, and vice versa." ], [ "FIELD OF THE INVENTION The present invention relates to a data processor apparatus, and in particular to a system and method for controlling access to a memory which is shared by two or more data processors or other devices.", "BACKGROUND OF THE INVENTION In a typical computer system having multiple computer processor units (CPUs) which require access to a common memory, the CPUs and memory are connected to a data communication bus for shared memory access.", "An example of a multi-CPU system is shown in FIG.", "1.The system 1 includes a number of microprocessors 3, 5 and other devices such as a Direct Memory Access (DMA) device 7 and an input/output (I/O) device 9 connected to a data communication bus 11, which is also connected to a number of shared memory blocks 13, 15 by respective memory interface units (MIU) 17, 19.One problem with this implementation is that only one memory can be accessed by only one microprocessor or other device at any one time through the data communication bus, which often leads to a bottle neck or congestion in data transfer.", "For example, if microprocessors 3, 5 both require access to a memory at the same time, and one of the microprocessors has priority over the other, the microprocessor having lower priority has to wait until memory access by the higher priority microprocessor is complete.", "This problem becomes greater as the number of devices connected to the data communication bus increases, so that, for example, access waiting times for other devices such as the DMA and input/output devices become significantly large.", "Another form of data processor is the single-instruction-multiple-data (SIMD) processor, which has multiple processor units each having its own associated memory space.", "The processor units are simple processors, unable to fetch or interpret instructions, and are controlled by a single control unit, so that the processor units act as slaves to the control unit, performing at its request, arithmatic-logic operations.", "A typical SIMD architecture is depicted in FIG.", "2.The data processor 21 has a number of processing units 23, 25 each coupled to an associated memory 27, 29.The data processor has a control unit (not shown) for controlling the processing units in parallel via a data communication bus 33 and other devices such as a DMA 35 and an input/output device 37, which are also connected to the data communication bus.", "One advantage of this system is that more memory and processor units can be easily added to the computer.", "However, a disadvantage of this system is that when a processor unit requires access to the memory space of another processor unit, the transfer of data is managed by the control unit, which therefore consumes control unit processing time or cycles, and during the time data is being moved around, the processor units remain idle.", "Another example of a SIMD processor is described in U.S. Pat.", "No.", "5,956,274 issued on 21 Sep., 1999 to Duncan G. Elliot, et al, and is shown schematically in FIG.", "3.In this architecture, the processing units 33 are placed within the memory, there being one processor unit per column of storage elements, each processor unit being directly coupled to the sense amplifier of each column, and whose output is coupled to the memory column decoder.", "While this architecture provides a large number of processor units, each tightly coupled to its own memory space, when the microprocessor requires access to memory, the processor elements must remain idle.", "A further disadvantage of this architecture is that the memory must be designed specifically to incorporate the processing elements.", "SUMMARY OF THE INVENTION According to one aspect of the present invention, there is provided a data processor apparatus comprising a memory having a plurality of storage elements arranged in a plurality of columns, a plurality of column decoders, a plurality of memory ports coupled to the decoders for at least one of outputting data from the memory and receiving data for the memory, and a plurality of processing elements, wherein each of the plurality of memory ports is coupleable to at least a respective one of the plurality of processor elements, such that each processor element is capable of accessing at least one column of storage elements.", "In this arrangement, the processor elements are coupleable to the external interface ports of the memory, rather than being embedded in the memory between the sense amplifiers and column decoder.", "Advantageously, this architecture enables a parallel data processor to be realized having a plurality of processing elements each having access to its own portion of memory, but without the requirement for knowledge of the internal memory structure, thereby considerably simplifying design, reducing design time, and offering designers the flexibility of using any suitable memory for the intended application.", "In one embodiment, the data processor apparatus includes switch means between at least one, and preferably each of the memory ports, and at least one, and preferably each of the processor elements, for selectively coupling and decoupling the memory port(s) to and from the processor element(s).", "Advantageously, this arrangement enables the processor elements to be decoupled from the memory, so that the memory can be accessed by another device.", "At the same time, this allows the processor elements to continue to perform operations, for example processing data which was previously read from the memory.", "In one embodiment, at least one storage element is provided for at least one and preferably each processor element for storing data read from the memory before being processed by the processing elements.", "In one embodiment, the storage elements can be decoupled from the memory, again to enable the memory to be accessed by another device while allowing the processor elements to process data stored in the storage element(s).", "According to another aspect of the present invention, there is provided a data processor apparatus comprising a memory having a plurality of memory ports for at least one of outputting data from the memory and receiving data for the memory, a processor coupleable to the memory ports, and a data bus coupleable to the memory ports, and a memory access controller for selectively coupling and decoupling the data bus to and from the memory ports.", "Advantageously, this arrangement allows the data bus to be decoupled from the memory, so that the data bus can be used to transfer data, for example between different devices connected to the data bus, while the memory is being accessed by the processor.", "According to another aspect of the present invention, there is provided a memory device comprising a memory having a plurality of memory ports for at least one of outputting data from the memory and receiving data for the memory, first and second data buses, each being coupleable to the memory ports, and memory access control means for selectively coupling one of the first and second data buses to the memory ports.", "Advantageously, this arrangement enables each of the data buses to be decoupled from the memory so that the decoupled data bus can continue to be used by other devices, while the other data bus is coupled to the memory.", "According to another aspect of the present invention, there is provided a memory device comprising a memory having a plurality of memory ports for at least one of outputting data from the memory and receiving data for the memory, a data bus having a plurality of bus lines, wherein the number of bus lines is different to the number of memory ports, and decoding means between the memory ports and the data bus for one of coupling selected ones of the memory ports to the bus lines, if the number of memory ports exceeds the number of bus lines, and coupling selected ones of the bus lines to the memory ports, if the number of bus lines exceeds the number of memory ports.", "Advantageously, this arrangement provides a decoder coupled between the memory ports and a data bus having a different number of serial bit lines to the number of memory ports, and controls the selection of which memory ports are coupled to which serial bus lines to enable any size of data bus full access to any size of memory, and vice versa.", "BRIEF DESCRIPTION OF THE DRAWINGS Examples of embodiments of the present invention will now be described with reference to the drawings, in which:— FIG.", "1 shows a block diagram of a multi-processor computer architecture according to the prior art; FIG.", "2 shows a block diagram of a single-instruction-multiple-data (SIMD) processor architecture, according to the prior art; FIG.", "3 shows a block diagram of another example of a SIMD processor architecture, according to the prior art; FIG.", "4 shows a block diagram of a data processor apparatus according to an embodiment of the present invention; FIG.", "5 shows a diagram of a data processor apparatus, according to another embodiment of the present invention; FIG.", "6 shows a diagram of a memory access controller according to an embodiment of the present invention; FIG.", "7 shows an example of a memory access controller according to another embodiment of the present invention; FIG.", "8 shows an example of a memory access controller according to another embodiment of the present invention, and FIG.", "9 shows a table of memory allocation for data received on two different buses, according to an embodiment of the present invention.", "DESCRIPTION OF EMBODIMENTS FIG.", "4 shows a data processor according to an embodiment of the present invention.", "The data processor 101 comprises a memory 103, e.g.", "a random access memory, having a plurality of storage elements 105 arranged in rows 107 and columns 109 (only a few of which are shown for clarity).", "The memory 103 includes a row decoder (or selector) 111, a plurality of sense amplifiers 113, one for each column 109 of storage elements, and a plurality of column decoders (or selectors) 115.Each sense amplifier 113 is arranged to amplify the signal either received on the memory column line to which it is connected (in the case of a memory read), or to amplify a received signal for outputting onto the column line (in the case of a memory write).", "Each column selector 115 has a plurality of input/output ports 117, one being connected to a respective input/output port of the sense amplifiers.", "In this embodiment, each column selector 115 is arranged to select from one of eight columns 109 of memory and to connect the selected column via a respective sense amplifier 113 to an I/O port 119.The memory columns, sense amplifiers and column selectors may extend laterally to include any number of memory columns, associated sense amplifiers and column selectors, to provide the required size of memory.", "In one embodiment, the memory 103 may comprise a memory bank containing a plurality of memory modules.", "The data processor 101 further includes a plurality of processing elements 121 each having an I/O port 123 connected to a respective I/O port 119 of the respective column selectors 115.In this embodiment, the processor elements are arranged in a one dimensional array, and there is one processor element per column selector 115, although in other embodiments, the data processor 101 may include a processor block having two or more processor elements coupleable to each memory I/O port.", "Advantageously, the processor architecture of the present embodiment, in which each processor element 121 is coupleable to a memory I/O port substantially simplifies the design process of integrating processor elements with a memory, e.g.", "RAM.", "One of the problems associated with the architecture described in U.S. Pat.", "No.", "5,956,274 (Elliot et al) is that by placing the processing elements between the sense amplifiers and the memory decoding circuits, access to the memory design is required.", "However, most high performance memory structure designs are strictly guarded company secrets, and therefore the only companies that are able to add structures within the memory column decoding circuits are the memory vendors themselves, and processor design is normally outside their scope of expertise, or would require too much time.", "In contrast, the present architecture allows any compileable memory structure to be used for the data processor, since the processor elements are coupled to the memory I/O ports, rather than to the sense amplifiers, and therefore detailed knowledge concerning the internal memory structure is not required.", "In the present embodiment, the connection between each processor element 117 and memory I/O port 119 can be regarded as a one bit line of a parallel data bus 127, which may have a width of any number of bits, e.g.", "16, 32, 64, 128, 192, 256 .", ".", ".", "etc., or any other number.", "FIG.", "5 shows a data processor according to another embodiment of the present invention.", "The data processor 201 comprises a memory 203, a parallel processing engine 205; an array controller 207, a memory arbitration unit 209, a data communication bus 211, a microprocessor 213 and one or more other devices 215, 217.The microprocessor 213 and the other devices 215, 217 are connected to the data communication bus 211.The memory arbitration unit 209 is coupled both to the data communication bus 211 and to the parallel processing engine 205, and is arranged to control access to the memory 203 by the microprocessor 213 or other device 215, 217 connected to the data communication bus 211, or by the parallel processing engine 205.The array controller 207 is also coupled to the data communication bus 211 and is arranged to control the parallel processing engine 205.The memory 203 has a plurality of I/O ports 219 (indicated schematically by the row of arrows between the memory 203 and the memory arbitration unit 209), which are coupleable through the memory arbitration unit to I/O ports 223 of the parallel processing engine 205 via a data bus 227.In one embodiment, the memory arbitration unit 209 is adapted to selectively permit data transfer between the memory and the data communication bus 211, or between the memory 203 and the parallel processing engine 205, where the data communication bus 211, which enables data communication to and from the microprocessor 213 (and other devices 215, 217), has a different number of single bit bus lines to the data bus 227, which enables data to be transferred between the memory 203 and the parallel processing engine 205.In another embodiment, the memory arbitration unit 209 is adapted to de-couple the parallel processing engine 205 from the memory 203, and to enable the processing engine 205 to continue to process data while at the same time permitting a device 213, 215, 217 coupled to the data communication bus 211 to access the memory 203.Embodiments of the arbitration unit 209 will now be described with reference to FIGS.", "6, 7 and 8.Referring to FIG.", "6, a data processing apparatus 301 comprises a memory 303, a first processor 305 and one or more optional peripheral devices 307, connected to a first data bus 309.The processing apparatus 301 further includes a second processor 311 and, optionally, one or more additional peripheral devices 313 connected to a second data communication bus 315.In this embodiment, the second data communication bus 315 has a greater number of single bit lines than the first communication bus 309, and in the particular embodiment shown in FIG.", "5, the first data communication bus has a single bit width of 32 (bit lines) and the second communication bus 315 has a single bit width of 192 (bit lines), although in other embodiments the first and second communication buses may have any other number of bit lines.", "A memory arbitration unit 317 includes a third data bus 319 having the same number of single bit lines (i.e.", "bit width) as the second data communication bus 315 (in this particular embodiment 192 bit lines), each coupled to an I/O port of the memory 303.The memory arbitration unit (MAU) 317 further includes a decoder 321, one side of which is coupled to the third communication bus 319 and the other side of the decoder being switchably coupled to the first communication bus 309 via a first switching unit 323.The third bus 319 of the MAU 317 is also switchably coupled to the second communication bus 315 via a second switching unit 325.The decoder 321 is capable of connecting each of the single bit lines of the first communication bus 309 to a selected single bit line of the third communication bus 319.For example, in read or write memory access, the decoder 321 may be controlled to connect the 32 bit lines of the first communication bus to the first 32 I/O ports of the memory via the first 32 of the 192 bit lines of the third communication bus 319, which allows, for example 4 bytes of data to be written to, or read from memory in parallel.", "In a subsequent operation, the decoder 321 may be controlled to connect each of the 32 bit lines of the first communication bus 309 to the next 32 I/O ports of the memory 303 via the next 32 bit lines of the 192 bit communication bus 319, thereby permitting a subsequent 4 bytes of data to be read from or written to the memory 303.The first switching unit 323 may comprise any suitable switching means which enables the first communication bus 309 to be switchably connected to and decoupled from the MAU bus 319.Preferably, the switching unit 323 is switchable between a closed position and an open (i.e.", "neutral or floating) e.g.", "tri-state position.", "In one embodiment, the switching unit may comprise a plurality of tri-stateable buffers, one connected in each single bit line between the decoder 321 and the first data communication bus 309.The second switching unit 325 may also comprise any suitable means which switchably connects and decouples the second data communication bus 315 to and from the MAU data communication bus 319.Preferably, the second switching unit is switchable between closed and open (i.e.", "neutral or floating) positions, and, as for the first switching unit 323, may comprise a plurality of switching elements, such as a tri-stateable buffer, one connected in each bit line between the second data communication bus 315 and the MAU data bus 319.The MAU 317 has a memory access controller 326 which controls access to the memory 303 by the device(s) coupled to the first and second data communication buses.", "The memory access controller may be arranged to arbitrate memory access between devices coupled to the same data communication bus 309, 311 and to arbitrate between devices coupled to different data communication buses 309, 315.In operation, the memory access controller may receive memory access requests from the various devices and may be arranged to control the connectivity between each device and memory based on predetermined rules, which may include different priorities assigned to different devices and round robin memory accesses for devices having equal priority.", "The memory access controller may be arranged to control the decoder 321, the first switching unit 323 to selectively connect and decouple the first data communication bus 309 to and from the memory 303, and/or the second switching unit 325 to selectively connect and decouple the second data communication bus 315 to and from the memory 303.Advantageously, the memory arbitration unit 317 allows data buses of different widths or capacity (i.e.", "having different numbers of single bit lines) to be selectively coupled to a memory, and therefore allows a memory to be shared between devices which handle different length words.", "The MAU 317 also enables a selected communication bus to be decoupled from the memory, so that the decoupled bus can continue to be used, for example, to transfer data between devices connected to the same bus.", "In one embodiment, the second processor 311 may include one or more registers for receiving data from the memory 303 prior to processing.", "Advantageously, this enables the processor to process data and at the same time the memory 303 to be accessed by another device, for example by the first processor 305, or by another peripheral device 307, 313.For example, while the second processor 311 is processing data, the result of a previous calculation by the second processor 311 stored in memory 303 may be output via the first data communication bus 309 to a device connected thereto, for example an output device.", "The second processor 311 may comprise a parallel processing engine containing a plurality of processor elements, similar to that described above with reference to FIGS.", "4 and 5.The processing engine may be arranged to perform parallel processing on a two-dimensional array of data representative of an image.", "While a calculation is being performed, for example on one image frame, the memory 303 may be accessed to output a previous image frame, calculated by the two-dimensional array processor and written to the memory 303.In another embodiment, the MAU 317 may be adapted to temporarily store data from the memory 303 prior to processing by the second processor 311, which again may permit the memory 303 to be accessed by another device while the processor accesses and/or processes the stored data.", "An example of a memory arbitration unit having a buffer or memory is shown in FIG.", "7.FIG.", "7 shows a data processor apparatus 301, which is similar to that shown in FIG.", "5, and like parts are designated by the same reference numerals.", "The data processor has a memory 303, a first processor 305 and optionally additional peripheral devices 307 connected to a data bus 309.The memory arbitration unit 317 includes a data communication bus 319, a decoder 321 and a switching unit 323, and the description of these components given above in connection with the embodiment of FIG.", "6 applies equally to the embodiment of FIG.", "7.The main difference between the embodiments of FIGS.", "6 and 7 is that, in the embodiment of FIG.", "7, the memory arbitration unit 317 includes a plurality of register units 327, one being connected to each single bit line of the MAU data communication bus 319.In this embodiment, each register unit 327 has first and second registers 329, 331 which are separately coupleable to a respective single bit line of the bus 319, and a two to one selector switch 333 for selectively connecting the output of one of the first and second registers to a single bit line 335, each of which is connected to an input of the second processor 311.The second processor may comprise a parallel processing engine, for example having a plurality of processing elements, each of which is capable of processing data received on a single bit line to which it is connected.", "For example, the parallel processing engine may be similar to that described above in connection with FIGS.", "4, 5 or 6.In this embodiment, the provision of register units 327 allows data to be written from the memory 303 into the registers for processing by the processor 311.Writing to the first and second registers of the register units 327 may be controlled by a write enable signal applied to the registers, as required.", "The registers also provide a means for decoupling the MAU bus 319 from the registers and the second processor 311, by disabling the write enable control signal.", "Thus, once data has been written to one or more of the first and second registers of each unit 327, the registers can be decoupled from the MAU bus 319, for example, by disabling the write enable control signal, so that the memory 303 can be accessed by another device, for example connected to the data communication bus 309.At the same time, data stored in one or more of the first and second registers can be accessed and processed by the second processor 311.In addition to controlling the switching operations of the decoder 321 and the switching unit 323, the memory access controller 326 may also be arranged to control write operations into each of the first and second registers 329, 331, and read operations from one or more of the registers into the second processor 311.In other embodiments, the register units 327 may have any number (i.e.", "one or more than one) registers, and the selector switch 333 may be omitted, for example, if the register unit contains a single register, and may be sized to switchably connect any of the registers to the second processor, if the register unit contains two or more registers.", "Advantageously, the more registers that are provided per single bit line, the greater the flexibility in controlling memory access scheduling, for example between the second processor 311 and other devices connected to the data communication bus 309.Furthermore, if more than one register is used, it is possible to design the MAU and the controller of the second processor 311 to schedule and perform memory reads during periods when the memory is less active.", "An embodiment of a data processing apparatus having a memory arbitration unit which controls write operations to memory from communication buses of different width is shown in FIG.", "8.The data processing apparatus 301 includes a memory 303, a first processor 305, and, optionally, one or additional devices 307 connected to a first data communication bus 309.The data processing apparatus also includes a second data processor 311 and, optionally, one or more further devices 313 connected to a second data communication bus 315.In this embodiment, the first communication bus comprises 32 single bit bus lines, and the second communication bus 315 has 192 single bit bus lines, although in other embodiments, the first and second data communication buses 309, 315 may have any other number of bit lines.", "The data processor 301 includes a memory arbitration unit 317, having a plurality of selector switches 351, each having an output port 353 and two input ports 357, 359.In this embodiment, each of the 192 single bit bus lines of the second data communication bus 315 maps onto a memory I/O port 355, and therefore the data processor apparatus includes 192 selector switches 351 (only two of which are shown), the output 355 of each of which is connected to a respective memory I/O port 355.One of the two input ports 357, 359 of each selector switch 351 is connected to a single bit line of the second data communication bus 315.The first communication bus 309 may be mapped onto the memory I/O ports in any desired configuration.", "In one embodiment, the first communication bus 309 is configured to enable byte length words or multiple byte length words to be written to memory.", "In one configuration, the 32 bit bus lines are divided into four groups of 8 bus lines, the first group of eight bus lines being coupled to the first inputs 357 of the first eight selector switches 351 for input to the first eight I/O ports of the memory, the second group of eight bit lines connected to the first input port 357 of the second group of eight selector switches 351, for connection to the next eight memory I/O ports, and so on, so that the third group of eight bit lines is connected to the third group of eight selector switches, and the fourth group of eight bit lines is connected to the fourth group of eight selector switches.", "As there are many more available I/O ports than there are bit lines on the 32 bit bus, the 32 bit lines may also be connected to the remaining I/O ports so that the bus has full access to the entire memory.", "In one embodiment, the first group of eight single bit lines of the first data communication bus 309 may be connected to the fifth group of eight selector switches, the second group of bit lines connected to the sixth group of eight selector switches, and so on, until the 32 bit bus has access to all memory I/O ports.", "The selector switches may be controlled to allow 32 bits of data to be written to memory in parallel.", "During a write enable, the other selector switches coupled to memory I/O ports to which the memory write is not required, are disabled (or masked), so that copies of the same data are not written to the memory, if this is the intention.", "The selector switches 351 may be enabled in groups of eight by a byte write enable signal, as shown in Table 1 of FIG.", "9.This allows the 32 bit data word to be divided into eight bit lengths, to allow a user to perform 8, 16 and 24 bit write operations.", "The selector switches may be controlled to permit byte lengths of a word having a length of two bytes or more either to be written into contiguous memory segments, or non-contiguous memory segments.", "In the embodiment of FIG.", "8, in which the second data bus has 192 bit lines, masked writes are not required since the bus width is the same as the width (i.e.", "number) of memory I/Os.", "Modifications and changes to the embodiments disclosed herein will be apparent to those skilled in the art." ] ]	Patent_10469526
[ [ "Stabilized biocompatible supported lipid membrane", "A lipid membrane is self-assembled and stabilized at a solid surface by depositing a lipid monolayer or a lipid multilayer on a substrate, otaining a supported lipid monolayer or a supported lipid multilayer; and in situ polymerizing the supported lipid monolayer or the supported lipid multilayer, thereby obtaining a polymerized membrane." ], [ "1.A method for the self-assembly and stabilization of a lipid membrane at a solid surface, comprising: depositing a lipid monolayer or a lipid multilayer on a substrate, thereby obtaining a supported lipid monolayer or a supported lipid multilayer; in situ polymerizing said supported lipid monolayer or said supported lipid multilayer, thereby obtaining a polymerized membrane.", "2.The method according to claim 1, wherein said polymerized membrane is at least partly cross-linked.", "3.The method according to claim 1, wherein said supported lipid monolayer or said supported lipid multilayer are formed by fusion of fluid, small unilamellar vesicles comprising a polymerizable lipid.", "4.The method according to claim 3, wherein said polymerizable lipid contains at least one of the polymerizable group selected from the group consisting of a styryl group, a dienyl group, a dienoyl group, a sorbyl group, an acryloyl group, a methacryloyl group, a vinyl ester group and a mixture thereof.", "5.The method according to claim 3, wherein said polymerizable lipid has a lipid tail having 14 to 22 carbon atoms.", "6.The method according to claim 3, wherein said lipid tail is an unsaturated or saturated linear tail or an unsaturated or saturated branched tail.", "7.The method according to claim 3, wherein a head group of said polymerizable lipid is selected from the group consisting of phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine and phosphatidylserine.", "8.The method according to claim 3, wherein said polymerizable lipid is terminated with a succinate group, a metal chelating group, a thioethanol group, a maleimido group, a pyridyldithio group, a biotinyl group, a succinimidyl ester group, a sulfo succinimidyl ester group, a alkyl halide group, a haloacetamide group, an ethylene glycol-based oligomer group or an ethylene glycol-based polymer group.", "9.The method according to claim 1, wherein said solid surface is a silicon dioxide surface, a silicon oxide surface, a noble metal surface, a mica surface, a polymer surface, an indium-tin oxide surface, a tin oxide surface, an indium oxide surface, a steel surface or a silicon surface.", "10.The method according to claim 1, wherein said in situ polymerizing is initiated by a redox initiator system.", "11.The method according to claim 10, wherein said redox initiator system is K2S2O8/NaHSO3.12.The method according to claim 1, wherein said in situ polymerizing occurs by irradiation with UV-rays, visible rays, near infrared rays or γ-rays.", "13.The method according to claim 12, wherein said UV-rays have a wavelength of between 230 and 350 nm.", "14.The method according to claim 12, wherein said VIS-rays have a wavelength of between 350 and 700 nm.", "15.The method according to claim 12, wherein said near infrared rays have a wavelength of between 700 and 1000 nm.", "16.The method according to claim 12, wherein said UV-rays, visible rays or near infrared rays are polarized or unpolarized.", "17.The method according to claim 3, wherein said polymerizable lipid is mixed with a non-polymerizable amphiphile.", "18.The method according to claim 17, wherein said non-polymerizable amphiphile is a lipid or a surfactant.", "19.The method according to claim 3, wherein a mixture of at least two polymerizable lipids is used.", "20.The method according to claim 1, wherein a membrane protein is incorporated into said polymerized membrane.", "21.The method according to claim 1, wherein water soluble protein is bonded to or adsorbed to said polymerized membrane.", "22.The method according to claim 1, wherein a structure of said polymerized membrane is preserved upon transfer into air and exposure to a surfactant solution or an organic solvent.", "23.A polymerized membrane obtained by the method according to claim 1.24.The polymerized membrane according to claim 23, wherein said polymerized membrane is at least partly cross-linked.", "25.The polymerized membrane according to claim 23, wherein said membrane is obtained using a mixture of a polymerizable lipid and a non-polymerizable amphiphile.", "26.The polymerized membrane according to claim 25, wherein said non-polymerizable amphiphile is a lipid or a surfactant.", "27.The polymerized membrane according to claim 23, wherein said membrane is obtained using a mixture of at least two polymerizable lipids.", "28.The polymerized membrane according to claim 23, wherein a membrane protein is incorporated into said polymerized membrane.", "29.The polymerized membrane according to claim 23, wherein a water soluble protein is bonded to or adsorbed to said polymerized membrane.", "30.The polymerized membrane according to claim 23, wherein a structure of said polymerized membrane is preserved upon transfer into air and exposure to a surfactant solution or an organic solvent.", "31.A spatially addressable, planar array of molecules deposited on the membrane according to claim 23.32.The array according to claim 31, wherein said membrane has a linearly polymerized portion and a cross-lined portion.", "33.A surface coated with the membrane according to claim 23.34.The surface according to claim 33, wherein said membrane comprises a protein.", "35.The surface according to claim 33 which is a silicon dioxide surface, a silicon oxide surface, a noble metal surface, a mica surface, a polymer surface, an indium-tin oxide surface, a tin oxide surface, an indium oxide surface, a steel surface or a silicon surface.", "36.The surface according to claim 33, wherein said polymerized membrane is at least partly cross-linked.", "37.The surface according to claim 33, wherein said membrane is obtained using a mixture of a polymerizable lipid and a non-polymerizable amphiphile.", "38.The surface according to claim 37, wherein said non-polymerizable amphiphile is a lipid or a surfactant.", "39.The surface according to claim 33, wherein said membrane is obtained using a mixture of at least two polymerizable lipids.", "40.The surface according to claim 33, wherein a membrane protein is incorporated into said polymerized membrane.", "41.The surface according to claim 33, wherein a water soluble protein is bonded to or adsorbed to said polymerized membrane.", "42.The surface according to claim 33, wherein a structure of said polymerized membrane is preserved upon transfer into air and exposure to a surfactant solution or an organic solvent.", "44.The surface according to claim 33, which is included in a medical implant material, an analytical fluid handling instrument, a biomedical device or a personal care product.", "45.A medical implant material, an analytical fluid handling instrument, a biomedical device or a personal care product, comprising: the membrane according to claim 23; and a solid surface.", "46.The medical implant material, the analytical fluid handling instrument, the biomedical device or the personal care product according to claim 44, wherein said solid surface is selected from the group consisting of a silicon dioxide surface, a silicon oxide surface, a noble metal surface, a mica surface, a polymer surface, an indium-tin oxide surface, a tin oxide surface, an indium oxide surface, a steel surface, a silicon surface and a combination thereof.", "47.The medical implant material, the analytical fluid handling instrument, the biomedical device or the personal care product according to claim 45, which contacts a biological sample or an organism.", "48.The medical implant material, the analytical fluid handling instrument, the biomedical device or the personal care product according to claim 45, wherein said personal care product is a razor blade." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to a self-assembled lipid membrane, in the form of a monolayer, bilayer, or multilayer, that is stabilized on a solid support.", "2.Discussion of the Background The development of durable, biomembrane-mimetic coatings for inorganic and polymeric surfaces that are resistant to nonspecific protein adsorption (protein resistant) is impacting numerous fields (Sackman, E., Science, 1996, 271, 43; Plant, A. L., Langmuir, 1999, 15, 5128; Marra, K. G.; Winger, T. M.; Hanson, S. R.; Chaikof, E. L., Macromolecules, 1997; 30, 6483; Wisniewski, N.; Reichert, M., Coll.", "Surf.", "B: Biointerfaces, 2000, 18, 197-219).", "One example is the design of a biosensor surface at which a ligand binding event must be detected in the presence of numerous other non-target proteins (Wisniewski, N.; Reichert, M., Coll.", "Surf.", "B: Biointerfaces 2000, 18, 197-219; Stelzle, M.; Weissmuller, G.; Sackman, E., J. Phys.", "Chem., 1993, 97, 2974; Duschl, C.; Liley, M.; Corradin, G.; Vogel, H., Biophys.", "J., 1994, 67, 1229; Song, X. D.; Swanson, B. I., Anal.", "Chem., 1999, 71, 2097; Parikh, A. N.; Beers, J. D.; Shreve, A. P.; Swanson, B. I., Langmuir, 1999, 15, 5369; Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E., Langmuir, 1993, 9, 136).", "In most optical and electrochemical sensors, the transducer is an oxide or noble metal surface to which dissolved proteins can irreversibly adsorb, “fouling” the sample/transducer interface.", "Planar lipid monolayer, bilayer, and multilayer structures have been used to coat such surfaces (Sackman, E., Science, 1996, 271, 43; Plant, A. L., Langmuir, 1999, 15, 5128; Song, X. D.; Swanson, B. I., Anal.", "Chem., 1999, 71, 2097; Parikh, A. N.; Beers, J. D.; Shreve, A. P.; Swanson, B. I., Langmuir, 1999, 15, 5369; Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E., Langmuir, 1993, 9, 136; Thompson, N. L.; Palmer, A. G., Comments Mol.", "Cell.", "Biophys., 1988, 5, 39; Watts, T. H.; Gaub, H. E.; McConnell, H. M., Nature, 1986, 320, 179; McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A.", "A., Biochim.", "Biophys.", "Acta., 1986, 864, 95; Meuse, C. W.; Krueger, S.; Majkrzak, C. F.; Dura, J.", "A.; Fu, J.; Connor, J. T.; Plant, A. L., Biophys.", "J., 1998, 74, 1388; Kalb, E.; Frey, S.; Tanun, L. K., Biochim.", "Biophys.", "Acta., 1992, 1103, 307; Edmiston, P. L.; Saavedra, S. S., Biophys.", "J., 1998, 74, 999; Majewski, J.; Wong, J. Y.; Park, C. K.; Seitz, M.; Israelachvili, J. N.; Smith, G. S., Biophys.", "J., 1998, 75, 2363; Hillebrandt, H.; Wiegrand, G.; Tanaka, M.; Sackmann, E., Langmuir, 1999, 15, 8451).", "Such lipid monolayers, bilayers, or multilayers offer the ability to minimize sensor “fouling”, i.e., the undesirable adsorption of non-target proteins and biomolecules invariably present in complex biological matrices, by exploiting the characteristic protein adsorption resistance associated with the phosphorylcholine (PC) lipid headgroup (Hayward, J.; Chapman, D., Biomaterials, 1984, 5, 135; Chapman, D., Langmuir, 1993, 9, 39; Malmsten, M. J., Colloid Interface Sci., 1995, 171, 106; Murphy, I. F.; Lu, J. R.; Lewis, L. L.; Brewer, J.; Russell, J.; Stratford, P., Macromolecules, 2000, 33, 4545).", "Additionally, their well-defined and controllable architecture may allow for favorable orientation and minimal denaturation of immobilized antigens or biomolecules such as fab antibody fragments, to maximize sensitivity of the device (Song, X. D.; Swanson, B. I., Anal.", "Chem., 1999, 71, 2097; Parikh, A. N.; Beers, J. D.; Shreve, A. P.; Swanson, B. I., Langmuir, 1999, 15, 5369; Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E., Langmuir, 1993, 9, 136; Viitala, T.; Vikholm, I.; Peltonen, J., Langmuir, 2000, 16, 4953-4961; Duschle, C.; Se(slash)vin-Landais, A. F.; Vogel, H., Biophys., 1996, 70, 1985-1995).", "Supported lipid membrane structures also provide the necessary environment for transmembrane receptor incorporation, which has been demonstrated by several authors through the fabrication of proteo-lipid structures with retained protein activity (Salafsky, J.; Groves, J. T.; Boxer, S. G., Biochem., 1996, 35, 14773-14781; Schmidt, E. X.; Liebermann, T.; Kreiter, M.; Jonczyk, A.; Naumann, R.; Offenhäusser, A.; Neumann, E.; Kukol, A.; Maelicke, A.; Knoll, W., Biosensors Bioelectronics, 1998, 13, 585-591; Naumann, R; Jonczyk, A.; Hampel, C.; Ringsdorf, H.; Knoll, W.; Bunjes, N., Gräber, P. Bioelectrochemistry and Bioenergetics, 1997, 42, 241-247; Fisher, M. L; Tjärnhage, T., Biosensors and Bioelectronics, 2000, 15, 463-471; Pun, G.; Gustafson, L; Artursson, E.; Ohlsson, P. A., Biosensors and Bioelectronics 1995, 10, 463-476; Puu, G.; Aartursson, E.; Gustafson, L; Lundsröm, M.; Jass, J., Biosensors and Bioelectronics, 2000, 15, 31-41; Graff, A.; Winterhalter, M.; Meier, W., Langmuir, 2001, 17, 919-923; Liley, M.; Bouvier, J.; Vogel, H. J., Coll.", "Inter.", "Sci., 1997, 194, 53-58; Naumann, R.; Schmidt, E. X.; Jonczyk, A.; Fendler, K.; Kadenback, B.; Liebermann, T.; Offenhäusser, A.; Knoll, W., Biosensors and Bioelectronics, 1999, 14, 651-662).", "Supported lipid monolayers, bilayers and multilayers can be self-assembled by fusion of fluid, unilamellar vesicles, an important issue for commercial application, onto a variety of optically or electrically active substrates.", "Furthermore, the recent development of micro-patterning techniques to modify planar, substrate supported thin films, including supported lipid bilayers, adds promise to the potential of biochips with parallel arrays of sensing elements for high throughput biological or pharmaceutical screening or sensing (Hovis, J. S.; Boxer, S. B., Langmuir, 2000, 16(3), 894-897; Hovis, J. S.; Boxer, S. B., Langmuir, 2001, 17(11), 3400-3405; Kam, L.; Boxer, S. G., J.", "Am.", "Chem.", "Soc., 2000, 122, 12901-12902; Toby, A.; Jenkins, A.; Boden, N.; Bushby, R. J.; Evans, S. D.; Knowles, P. F.; Miles, R. E.; Ogier, S. D.; Schönherr, H.; Vancso, J. G., J.", "Am.", "Chem.", "Soc., 1999, 121, 5271-5280; Groves, J. T.; Mahal, L. K.; Bertozzi, C. R., Langmuir, 2001; Srinivasan, M. P.; Ratto, T. V.; Stroeve, P.; Longo, M. L., Langmuir, 2001, 17, 7951-7954; Morigaki, K.; Baumgart, T.; Offenhäusser, A.; Knoll, W., Angew.", "Chem., Int.", "Ed., 2001, 40, 172).", "The key problem associated with implementing lipid structures in commercial molecular devise applications is the inherent lack of stability that arises from the exclusively non-covalent forces that are responsible for lipid lamellar assembly.", "As a result, partial or complete lamellar-structure loss is realized upon exposure to surfactants, organics, or removal of the film from aqueous environments.", "Finite aqueous lifetimes have also been observed, and lipid layer damage can occur upon fluid exchange, in the presence of soluble lipophilic proteins, or upon pH or temperature changes (Hui, S. W.; Viswanathan, R.; Zasadzinski, J.", "A.; Israelachvili, J. N., Biophys.", "J., 1995, 68, 171-178; Winger, T. M.; Ludovice, P. J.; Chaikof, E. L., Langmuir, 1999, 15, 3866-3874).", "These shortcomings prevent washing and reusing of a biosensor and seriously compromise the storage/shelf-life, reliability, and thus applicability of the device.", "Covalently bound self-assembled monolayers (SAMS) featuring oligo(ethylene glycol) (Yang, Z.; Galloway, J.", "A.; Yu, H., Langmuir, 1999, 15); or other protein adsorption resistant headgroups (Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G. M., J.", "Am.", "Chem.", "Soc., 2000, 122, 8303-8304) address the stability issue of biosensor coatings but are not without shortcomings, including an increased difficulty in functionalizing these films with water-soluble proteins in a well-defined manner, and not providing a suitable environment for transmembrane receptor proteins.", "Therefore, interest in stabilizing lipid films on solid supports continues to receive scientific attention.", "An alternate method for incorporating phosphorylcholine groups into a substrate supported polymer film is copolymer synthesis followed by direct grafting to the substrate surface (Murphy, E. F.; Lu, J. R.; Lewis, A. L.; Brewer, J.; Russell, J.; Stratford, P., Macromolecules, 2000, 33, 4545).", "However, the molecular architecture of this assembly is more difficult to control than that of a lipid-based film, and is not amenable to functionalization with transmembrane proteins (Murphy, E. F.; Lu, J. R.; Lewis, A. L.; Brewer, J.; Russell.", "J.; Stratford, P., Macromolecules, 2000, 33, 4545; Sackman, E., Science, 1996, 271, 43; Watts, T. H.; Gaub, H. E.; McConnell, H. M., Nature, 1986, 320, 179; McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A.", "A., Biochim.", "Biophys.", "Acta, 1986, 864, 95; Salafsky, J.; Groves, J. T.; Boxer, S. G., Biochemistry, 1996, 35, 14773; Brian, A.", "A.; McConnell, H. M., Proc.", "Natl.", "Acad.", "Sci., 1984, 81, 6159).", "Although the results achieved using supported lipid membranes as sensor coatings have been encouraging with respect to protein resistance, these structures lack the chemical and thermal stability required for technological implementation (e.g.", "as a non-fouling coating for a reusable biosensor).", "This is because the low molecular mass lipids in the bilayer are self-organized by relatively weak, noncovalent forces that are insufficient to maintain the bilayer structure when the membrane is, for example, removed from water.", "Strategies employed to stabilize planar lipid structures under water include: i) incorporation of template molecules, covalently attached either directly to the substrate or to a thin hydrophilic polymer, around which free lipids self-organize to form a bilayer (Duschl, C.; Liley, M.; Corradin, G.; Vogel, H., Biophys.", "J., 1994, 67, 1229; Yang, Z.; Yu, H., Langmuir, 1999, 15, 1731; Bunjes, N.; Schmidt, E. K.; Jonczyk, A.; Rippmann, F.; Beyer, D.; Ringsdorf, H.; Graber, P.; Knoll, W.; Naumann, R., Langmuir, 1997, 13, 6188) and ii) derivatization of a metal or silica surface with an alkyl self-assembled monolayer, followed by deposition of a lipid monolayer, creating a hybrid bilayer (Plant, A. L., Langmuir, 1999, 15, 5128; Stelzle, M.; Weissmuller, G.; Sackman, E. J., Phys.", "Chem., 1993, 97, 2974; Song, X. D.; Swanson, B. I., Anal.", "Chem., 1999, 71, 2097; Parikh, A. N.; Beers, J. D.; Shreve, A. P.; Swanson, B. I., Langmuir, 1999, 15, 5369; Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E., Langmuir, 1993, 9, 136; Meuse, C. W.; Krueger, S.; Majkrzak, C. F.; Dura, J.", "A.; Fu, J.; Connor, J. T.; Plant, A. L., Biophys.", "J., 1998, 74, 1388).", "Both strategies increase the stability of the structure in water while maintaining some degree of lateral lipid mobility.", "However, the integrity of these structures is compromised by lipid loss upon exposure to harsher environments, such as organic solvents, surfactant solutions, or transfer across the water/air interface.", "A considerable body of work has shown that the stability and permeability of lipid bilayer vesicles (liposomes) can be significantly altered by polymerization of lipids containing reactive moieties (O'Brien, D. F.; Armitage, B.; Benedicto, A.; Bennett, D.; Lamparski, H. G.; Lee, Y. S.; Srisiri W.; Sisson, T. M., Acc.", "Chem.", "Res., 1998, 31, 861; Regen, S. L.; Singh, A.; Oehme, G.; Singh, M. J., Amer.", "Chem.", "Soc., 1982, 104; 791; Sisson, T. M.; Lamparski, H. G.; Kolchens, S.; Elyadi, A.; O'Brien, D. F., Macromolecules, 1996, 29, 8321).", "For example, unilamellar vesicles composed of bis-substituted lipids can be polymerized to form cross-linked vesicles that are insoluble in surfactant solutions and organic solvents (Sisson, T. M.; Lamparski, H. G.; Kolchens, S.; Elyadi, A.; O'Brien, D. F., Macromolecules, 1996, 29, 8321).", "Several groups have prepared polymerized, multilamellar supported lipid films composed of commonly used diacetylenic PC lipids which can be stabilized by UV photopolymerization (Hayward, J.; Chapman, D., Biomaterials, 1984, 5, 135; Chapman, D., Langmuir, 1993, 9, 39; 21).", "However, to be efficiently polymerized, these lipids must be in the solid analogous phase (L β ), which is incompatible with the self-assembly methods of the present invention and does not produce a high percentage of monomer to polymer conversion (Hayward, J.; Chapman, D., Biomaterials, 1984, 5, 135; Chapman, D., Langmuir, 1993, 9, 39; Albrecht, O.; Johnston, D. S.; Villayerde, C.; Chapman, D., Biochim.", "Biophys.", "Acta, 1982, 687, 165; Binder, H.; Anikin, A.; Kohlstrunk, B. J., Phys.", "Chem., 1999, 103, 450-460).", "At least two research groups have used the polymerization strategy to stabilize lipid mono- and bilayers on solid supports.", "Regen and coworkers adsorbed films of mono- and di-acrylate functionalized lipids on poly(ethylene), followed by UV-photo-polymerization to form a supported polymerized lipid film of near monolayer thickness (Regen, S. L.: Kirszensztejn, P.; Singh, A., Macromolecules, 1983, 16, 338; Foltynowicz, Z.; Yamaguchi, K.; Czajka, B,.", "Regen, S. L., Macromolecules, 1985, 18, 1394).", "Their water contact angle data were indicative of a surface more hydrophobic than expected for a uniform array of PC groups, suggesting incomplete coverage and/or significant film disorder.", "However, the analytical tools (e.g.", "atomic force microscopy) needed to characterize film morphology and uniformity were not available at that time.", "More recently, Chaikof and coworkers formed a hybrid bilayer by fusing vesicles (Marra, K. G.; Winger, T. M.; Hanson, S. R.; Chaikof, E. L., Macromolecules, 1997; 30, 6483; Orban, J. M.; Faucher, K. M.: Dluhy, R. A.; Chaikof, E. L., Macromolecules, 2000, 33, 4205) composed of mono-acrylate lipids onto a support coated with an alkylsilane monolayer; in situ polymerization produced linear polymers in the upper leaflet of this structure.", "Although enhanced stability during extended incubation in water was observed, significant lipid desorption occurred when the assembly was exposed to surfactant." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It is therefore an object of the present invention to provide a lipid membrane which is a monolayer, bilayer, or multilayer that is self-assembled and stabilized at a solid surface.", "It is another object of the present invention to provide a solid supported lipid film that is stable to transfer into air and exposure to surfactant solutions and organic solvents, yet retains the protein resistance characteristic of a fluid lipid bilayer.", "It is yet another object of the present invention to include non-polymerizable amphiphilic molecules into a stabilized lipid membrane.", "It is another object of the present invention to provide a stabilized lipid membrane that is an appropriate environment for reconstitution of a transmembrane protein and/or a water-soluble protein with retention of native protein structure and activity.", "This and other objects have been achieved by the present invention the first embodiment which includes a method for the self-assembly and stabilization of a lipid membrane at a solid surface, comprising: depositing a lipid monolayer or a lipid multilayer on a substrate, thereby obtaining a supported lipid monolayer or a supported lipid multilayer; in situ polymerizing said supported lipid monolayer or said supported lipid multilayer, thereby obtaining a polymerized membrane." ], [ "RELATED APPLICATIONS This application claims priority to provisional U.S. patent application 60/274,591, filed Mar.", "9, 2001, and provisional U.S. patent application entitled “Stabilized, Biocompatible Supported Lipid Membrane,” filed Mar.", "8, 2002, both of which, and all references and patent applications cited therein are incorporated herein by reference.", "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to a self-assembled lipid membrane, in the form of a monolayer, bilayer, or multilayer, that is stabilized on a solid support.", "2.Discussion of the Background The development of durable, biomembrane-mimetic coatings for inorganic and polymeric surfaces that are resistant to nonspecific protein adsorption (protein resistant) is impacting numerous fields (Sackman, E., Science, 1996, 271, 43; Plant, A. L., Langmuir, 1999, 15, 5128; Marra, K. G.; Winger, T. M.; Hanson, S. R.; Chaikof, E. L., Macromolecules, 1997; 30, 6483; Wisniewski, N.; Reichert, M., Coll.", "Surf.", "B: Biointerfaces, 2000, 18, 197-219).", "One example is the design of a biosensor surface at which a ligand binding event must be detected in the presence of numerous other non-target proteins (Wisniewski, N.; Reichert, M., Coll.", "Surf.", "B: Biointerfaces 2000, 18, 197-219; Stelzle, M.; Weissmuller, G.; Sackman, E., J. Phys.", "Chem., 1993, 97, 2974; Duschl, C.; Liley, M.; Corradin, G.; Vogel, H., Biophys.", "J., 1994, 67, 1229; Song, X. D.; Swanson, B. I., Anal.", "Chem., 1999, 71, 2097; Parikh, A. N.; Beers, J. D.; Shreve, A. P.; Swanson, B. I., Langmuir, 1999, 15, 5369; Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E., Langmuir, 1993, 9, 136).", "In most optical and electrochemical sensors, the transducer is an oxide or noble metal surface to which dissolved proteins can irreversibly adsorb, “fouling” the sample/transducer interface.", "Planar lipid monolayer, bilayer, and multilayer structures have been used to coat such surfaces (Sackman, E., Science, 1996, 271, 43; Plant, A. L., Langmuir, 1999, 15, 5128; Song, X. D.; Swanson, B. I., Anal.", "Chem., 1999, 71, 2097; Parikh, A. N.; Beers, J. D.; Shreve, A. P.; Swanson, B. I., Langmuir, 1999, 15, 5369; Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E., Langmuir, 1993, 9, 136; Thompson, N. L.; Palmer, A. G., Comments Mol.", "Cell.", "Biophys., 1988, 5, 39; Watts, T. H.; Gaub, H. E.; McConnell, H. M., Nature, 1986, 320, 179; McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A.", "A., Biochim.", "Biophys.", "Acta., 1986, 864, 95; Meuse, C. W.; Krueger, S.; Majkrzak, C. F.; Dura, J.", "A.; Fu, J.; Connor, J. T.; Plant, A. L., Biophys.", "J., 1998, 74, 1388; Kalb, E.; Frey, S.; Tanun, L. K., Biochim.", "Biophys.", "Acta., 1992, 1103, 307; Edmiston, P. L.; Saavedra, S. S., Biophys.", "J., 1998, 74, 999; Majewski, J.; Wong, J. Y.; Park, C. K.; Seitz, M.; Israelachvili, J. N.; Smith, G. S., Biophys.", "J., 1998, 75, 2363; Hillebrandt, H.; Wiegrand, G.; Tanaka, M.; Sackmann, E., Langmuir, 1999, 15, 8451).", "Such lipid monolayers, bilayers, or multilayers offer the ability to minimize sensor “fouling”, i.e., the undesirable adsorption of non-target proteins and biomolecules invariably present in complex biological matrices, by exploiting the characteristic protein adsorption resistance associated with the phosphorylcholine (PC) lipid headgroup (Hayward, J.; Chapman, D., Biomaterials, 1984, 5, 135; Chapman, D., Langmuir, 1993, 9, 39; Malmsten, M. J., Colloid Interface Sci., 1995, 171, 106; Murphy, I. F.; Lu, J. R.; Lewis, L. L.; Brewer, J.; Russell, J.; Stratford, P., Macromolecules, 2000, 33, 4545).", "Additionally, their well-defined and controllable architecture may allow for favorable orientation and minimal denaturation of immobilized antigens or biomolecules such as fab antibody fragments, to maximize sensitivity of the device (Song, X. D.; Swanson, B. I., Anal.", "Chem., 1999, 71, 2097; Parikh, A. N.; Beers, J. D.; Shreve, A. P.; Swanson, B. I., Langmuir, 1999, 15, 5369; Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E., Langmuir, 1993, 9, 136; Viitala, T.; Vikholm, I.; Peltonen, J., Langmuir, 2000, 16, 4953-4961; Duschle, C.; Se(slash)vin-Landais, A. F.; Vogel, H., Biophys., 1996, 70, 1985-1995).", "Supported lipid membrane structures also provide the necessary environment for transmembrane receptor incorporation, which has been demonstrated by several authors through the fabrication of proteo-lipid structures with retained protein activity (Salafsky, J.; Groves, J. T.; Boxer, S. G., Biochem., 1996, 35, 14773-14781; Schmidt, E. X.; Liebermann, T.; Kreiter, M.; Jonczyk, A.; Naumann, R.; Offenhäusser, A.; Neumann, E.; Kukol, A.; Maelicke, A.; Knoll, W., Biosensors Bioelectronics, 1998, 13, 585-591; Naumann, R; Jonczyk, A.; Hampel, C.; Ringsdorf, H.; Knoll, W.; Bunjes, N., Gräber, P. Bioelectrochemistry and Bioenergetics, 1997, 42, 241-247; Fisher, M. L; Tjärnhage, T., Biosensors and Bioelectronics, 2000, 15, 463-471; Pun, G.; Gustafson, L; Artursson, E.; Ohlsson, P. A., Biosensors and Bioelectronics 1995, 10, 463-476; Puu, G.; Aartursson, E.; Gustafson, L; Lundsröm, M.; Jass, J., Biosensors and Bioelectronics, 2000, 15, 31-41; Graff, A.; Winterhalter, M.; Meier, W., Langmuir, 2001, 17, 919-923; Liley, M.; Bouvier, J.; Vogel, H. J., Coll.", "Inter.", "Sci., 1997, 194, 53-58; Naumann, R.; Schmidt, E. X.; Jonczyk, A.; Fendler, K.; Kadenback, B.; Liebermann, T.; Offenhäusser, A.; Knoll, W., Biosensors and Bioelectronics, 1999, 14, 651-662).", "Supported lipid monolayers, bilayers and multilayers can be self-assembled by fusion of fluid, unilamellar vesicles, an important issue for commercial application, onto a variety of optically or electrically active substrates.", "Furthermore, the recent development of micro-patterning techniques to modify planar, substrate supported thin films, including supported lipid bilayers, adds promise to the potential of biochips with parallel arrays of sensing elements for high throughput biological or pharmaceutical screening or sensing (Hovis, J. S.; Boxer, S. B., Langmuir, 2000, 16(3), 894-897; Hovis, J. S.; Boxer, S. B., Langmuir, 2001, 17(11), 3400-3405; Kam, L.; Boxer, S. G., J.", "Am.", "Chem.", "Soc., 2000, 122, 12901-12902; Toby, A.; Jenkins, A.; Boden, N.; Bushby, R. J.; Evans, S. D.; Knowles, P. F.; Miles, R. E.; Ogier, S. D.; Schönherr, H.; Vancso, J. G., J.", "Am.", "Chem.", "Soc., 1999, 121, 5271-5280; Groves, J. T.; Mahal, L. K.; Bertozzi, C. R., Langmuir, 2001; Srinivasan, M. P.; Ratto, T. V.; Stroeve, P.; Longo, M. L., Langmuir, 2001, 17, 7951-7954; Morigaki, K.; Baumgart, T.; Offenhäusser, A.; Knoll, W., Angew.", "Chem., Int.", "Ed., 2001, 40, 172).", "The key problem associated with implementing lipid structures in commercial molecular devise applications is the inherent lack of stability that arises from the exclusively non-covalent forces that are responsible for lipid lamellar assembly.", "As a result, partial or complete lamellar-structure loss is realized upon exposure to surfactants, organics, or removal of the film from aqueous environments.", "Finite aqueous lifetimes have also been observed, and lipid layer damage can occur upon fluid exchange, in the presence of soluble lipophilic proteins, or upon pH or temperature changes (Hui, S. W.; Viswanathan, R.; Zasadzinski, J.", "A.; Israelachvili, J. N., Biophys.", "J., 1995, 68, 171-178; Winger, T. M.; Ludovice, P. J.; Chaikof, E. L., Langmuir, 1999, 15, 3866-3874).", "These shortcomings prevent washing and reusing of a biosensor and seriously compromise the storage/shelf-life, reliability, and thus applicability of the device.", "Covalently bound self-assembled monolayers (SAMS) featuring oligo(ethylene glycol) (Yang, Z.; Galloway, J.", "A.; Yu, H., Langmuir, 1999, 15); or other protein adsorption resistant headgroups (Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G. M., J.", "Am.", "Chem.", "Soc., 2000, 122, 8303-8304) address the stability issue of biosensor coatings but are not without shortcomings, including an increased difficulty in functionalizing these films with water-soluble proteins in a well-defined manner, and not providing a suitable environment for transmembrane receptor proteins.", "Therefore, interest in stabilizing lipid films on solid supports continues to receive scientific attention.", "An alternate method for incorporating phosphorylcholine groups into a substrate supported polymer film is copolymer synthesis followed by direct grafting to the substrate surface (Murphy, E. F.; Lu, J. R.; Lewis, A. L.; Brewer, J.; Russell, J.; Stratford, P., Macromolecules, 2000, 33, 4545).", "However, the molecular architecture of this assembly is more difficult to control than that of a lipid-based film, and is not amenable to functionalization with transmembrane proteins (Murphy, E. F.; Lu, J. R.; Lewis, A. L.; Brewer, J.; Russell.", "J.; Stratford, P., Macromolecules, 2000, 33, 4545; Sackman, E., Science, 1996, 271, 43; Watts, T. H.; Gaub, H. E.; McConnell, H. M., Nature, 1986, 320, 179; McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A.", "A., Biochim.", "Biophys.", "Acta, 1986, 864, 95; Salafsky, J.; Groves, J. T.; Boxer, S. G., Biochemistry, 1996, 35, 14773; Brian, A.", "A.; McConnell, H. M., Proc.", "Natl.", "Acad.", "Sci., 1984, 81, 6159).", "Although the results achieved using supported lipid membranes as sensor coatings have been encouraging with respect to protein resistance, these structures lack the chemical and thermal stability required for technological implementation (e.g.", "as a non-fouling coating for a reusable biosensor).", "This is because the low molecular mass lipids in the bilayer are self-organized by relatively weak, noncovalent forces that are insufficient to maintain the bilayer structure when the membrane is, for example, removed from water.", "Strategies employed to stabilize planar lipid structures under water include: i) incorporation of template molecules, covalently attached either directly to the substrate or to a thin hydrophilic polymer, around which free lipids self-organize to form a bilayer (Duschl, C.; Liley, M.; Corradin, G.; Vogel, H., Biophys.", "J., 1994, 67, 1229; Yang, Z.; Yu, H., Langmuir, 1999, 15, 1731; Bunjes, N.; Schmidt, E. K.; Jonczyk, A.; Rippmann, F.; Beyer, D.; Ringsdorf, H.; Graber, P.; Knoll, W.; Naumann, R., Langmuir, 1997, 13, 6188) and ii) derivatization of a metal or silica surface with an alkyl self-assembled monolayer, followed by deposition of a lipid monolayer, creating a hybrid bilayer (Plant, A. L., Langmuir, 1999, 15, 5128; Stelzle, M.; Weissmuller, G.; Sackman, E. J., Phys.", "Chem., 1993, 97, 2974; Song, X. D.; Swanson, B. I., Anal.", "Chem., 1999, 71, 2097; Parikh, A. N.; Beers, J. D.; Shreve, A. P.; Swanson, B. I., Langmuir, 1999, 15, 5369; Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E., Langmuir, 1993, 9, 136; Meuse, C. W.; Krueger, S.; Majkrzak, C. F.; Dura, J.", "A.; Fu, J.; Connor, J. T.; Plant, A. L., Biophys.", "J., 1998, 74, 1388).", "Both strategies increase the stability of the structure in water while maintaining some degree of lateral lipid mobility.", "However, the integrity of these structures is compromised by lipid loss upon exposure to harsher environments, such as organic solvents, surfactant solutions, or transfer across the water/air interface.", "A considerable body of work has shown that the stability and permeability of lipid bilayer vesicles (liposomes) can be significantly altered by polymerization of lipids containing reactive moieties (O'Brien, D. F.; Armitage, B.; Benedicto, A.; Bennett, D.; Lamparski, H. G.; Lee, Y. S.; Srisiri W.; Sisson, T. M., Acc.", "Chem.", "Res., 1998, 31, 861; Regen, S. L.; Singh, A.; Oehme, G.; Singh, M. J., Amer.", "Chem.", "Soc., 1982, 104; 791; Sisson, T. M.; Lamparski, H. G.; Kolchens, S.; Elyadi, A.; O'Brien, D. F., Macromolecules, 1996, 29, 8321).", "For example, unilamellar vesicles composed of bis-substituted lipids can be polymerized to form cross-linked vesicles that are insoluble in surfactant solutions and organic solvents (Sisson, T. M.; Lamparski, H. G.; Kolchens, S.; Elyadi, A.; O'Brien, D. F., Macromolecules, 1996, 29, 8321).", "Several groups have prepared polymerized, multilamellar supported lipid films composed of commonly used diacetylenic PC lipids which can be stabilized by UV photopolymerization (Hayward, J.; Chapman, D., Biomaterials, 1984, 5, 135; Chapman, D., Langmuir, 1993, 9, 39; 21).", "However, to be efficiently polymerized, these lipids must be in the solid analogous phase (Lβ), which is incompatible with the self-assembly methods of the present invention and does not produce a high percentage of monomer to polymer conversion (Hayward, J.; Chapman, D., Biomaterials, 1984, 5, 135; Chapman, D., Langmuir, 1993, 9, 39; Albrecht, O.; Johnston, D. S.; Villayerde, C.; Chapman, D., Biochim.", "Biophys.", "Acta, 1982, 687, 165; Binder, H.; Anikin, A.; Kohlstrunk, B. J., Phys.", "Chem., 1999, 103, 450-460).", "At least two research groups have used the polymerization strategy to stabilize lipid mono- and bilayers on solid supports.", "Regen and coworkers adsorbed films of mono- and di-acrylate functionalized lipids on poly(ethylene), followed by UV-photo-polymerization to form a supported polymerized lipid film of near monolayer thickness (Regen, S. L.: Kirszensztejn, P.; Singh, A., Macromolecules, 1983, 16, 338; Foltynowicz, Z.; Yamaguchi, K.; Czajka, B,.", "Regen, S. L., Macromolecules, 1985, 18, 1394).", "Their water contact angle data were indicative of a surface more hydrophobic than expected for a uniform array of PC groups, suggesting incomplete coverage and/or significant film disorder.", "However, the analytical tools (e.g.", "atomic force microscopy) needed to characterize film morphology and uniformity were not available at that time.", "More recently, Chaikof and coworkers formed a hybrid bilayer by fusing vesicles (Marra, K. G.; Winger, T. M.; Hanson, S. R.; Chaikof, E. L., Macromolecules, 1997; 30, 6483; Orban, J. M.; Faucher, K. M.: Dluhy, R. A.; Chaikof, E. L., Macromolecules, 2000, 33, 4205) composed of mono-acrylate lipids onto a support coated with an alkylsilane monolayer; in situ polymerization produced linear polymers in the upper leaflet of this structure.", "Although enhanced stability during extended incubation in water was observed, significant lipid desorption occurred when the assembly was exposed to surfactant.", "SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a lipid membrane which is a monolayer, bilayer, or multilayer that is self-assembled and stabilized at a solid surface.", "It is another object of the present invention to provide a solid supported lipid film that is stable to transfer into air and exposure to surfactant solutions and organic solvents, yet retains the protein resistance characteristic of a fluid lipid bilayer.", "It is yet another object of the present invention to include non-polymerizable amphiphilic molecules into a stabilized lipid membrane.", "It is another object of the present invention to provide a stabilized lipid membrane that is an appropriate environment for reconstitution of a transmembrane protein and/or a water-soluble protein with retention of native protein structure and activity.", "This and other objects have been achieved by the present invention the first embodiment which includes a method for the self-assembly and stabilization of a lipid membrane at a solid surface, comprising: depositing a lipid monolayer or a lipid multilayer on a substrate, thereby obtaining a supported lipid monolayer or a supported lipid multilayer; in situ polymerizing said supported lipid monolayer or said supported lipid multilayer, thereby obtaining a polymerized membrane.", "BRIEF DESCRIPTION OF DRAWINGS FIG.", "1 shows types of polymerizable groups that can be used in polymerizable lipids.", "FIG.", "2 shows examples of mono-substituted polymerizable lipids.", "FIG.", "3 shows examples of bis-substituted polymerizable lipids.", "FIG.", "4 shows examples of heterobifunctional polymerizable lipids.", "FIG.", "5 shows examples of polymerizable lipids that differ in the length of the lipid tail (can be 14 to 22 atoms) and the extent and location of unsaturation and/or branching in the lipid tail(s).", "FIG.", "6 shows some examples of the different types of head groups for polymerizable lipids.", "FIG.", "7 shows a schematic of the vesicle fusion process, forming a fluid supported lipid bilayer (1,2), followed by redox-initiated, radical polymerization (3) to produce a cross-linked bilayer (4).", "FIG.", "8 shows AFM images and linescans of a polymerized bis-SorbPC (redox) bilayer in air (left) and under water (center).", "On the right is an image of a region of the film that was deliberately damaged by repeated high force scanning.", "FIG.", "9 shows a bar graph of relative bovine serum albumin (BSA) adsorption to various films.", "The diagram illustrates the principle of total internal reflectance fluorescence (TIRF), which is used to measure adsorption of rhodamine labeled BSA molecules to the various films.", "FIG.", "10 shows TIRF generated BSA adsorption isotherms for various films on quartz substrates.", "The dried and rehydrated polymerized bis-SorbPC (redox) film demonstrates equivalent adsorption resistance at a BSA solution concentration of 1.5×10−5M.", "FIG.", "11 shows AFM images and linescans of a blank silicon substrate and a polymerized bis-SorbPC (redox) supported bilayer before and after exposure to a 15 μM BSA solution.", "FIG.", "12 shows an AFM image and a linescan of a dried, poly-diacetylenic PC lipid bilayer deposited by the Langmuir-Schaefer technique and polymerized by direct UV irradiation.", "FIG.", "13 shows show an AFM image and linescan of a dried, polymerized bis-SorbPC bilayer deposited by vesicle fusion and polymerized by direct UV irradiation.", "FIG.", "14 shows an AFM image of a dried, redox polymerized bilayer deposited by vesicle fusion and composed of 70% bis-SorbPC monomer and 30% non-polymerizable lipid DOPC.", "FIG.", "15 shows an AFM image and linescan of a dried, redox polymerized mono-SorbPC bilayer deposited by vesicle fusion.", "FIG.", "16 shows an AFM image and linescan of a dried, redox polymerized bis-DenPC bilayer deposited by vesicle fusion.", "FIG.", "17 shows an AFM image and linescan of a dried, redox polymerized DenSorbPC bilayer deposited by vesicle fusion.", "FIG.", "18 shows an AFM image (left) of biotin-BSA microcontact printed on a polymerized bis-SorbPC (redox) bilayer.", "The schematic on the right depicts binding of rhodamine labeled avidin to the patterned regions of biotin-BSA.", "FIG.", "19 shows an AFM image (left) of a UV polymerized, bis-SorbPC film patterned by microcontact printing.", "Printing removed portions of the supported fluid bilayer (dark stripes); UV polymerization then stabilized the remaining regions (light stripes).", "The illustration on the right depicts the procedure graphically.", "FIG.", "20 shows schematic of TIRF spectroscopy instrumentation, a) fused silica slide, b) quart prism, c) Teflon™ block and Viton™ o-ring, d) 4× microscope objective, e) long pass filter, f) PMT, g) lock-in amplifier, h) frequency generator, i) data acquisition computer, and j) reference photo diode.", "FIG.", "21 shows kinetic data for the UV polymerization of bis-SorbPC bilayers which was obtained by measuring the depletion of the monomer absorbance as a function of time.", "Inset: absorbance spectrum of the monomeric bis-SorbPC prior to polymerization.", "FIG.", "22 shows AFM images for (a) a dried bis-SorbPC bilayer film, and (b) the same film imaged under water to Example for UV polymerized filer.", "The film was deposited using the Langmuir-Schaefer method and polymerized with UV light.", "FIG.", "23 shows adsorption isotherms of FITC labeled BSA to a POPC monolayer, (a hydrophobic surface, solid line), a dehydrated bis-SorbPC bilayer (dashed line), and a POPC bilayer (dash-dot line).", "The lines through the data in each case represent the fitting the data to a Langmuir adsorption isotherm.", "FIG.", "24 shows the structures of several cyanine dyes that can be used for photosensitized polymerization of supported lipid films.", "DETAILED DESCRIPTION OF THE INVENTION The present inventors have found a novel and successful strategy for the self-assembly and stabilization of a lipid bilayer, particularly a phospholipid bilayer, at a solid surface.", "After deposition of a lipid bilayer on a substrate, in situ polymerization of the supported bilayer produces a cross-linked membrane that is stable to transfer into air and exposure to surfactant solutions and organic solvents, yet retains the protein resistance characteristic of a fluid phosphatidylcholine (PC) bilayer.", "In a first embodiment of the present invention, a self-assembled, supported fluid membrane is formed by fusion of fluid, small unilamellar vesicles (SUVs) composed of a polymerizable lipid to a clean surface in a buffered aqueous solution or deionized water.", "The buffer solution or water used may also include added mono-, di-, or trivalent metal salts.", "Upon adsorption at a substrate/buffer solution interface, fluid bilayer SUVs spontaneously unroll to produce an extended, continuous lipid monolayer or bilayer (FIG.", "7).", "In contrast, pre-polymerized phospholipid vesicles do not fuse to surfaces.", "The supported lipid film is then transferred to a redox polymerization medium to initiate polymerization without exposing the film to air.", "Preferably, after incubating the film in the redox polymerization medium, the film is removed, cleaned, and dried under an inert gas atmosphere.", "Polymerizable lipids that are useful for this invention include those which contain at least one of the polymerizable groups shown in FIG.", "1, e.g.", "styryl, dienyl, dienoyl, sorbyl, acryloyl, methacryloyl, vinyl ester, among others.", "These groups can be located anywhere along the lipid tails as indicated by the examples shown in the following FIGS.", "2-6.These examples include mono- and bis-substituted lipids, shown in FIGS.", "2 and 3 respectively as phosphatidylcholines, which are ester lipids based on a glycerol backbone.", "The lipid backbone is not limited to glycerol, but could also be 1-aminopropane-2,3-diol, glutamic acid, aspartic acid, among others.", "In the lipid examples shown, the lipid tail is linked to the glycerol backbone through an ester bond.", "It is also possible to prepare similar polymerizable lipids with an ether bond.", "The polymerizable lipid can have two identical reactive groups in each lipid tail, or two different reactive groups in the same lipid tail, which are heterobifunctional lipids (FIG.", "4).", "In order to control the bilayer fluidity, the main phase transition temperature of the lipid can be controlled through the choice of the length of the lipid tail from 14 to 22 atoms, and the extent and location of unsaturation and/or branching in the lipid tail(s) as shown in FIG.", "5.The lipid head group can vary widely from phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylserine (PS), to PE-like lipids with associated groups such as succinate or chelating groups for the conjugation of functional compounds and metals to the lipid membrane surface (FIG.", "6).", "Numerous other functionalized lipid headgroups (not shown) could be used, including headgroups terminated with thioethanol, maleimido, pyridyldithio, biotinyl, succinimidyl ester, sulfo succinimidyl ester, alkyl halide, or haloacetamide groups, as well as lipids functionalized with ethylene glycol-based oligomers and polymers.", "Preferably, the lipid solutions are prepared as follows: Lipids from stock chloroform or benzene solutions or any other organic solvent in which the lipid is soluble are dried under a flowing inert gas such as Ar or N2 to remove storage solvents.", "The lipids are then resuspended in deionized water (18 MΩ) or aqueous buffer.", "The lipid concentration is in the range of from 0.01 mg/l to 5 mg/l, and preferably in the order of 0.5 mg/ml.", "The lipid concentration includes all values and subvalues therebetween, especially including 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 and 4.5 mg/l.", "The lipid suspension is then mechanically treated, for example, vortexed and sonicated to clarity, forming SUVs (eg., Barenholz, Y.; Gibbes, D.; Litman, B; Goll, J.; Thomson, T.; Carlson, F., Biochemistry, 1977, 16, 2806).", "Temperature control is preferably maintained at more than 10 degrees above the reported lipid transition temperature.", "The SUVS are preferably used within 30 minutes of preparation, more preferably within 20 minutes after preparation and most preferably within 10 minutes after preparation.", "Other methods to prepare unilamellar vesicles include, but are not limited to, extrusion of lipids through porous membranes (eg., MacDonald, R.; MacDonald, R. I.; Menco, B.; Takeshita, K.; Subbarao, N.; Lan-rong, H., Biochimica et Biophysica Acta, 1991, 1061, 297) and surfactant dialysis (eg., Mimms, L. T.; Zampighi, G.; Nozaki, Y.; Tanford, C.; Reynolds, J.", "A., Biochemsitry, 1981, 20, 833).", "Both methods have been successfully used to prepare vesicles for subsequent use in preparation of supported fluid lipid bilayers by vesicle fusion (Cremer, P. S.; Boxer, S. G., J. Phys.", "Chem.", "B, 1999, 103, 2554; Puu, G.; Gustafson, I., Biochim.", "Biophys.", "Acta, 1997, 1327, 149; Noller, P.; Kiefer, H.; Jähnig, F., Biophysical J., 1995, 69, 1447.)", "Briefly, extrusion involves resuspension of dried lipids in appropriate solutions, as described above.", "A repeated freeze, thaw cycle may or may not be applied to produce multilamellar vesicles before the suspension is repeatedly passed through a porous size exclusion membrane.", "Unilamellar vesicles with a mean diameter ranging from 50 to 1000 nm are created depending on the size of the pores in the membrane used.", "The diameter includes all values and subvalues therebetween, especially including 100, 200, 300, 400, 500, 600, 700, 800 and 900 nm.", "Surfactant dialysis, also known as detergent depletion, occurs when a suspension of lipid and detergent, (present together in aqueous solution at a concentration above the detergent critical micelle concentration) is dialyzed against another aqueous solution.", "The detergent passes through the dialysis membrane and is removed from the compartment containing the lipid, whereupon the remaining lipid spontaneously forms unilamellar vesicles.", "Supported lipid films are prepared by vesicle fusion (FIG.", "7), while avoiding exposure of the unpolymerized films to air, or excessive mechanical shocks.", "Care must be taken to avoid light exposure to polymerizable lipids or lipid films.", "Thus, they are handled under yellow light.", "Vesicle fusion to solid supports is a well documented, and commonly used practice to form substrate supported fluid lipid bilayers.", "The rate of fusion and bilayer spreading is controlled by a ‘subtle balance’ of van der Waals, electrostatic, hydration, and steric forces, but it is of yet, poorly understood what relation these forces play in the process.", "(Cremer, P. S.; Boxer, S. G., J. Phys.", "Chem.", "B, 1999, 103, 2554).", "Vesicle fusion of liposomes containing no net charge (eg., phosphorylcholine headgroups) to glass supports has no observable pH dependence over a range of 2.5-12.3, nor a dependency upon ionic strength.", "(Cremer, P. S.; Boxer, S. G., J. Phys.", "Chem.", "B 1999, 103, 2554) The concentration of suspended vesicles in the aqueous solution plays a role in the kinetics of bilayer formation, but not in the physical structure of the final supported film.", "Preferably, a concentration is used that will allow timely formation of the bilayer, for example, on oxidized silicon, this is a lipid concentration of typically greater than 0.1 mg/ml, but it is noted that lower and higher concentrations will produce supported films.", "Preferably, the lipid concentration is greater than 0.5 mg/ml, particularly preferably greater than 1 mg/ml.", "Alternatively lipid films can be formed using standard Langmuir-Schaefer techniques according to reference procedures (Morigaki, K.; Baumgart, T.; Offenhausser, A.; Knoll, W., Angew.", "Chem., Int.", "Ed., 2001, 40, 172).", "The substrate surface is preferably cleaned using a plasma cleaner, a sonicator, UV light, an organic solvent such as alcohol or chloroform, a strong acid solution such as a pirhana solution, an aqueous or alcoholic solution of H2O2, or an aqueous or alcoholic solution of a hydroxide of an alkali earth metal, such as NaOH or KOH.", "Surfaces are preferably used within 1 hours of cleansing, preferably within 30 minutes, more preferably within 20 minutes and most preferably within 10 minutes.", "Preferred surfaces of the solid support are silicon dioxide (SiO2), silicon oxide (SiOx), a noble metal such as gold, silver, platinum; mica, a polymer surface, a thin polymer film coated substrate, indium-tin oxide (ITO), tin oxide, indium oxide and silicon.", "The surface can be planar or non-planar.", "A preferred buffer solution is phosphate.", "A preferred pH of the buffer solution is 7.4.The pH of the solution can be any value from pH 5.6 to pH 8.The buffer can be prepared with any chemical compound having a pKa between 5 and 9.The solution can also contain added metal salts, including monovalent, divalent, and trivalent metal salts.", "Preferred concentrations are from 0 up to and including 500 mM.", "The concentration includes all values and subvalues therebetween, especially including 1, 10, 50, 100, 150, 200, 250, 300, 350, 400 and 450 mM.", "The redox initiator system is preferably K2S2O8/NaHSO3 (FIG.", "7).", "A preferred concentration of the persulfate is 1 mM to 1 M. The concentration of the persulfate includes all values and subvalues therebetween, especially including 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800 and 900 mM.", "A preferred oxidant to reductant ratio is from 1:1 to 1:10.The oxidant to reductant ratio includes all values and subvalues therebetween, especially including 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 and 1:9.At all concentrations above 0.01M, regardless of the oxidant/reductant ratios used, polymerized lipid films are indistinguishable by AFM and ellipsometry.", "Many other redox initiator systems can also be used.", "Examples of suitable oxidants include H2O2, KrBrO3, CuCl, Cs(SO4)2.Examples of suitable reductants include L-cysteine, H2N2H2, ascorbic acid, HCOOH, R3N (where R is hydrogen or any group that contains carbon), and salts of Fe+2, Ag+, SO3−.", "In all cases, a preferred concentration of the oxidant is 1 mM to 1 M. The oxidant concentration includes all values and subvalues therebetween, especially including 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800 and 900 mM.", "A preferred oxidant to reductant ratio is from 1:1 and 1:10.The oxidant to reductant ratio includes all values and subvalues therebetween, especially including 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 and 1:9.In a preferred case oxygen is excluded by deoxygenating the reaction solutions with a flowing inert gas such as Ar or N2.The gas flow can occur before the polymerization and can continue throughout the polymerization.", "The film is preferably incubated in the redox polymerization medium for 1 minute to five hours.", "The incubation time includes all values and subvalues therebetween, especially including 5 min, 10 min., 20 min., 40 min., 60 min., 80 min., 100 min., 120 min., 140 min., 160 min., 180 min., 200 min., 220 min., 240 min., 260 min.", "and 280 min.", "After incubation, the film is preferably rinsed with water or an aqueous solution of an organic solvent, such as a lower alcohol in water.", "Water is preferably purified to 18 MOhms and made organic free.", "The inert gas for drying is preferably Ar or N2.Polymerized lipid bilayers have been prepared from bis-SorbPC on SiO2 (FIG.", "3).", "Redox generated radical polymerization resulted in dried bilayer films of bis-SorbPC (hereinafter referred to as “bis-SorbPC(redox)”) with a thickness of about 45 Å and a sessile water contact angle of about 32 degrees.", "The contact angle of 32 degrees for the bis-SorbPC(redox) film is very similar to the value of 28 degrees reported by Cooper et al.", "(Tegoulia, V.; Rao, W.; Kalamber, A.; Rabolt, J.; Cooper, S., Langmuir, 2001, 17, 4396), for a phosphorylcholine terminated SAM film on gold.", "This is strong evidence that the polymeric bis-SorbPC (redox) bilayer film remains structurally similar to a fluid bilayer, presenting polar, zwitterionic head groups at the film/air interface.", "The images in FIG.", "8, acquired using tapping mode atomic force microscopy (AFM), show that the polymerized bilayer surface is very smooth.", "The root mean square roughness of the image acquired in air (left) is 1.25 Å, which is comparable to the roughness of the bare silicon substrate (rms of 1.1 to 1.3 Å).", "No discernible change in film morphology or surface roughness was observed when a previously dried region of a film was re-imaged under water (center image).", "The bilayer surface morphology was surprisingly uniform; the left and center images are representative of images acquired at numerous locations over a ca.", "1 cm2 sample area.", "No topographical features greater than 1 nm in height (peak-to-peak) were detected.", "Thus any defects at which bare substrate was exposed were too narrow to image by AFM.", "Polymerized bilayers can be deliberately damaged by repeated, high force scanning (right image in FIG.", "8); a line scan across a film containing such a ‘trough’ yielded an apparent film thickness of 39-47 Å, consistent with the ellipsometry data.", "The phospholipid bilayer of the present invention is stable in organic solvents, particularly to chlorinated hydrocarbons such as chloroform, ethers such as tetrahydrofuran, alcohols such as methanol and ethanol, sulfur-containing solvents such as DMSO, ketones such as acetone, and aromatic solvents such as toluene, benzene.", "It is also stable when exposed to solutions of anionic, cationic, non-ionic, or polymeric surfactants.", "Exposure to organic solvents or surfactant solutions does not alter the ellipsometric thickness or the AFM images of the stabilized bilayers.", "The polymerized phospholipid bilayer according to the first embodiment of the present invention exhibits resistance to nonspecific protein adsorption even after polymerization of the hydrophobic tails of the lipid monomers, which provides evidence that the “headgroup out” structure of the bilayer is preserved after drying and rehydration.", "In fact, the resistance of the bis-SorbPC bilayer of the present invention for BSA (bovine serum albumin) is comparable to that of a fluid 1-palmitoyl-2-oleolyl-PC(POPC) bilayer as demonstrated by the comparative data shown in FIGS.", "9-11.Lipids in addition to bis-SorbPC have also been used in the present invention.", "The above described vesicle fusion, Langmuir-Schaefer, redox-initiated polymerization, or the UV polymerization methods may be used as described above.", "Supported lipid bilayers have been prepared using both bis-DenPC (FIG.", "3) and DenSorbPC (FIG.", "4).", "A DenSorbPC lipid bilayer formed by vesicle fusion and redox polymerization was indistinguishable from a bilayer of bis-SorbPC (redox) as judged by AFM (FIG.", "17).", "Ellipsometric thickness were nearly equivalent as well, and upon bath sonication in surfactant, only a minute thickness change was observed.", "The redox polymerization of bis-DenPC lipids after vesicle fusion to form a supported bilayer resulted in an ellipsometric thickness of 52 Å, however upon bath sonication in the surfactant Triton-X-100, a significant decrease in film thickness was recorded.", "AFM images (FIG.", "16) of the film surface reveal the surface to contain defects located uniformly throughout the film.", "Examination of the line scans suggest that the defects do not reach the substrate but instead are losses of lipid from the outer monolayer of the film since the depth of the holes is less than 3 nm.", "These differences in film structure arise from differences in the location of the polymerizable moiety in the lipid.", "Another example, shown in FIG.", "15, is an AFM image of a dried, redox-polymerized bilayer composed of mono-SorbPC (FIG.", "2) that was deposited by vesicle fusion.", "The incomplete structure of the film is ascribed to the absence of cross-linking, which is precluded when using mono-functionalized lipid at a mole fraction of 1.For comparison, protein adsorption data to other surfaces, including bare silica and a hydrophobic monolayer of arachidic acid, are also shown in FIGS.", "9-11.In addition, data are presented for a supported bilayer formed from a commercially available diacetylenic PC lipid (1,2-bis(10,12-tricosadiynyl)-sn-glycero-3-phosphocholine; Avanti Polar Lipids) that was deposited by the Langmuir-Schaefer technique and photopolymerized using UV light.", "This type of bilayer exhibits considerably more protein adsorption than a bis-SorbPC (redox) bilayer (comparison data shown in FIG.", "9).", "The difference is attributable to the large number of defects in the diacetylenic PC lipid bilayer (AFM image shown in FIG.", "12).", "Thus clearly the performance of the present invention is superior to existing technology.", "In a second embodiment of the present invention, the lipid bilayers are prepared by the vesicle fusion method, or using Langmuir-Blodgett and/or Langmuir-Schaefer technique, and polymerized by direct photo-irradiation with V, visible or near infrared light or γ-rays.", "The rays can be polarized or unpolarized.", "Preferred polymerizable lipids are those described above in the first embodiment and shown in FIGS.", "1-6.Direct UV polymerization is performed by exposing the lipid bilayer films to UV radiation at a wavelength of between 230 and 350 nm, preferably at 260 nm and more preferably at 254 μm.", "The wavelength includes all values and subvalues therebetween, especially including 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, and 340 nm.", "The UV light may be polarized or unpolarized.", "Both, direct UV-photoinitiation and redox-initiated radical polymerization stabilize films of the lipid to surfactant dissolution suggesting the formation of a cross-linked polymeric network.", "However, a difference in the degrees of polymerization occurs for the two initiation methods.", "For example, redox initiated polymers of bis-SorbPC are larger (Xn approx 50+) than UV photopolymerized polymers (Xn<10), which suggests different propagation mechanisms for the polymerizations.", "The UV-irradiation proceeds for 1 second to 1 hour at photon fluxes ranging from 1×1013 to 1×1017 photons/second.", "The irradiation time includes all values and subvalues therebetween, especially including 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260 and 280 seconds, 10 min., 15 min., 20 min., 25 min., 30 min., 35 min., 40 min., 45 min., 50 min., and 55 min.", "The photon flux includes all values and subvalues therebetween, especially including 5×1013, 1×1014, 5×1014, 1×1015, 5×1015, 1×1016 and 5×1016 photons/second.", "The thickness of the bis-SorbPC (UV) films deposited by vesicle fusion and UV polymerized are about 29 Å and the surface is usually more hydrophobic than redox polymerized bis-SorbPC films (redox) with a contact angle of 52 degrees.", "Furthermore, AFM images presented in FIG.", "13 illustrate that by comparison to bis-SorbPC(redox) films (rms roughness of 0.15 nm), the UV polymerized films (bis-SorbPC(UV)) are much rougher (rms roughness=0.35 nm), and have discernable features or domains approximately 1.5 to 2 nm thick.", "These features are very uniformly distributed on the film surface.", "No regions were found on any of the UV polymerized film that were devoid of polymer film, or where the domains differed appreciably in size.", "The ellipsometric thickness, combined with the depth of the features suggest that they are likely regions of film where the surrounding lipid-polymer has been removed upon drying and rinsing and likely do not extend to the substrate because partial coverage of a 1.5 to 2 mm film would be inconsistent with the ellipsometric thickness of 29 Å.", "The UV-polymerizations are usually not sensitive to the presence of oxygen, nor has the rate of polymerization a noticeable effect on the film properties.", "The rate of polymerization can be affected by altering the intensity of the light used to photopolymerize the film.", "UV-Vis spectroscopy of polymerized bilayers reveals an equivalent degree of conversion for both UV and redox-initiated polymerizations.", "The degree of conversion is >90%, preferably >95% and most preferably >99%.", "Because the polymerization by redox initiators and UV light produce the same polymer product, the difference in acyl-chain structure is not likely the reason for the difference in film properties.", "Evidence from protein adsorption studies on UV polymerized films before they are subjected to drying suggest that the polymerization does not significantly alter the structure of the film.", "Therefore, the defects appearing in the bis-SorbPC(UV) films may be due to a decrease in the stabilization of lower molecular weight polymer fragments produced by direct photopolymerization.", "Polymerizations are not monodisperse, therefore a range of molecular weights exist in the polymer film and it is possible that the smaller polymer fragment population accounts for the material lost upon drying.", "The fact that the UV polymerization resulted in the presence of any film after drying at all represents a significant increase in the stability of an unpolymerized fluid lipid film, which by comparison returned negligible ellipsometric thickness.", "AFM images of surfaces of unpolymerized fluid lipid films on silica were basically indistinguishable from images of a blank silica surface.", "This is consistent with the observation of several authors that lipid film loss and/or disruption to the lamellar structure occurs upon drying fluid supported phospholipid bilayers (Cremer, P. S., Boxer, S. G., J. Phys.", "Chem.", "B, 1999, 103, 2554).", "The many variables under which lipid bilayers are polymerized by direct UV irradiation have not been exhaustively investigated.", "Considering the independence of the degree of polymerization observed in vesicles to temperature or polymerization rate, it is likely that the mechanism of polymerization for bis-SorbPC may limit the polymer product to low molecular weights.", "However, it is expected that other types of polymerizable lipids, such as those shown in FIGS.", "1-6, may be converted by direct UV irradiation to polymer in higher yields than bis-SorbPC, resulting in lipid films of quality comparable to bis-SorbPC (redox).", "In addition, further optimization is anticipated by systematically varying other variables, such as irradiation time and photon flux.", "Lipid polymerization can also be initiated by a dye-sensitized process (Clapp, P. J.; B.", "A. Armitage, B.", "A.; O'Brien, D. F. Macromolecules, 1997, 29, 32).", "Here a membrane-bound cyanine dye that absorbs in the visible or near-infrared spectral regions is incorporated into the membrane.", "Irradiation at a wavelength at which the dye absorbs, in the presence of oxygen, is thought to generate hydroxyl radicals which initiate lipid polymerization.", "The dye can be added to the lipid solution either before or after formation of SUVs, prior to using the vesicles to perform vesicle fusion.", "For Langmuir-Blodgett or Langmuir-Schaefer deposition, the dye is added to the lipid before it is spread as a monolayer film on a Langmuir trough.", "The preferred molar ratio of lipid to dye ranges from 5:1 up to 30:1.The preferred pH range is 6.0 to 9.5.The preferred temperature is 15° C. to 45° C. The preferred wavelength of incident light is 350-800 nm.", "The irradiation proceeds for 1 second to 5 hours at preferred incident photon flux ranges from 0.036×1018 to 2×1018 photons/second.", "In the preferred method, ambient oxygen is present in the solution and the gas surrounding the solution.", "A number of different dyes can be used to initiate the polymerization of the types of lipids shown in FIGS.", "1-6, including but not limited to the cyanine dyes shown in FIG.", "24.Supported lipid membranes polymerized using the dye-sensitized process have been prepared in our laboratories, with results similar to that obtained using direct UV photopolymerization (described above).", "Further optimization of the dye-sensitized process is anticipated by systematically varying the numerous variables involved, including dye:lipid ratio, irradiation time and photon flux, type of dye used, type of lipid used, temperature, oxygen concentration, lipid film deposition method.", "A third embodiment relates to the incorporation of non-polymerizable amphiphiles (e.g.", "surfactants or lipids) or any other molecule that will insert in the stabilized lipid membranes.", "Phospholipid bilayer films according to the present invention may be formed using a mixture of polymerizable lipid and non-polymerizable lipid by the above described methods.", "The amount of non-polymerizable lipid in the mixture is in the range of from 0.01 to 50%, preferably not more than 30%, more preferably not more than 10% and most preferably not more than 2%.", "The amount of non-polymerizable lipid in the mixtures includes all values and subvalues therebetween, especially including 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40 and 45%.", "The polymerizable lipid in these films can be any of the lipids or lipid types shown in FIGS.", "1-6, and mixtures thereof, in any molar desired ratio.", "The above described vesicle fusion, Langmuir-Schaefer, redox-initiated polymerization, and light-driven (UV, visible, or near-infrared) methods may be used as described above to deposit and polymerize the polymerizable lipids in the membrane.", "In the preferred implementation, the non-polymerizable molecules are mixed with the polymerizable lipids prior to vesicle preparation.", "Vesicles composed of non-polymerizable molecules and polymerizable lipids are prepared and fused to appropriate substrates (as described above) to form supported lipid membranes that are subsequently polymerized.", "Alternately, the polymerized, supported lipid membrane is prepared and then a solution of non-polymerizable molecules is brought into contact with the membrane, which causes the non-polymerizable molecules to bind to and insert into the membrane.", "The non-polymerizable molecules incorporated into the membrane are typically amphiphilic, e.g.", "a single-chain surfactant or a non-polymerizable lipid, that will bind to and associate with a membrane.", "The nature of the association reaction can be either non-covalent or covalent.", "Preferred non-polymerizable molecules are those which impart a functional property to the membrane, i.e.", "a surfactant or lipid bearing a headgroup that is functionally distinct from the headgroups on the polymerizable lipids in the membrane.", "Examples include single-chain and double-chain surfactants having anionic or cationic headgroups, headgroups functionalized with ethylene glycol-based oligomers and polymers, headgroups designed to chelate metal ions, headgroups functionalized with dyes that absorb light and/or emit fluorescence in the UV, visible, and/or near-infrared spectral regions, and headgroups designed to react with other molecules.", "Examples of the latter category include headgroups terminated with thioethanol, maleimido, pyridyldithio, biotinyl, succinimidyl ester, sulfo succinimidyl ester, alkyl halide, or haloacetamide groups.", "Another preferred implementation is the use of a non-polymerizable lipid, e.g.", "DOPC, mixed with a polymerizable lipid in a fluid vesicle or fluid supported lipid membrane.", "Upon polymerization, the non-polymerizable lipid will spatially segregate from the domains of polymerized lipid, forming a lipid membrane that contains spatially defined and distinct fluid and polymerized regions.", "An example is shown in FIG.", "14, which shows an AFM image of a dried, redox polymerized, supported lipid bilayer deposited by vesicle fusion and composed of 70% bis-SorbPC and 30% non-polymerizable lipid DOPC.", "In the fourth embodiment, supported phospholipid membranes are produced from mixtures of polymerizable lipids.", "Phospholipid bilayer films according to the present invention may be formed using a mixture of different types of polymerizable lipids by the above described methods.", "The amount of each different type of lipid in the mixture is in the range of from 0.01 to 99.99%, including all values and subvalues therebetween.", "The polymerizable lipids can be any of the lipids or lipid types shown in FIGS.", "1-6, and mixtures thereof, in any molar desired ratio.", "The above described vesicle fusion, Langmuir-Schaefer, redox-initiated polymerization, and light-driven (UV, visible, or near-infrared) methods may be used to deposit and polymerize the membrane.", "In the preferred implementation, the different types of polymerizable lipids are mixed prior to vesicle preparation.", "Vesicles are then prepared and fused to appropriate substrates (as described above) to form supported lipid membranes that are subsequently polymerized.", "In one preferred implementation, two types of polymerizable lipid molecules are present in the membrane.", "One type of lipid molecule, present in an amount less than 50%, preferably less than 30%, imparts a functional property to the membrane, i.e.", "it bears a headgroup that is functionally distinct from the headgroups on the other type of lipid in the membrane.", "Examples include functional lipids having anionic or cationic headgroups, having headgroups functionalized with ethylene glycol-based oligomers and polymers, having headgroups designed to chelate metal ions, or having headgroups designed to react with other molecules.", "Examples of the latter category include headgroups terminated with thioethanol, maleimido, pyridyldithio, biotinyl, succinimidyl ester, sulfo succinimidyl ester, alkyl halide, or haloacetamide groups.", "The second type of lipid molecule in the membrane is selected to be protein resistant, e.g.", "bis-SorbPC.", "In another preferred implementation, the lipid membrane is composed of a mixture of complementary mono- and bisfunctionalized polymerizable lipids, e.g.", "mono-SorbPC and bis-SorbPC.", "Prior to polymerization, such lipids mix homogeneously in a fluid supported lipid membrane.", "Thus by varying the percentage of each, the density of cross-links in the polymerized bilayer is systematically adjusted.", "A lower cross-link density generates a more flexible yet still polymeric membrane.", "As long as the mole fraction of bis-substituted lipid exceeds 0.30±0.05, the polymerized bilayer will be still be cross-linked (Sisson, T. M.; Lamparski, H. G.; Kolchens, S.; Elyadi, A.; O'Brien, D. F., Macromolecules, 1996, 29, 8321).", "The fifth embodiment of the present invention relates to the incorporation of membrane proteins into polymerized, supported lipid membranes.", "Incorporating protein receptors into a lipid membrane confers a biorecognition function to the membrane.", "Any membrane-associated protein can be incorporated into a polymerized, supported lipid membrane.", "In all cases, a preferred surface coverage of receptors is 0.1% to 50% of the coverage equivalent to a one monolayer of receptor.", "Receptor incorporation in an appropriate manner and orientation that maintains receptor activity can be assayed by the observation of the specific binding to complementary partners.", "Membrane proteins, especially transmembrane proteins, require a lipid bilayer environment to preserve their structure and support their specific bioactivity.", "Reconstitution of transmembrane receptors into fluid, supported lipid membranes has been described (Z. Salamon, S. Cowell, E. Varga, H. I. Yamamura, V. J. Hruby and G. Tollin, Biophys.", "J., 2000, 79, 2463; J. D. Burgess, M. C. Rhoten and F. M. Hawkridge, Langmuir, 1998, 14, 2467; Heyse, S.; Ernst, O. P.; Dienes, Z.; Hofmann, K. P.; Vogel, H. Biochemistry, 1998, 37, 507; ReBieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nature Biotechnology 1999, 17, 1105; Salamon, Z.; Tollin, G. Biophysical Journal, 1996, 71, 858; Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 14773; McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A.", "A. Biochim.", "Biophys.", "Acta, 1986, 864, 95; J. K. Cullison, F. M. Hawkridge, N. Nakashima, and S. Yoshikawa, Langmuir, 1994, 10, 877.)", "Typically, the receptor is solubilized in an aqueous buffer containing a surfactant above its critical micelle concentration (cmc).", "In the presence of fluid, unilamellar bilayer vesicles, removal of the surfactant from the solution (usually performed by dialysis) causes spontaneous insertion of the receptor into the bilayer, forming proteo-liposomes.", "Fusion of the proteo-liposomes to a solid support results in formation of a fluid, supported membrane containing receptor molecules (Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 14773).", "Retention of bioactivity for receptors reconstituted in this manner has been observed.", "However, with respect to use as a protein-resistant coating in, for example, a receptor-based biosensor, a fluid lipid bilayer lacks the required physical and chemical stability, such as removal from water.", "Polymerization of the lipid monomers to create a stabilized membrane, as described above, is a logical solution to this problem.", "To demonstrate the feasibility of this strategy, two different types of transmembrane receptors, cytochrome c oxidase (CcO) and human delta opioid receptor (d-OR) have been successfully reconstituted into polymerized, supported lipid membranes composed of bis-SorbPC, as described in the example section (see below).", "The receptor is incorporated into the fluid, supported lipid membrane prior to carrying out polymerization step.", "There are two preferred methods for incorporation: surfactant dialysis followed by vesicle fusion (described briefly above and extensively in the literature; e.g.", "Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 14773), and insertion into a pre-formed supported membrane (Z. Salamon, S. Cowell, E. Varga, H. I. Yamamura, V. J. Hruby and G. Tollin, Biophys.", "J., 2000, 79, 2463).", "In the second method, SUVs composed of polymerizable lipids are fused on a support to form a fluid, supported lipid membrane that does not contain protein (as described above).", "Small aliquots of a concentrated solution of the receptor solubilized in a surfactant, e.g.", "octylglucoside, present above its cmc are added to the aqueous buffer solution in contact with the supported membrane.", "This dilutes the surfactant to a final concentration below its cmc, which results in spontaneous transfer of the receptor from the surfactant micelles to the supported membrane.", "For both incorporation methods, there are many experimental variables that are specific to the type of transmembrane protein receptor being used.", "These variables include buffer concentration and pH, presence and concentration of added salts, protein concentration, lipid concentration, presence and concentration of charged lipid headgroups, surfactant concentration, and temperature.", "Values of these variables that are appropriate for incorporation of different transmembrane proteins into fluid supported lipid membranes have been published (Z. Salamon, S. Cowell, E. Varga, H. I. Yamamura, V. J. Hruby and G. Tollin, Biophys.", "J., 2000, 79, 2463; J. D. Burgess, M. C. Rhoten and F. M. Hawkridge, Langmuir, 1998, 14, 2467; Heyse, S.; Ernst, O. P.; Dienes, Z.; Hofmann, K. P.; Vogel, H., Biochemistry, 1998, 37, 507; ReBieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H., Nature Biotechnology 1999, 17, 1105; Salamon, Z.; Tollin, G., Biophys.", "J., 1996, 71, 858; Salafsky, J.; Groves, J. T.; Boxer, S. G., Biochemistry 1996, 35, 14773; McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A.", "A., Biochim.", "Biophys.", "Acta, 1986, 864, 95; J. K. Cullison, F. M. Hawkridge, N. Nakashima, and S. Yoshikawa, Langmuir, 1994, 10, 877.)", "Any of these conditions is also appropriate for transmembrane protein incorporation into a fluid supported membrane composed of polymerizable lipids.", "The difference between prior art and the present invention is the use of polymerizable lipids, such as those shown in FIGS.", "1-6.In addition to transmembrane proteins, the present invention is also advantageous for biofunctional presentation of water-soluble protein receptors.", "Attaching water-soluble proteins to the surface of a polymerized, supported lipid membrane confers a biorecognition function to the membrane.", "Any water-soluble protein can be attached to the supported membranes described herein, either before or after polymerization has been effected, but preferably after.", "In all cases, a preferred surface coverage of receptors is 0.1% to 100% of the coverage equivalent to a one monolayer of receptor.", "Attachment in an appropriate manner and orientation that maintains receptor activity can be assayed by the observation of the specific binding to complementary partners.", "Numerous methods to attach water-soluble proteins to fluid and gel phase supported lipid bilayers have been described in the literature and are applicable to polymerized membranes described above as well.", "Three preferred methods are listed here: a) Biospecific binding between a protein ligand attached to a lipid headgroup in the membrane and a binding site for the ligand on the protein.", "For example, streptavidin can be attached to a supported lipid membrane that contains biotin-conjugated lipids (Edmiston, P. L.; Saavedra, S. S., J. Amer.", "Chem.", "Soc., 1998, 120, 1665).", "b) Covalent linkage between a functional group on the protein, e.g an amino or a thiol group, and a lipid bearing a reactive headgroup, e.g.", "a maleimido, pyridyldithio, or succinimidyl ester group.", "For example, yeast cytochrome c can be attached to a supported lipid membrane that contains pyridyldithio-conjugated lipids (Edmiston, P. L.; Saavedra, S. S., Biophys.", "J., 1998, 74, 999).", "c) Electrostatic adsorption of a charged protein to an oppositely charged membrane surface.", "For example, horse cytochrome c, which is positively charged at neutral pH, can be adsorbed to the surface of a lipid membrane that contains lipids having negatively charged headgroups such as phosphatidic acid and/or phosphatidylserine (Pachence, J. M.; Amador, S.; Maniara, G.; Vanderkooi, J.; Dutton, P. L.; Blasie, J. K., Biophys.", "J., 1990, 58, 379).", "For any of these methods, there are many experimental variables that are specific to the type of protein being attached.", "These variables include buffer concentration and pH, presence and concentration of added salts, protein concentration, presence and concentration of reactive lipid headgroups, presence and concentration of charged lipid headgroups, and temperature.", "Any of the conditions used in published methods is also appropriate for protein attachment to a polymerized, supported lipid membrane.", "The difference between prior art and the present invention is the use of polymerizable lipids, such as those shown in FIGS.", "1-6.Regardless of the method and conditions used to insert or attach receptors into or to the surface of a fluid supported membrane composed of polymerizable lipids, the above described redox-initiated or light-driven (UV, visible, or near-infrared) methods may then be used to polymerize the membrane.", "A preferred strategy to preserve receptor activity during the polymerization step is pre-incubation of receptors with a solution of their respective ligand or agonist or antagonist at a concentration sufficiently high to saturate the binding sites on the receptors.", "Occupancy of the binding sites before polymerization provides a degree of steric ‘protection’ during the subsequent polymerization step.", "After polymerization is effected, the bound ligands can be dissociated from the receptors by standard methods to generate a membrane with unoccupied ligand binding sites.", "The sixth embodiment of the present invention relates to the fabrication of spatially addressable, planar arrays of biomolecules.", "Techniques for such processes are currently being developed in numerous laboratories, based on projected applications for these arrays in rapid screening assays and multianalyte biosensors.", "For example, a method to generate an array of protein molecules adsorbed to a substrate using microcontact printing is disclosed in Bernard, A.; Renault, J. R.; Michel, B.; Bosshard, H. R.; Delamarche, E., Adv.", "Mater,.", "2000, 12, 1067-1070.Boxer and coworkers have pioneered the development of methods to generate micro-patterned fluid lipid bilayers.", "Hovis, J. S.; Boxer, S. G., Langmuir, 2000, 16, 894-897 disclose patterning barriers to lateral bilayer membranes by blotting and stamping.", "Kung, L. A.; Hovis, J. S.; Boxer, S. G., Langmuir, 2000, 16, 6773-6776, disclose patterning hybrid surfaces of proteins and supported lipid bilayers.", "Based on their work, the potential for creating arrays of membrane-associated receptors at a biocompatible surface which mimics many of the properties of a native cell membrane is clear.", "However, the fact that these patterned bilayers cannot be removed from water is a serious impediment to their practical implementation which can be overcome by the stabilized membranes of the present invention.", "Specifically, the present invention relates to a) an array of protein molecules deposited on a uniformly polymerized lipid membrane; and b) an array of fluid (or partially polymerized) lipid domains in the membrane, separated by a regular array of domains in which the lipids are highly cross-linked.", "Exploiting the air stability and biocompatibility of polymerized lipid membranes, microcontact printing (μCP) can be used to generate arrays of protein molecules attached to membrane surfaces in a manner designed to maximize specific activity.", "In μCP, a poly(dimethylsiloxane) (PDMS) stamp is linked with the molecule of interest, which is then transferred to a planar substrate by stamping.", "Two recent reviews of μCP and related soft lithography techniques are: (a) Xia, Y.; Whitesides, G. M.; Annu.", "Rev.", "Mater.", "Sci., 1998, 28, 153-184 and (b) Xia, Y.; Rogers, J.", "A.; Paul, K. E.; Whitesides, G. M., Chem.", "Rev., 1999, 99, 1823-1848.To date, μCP of proteins has been performed on high energy substrate surfaces (e.g.", "silica), to which the proteins bind by strong nonspecific interactions (St. John, P. M.; Davis, R.; Cady, N.; Czajka, J.; Batt, C. A.; Craighead, H. G., Anal.", "Chem., 1998, 70, 1108-1111; Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H., Langmuir, 1998, 14, 2225-2229; Bernard, A.; Renault, J. R.; Michel, B.; Bosshard, H. R.; Delamarche, E., Adv.", "Mater., 2000, 12, 1067-1070).", "Although some of the adsorbed molecules retain bioactivity, this method is clearly inefficient since a significant fraction of the adsorbed proteins are likely to be inactivated due to surface-induced denaturation, which is known to occur for proteins immobilized on high energy (e.g.", "silica) and hydrophobic (e.g.", "polystyrene) surfaces.", "Furthermore, when the printed array is subsequently brought into contact with a solution of dissolved proteins (e.g.", "during bioassay), the regions of the substrate not coated with printed protein will be subject to nonspecific protein adsorption interactions.", "Thus it is desirable to prepare protein arrays on substrates that are inherently protein-resistant, such as a polymerized supported lipid bilayer.", "In one preferred implementation of the present invention, a spatial array of protein molecules is deposited on a uniformly polymerized lipid membrane.", "The present inventors have found that arrays of proteins can be deposited by μCP on a uniformly polymerized, supported lipid bilayer when the bilayer surface is dried.", "Upon subsequent immersion into aqueous solution, the printed proteins remain adhered to the printed areas on the bilayer.", "Furthermore, the printed proteins retain the capability to bind to other dissolved proteins that are subsequently incubated with the patterned surface.", "An example is shown in FIG.", "18.A bis-SorbPC (redox) bilayer was prepared on a SiO2 substrate as described above and dried under Ar.", "A PDMS stamp was inked with a solution of biotin-BSA (BSA molecules bearing covalently attached biotin groups.", "Stamping was used to create a pattern of biotin-BSA on the lipid bilayer.", "An AFM image of the biotin-BSA stripes on the dried lipid bilayer is shown on the left side of FIG.", "18.The printed bilayer was then immersed in a solution of rhodamine-conjugated avidin (schematic on right side of FIG.", "18).", "The avidin bound to the exposed biotin groups in the regions where biotin-BSA had been printed, but did not adsorb to the non-printed regions, as expected since the bare bis-SorbPC (redox) bilayer is highly protein resistant.", "An epifluorescence micrograph (inset at center of FIG.", "18) shows the emission pattern of rhodamine-conjugated avidin bound to the lipid bilayer, and confirms that binding occurred only in the regions where biotin-BSA had been printed.", "At this time, it is not known why proteins adsorb strongly and nonspecifically to a dried bilayer, whereas a hydrated bilayer is protein resistant.", "Accordingly patterns of proteins can be created on dried, uniformly polymerized, supported lipid membranes.", "The microcontact printed protein adheres strongly to the printed regions, remains so when the membrane is rehydrated, and retains the capability to specifically bind other ligands, including other proteins.", "Furthermore, the remaining regions of the membrane retain their characteristic protein resistance.", "This implementation can also be performed on polymerized lipid membranes containing any of the types of lipids shown in FIGS.", "1-6, or mixtures thereof.", "Particularly, uCP of proteins can be performed on membranes containing functional lipids having anionic or cationic headgroups, headgroups designed to chelate metal ions, or headgroups designed to covalently react with other molecules.", "Examples of the latter category include headgroups terminated with thioethanol, maleimido, pyridyldithio, biotinyl, succinimidyl ester, sulfo succinimidyl ester, alkyl halide, or haloacetamide groups.", "When any of these lipids types is mixed with a second lipid type to form a membrane, the second type of lipid molecule is usually selected to be protein resistant, e.g.", "bis-SorbPC.", "The first lipid type is usually selected so that it reacts with the protein molecules that are being printed on the membrane; the objective being to maximize the adherence of the protein to the printed regions on the membrane.", "For example, uCP of a protein onto a polymerized lipid bilayer containing succinimidyl ester-conjugated lipids will result in formation of a covalent bond between these lipids and the lysine groups on the surface of the protein, thereby firmly attaching the protein to the bilayer surface.", "In a second preferred implementation of the present invention, a supported lipid membrane is composed of an array of fluid (or partially polymerized) lipid domains that are separated by a regular array of domains in which the lipids are highly cross-linked.", "Transmembrane proteins can be reconstituted into polymerized bilayers as described above.", "However, to maintain bioactivity, some transmembrane proteins require a fluid membrane environment.", "Thus, it may be necessary to preserve a domain of fluid lipids in the immediate vicinity of an incorporated protein, while the remainder of the bilayer is polymerized to generate a stabilized membrane.", "These pattern are preferably created using uCP or photolithographic methods.", "Specifically, membrane proteins can be reconstituted into microfluid domains within a supported lipid membrane that has undergone patterned polymerization to effect overall stability.", "For example, a patterned polymerized supported lipid bilayer is shown in FIG.", "19.Here, the pattern was obtained using a uCP method developed by Boxer's group (Hovis, J. S.; Boxer, S. G., Langmuir, 2000, 16, 894-897, Kung, L. A.; Hovis, J. S.; Boxer, S. G., Langmuir, 2000, 16, 6773-6776).", "A PDMS stamp was pressed against and then removed from a fluid bis-SorbPC lipid bilayer on SiO2 under water; the contacted regions of the bilayer adhered to the stamp and were removed, leaving the underlying glass surface exposed (shown on the right in FIG.", "19).", "UV polymerization of the remaining regions of the bilayer then yielded an air-stable structure from which the AFM image shown on the left in FIG.", "19 was acquired.", "The importance of this result is that it is feasible to regenerate fluid domains between the polymerized domains by incubating the patterned surface with fluid lipid vesicles, which will fuse to the exposed substrate surface between the polymerized regions (Hovis, J. S.; Boxer, S. G., Langmuir, 2000, 16, 894-897) producing a continuous bilayer.", "Patterned polymerization is achievable using UV exposure to initiate cross-linking, either through an optical mask or using holography.", "Here, the UV-light may be polarized or unpolarized.", "Following polymerization, the unreacted lipids can be dissolved away from the substrate, yielding a pattern of substrate exposed and polymeric bilayer-coated regions.", "Vesicle fusion can then be used to form fluid bilayer domains between the polymerized regions.", "Alternatively, “incompletely” polymerized domains of lipids can be created between the highly cross-linked domains.", "Incomplete polymerization can be achieved, for example, using an appropriate molar ratio of a non-polymerizable lipid and mono-SorbPC and/or bis-SorbPC.", "Accordingly, patterned bilayers composed of polymerized and fluid domains can be obtained by uCP printing and UV lithography.", "The seventh embodiment relates to the use in sensors.", "In a seventh embodiment of the present invention, polymerized, supported lipid membranes, with and without associated proteins, are used as nonfouling coatings for chemical sensing and biosensing devices.", "In a biosensing device, the characteristic selectivity of biorecognition is exploited in the form of an integrated device that couples a biological binding element, e.g.", "a protein receptor, to a physical transducer, to perform highly selective analysis of one component (or class of components) in a complex sample matrix (Biosensors: Fundamentals and Applications; A. P. F. Turner, I. Karube, and G. S. Wilson, Eds.", "; Oxford: New York, 1987; M. A. Arnold and M. E. Meyerhoff, CRC Crit.", "Rev.", "Anal.", "Chem., 1998, 20,149-196).", "A biochip is a biosensor that presents a spatially defined array of different recognition elements to a sample, permitting parallel analysis of multiple analytes in a single sample (Vo-Dinh, T.; Cullum, B. M.; Stokes, D. L., Sensors and Actuators B, 2001, 74, 2-11).", "The potential for widespread application of these devices in numerous areas, including drug screening, is well accepted.", "Supported lipid membranes are useful as transducer coatings for biosensing devices because: a) they preserve the bioactivity of incorporated and/or attached proteins (e.g.", "P. L. Edmiston and S. S. Saavedra, Biophys.", "J., 1998, 74, 999-1006; P. L. Edmiston and S. S. Saavedra, J. Amer.", "Chem.", "Soc., 1998, 120, 1665-1671; Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E., Langmuir, 1993, 9, 136-140; Salafsky, J.; Groves, J. T.; Boxer, S. G., Biochemistry, 1996, 35, 14773-14781; Z. Salamon, S. Cowell, E. Varga, H. I. Yamamura, V. J. Hruby and G. Tollin, Biophys.", "J., 2000, 79, 2463-2474.", "); and b) they resist nonspecific adsorption of non-target proteins that may present in the sample matrix in addition to the analyte (Wisniewski, N.; Reichert, M., Colloids and Surfaces B-Biointerfaces, 2000, 18, 197-219; Eric E. Ross, Bruce Bondurant, Tony Spratt, John C. Conboy, David F. O'Brien, and S. Scott Saavedra, Langmuir, 2001, 17, 2305-2307; Hayward, J.", "A.; Chapman, D., Biomaterials, 1984, 5, 135-142; Sackman, E. Science, 1996, 271, 43-48).", "Polymerized, supported lipid membranes can be used in many types of biosensing devices, including devices based on electrochemical, spectro-electrochemical, or optical (absorbance, luminescence, reflectivity, or scattering) transduction methods.", "In all cases, a polymerized, supported lipid membrane containing receptors, either water-soluble or membrane-associated receptor proteins or nucleic acids, is present between the physical transducer and the sample solution.", "The sample solution contains the analyte of interest.", "Binding of the analyte molecules to the membrane-incorporated receptors is detected at the transducer using, for example, electrochemical, spectro-electrochemical, or optical (absorbance, luminescence, reflectivity, or scattering) methods.", "The protein resistant properties of the lipid membrane prevent binding of other molecules present in the sample matrix, especially other proteins.", "In a preferred implementation, a supported, lipid membrane that contains transmembrane protein receptors is deposited on the transducer surface and polymerized using the preparation methods described above.", "Binding of ligands to receptors, where the ligands are also analytes, is detected optically as a change in absorbance, luminescence, reflectivity, or scattering at the transducer surface.", "More preferably the binding is detected using fluorescence methods or surface plasmon resonance methods.", "Accordingly, a self-assembled, supported lipid bilayer formed from the types of lipids shown in FIGS.", "1-6 can be stabilized to surfactants, organic solvents, and transfer across the water/air interface by cross-linking polymerization of moieties in the acyl chains.", "The self-assembled, supported lipid membrane of the present invention can be utilized as a protein-resistant coating for molecular devices.", "Furthermore, the stabilized lipid membrane of the present invention are suitable as a non-fouling coating for medical implant materials or analytical fluid handling instruments or biomedical devices requiring a non-fouling coating.", "In addition, they find application as a cell-membrane mimetic for supporting surface-associated and transmembrane proteins in their native state in various biological detection devices (e.g.", "biosensors).", "The stabilized phospholipid bilayers of the present invention can also be used as a general non-fouling coating for mass produced commercial items, for example razor blades.", "Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.", "EXAMPLES General Procedures Materials: Bis-sorbyl phosphatidylcholine (bis-SorbPC) was prepared by a modification of the procedure reported by Lamparski, H.; Liman, U.; Frankel, D. A.; Barry, J.", "A.; Ramaswami, V.; Brown, M. F.; O'Brien, D. F., Biochemistry, 1992, 31, 685-694.The synthesis of bis-dienoyl phosphatidylcholine (bis-DenPC) was adapted from that reported by Dom, K.; Klingbiel, R. T.; Specht, D. P.; Tyminski, P. N.; Ringsdorf, H.; O'Brien, D. F., J.", "Am.", "Chem.", "Soc., 1984, 106, 1627-1633.The synthesis of mono-sorbyl phosphatidylcholine (mono-SorbPC) is described in Lamparski, H., and D. F. O'Brien, Macromolecules, 1995, 28, 1786-1794.The synthesis of dienoyl sorbyl phosphatidylcholine (DenSorbPC) is described in Liu, S.; Sisson, T. M.; O'Brien, D. F., Macromolecules, 2001, 34, 465-473.Lipid structures were established by 1H NMR and HRMS.", "In addition the purity was assessed by the presence of only one spot on TLC.", "All other lipids were purchased from either Avanti Polar Lipids, Inc. (Alabaster, Ala.) or Sigma Chemical.", "Potassium persulfate and sodium bisulfate were purchased from Aldrich and used as received.", "Bovine serum albumin labeled with fluorescein (FITC-BSA, labeling ratio of 11.2:1) and tetramethylrhodamine (TMR-BSA, ratio of 1:0.9) were obtained from Sigma and used without any further purification.", "Fluorescein labeled dextran (10,000 MW, 2.9:1 labeling ratio) and rhodamine labeled dextran were purchased from Molecular Probes.", "All other chemicals and solvents were purchased from standard commercial suppliers and used without further purification.", "Single crystal (111) silicon wafers having a 20±5 Å thick native oxide layer were purchased from Wacker.", "Fused silica slides were purchased from Dynasil Corp. Deionized water (18 MOhms and made organic free (<10 ppb)) was obtained from a Barnstead Nanopure water system.", "Substrate preparation: Si wafers and fused silica slides were soaked for 30 minutes in pirhana solution (70% H2SO4/30% H2O2), followed by extensive rinsing and sonication in deionized water.", "Unless otherwise noted, substrates were stored in deionized water until used, within 1 hour of cleansing.", "Self-assembly of supported lipid bilayers by vesicle fusion: Lipids from stock chloroform or benzene solutions were dried under flowing Ar to remove storage solvents and were then dried overnight under vacuum in ½ dram vials.", "The lipids were then resuspended in deionized water or in buffer (100 mM NaCl, 10 mM phosphate, pH 7.4) to a final lipid concentration of 0.5 mg/ml.", "The lipid suspension was then vortexed and sonicated to clarity in a Branson Sonicator fitted with a cup horn (Barrow, D. A.; Lentz, B. R Biochim Biophys Acta 1980, 597, 92-99) to form SUVs.", "Temperature control was maintained with a water bath and was performed at more than 10 degrees above the reported lipid transition temperature.", "The SUVs were used within 30 minutes of preparation.", "Clean Si substrates (or fused silica) were dried by N2 immediately prior to fusion.", "A few drops of lipid vesicle solution (SUVs) were deposited on the Si substrate (or fused silica).", "Lipids were fused at a temperature equal to or greater than their respective main phase transition temperature for at least ten minutes.", "The surfaces were then either transferred to test tubes for redox polymerization, or to shallow crystallization dishes to be polymerized by direct UV irradiation.", "Care was taken to not expose the unpolymerized films to air, or excessive mechanical shocks.", "Langmuir-Blodgett Schaefer deposition of supported lipid monolayers and bilayers: Supported lipid films were formed using standard Langmuir-Blodgett-Schaefer techniques according to reference procedures (e.g.", "Morigaki, K.; Baumgart, T.; Offenhäusser, A.; Knoll, W., Angew.", "Chem., Int.", "Ed., 2001, 40, 172; Conboy, J. C.; McReynolds, K. D.; Gervay-Hague, J.; Saavedra, S. S. J. Amer.", "Chem.", "Soc., 2002, 124, 968-977; McConnell, H. M.; Watts, T. H.; Weis, R. M. Biochim.", "Biophys.", "Acta 1984, 864, 95-106) on a Nima Model 611D Langmuir-Blodgett trough.", "Care was taken to avoid exposure of polymerizable lipids to visible light that could potentially cause photodegradation; thus prior to polymerization, all manipulations were performed under yellow light.", "The first layer of the bilayer was deposited vertically.", "Langmuir lipid monolayers were spread on a Nima Model 611D Langmuir-Blodgett trough using benzene as the spreading solvent and deionized water as the subphase.", "Film depositions were performed at a surface pressure of 35-40 mN/m, corresponding to approximately 60 Å2/molecule.", "The inner leaflet of the bilayer was deposited by withdrawing the substrate from the subphase at a rate of 10 mm/min.", "Transfer ratios of approximately 98.5% were repeatedly obtained.", "The second leaflet of the bilayer structure was deposited using the Langmuir-Schaefer horizontal transfer technique.", "The substrate with the previously deposited lipid monolayer was passed horizontally through the air-water interface at constant pressure (35-40 mN/m).", "After formation, the unpolymerized bilayer was maintained in an aqueous environment at all times.", "All depositions were carried out at 25° C. Redox polymerization: Redox initiated, radical polymerization was performed with deoxygenated solutions of potassium persulfate and sodium bisulfate.", "The concentration ratio was 100 mM K2S2O8/10 mM NaHSO3.Polymerizations were also performed at other K2S2O8/NaHSO3 concentrations, ranging from 0.001 to 1.0 M. After deposition by vesicle fusion or Langmuir-Blodgett-Schaefer techniques, the supported lipid bilayer is transferred to the Ar-saturated polymerization solution without exposing the bilayer to air, incubated for two hours under flowing Ar, then rinsed extensively with deionized water, and dried under a stream of N2.A two hour incubation period was determined to be sufficient to achieve near quantitative polymerization of bis-SorbPC bilayers, based on the near quantitative disappearance of the monomer absorbance band at 260 nm during the incubation period.", "The disappearance of the band was monitored by UV transmission spectroscopy performed (as described in Example 4) on 4 bis-SorbPC bilayers prepared by vesicle fusion as described above.", "UV polymerization: UV-induced polymerization of supported lipid films was performed by exposure to UV radiation from a low-pressure mercury pen lamp (Fisher Scientific) with a rated intensity of 4500 mW/cm2 at 254 nm.", "A 1.0 mm thick UV band pass filter from Scott Glass (UG5) was used to remove the short wavelength UV (<230 nm) that can fragment polymer chains into oligomers.", "In cases where oxygen was to be excluded, the solution in contact with the lipid film was deoxygenated with flowing Ar for at least 30 minutes prior to and throughout the polymerization.", "Ellipsometry: The thickness of dried lipid films deposited on Si substrates was determined by ellipsometry.", "Measurements were made with a Rudolph Research model 43603-200E ellipsometer using a 632.8 nm He—Ne laser at an incident angle of 70 degrees.", "Initial readings were taken on the bare Si substrates to establish the substrate optical constants and oxide layer thickness prior to any film formation.", "A refractive index of 1.46 was assumed for all lipid layers.", "The ellipsometry data were used to calculate the corresponding thickness values using DafIBM version 2.0, a computer program supplied by Rudolph Research and implemented on a DOS-based PC system.", "Contact angle measurements: Contact angles of deionized water deposited on supported lipid films were measured using the sessile drop method.", "In some case, images of multiple 3 mL water droplets on each surface were taken using a Pulnex TM-7CN video camera and Video Snapshot Snappy and were the average of at least three samples.", "Images were converted into tagged image format using corresponding software, and angles were measured using Image-Pro Plus 1.3 software (Media Cybernetics).", "In other cases, water droplets on surfaces were photographed using a TE-cooled CCD camera (Princeton Instruments Model 512TK) and the contact angle retrieved via imaging analysis software (Scion Image).", "Both methods gave equivalent results.", "Atomic force microscopy: The surface morphology of supported lipid films was examined by atomic force microscopy (AFM), performed in tapping mode on a Digital Instruments Multimode III microscope.", "Oxide sharpened silicon nitride tips (TESP-7) were purchased from Digital Instruments, and were tuned to between 300 and 400 kHz.", "For water immersion studies, measurements were performed in a fluid cell (Digital Instruments) in tapping mode with contact tips tuned to 33 kHz as per supplemental Digital Instrument instructions.", "Samples were immersed in deionized water for 0.5-1.5 hours before image acquisition commenced.", "Images were acquired at several areas on each substrate, and images presented in this document are representative of scans from different locations on each sample, different samples, and with different tips used to image the surfaces.", "Deviations amongst different scan areas on a given film were extremely rare.", "The samples were not altered by the AFM measurement, as noted by the invariance of successive AFM scans.", "Total internal reflection fluorescence (TIRF) spectroscopy: Protein adsorption studies were performed by TIRF spectroscopy.", "The experimental approach is described in Conboy, J. C.; McReynolds, K. D.; Gervay-Hague, J.; Saavedra, S. S., J. Amer.", "Chem.", "Soc., 2002; 124, 968-977.Protein adsorption was measured to fused silica slides coated with lipid films and other types of organic layers, as well as to clean fused silica.", "The optical arrangement (FIG.", "20) consists of two right-angle quartz prisms mounted in a TIRF flow cell.", "One prism is used to couple the excitation light from an Ar-ion laser into the cell; the light then propagates by total internal reflection down the fused silica slide.", "The other prism is used to outcouple the excitation light, thereby reducing scattered light in the cell volume.", "Index matching fluid (1.463 nd, Cargille) was used to allow for efficient incoupling and outcoupling of the incident laser light.", "The flow cell was mounted on a Nikon Diaphot inverted microscope.", "Excitation wavelengths were 488 nm (for measuring fluorescein emission) or 514 nm (for measuring rhodamine emission).", "Fluorescence emission was back-collected through the quartz slide with a 4× or 10× objective, optically filtered, and detected with a photomultiplier tube.", "The incident excitation light was modulated at a frequency of 2.5 kHz.", "Phase sensitive detection was used to retrieve the fluorescence intensity.", "The experiment was interfaced to a PC for data collection.", "All experiments were performed at 25° C. To determine an equilibrium binding constant for protein adsorption to the surfaces under investigation, solutions of fluorescently-tagged BSA were injected into the flow cell and allowed to equilibrate for 30 min prior to each measurement, which was determined experimentally to be a sufficient time for a steady-state fluorescence intensity to be measured.", "A Langmuir model was used to extract binding constants from the measured fluorescence intensities, according to published procedures.", "A modified form of a numerical quantitation method (Hlady, V.; Reinecke, D. R.; Andrade, J. D. J. Colloid Interface Sci.", "1986, 111, 555-569) was used to determined the surface coverage of protein molecules from the fluorescence data.", "Surface coverages were determined relative to reference surfaces known to strongly adsorb all classes of protein molecules (e.g.", "clean fused silica).", "A detailed description of the procedures used for determination of equilibrium binding constants and surface coverages has been published and is incorporated herein by reference (Conboy, J. C.; McReynolds, K. D.; Gervay-Hague, J.; Saavedra, S. S., J. Amer.", "Chem.", "Soc., 2002; 124, 968-977).", "X-ray photoelectron spectroscopy (XPS): XPS was used to determine the chemical composition of supported lipid films.", "XPS measurements were made on polymerized bilayers supported on silicon wafers using a Kratos 165 Ultra Imaging XPS equipped with a 165 mm mean radius hemispherical analyzer and an eight channeltron detection system.", "The base pressure in the analyzer chamber was ca.", "5×10−9 Torr.", "X-rays from the Al Kα line at 1486.6 eV were used for excitation.", "Electrons were collected in the constant analyzer energy (CAE) mode with a pass energy of 50 keV.", "Integration times were 0.25 s, co-added four times, for a total of 1.0 s at an interval of 0.1 eV.", "The areas under the XPS peaks were measured by numerical integration after baseline correction.", "Relative peak area ratios were calculated using previously published photoionization cross-sections (Schofield, J. J. Electron Spectrosc.", "Relat.", "Phenom.", "1976, 8, 129-137) after accounting for the transmission properties of the analyzer.", "Example 1 Polymeric bis-SorbPC Films Self-Assembled by Vesicle Fusion; Comparison to Polydiacetylene Lipid Films.", "Supported lipid bilayer films composed of bis-SorbPC were self-assembled by vesicle fusion and polymerized by redox initiation as described above.", "Assuming an index of refraction of 1.46 for the lipid film, the ellipsometric thickness of the dried, polymerized bis-SorbPC bilayer was found to be 46±3 Å. X-ray reflectometry was used to measure the electron density of a dried, polymerized bis-SorbPC bilayer supported on a quartz substrate along the axis normal to the bilayer plane.", "X-ray reflectivity measurements (kindly perfomed at the National Institute for Standards and Technology by Dr. Jarek Majewski of Los Alamos National Laboratory) yielded a thickness of 45±1.4 Å.", "Both thickness measurements agree well with the expected thickness for a bilayer composed of fully extended bis-SorbPC.", "The acyl chains in a bis-SorbPC molecule are shorter by one bond than the acyl chains in a DOPC molecule.", "The thickness of a bis-SorbPC bilayer should therefore be slightly less than that of a DOPC bilayer, which has been determined to be about 45 Å (Wiener, M. C.; White, S. H., Biophys.", "J., 1992, 61, 434).", "Thus the thickness data provide strong evidence that the overall structure of bilayer structure is preserved upon transfer through the water/air interface.", "The contact angle of a sessile water drop on a polymerized bis-SorbPC bilayer was 31±4 degrees, consistent with a surface composed of outward facing phosphorylcholine headgroups.", "For comparison, the water contact angle measurements on a freshly cleaned Si wafer and on a Langmuir-Blodgett transferred monolayer of bis-SorbPC were <5 and 63±5 degrees, respectively.", "The contact angle for the bis-SorbPC monolayer is lower than that expected for a surface composed of saturated alkyl chains, and reflects the presence of ester groups near the chain termini.", "Evidence for extensive cross-linking in polymerized, supported bis-SorbPC bilayers is given by the insolubility of these structures in surfactant solution.", "The ellipsometric thickness did not change upon bath sonication in a 1% solution of Triton X-100 for ten minutes or immersion in chloroform or acetone for 10 seconds (both conditions at room temperature), which suggests that the polymer size in these films is sufficiently large to render them insoluble.", "The image in FIG.", "8, acquired using tapping mode atomic force microscopy (AFM) in air, shows that the surface of a polymerized, supported bis-SorbPC bilayer is very smooth.", "The rms of the image in FIG.", "8 (left) is 1.25 Å, which is comparable to the bare silicon substrate (rms roughness of 1.1 to 1.3 Å).", "The bilayer surface morphology was surprisingly uniform; the image shown in FIG.", "8 is representative of images acquired at numerous locations over a ca.", "1 cm2 sample area.", "No topographical features greater than 1 nm in height (peak-to-peak) were detected.", "Thus any defects at which bare substrate was exposed were too narrow to be detected by AFM.", "Polymerized films could be deliberately damaged by repeated, high force scanning; a line scan across a “trough” produced in a film in this manner showed an apparent film thickness of 39-47 Å, consistent with the thickness measurements described above.", "No discernible chance in film morphology was observed when a previously dried region of a film was rehydrated and then re-imaged under water (FIG.", "8 (center)).", "Supported bilayers composed of a mixture of bis-SorbPC and the non-polymerizable lipid DOPC at a molar ratio of 7 to 3 were also prepared by vesicle fusion and polymerized by redox initiation as described above.", "These films are observed to contain numerous defects as revealed by AFM (FIG.", "14).", "This result shows that polymerized bilayer films that contain appreciable amounts of unpolymerized lipids are not stable to removal from water.", "Supported bis-SorbPC bilayers, self-assembled by vesicle fusion as described above, were also polymerized by direct UV irradiation, as described above, and characterized by ellipsometry, AFM, and contact angle measurements, etc.", "In comparison to redox polymerized bis-SorbPC films, the UV polymerized bis-SorbPC films were thinner and less hydrophilic.", "Specifically, the film thickness was approximately 29 Å and the water contact angle was 52 degrees.", "AFM images shown in FIG.", "13 illustrate that relative to redox polymerized bis-SorbPC films (rms roughness of 0.13 nm), the UV polymerized films are rougher (rms roughness=0.35 mm), and have discernable features or domains approximately 1.5 to 2 nm thick.", "These features are very uniformly distributed on the film surface; no regions were found that were devoid of polymer film, or where the domains differed appreciably in size.", "For comparison purposes, supported lipid bilayers were also prepared using a commercially available, polymerizable diacetylenic PC lipid (1,2-bis(10,12-tricosadionyl)sn-glycero-3-phosphocholine (DAPC); Avanti Polar Lipids).", "Supported bilayers of DAPC were prepared by Langmuir-Blodgett-Schaefer deposition, as described above, and photopolymerized using UV light (procedures for this lipid are described in detail in Morigaki, K.; Baumgart, T.; Offenhausser, A.; Knoll, W., Angew.", "Chem., Int.", "Ed., 2001, 40, 172).", "Polymerization of DAPC films could not be induced with oxygen present in the solution contacting the bilayer; negligible film thickness resulted if the solution was not purged thereof before and during polymerization.", "After polymerization and drying, supported DAPC bilayers produced an ellipsometric thickness of 55 Å.", "However, AFM imaging showed that these films were highly nonuniform.", "The example shown in FIG.", "12 contains relatively large defects, some of which extended down to the substrate.", "Line scans generally showed two defect depths of defects, 2.5-3 nm and 4.5-6 nm.", "Thus the performance of the present invention is superior to existing technology, as a comparison of FIGS.", "8 and 12 clearly shows.", "Example 2 Polymeric Lipid Bilayers Self-Assembled by Vesicle Fusion From Other Sorbyl and Dienoyl Lipids.", "Supported bilayers composed of mono-SorbPC were also self-assembled by vesicle fusion and polymerized by redox initiation as described above.", "The quality of the resulting films was generally poorer that the corresponding bis-SorbPC films.", "The ellipsometric thickness was measured to be 31 Å, and the AFM images (e.g.", "FIG.", "15) revealed domain-like features similar to those observed for UV polymerized bis-SorbPC films.", "This result is consistent with the observation in vesicle studies that a cross-linked lipid polymer is more stable to solvent and surfactant dissolution than a linearly polymerized lipid polymer.", "Supported bilayers composed of DenSorbPC were self-assembled by vesicle fusion and polymerized by redox initiation as described above.", "Polymerized DenSorbPC bilayers were indistinguishable from polymerized bis-SorbPC films by AFM.", "(FIG.", "17).", "The measured ellipsometric thickness of 45 Å was nearly identical as well, and upon bath sonication in surfactant, only a minute thickness change was observed.", "The sessile water contact angle was measured to be 42 degrees, which is slightly less hydrophilic in comparison to a redox polymerized bis-SorbPC bilayer.", "The redox polymerization of supported bis-DenPC lipid bilayers self-assembled by vesicle fusion also produced relatively thick films.", "The ellipsometric thickness measured after drying the film was 52 Å; however upon bath sonication in the surfactant Triton-X-100, a significant decrease in film thickness was observed.", "AFM images of the film after sonication in surfactant reveal the surface to contain defects located uniformly throughout the film (e.g.", "FIG.", "16).", "Linescans across the defects indicate that they do not reach the substrate (depth less than 3 nm); this is consistent with lipid loss from only the outer leaflet of bilayer.", "From the domain-like structure of the films shown FIGS.", "13-16, it is clearly feasible to generate a partially polymerized film that contains a regular array of microscopic voids.", "By performing vesicle fusion on these films, it should be possible to fill the voids with a second type of lipid, either polymerizable or non-polymerizable, and thus generate a mixed film containing a non-uniform spatial distribution of lipid types.", "Example 3 Extent of BSA Adsorption to Polymerized, Supported Lipid Films and Reference Surfaces.", "To examine the effect that cross-linking has on the nonspecific protein adsorption properties of a fluid PC bilayer, the degree of BSA adsorption to both UV and redox polymerized bis-SorbPC bilayers was measured using TIRF spectroscopy, and compared to BSA adsorption to a fluid 1-palmitoyl-2-oleolylPC(POPC) bilayer.", "Redox polymerized and UV polymerized bis-SorbPC bilayers were self-assembled by vesicle fusion on fused silica substrates according to Example 1, rinsed and dried under nitrogen, mounted in the TIRF flow cell (FIG.", "20), and rehydrated.", "POPC bilayers were fused to silica substrates that were preassembled in the cell, to avoid exposure of the fluid bilayer to air.", "The extent of BSA adsorption was also measured for several reference surfaces: (1) a supported DAPC bilayer, prepared and UV polymerized on fused silica as described in Example 1, then rehydrated in the TIRF flow cell; (2) a clean quartz substrate, which served as a model of a hydrophilic surface at which nonspecific protein adsorption is highly favored; (3) a ‘tail-group out’ monolayer of arachidic acid (AA), which served as a model of a hydrophobic surface at which nonspecific protein adsorption is highly favored.", "AA monolayers were deposited using the Langmuir-Blodgett method on fused silica substrates, which were then mounted in the flow cell and hydrated.", "Each type of surface was equilibrated with TMR-BSA (bovine serum albumin labeled with tetramethylrhodamine isothiocyanate) solution (1 mg/ml, containing 50 mM phosphate buffer, pH 7.4) for 30 minutes before the flow cell was flushed with buffer and TIRF emission from the adsorbed protein film was measured.", "Relative TMR-BSA surface coverages were determined using the calibration procedures described above (Hlady, V.; Reinecke, D. R.; Andrade, J. D. J. Colloid Interface Sci.", "1986, 111, 555-569; Conboy, J. C.; McReynolds, K. D.; Gervay-Hague, J.; Saavedra, S. S., J. Amer.", "Chem.", "Soc., 2002; 124, 968-977); the calibration solutions had known concentrations of dissolved (i.e.", "non-adsorbed) TMR-BSA.", "The bar graph in FIG.", "9 shows the relative TMR-BSA adsorption to all surfaces listed above.", "The BSA surface coverages on the redox polymerized bis-SorbPC and fluid POPC bilayers were 6±3% and 6±6%, respectively, of that obtained on the hydrophobic AA monolayer (100±24%; estimated to be ca.", "one monolayer).", "The statistical equivalence of the BSA surface coverage on the bis-SorbPC (redox) and fluid POPC bilayers demonstrates that the native resistance of the fluid bilayer to nonspecific protein adsorption is retained upon polymerization of the hydrophobic tails of the bis-SorbPC monomers, and provides further evidence that the “headgroup out” structure of the bis-SorbPC bilayer is preserved after drying and rehydration.", "Furthermore, approximately 70% of the TMR-BSA adsorbed to the bis-SorbPC (redox) bilayer could be removed by flushing the cell with a 1% Triton X-100 solution.", "No increase in the amount of adsorbed TMR-BSA was observed when the surface was re-exposed to 1 mg/ml TMR-BSA, which demonstrates the stability of the polymeric bis-SorbPC bilayer to surfactant solutions.", "The relative protein adsorption on the DAPC bilayer (40%) was slightly less than the 47% measured on clean fused silica (which is labeled as quartz in FIG.", "9).", "The relative adsorption on UV photopolymerized bis-SorbPC bilayers was 24%, intermediate between bis-SorbPC (redox) and DAPC.", "TIRF isotherms for TMR-BSA adsorption to several of these surfaces were measured over a protein concentration range of 5.0×10−9 M to 1.5×10−6 M and are plotted in FIG.", "10.The raw data were calibrated as described above, allowing the magnitude of the normalized fluorescence intensities plotted in FIG.", "10 to be directly compared.", "The shape of the adsorption isotherms and relative measured intensities show that the BSA interacts most strongly with the hydrophobic AA monolayer surface.", "Relative protein adsorption to the bis-SorbPC (redox) and POPC bilayers is very similar.", "AFM images and line scans of a silicon wafer and a wafer coated with a bis-SorbPC (redox) bilayer are shown in FIG.", "11.The surfaces were imaged both before and after incubation in a 1 ml/mg BSA solution (conditions given above).", "Consistent with the TIRF data, a significant increase in measured roughness occurs on the SiO2 surface, which is due to the considerable protein adsorption that occurs on clean SiO2.In contrast, a negligible change is observed for the bis-SorbPC (redox) bilayer, consistent with its demonstrated protein resistance.", "These results compare favorably with published data.", "At a dissolved BSA concentration of ca.", "0.05 g/L, Yang et al.", "(Yang, Z.; Galloway, J.", "A.; Yu, H., Langmuir, 1999, 15, 8405) reported a BSA surface coverage of ca.", "6% on methoxy-terminated polyethylene glycol) self-assembled monolayers, relative to the coverage measured on glass.", "At dissolved BSA concentrations of 0.05 and 2 g/L, Murphy and Lu (Murphy, E. F.; Lu, J. R.; Lewis, A. L.; Brewer, J.; Russell, J.; Stratford, P., Macromolecules, 2000, 33, 4545) measured BSA surface coverages on hydrogel polymers with incorporated phosphorylcholine groups of 20% and 36%, respectively, relative to SiO2.Thus the degree of non-specific BSA adsorption on a redox polymerized bis-SorbPC membrane is comparable to or better than that reported for other protein resistant surfaces.", "Example 4 bis-SorbPC and bis-DenPC Bilayers Formed by Langmuir-Blodgett Techniques and UV Polymerized.", "Preparation of supported lipid films: Substrates (either Si wafers or fused silica slides) were first sonicated in 50% isopropyl alcohol/50% water (v/v), rinsed in deionized water, and then cleaned in piranha solution as described above.", "The cleaned substrates were then sonicated in a 0.1 M solution of AlCl3 for 30 minutes, rinsed repeatedly with deionized water, sonicated for 15 minutes in deionized water, and then rinsed again.", "This procedure resulted in hydrophilic substrates having with a sessile water contact angle of 10±3.5 degrees.", "Planar supported lipid bilayers (PSLBs) were deposited on substrates using Langmuir-Blodgett-Schaefer techniques and maintained under water until after polymerization was performed.", "UV Polymerization: The low-pressure mercury pen lamp was held 7.5 cm from the PSLB-coated substrate and illuminated for 4 minutes.", "The water solution contacting the PSLB was purged with Ar for 30 minutes prior to polymerization.", "After UV exposure, the PSLB was removed from solution, rinsed several times with deionized water and dried with a stream of nitrogen.", "Kinetics of Polymerization: Kinetic experiments were performed on a Spectral Instruments 440 UV-Vis spectrometer.", "Bilayer films were deposited on four individual quartz slides which were mounted together in a fluid cell and kept equidistant by the presence of 2 mm thick, 25 mm OD Viton o-rings.", "The two slides in the center of the cell had bilayers on each side, whereas the slides on the outside of the cell had one bilayer on the inner (hydrated) surface.", "This arrangement allowed measurements to be performed simultaneously on six lipid bilayers that were maintained under water; thus sufficient sensitivity was obtained in a transmission geometry.", "Absorbance spectra were collected at various time intervals after exposure to polymerizing UV irradiation.", "The kinetic data were retrieved from the absorption spectra by integrating the absorbance peak at 260 nm after baseline correction.", "Protein Adsorption Studies: Increasing concentrations of FITC-labeled BSA in 150 mM phosphate buffered saline (50 mM.", "phosphate, 150 mM NaCl, pH 7.4) were injected into the TIRF flow cell (FIG.", "20) and allowed to equilibrate for 30 min prior to each measurement of fluorescence intensity.", "Calibration to determine the surface coverage of adsorbed protein was performed by measuring the fluorescence from several standard solutions of FITC-labeled dextran injected into the flow cell, as described in Conboy, J. C.; McReynolds, K. D.; Gervay-Hague, J.; Saavedra, S. S., J. Amer.", "Chem.", "Soc., 2002; 124, 968-977.Surface coverages were measured relative to the coverage on a reference surface, here a Langmuir-Blodgett deposited monolayer of POPC.", "The tail group-out” orientation of the POPC molecules in the monolayer makes this surface highly hydrophobic, and consequently it nonspecifically adsorbs protein strongly.", "This calibration procedure also allows for normalization of fluorescence adsorption isotherms measured for different samples.", "Results: The kinetics of polymer formation during UV irradiation of bis-SorbPC bilayers was measured by UV-vis absorbance spectroscopy.", "The bis-SorbPC monomer has an absorption maximum at 260 nm (Lamparski, H.; O'Brien, D. F., Macromolecules, 1995, 28, 1786-1794), as shown in the inset in FIG.", "21.By monitoring the depletion of the monomer absorbance as a function of exposure time to filtered UV light from the low-pressure mercury lamp, the rate of polymerization was determined.", "Example data are shown in FIG.", "21.Complete disappearance of the monomer absorbance is observed at times greater than 2 minutes, which was taken as complete polymerization of the bilayer.", "The decay of the integrated monomer absorbance occurs at a rate of 18.9±0.96 per second.", "Irradiation of bilayer bis-SorbPC films for times greater than 2 minutes was found not to alter the film structure or morphology as observed by AFM and ellipsometry (described below).", "However, irradiation times below 2 minutes result in substantially reduced degrees of polymerization.", "Static water contact angle and ellipsometry measurements made on bilayers of UV polymerized bis-SorbPC are listed in Table 1.Also tabulated for comparison are the contact angle and ellipsometric thickness of a bis-SorbPC monolayer polymerized under the same conditions as the bis-SorbPC bilayers as well as a bis-DenPC bilayer.", "The measured thickness of 48.4 Å for bis-SorbPC is consistent with a fully extended lipid bilayer structure.", "A static water contact angle of 41.9?3.1 degrees is indicative of a hydrophilicity intermediate between SiO2 (about 10 degrees) and bis-SorbPC monolayer (60.4 degrees), which has a “tail group out” orientation.", "TABLE 1 Film Contact Angle Ellipsometry bis-Sorb PC (monolayer) 60.4 ± 3.9 26.2 ± 3.1 bis-Sorb PC (bilayer) 41.9 ± 3.1 48.4 ± 4.2 bis-DenPC (bilayer) 65.2 ± 2.4 25.2 ± 4.9 Contact angle and elipsometric data for a polymerized bis-SorbPC monolayer and bilayer.", "Also shown for comparison is the data for a polymerized bis-DenPC bilayer.", "In contrast, polymerization and drying of a bis-DenPC bilayer yields a film of only monolayer thickness, with a contact angle similar to that measured for a bis-SorbPC monolayer.", "The fact that only a monolayer is observed is a consequence of the structure of bis-DenPC (FIG.", "3) which precludes the possibility of covalent bonding between the two lipid monolayers in a polymeric lipid bilayer.", "In contrast, interlayer bonding is likely in a bis-SorbPC bilayer since the reactive moieties are located at the chain termini, and is probably required to create an air-stable bilayer.", "The presence of the polymerized, supported bis-SorbPC film was also confirmed by XPS.", "A carbon to nitrogen (C/N) elemental ratio of approximately 42±6.3:1 was measured.", "This result indicates that the chemical composition of the surface layer is consistent with that of a bis-Sorb PC lipid layer (calculated C/N ratio of 38:1) within the error inherent in the XPS data (typically 15%).", "AFM was used to characterize the morphology of polymerized bis-SorbPC bilayers.", "AFM images of a dehydrated and hydrated (i.e.", "immersed in deionized water) polymerized bis-SorbPC bilayer are displayed in FIG.", "22a and FIG.", "22b respectively.", "Surprisingly different morphologies are seen for the water-immersed surface versus the same film in air.", "In the dry state, the surface of the bilayer appears as a uniformly coated surface with small irregularly shaped circular domains roughly 10-50 Å in diameter, ranging in height from 5-10 Å.", "Larger voids are also apparent on the surface, 60?15 nm in diameter with depths ranging from 15-25 nm.", "The rms roughness for the dehydrated surface, FIG.", "22a, is 5.2±1.4 Å.", "The roughness of the underlining silicon substrate was measured as 2.1±1.6 Å.", "The topographical depth determined by AFM is 48-52 Å, which is comparable to the thickness determined by ellipsometry.", "Upon immersion in water the surface morphology changes considerably, as shown in FIG.", "22b.", "The previously “cracked” surface becomes much more uniform and the calculated surface roughness declines to 3.5±0.8 Å.", "The large voids, which were present in the dried sample, are still apparent although the mean size decreases to roughly 40 nm in diameter with the void depth remaining constant at 20±5 nm.", "Analysis of the hydrated AFM image shows that approximately 36±8% of the surface area corresponds to large and smaller voids within film which extend to a depth of 15-20 Å.", "The probable origin of these voids is loss of unreacted lipid monomers or low molecular weight oligomers from the upper leaflet of the bilayer upon removal of the structure from water.", "To assess the biocompatibility of UV polymerized lipid bilayers and more specifically to determine if the protein resistance of a fluid lipid bilayer is preserved upon lipid polymerization, protein adsorption studies were performed.", "Nonspecific adsorption of fluorescein labeled bovine serum albumin (FTIC-BSA), measured using TIRF spectroscopy, was used to quantitatively compare the protein resistance of bis-SorbPC bilayers to fluid POPC (1-palmitoyl-2-oleolylphosphatidylcholine) lipid bilayers.", "UV polymerized bis-SorbPC bilayers on fused silica were prepared as described, dried, and then rehydrated after mounting in the TIRF flow cell.", "POPC bilayers were deposited on fused silica slides using Langmuir-Blodgett-Schaefer techniques and mounted in the TIRF flow cell without exposure to air.", "Measurements were also made on a hydrophobic reference surface, which was a Langmuir-Blodgett deposited, “tail group out” POPC monolayer.", "Representative adsorption isotherms are plotted in FIG.", "23.The binding affinities were extracted from the adsorption isotherms using a Langmuir model and are summarized in Table 2.The surface coverage data were normalized by assuming that protein adsorption was minimal on POPC bilayers and that monolayer coverage occurred on POPC monolayers.", "TABLE 2 % surface Surface Ka Fmax coverage POPC (monolayer) 9.8 ± 2.9 × 106 1.0 ± 0.067 100 ± 6.7 bis-SorbPC 9.1 ± 2.1 × 105 0.51 ± 0.049 35 ± 3.4 (polymerized, dried, and rehydrated) POPC (bilayer) 4.8 ± 0.32 × 105 0.16 ± 0.0046 0 Comparison of BSA adsorption to POPC monolayer, POPC bilayer and UV polymerized bis-SorbPC bilayer films.", "Both the binding affinity and surface coverage data show that the protein resistance of a UV polymerized bis-Sorb Bilayer falls in between that of a POPC monolayer and a POPC bilayer.", "The TIRF adsorption isotherm for a POPC bilayer and a re-hydrated bis-SorbPC bilayer are similar in shape, with an increase in protein adsorption observed for the polymer bilayer, as indicated by an increase in total fluorescence and an increase in the binding affinity.", "Both effects are attributed to the non-uniformity of the polymer films which have exposed hydrophobic domains as seen in the AFM images.", "A more quantitative examination of protein adsorption reveals a direct correlation between exposed hydrophobic domains on the polymer surface and the amount of adsorbed BSA.", "The relative percent surface concentrations of adsorbed BSA were determined by using the fluorescent intensity, Fmax, obtained by the nonlinear least squares fit to the adsorption data for each case.", "The measured percentage of void space on the polymer bilayers in the hydrated state, as determined by AFM, is approximately 36%.", "This correlates well with the 41±4.5% monolayer coverage of BSA measured for this same surface by TIRF, after correcting for finite amount of nonspecific protein adsorption to the fluid POPC bilayer.", "In summary, this example shows that air-stable, supported lipid bilayers can be formed by Langmuir-Blodgett-Schaefer deposition and UV-induced polymerization.", "The performance of these films (stability and protein resistance) is better than that of films formed from commercially available polymerizable lipids (i.e.", "DAPC) but is less optimal as compared to bis-SorbPC (redox) bilayers formed by vesicle fusion (see example 1).", "Example 4 Reconstitution of Transmembrane Proteins into Polymerized Lipid Bilayers.", "This example shows that transmembrane protein activity can be supported in the polymerized bis-SorbPC films.", "Two transmembrane proteins, cytochrome c oxidase (CcO), and human delta opiod receptor (d-OR), were used in these experiments.", "CcO was isolated from fresh beef hearts and purified according to published procedures (T. Soulimane and G. Buse, Eur.", "J.", "Biochem., 227(1995) 588-595).", "d-OR was expressed, isolated, and purified from a transfected cell line, also according to published procedures (Salamon, S. Cowell, E. Varga, H. I. Yamamura, V. J. Hruby and G. Tollin, Biophys.", "J., 2000, 79, 2463).", "Detergent dialysis (described above) was used to insert each of these proteins into bilayer vesicles, forming proteo-vesicles, following standard procedures (Mimms, L. T.; Zampighi, G.; Nozaki, Y.; Tanford, C.; Reynolds, J.", "A. Biochemistry 1981, 20, 833-840).", "The surfactant used was octyl glucoside.", "The surfactant concentration was initially 40 mM, well above the reported cmc of about 20 mM, and the lipid to protein ratio was 1000:1.Proteo-vesicles were formed using either pure bis-SorbPC or pure DOPC, and then fused to silica substrates to form planar supported proteo-lipid bilayers.", "Supported bilayers containing bis-SorbPC were redox polymerized as described above.", "TIRF spectroscopy was used to measure the specific binding of ligands to both types of planar supported proteo-lipid bilayers; the experimental design was equivalent to that used to measure nonspecific BSA adsorption to lipid membrane surfaces, as described above.", "CcO binds cytochrome c (Cyt c) in low ionic strength solutions; raising the ionic strength dissociates the complex.", "TMR-Cyt c binding to bis-SorbPC and POPC bilayers containing CcO was compared.", "Although Cyt c nonspecifically adsorbs to lipid bilayers to some extent, this can be distinguished from specific binding to membrane-bound CcO by the difference in ionic strength dependence (i.e.", "rinsing with a high ionic strength buffer solution dissociates specifically bound Cyt c).", "After incubating the CcO-bis-SorbPC bilayer with TMR-Cyt c for 30 minutes, the flow cell was flushed with low ionic strength buffer and the fluorescence corresponding to adsorbed TMR-Cyt c was measured.", "Flushing the cell with a high ionic strength (0.5M NaCl) solution removed specifically bound TMR-Cyt c, which was 80% of the total adsorbed Cyt c. This high removal percentage indicates that a significant population of CcO molecules are properly oriented and retain specific binding activity after polymerization of bis-SorbPC membrane.", "By comparison, on bis-SorbPC films with no incorporated CcO, less than 20% of the adsorbed Cyt c was removed by the NaCl rinse.", "Furthermore, if the polymerized bilayer was dried and then rehydrated before assaying the binding activity, the removal percentage decreased only slightly to 74%.", "Using a TIRF calibration procedure (described above) with TMR-labeled dextran as the calibrant, the surface coverage of the specifically bound TMR-Cyt c was determined to be 9.8±4.5×10−14 mol/cm2.A comparable value, 8.1±3.8×10−14 mol/cm2, was measured for CcO-functionalized fluid DOPC bilayers.", "Both of these surface coverages are within reasonable range of the theoretically calculated CcO surface coverage of 5.5×10−3 mol/cm2, assuming a 1000 to 1 lipid to protein ratio in the film.", "In summary, CcO binding activity equivalent to a CcO-functionalized DOPC bilayer was retained in bis-SorbPC bilayers even after redox polymerization, drying and rehydration.", "d-OR selectively binds many opioid peptides, among them the ligand enkephalin analogue [D-Pen2, D-Pen2]enkephalin (DPDPE) (Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Yamamura, H. I.; Galligan, J. J.; Burks, T. F. Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 1983, 80, 5871-5874.)", "Analagous to the procedure for CcO, d-OR was incorporated into fluid DOPC and polymerized bis-SorbPC lipid films and assayed for binding activity using a fluorescently labeled ligand, TMR-DPDPE.", "After the labeled ligand was incubated with each type of d-OR functionalized lipid film, the film was rinsed.", "Competitive desorption was effected by subsequent incubation with unlabeled ligand, DPDPE, and revealed the fraction of the TMR-DPDPE that was specifically associated with each proteo-lipid film.", "40% and 60% of the adsorbed TMR-DPDPE was competitively desorbed by DPDPE on the polymerized bis-SorbPC and fluid DOPC films respectively (both films were functionalized with d-OR).", "Thus in both cases, a significant population of active opioid receptors was present in the bilayer.", "These data indicate that polymerization does not significantly affect receptor activity.", "Example 5 Patterning Polymerized Lipid Bilayers and Proteins on Polymerized Lipid Bilayers.", "In this example, results are presented for two types of patterned arrays created by microcontact printing (μCP): (a) Protein films are patterned on polymerized bis-SorbPC bilayers.", "(b) Patterned regions of polymerized bis-SorbPC are created by selective removal of portions of the fluid bilayer prior to the polymerization step.", "Poly(dimethylsiloxane) (PDMS) stamps were made by curing Sylgard 184 (Dow Corning) on a silicon master with line features (i.e.", "stripes) approximately 10 microns wide separated by 15 micron wide spaces.", "The PDMS stamp was then removed from the master, rinsed in deionized water.", "To create pattern type (a), the stamp was immersed in an aqueous solution of 0.05 mg/ml BSA in 50 mM, pH 7.4, phosphate buffer for 30 minutes.", "The protein-coated stamp was rinsed with buffer and water, and then placed upon a dried, bis-SorbPC (redox) bilayer supported on a Si wafer.", "Light pressure (50-100 g over a 1 cm2 area) was applied to the stamp; then it was removed after 20 seconds.", "The bilayer was then rinsed with deionized water, dried, and imaged by atomic force microscopy.", "FIG.", "18 shows an AFM image of the pattern of protein “stripes” that was transferred to the bilayer surface from the stamp.", "The protein stripes are quite uniform and the protein appears to be located exclusively in the patterned regions.", "In another experiment, biotin-labeled BSA was printed onto a dried, bis-SorbPC (redox) bilayer supported on a fused silica slide.", "Subsequently, the slide was immersed into an aqueous solution of TMR-labeled avidin.", "TMR-avidin bound specifically to the biotin-BSA stripes, with minimal adsorption observed in the unprinted regions of the film, which shows that these regions retain their characteristic protein resistance.", "The inset in FIG.", "18, an epifluorescence micrograph taken of the bilayer after rinsing away the unbound TMR-avidin, shows the fluorescent stripes of TMR-avidin bound to the printed surface.", "These experiments demonstrate the feasibility of printing arrays of proteins onto dried lipid polymer films.", "The microcontact printed protein adheres strongly to the dried bilayer, remains in place when the membrane is rehydrated, and retains the capability to specifically bind other ligands, including other proteins.", "To create pattern type (b), a μCP printing technique developed to create patterns in hydrated, fluid lipid bilayers (Hovis, J. S.; Boxer, S. B., Langmuir, 2000, 16(3), 894-897; Hovis, J. S.; Boxer, S. B., Langmuir, 2001, 17(11), 3400-3405) was adapted to create polymerized lipid bilayer patterns.", "A schematic of the process is shown in FIG.", "19 (right).", "A fluid bilayer of bis-SorbPC was formed by vesicle fusion on a clean Si wafer according to the procedures described above.", "The PDMS stamp was made to briefly (5 seconds) contact the bilayer while both are immersed in water.", "Withdrawing the stamp from the bilayer surface removed those portions of the fluid bilayer that were in contact with the stamp (i.e.", "15 μm wide stripes).", "The remaining, 10 μm wide stripes of fluid bis-SorbPC bilayer were then UV polymerized as described in Example 1.The AFM image shown on the left side of FIG.", "19 was obtained on the dried sample.", "The bright lines are polymerized bilayer; between them are wider, darker lines, which are the regions where the bilayer was removed.", "Much thinner lines of polymerized material are visible in the dark regions; this was caused by incomplete removal of lipid, which was probably due to imperfections on the surface of the stamp.", "From the array-like structure of the film shown FIG.", "19, it is clearly feasible to generate a polymerized film that contains a regular array of “bare areas.” By performing vesicle fusion on such a film, it should be possible to fill the bare areas vacated by the patterning process with a second type of lipid, either polymerizable or non-polymerizable, and thus generate a mixed film containing a defined spatial array of different types of lipids.", "Numerous modifications and variations on the present invention are possible in light of the above teachings.", "It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein." ] ]	Patent_10469539
[ [ "Substituted 2-oxy-3,5-dicyano-4-aryl-6-aminopyridines and use thereof", "The use of compounds of formula (I) as medicaments, novel compounds of formula (I) and a method for production thereof are disclosed.", "Compounds of formula (I) are effective as adenosine receptor ligands." ], [ "1.A compound of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C6)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano or —NH—C(O)—R7, in which R7 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S, which may be substituted by (C1-C4)-alkyl or oxo, R4 and R5 independently of one another represent hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl or 5- to 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C8)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted by (C3-C7)-cycloalkyl, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, or a salt, a hydrate, a hydrate of a salt or a solvate thereof for the prophylaxis and/or treatment of disorders.", "2.A compound of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C6)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano or —NH—C(O)—R7, in which R7 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S, which may be substituted by (C1-C4)-alkyl or oxo, R4 and R5 independently of one another represent hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl or 5- to 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C8)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted by (C3-C7)-cycloalkyl, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, or a salt, a hydrate, a hydrate of a salt or a solvate thereof, but except for the following compounds of the formula (I) in which the radicals R1, R2, R3, R4, R5 and R6 are as defined below: R1=R2=R3=R4=R5=H; R6=ethyl R1=4-methyl; R2=R3=R4=R5=H; R6=ethyl R1=3-methyl; R2=R3=R4═R5=H; R6=ethyl R1=4-methoxy; R2=R3=R4=R5=H; R6=ethyl R1=4-methoxy; R2=3-methoxy; R3=5-methoxy; R4=R5=H; R6=ethyl R1=2-chlorine; R2=R3=R4=R5=H; R6=ethyl R1=4-chlorine; R2=R3=R4=R5=H; R6=ethyl R1=3-methyl; R2=R3=R4=R5=H; R6=ethyl R1=R2=R3=R4=R5=H; R6=methyl R1=R2=R3=R4=R5=H; R6=propyl R1=R2=R3=R4=R5=H; R6=isopropyl R1=2-hydroxy; R2=R3=R4=R5=H; R6=ethyl R1=4-fluorine; R2=R3=R4=R5=H; R6=methyl R1=4-methoxy; R2=R3=R4=R5=H; R6=methyl R1=R2=—O—CH2—O—; R3=R4=R5=H; R6=methyl.", "3.A compound as claimed in claim 2 in which R1, R2 and R3 independently of one another represent hydrogen, hydroxyl, (C1-C6)-alkyl, trifluoromethyl, trifluoromethoxy, fluorine, chlorine, (C1-C4)-alkoxy, which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C2-C4)-alkenyl, —NH—C(O)—CH3 or —NH—C(O)—C2H5, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O— or —O—CH2—CH2—O—, R4 and R5 independently of one another represent hydrogen, (C1-C6)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or cyclopropyl, cyclopropyl, benzyl or pyridylmethyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain a further heteroatom from the group consisting of N, O or S in the ring and which may be mono- to trisubstituted, independently of one another, by hydroxyl, (C1-C4)-alkyl or (C1-C4)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C6)-alkyl, which is substituted by (C3-C7)-cycloalkyl, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, phenyl or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where phenyl and heteroaryl for their part may be substituted by fluorine, chlorine, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, or unsubstituted (C4-C6)-alkyl or a salt, a hydrate, a hydrate of a salt or a solvate thereof.", "4.A compound as claimed in claim 2, in which R1 and R2 independently of one another represent hydrogen, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or —NH—C(O)—CH3, where the alkoxy radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or cyclopropyl, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O—, R3 represents hydrogen, R4 represents hydrogen, methyl, ethyl, n-propyl, isopropyl, where the alkyl radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or cyclopropyl, or cyclopropyl, R5 represents hydrogen or a methyl group, and R6 represents methyl or ethyl which are substituted by pyridyl, phenyl which for its part may be substituted by cyano, nitro, methyl, ethyl, propyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or amino, hydroxyl or methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, or a salt, a hydrate, a hydrate of a salt or a solvate thereof.", "5.A compound of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C6)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano or —NH—C(O)—R7, in which R7 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S, which may be substituted by (C1-C4)-alkyl or oxo, R4 represents hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl or 5- to 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C8)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, and R5 represents (C1-C8)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl or 5- to 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C8)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted by (C3-C7)-cycloalkyl, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, or a salt, a hydrate, a hydrate of a salt or a solvate thereof.", "6.A process for preparing compounds of the formula (I) as defined in claim 2, characterized in that either [A] compounds of the formula (II) in which R1, R2, R3 and R6 are as defined in claim 2 and X represents a leaving group, are reacted with compounds of the formula (III) R4—NH—R5 (III) in which R4 and R5 are as defined in claim 2, or [B] compounds of the formula (IV) in which R1, R2 and R3 are as defined in claim 2 and X1 and X2 are identical or different and represent leaving groups, are initially converted with compounds of the formula (III) into compounds of the formula (V) in which R1, R2, R3, R4 and R5 are as defined in claim 2 and X2 represents a leaving group and these are then reacted with compounds of the formula (VI) R6—OH (VI) in which R6 is as defined in claim 2, or [C] if, in compounds of the formula (I), R4 and R5 each represent hydrogen, compounds of the formula (VII) in which R1, R2 and R3 are as defined in claim 2 are reacted in the presence of a base with malonitrile and compounds of the formula (VI).", "7.A compound of the formula (I) as defined in claim 3 for the prophylaxis and/or treatment of disorders.", "8.A compound of the formula (I) as defined in claim 4 for the prophylaxis and/or treatment of disorders.", "9.A composition, comprising at least one compounds of the formula (I) as defined in claim 1 and at least one further auxiliary.", "10.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the cardiovascular system (cardiovascular disorders).", "11.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the urogenital system and cancer.", "12.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of inflammatory and neuroinflammatory disorders, neurodegenerative disorders and pain.", "13.The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the respiratory tract, of liver fibrosis and liver cirrosis and diabetes." ], [ "The present invention relates to the use of substituted 2-oxy-3,5-dicyano-4-aryl-6-aminopyridines as medicaments and to novel 2-oxy-3,5-dicyano-4-aryl-6-aminopyridines and to a process for their preparation.", "Adenosine, a nucleoside consisting of adenine and D-ribose, is an endogenous factor having cell-protective activity, in particular under cell-damaging conditions with limited oxygen and substrate supply, such as, for example, in the case of ischemia in various organs (for example heart and brain).", "Adenosine is formed intracellularly as an intermediate during the degradation of adenosine-5′-monophosphate (AMP) and S-adenosylhomocysteine, but it can be released from the cell, in which case it acts as a hormone-like substance or neurotransmitter by binding to specific receptors.", "Under normoxic conditions, the concentration of free adenosine in the extracellular space is very low.", "However, under ischemic or hypoxic conditions, the extracellular concentration of adenosine in the affected organs is increased dramatically.", "Thus, it is known, for example, that adenosine inhibits platelet aggregation and increases the blood supply to the coronary arteries.", "Furthermore, it acts on the heart rate, on the release of neurotransmitters and on lymphocyte differentiation.", "The aim of these actions of adenosine is to increase the oxygen supply of the affected organs and/or to reduce the metabolism of these organs in order to adjust the metabolism of the organ to the blood supply of the organ under ischemic or hypoxic conditions.", "The action of adenosine is mediated via specific receptors.", "To date, subtypes A1, A2a, A2b and A3 are known.", "The actions of these adenosine receptors are mediated intracellularly by the messenger cAMP.", "In the case of the binding of adenosine to the A2a or A2b receptors, the intracellular cAMP is increased via activation of the membrane-bound adenylate cyclase, whereas binding of adenosine to A1 or A3 receptors results in a decrease of the intracellular cAMP concentration via inhibition of adenylate cyclase.", "According to the invention, “adenosine-receptor-selective ligands” are substances which bind selectively to one or more subtypes of the adenosine receptors, thus either mimicking the action of adenosine (adenosine agonists) or blocking its action (adenosine antagonists).", "According to their receptor selectivity, adenosine-receptor-selective ligands can be divided into different categories, for example ligands which bind selectively to the A1 or A2 receptors of adenosine and in the case of the latter also, for example, those which bind selectively to the A2a or the A2b receptors of adenosine.", "Also possible are adenosine receptor ligands which bind selectively to a plurality of subtypes of the adenosine receptors, for example ligands which bind selectively to the A1 and the A2, but not to the A3 receptors of adenosine.", "The abovementioned receptor selectivity can be determined by the effect of the substances on cell lines which, after stable transfection with the corresponding cDNA, express the receptor subtypes in question (see the publication M. E. Olah, H. Ren, J. Ostrowski, K. A. Jacobson, G. L. Stiles, “Cloning, expression, and characterization of the unique bovine A1 adenosine receptor.", "Studies on the ligand binding site by site-directed mutagenesis.” in J. Biol.", "Chem.", "267 (1992) pages 10764-10770, the disclosure of which is hereby fully incorporated by way of reference).", "The effect of the substances on such cell lines can be monitored by biochemical measurement of the intracellular messenger cAMP (see the publication K. N. Klotz, J. Hessling, J. Hegler, C. Owman, B. Kull, B.", "B. Fredholm, M. J. Lohse, “Comparative pharmacology of human adenosine receptor subtypes—characterization of stably transfected receptors in CHO cells” in Naunyn Schmiedebergs Arch.", "Pharmacol.", "357 (1998) pages 1-9, the disclosure of which is hereby fully incorporated by way of reference).", "The “adenosine-receptor-specific” ligands known from the prior art are mainly derivatives based on natural adenosine (S.-A.", "Poulsen and R. J. Quinn, “Adenosine receptors: new opportunities for future drugs” in Bioorganic and Medicinal Chemistry 6 (1998) pages 619 to 641; K. J. Broadley, “Drugs modulating adenosine receptors as potential therapeutic agents for cardiovascular diseases” in Exp.", "Opin.", "Ther.", "Patents 10 (2000) pages 1669-1692).", "However, most of the adenosine ligands known from the prior art have the disadvantage that their action is not really receptor-specific, that their activity is less than that of natural adenosine or that they have only very weak activity after oral administration.", "Thus they are mainly used only for experimental purposes.", "It is an object of the present invention to find or provide pharmacologically active substances suitable for the prophylaxis and/or treatment of various disorders, in particular disorders of the cardiovascular system (cardiovascular disorders), the substances preferably acting as adenosine-receptor-selective ligands.", "The present invention provides the use of compounds of the formula (1) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C6)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano or —NH—C(O)—R7, in which R7 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S, which may be substituted by (C1-C4)-alkyl or oxo, R4 and R5 independently of one another represent hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl or 5- to 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C8)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted by (C3-C7)-cycloalkyl, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and their salts, hydrates, hydrates of the salts and solvates, for the prophylaxis and/or treatment of disorders.", "Some of the substances mentioned above which, according to the present invention, can be used for the prophylaxis and/or treatment of disorders are known from the literature (see Alvarez-Insua, Lora-Tamayo, Soto, Journal of Heterocyclic Chemistry 7, pages 1305-1309, (1970); Quintela et al., Eur.", "J. Med.", "Chem.", "Chim.", "Ther.", "19, 555-557 (1984); Ballantyne, Drug Chem.", "Toxicol.", "8, 171-173 (1985); Seada et al., Orient.", "J. Chem.", "5, 273-280 (1989); Mishriki et al., Recl.", "Trav.", "Chim.", "Pays-Bas 113, 35-39 (1994); Quintela et al., Tetrahedron 52, 10497-10506 (1996)).", "However, a therapeutic use for the known compounds has hitherto not been described in the literature.", "For the first time, this is done in the context of the present invention.", "Depending on the substitution pattern, the compounds of the formula (I) can exist in stereoisomeric forms which are either like image and mirror image (enantiomers) or not like image and mirror image (diastereomers).", "The invention relates both to the enantiomers or diastereomers and to their respective mixtures.", "The racemic forms, like the diastereomers, can be separated in a known manner into the stereoisomerically uniform components.", "Likewise, the present invention also relates to the tautomers of the compounds of the formula (I).", "Salts of the compounds of the formula (I) can be physiologically acceptable salts of the compounds according to the invention with mineral acids, carboxylic acids, or sulfonic acids.", "Particular preference is given, for example, to salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, trifluoroacetic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.", "Salts which may be mentioned include salts with customary bases, such as, for example, alkali metal salts (for example sodium salts or potassium salts), alkaline earth metal salts (for example calcium salts or magnesium salts) or ammonium salts, derived from ammonia or organic amines, such as, for example, diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabietylamine, 1-ephenamine or methylpiperidine.", "According to the invention, hydrates or solvates are those forms of the compounds of the formula (I) which, in solid or liquid state, form, by hydration with water or coordination with solvent molecules, a molecule compound or a complex.", "Examples of hydrates are sesquihydrates, monohydrates, dihydrates or trihydrates.", "Likewise, the hydrates or solvates of salts of the compounds according to the invention are also suitable.", "Moreover, the invention also includes prodrugs of the compounds according to the invention.", "According to the invention, prodrugs are forms of compounds of the formula (I) which for their part may be biologically active or inactive, but which can be converted under physiological conditions (for example metabolically or solvolytically) into the corresponding biologically active form.", "In the context of the present invention, the substituents have, unless defined otherwise, the following meanings: Halogen generally represents fluorine, chlorine, bromine or iodine.", "Preference is given to fluorine, chlorine or bromine.", "Very particularly preferred are fluorine or chlorine.", "(C1-C8)-Alkyl (C1-C6)-alkyl and (C1-C4)-alkyl generally represent a straight-chain or branched alkyl radical having 1 to 8, 1 to 6 and 1 to 4 carbon atoms, respectively.", "Preference is given to a straight-chain or branched alkyl radical having 1 to 6 carbon atoms.", "Particular preference is given to a straight-chain or branched alkyl radical having 1 to 4 carbon atoms.", "Examples which may be mentioned are: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl.", "(C2-C4-Alkenyl generally represents a straight-chain or branched alkyl radical having 2 to 4 carbon atoms.", "Examples which may be mentioned are: vinyl, allyl, isopropenyl and n-but-2-en-1-yl.", "(C2-C4)-Alkynyl generally represents a straight-chain or branched alkynyl radical having 2 to 4 carbon atoms.", "Examples which may be mentioned are: ethynyl, n-prop-2-yn-1-yl and n-but-2-yn-1-yl.", "(C1-C8)-Alkoxy, (C1-C6)-alkoxy and (C1-C4)-alkoxy generally represent a straight-chain or branched alkoxy radical having 1 to 8, 1 to 6 and 1 to 4 carbon atoms, respectively.", "Preference is given to a straight-chain or branched alkoxy radical having 1 to 6 carbon atoms.", "Particular preference is given to a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms.", "Examples which may be mentioned are: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy.", "(C1-C4)-Alkoxycarbonyl generally represents a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms which is attached via a carbonyl group.", "Examples which may be mentioned are: methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl and t-butoxycarbonyl.", "In the context of the invention, mono- or di-(C1-C4)-alkylamino represents an amino group having one or two identical or different straight-chain or branched alkyl substituents each having 1 to 4 carbon atoms.", "Examples which may be mentioned are: methylamino, ethylamino, n-propylamino, isopropylamino, t-butylamino, N,N-dimethylamino, N,N-diethylamino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino, N-isopropyl-N-n-propylamino and N-t-butyl-N-methylamino.", "(C3-C7)-Cycloalkyl and (C3-C6)-cycloalkyl generally represent a cyclic alkyl radical having 3 to 7 and 3 to 6 carbon atoms, respectively.", "Preference is given to cyclic alkyl radicals having 3 to 6 carbon atoms.", "Examples which may be mentioned are: cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.", "(C6-C10)-Aryl generally represents an aromatic radical having 6 to 10 carbon atoms.", "Preferred aryl radicals are phenyl and naphthyl.", "(C6-C10)-Aryloxy generally represents an aromatic radical as defined above which is attached via an oxygen atom.", "5- to 10-membered heteroaryl having up to 3 heteroatoms from the group consisting of N, O and/or S generally represents a mono- or bicyclic, optionally benzo-fused heteroaromatic which is attached via a ring carbon atom of the heteroaromatic, if appropriate also via a ring nitrogen atom of the heteroaromatic.", "Examples which may be mentioned are: pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, thienyl, furyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl, oxazolyl, oxdiazolyl, isoxazolyl, benzofuranyl, benzothienyl or benzimidazolyl.", "The corresponding heteroaromatics having fewer heteroatoms, such as, for example, those having one or 2 heteroatoms from the group consisting of N, O and/or S, or those having a smaller ring size, such as, for example, 5- or 6-membered heteroaryl, are derived analogously from this definition.", "In general, preference is given to 5- or 6-membered aromatic heterocycles having one or 2 heteroatoms from the group consisting of N, O and/or S. Examples which may be mentioned are: pyridyl, pyrimidyl, pyridazinyl, furyl, imidazolyl or thienyl.", "5- to 7-membered heterocycle generally represents a saturated or partially unsaturated, optionally benzo-fused heterocycle having up to 3 heteroatoms from the group consisting of N, O and/or S. Examples which may be mentioned are: tetrahydrofuryl, pyrrolidinyl, pyrrolinyl, dihydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, hexahydropyranyl.", "The corresponding heterocycles having fewer heteroatoms, such as, for example, one or 2 heteroatoms from the group consisting of N, O and/or S, or a smaller ring size, such as, for example, 5- or 6-membered heterocyclyl, are derived analogously from this definition.", "Preference is given to saturated heterocycles having up to 2 heteroatoms from the group consisting of N, O and/or S, in particular piperidinyl, piperazinyl, morpholinyl and pyrrolidinyl.", "Moreover, the present invention relates to novel compounds of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C6)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano or —NH—C(O)—R7, in which R7 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S, which may be substituted by (C1-C4)-alkyl or oxo, R4 and R5 independently of one another represent hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl or 5- to 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C8)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted by (C3-C7)-cycloalkyl, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and their salts, hydrates, hydrates of the salts and solvates, but except for the following compounds of the formula (I) in which the radicals R1, R2, R3, R4, R5 and R6 are as defined below: R1=R2=R3=R4=R5=H; R6=ethyl R1=4-methyl; R2=R3=R4=R=H; R4=ethyl R1=3-methyl; R2=R3=R=R=H; R6=ethyl R1=4-methoxy; R2=R3=R4=R=H; R6=ethyl R1=4-methoxy; R2=3-methoxy; R3=5-methoxy; R4=R5=H; R6=ethyl R1=2-chlorine; R2=R3=R4=R5=H; R6=ethyl R1=4-chlorine; R1=R3=R4=R5=H; R6=ethyl R1=3-methyl; R2=R3=R4=R5=H; R6=ethyl R1=R2=R3=R4=R5=H; R6=methyl R1=R2<R3=R4=R5=H; R6=propyl R1=R2=R3=R4=R5=H; R6=isopropyl R1=2-hydroxy; R2=R3=R4=R5=H; R6=ethyl R=4-fluorine; R2=R3=R4=R5=H; R6=methyl R1=4-methoxy; R2=R3=R4=R5=H; R6=methyl R1=R2=—O—CH2—O—; R3=R4=R5=H; R6=methyl.", "Preference is given to compounds of the formula (I) in which R1, R2 and R3 independently of one another represent hydrogen, hydroxyl, (C1-C6)-alkyl, trifluoromethyl, trifluoromethoxy, fluorine, chlorine, (C1-C4)-alkoxy, which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C2-C4)-alkenyl, —NH—C(O)—CH3 or —NH—C(O)—C2H5, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O— or —CH2—CH2-O—, R4 and R5 independently of one another represent hydrogen, (C1-C6)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or cyclopropyl, cyclopropyl, benzyl or pyridylmethyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain a further heteroatom from the group consisting of N, O or S in the ring and which may be mono- to trisubstituted, independently of one another, by hydroxyl, (C1-C4)-alkyl or (C1-C4)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C6)-alkyl, which is substituted by (C3-C7)-cycloalkyl, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, phenyl or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where phenyl and heteroaryl for their part may be substituted by fluorine, chlorine, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, or unsubstituted (C4-C6)-alkyl and their salts, hydrates, hydrates of the salts and solvates.", "Particular preference is given to compounds of the formula (I) in which R1 and R2 independently of one another represent hydrogen, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or —NH—C(O)—CH3, where the alkoxy radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or cyclopropyl, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O—, R3 represents hydrogen, R4 represents hydrogen, methyl, ethyl, n-propyl, isopropyl, where the alkyl radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or cyclopropyl, or cyclopropyl, R5 represents hydrogen or a methyl group, and R6 represents methyl or ethyl which are substituted by pyridyl, phenyl which for its part may be substituted by cyano, nitro, methyl, ethyl, propyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or amino, hydroxyl or methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and their salts, hydrates, hydrates of the salts and solvates.", "Particular preference is also given to compounds of the formula (I) in which R3 represents hydrogen.", "Particular preference is also given to compounds of the formula (I) in which R2 and R3 represent hydrogen.", "Particular preference is also given to compounds of the formula (I) in which R4 represents hydrogen.", "Particular preference is also given to compounds of the formula (I) in which R1 and R2 are attached to adjacent phenyl ring atoms located in the para- and meta-positions to the point of attachment of the pyridine ring and represent a group —O—CH2—O—.", "Preference is also given to compounds of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C6)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano or —NH—C(O)—R7, in which R7 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S, which may be substituted by (C1-C4)-alkyl or oxo, R4 represents hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl or 5- to 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C8)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, and R5 represents (C1-C8)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl or 5- to 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C8)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted by (C3-C7)-cycloalkyl, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and their salts, hydrates, hydrates of the salts and solvates.", "Preference is also given to compounds of examples 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 17, 18, 19, 20 and their salts, hydrates, hydrates of the salts and solvates.", "The general or preferred radical definitions or illustrations given above can be combined with one another as desired, i.e.", "including combinations between the respective ranges and preferred ranges.", "They apply both to the end products and, correspondingly, to precursors and intermediates.", "The present invention furthermore relates to a process for preparing the compounds of the formula (I), characterized in that either [A] compounds of the formula (II) in which R1, R2, R3 and R6 are as defined above and X represents a suitable leaving group, for example chlorine, bromine, methylthio or phenylthio, are reacted in an inert solvent with compounds of the formula (III) R4—N—R5 (III) in which R4 and R5 are as defined above, or [B] compounds of the formula (IV) in which R1, R2 and R3 are as defined above and X1 and X2 independently of one another represent suitable leaving groups having the definition of X defined above, are initially converted in an inert solvent with compounds of the formula (III) into to compounds of the formula (V) in which R1, R2, R3, R4, R5 and X2 are as defined above, and these are then reacted in the presence of a base, if appropriate in an inert solvent, with compounds of the formula (VI) R6—OH (VI) in which R6 is as defined above, or [C] if, in compounds of the formula (I), R4 and R5 each represent hydrogen, compounds of the formula (VI) in which R1, R2 and R3 are as defined above are reacted in the presence of a base, if appropriate with addition of an inert solvent, with malonitrile and compounds of the formula (VI).", "The process according to the invention can be illustrated in an exemplary manner by the formula scheme below: Suitable solvents for the process step [A]: (II)+(III)→(I) are organic solvents which do not change under the reaction conditions.", "These include alcohols, such as methanol, ethanol and isopropanol, ketones, such as acetone and methyl ethyl ketone, acyclic and cyclic ethers, such as diethyl ether and tetrahydrofuran, esters, such as ethyl acetate, or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide (DMSO).", "Another suitable solvent is water.", "It is also possible to use mixtures of the solvents mentioned above.", "Preference is given to tetrahydrofuran.", "The reaction is generally carried out in a temperature range of from −78° C. to +150° C., preferably in the range from +20° C. to +80° C., in particular from +20° C. to +40° C. The reaction can be carried out under atmospheric, elevated or reduced pressure, for example in the range of from 0.5 to 5 bar.", "In general, the reaction is carried out at atmospheric pressure.", "In general, the reaction is carried out using an excess of compound (III), preferably in a ratio of from 2 to 8 mol of the compound (III) per mole of the compound (II).", "The compounds of the general formula (II) are known to the person skilled in the art or can be prepared analogously to methods known from the literature [see, for example, J. M. Quintela, J. L. Soto, Anales de Quimica 79, 368-372 (1983)].", "The compounds of the general formula (III) are commercially available, known to the person skilled in the art or can be prepared by methods from the literature.", "Suitable solvents for the first reaction step [B]: (IV)+(III)→(V) are organic solvents which are inert under the reaction conditions.", "These include ketones, such as acetone and methyl ethyl ketone, acyclic and cyclic ethers, such as diethyl ether, 1,2-dimethoxyethane or tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide (DMSO).", "It is also possible to use mixtures of the solvents mentioned above.", "Preference is given to 1,2-dimethoxyethane or tetrahydrofuran.", "The reaction is generally carried out in a temperature range of from −78° C. to +120° C., preferably in the range from +20° C. to +60° C., in particular from +20° C. to +40° C. The reaction can be carried out under atmospheric, elevated or reduced pressure, for example in the range of from 0.5 to 5 bar.", "In general, the reaction is carried out at atmospheric pressure.", "In general, the reaction is carried out using an equivalent amount or an excess of compound (III), preferably in a ratio of from 1 to 8 mol of the compound (III), particularly preferably in a ratio of from 1 to 2 mol of the compound (III), per mole of the compound (IV).", "The reaction can also be carried out in the presence of auxiliary bases, such as, for example, trialkylamines, such as triethylamine or diisopropylethylamine, alkali metal carbonates, such as sodium carbonate or potassium carbonate, or amidines, such as DBN (1,5-diazabicyclo[4.3.0]non-5-ene) or DBU (1,8-diazabicyclo[5.4.0]-undec-7-ene).", "The compounds of the general formula (IV) are known or can be prepared analogously to methods known from the literature [see, for example, Quintela et al., Heterocycles 38, 1299-1305 (1994)].", "The compounds of the general formula (V) are known or can be prepared analogously to methods known from the literature [see, for example, Quintela et al., Heterocycles 38, 1299-1305 (1994), Kambe et al., Synthesis, 531-533 (1981), Elnagdi et al., Z. Naturforsch.", "47b, 572-578 (1991)].", "The second process step [B]: (V)+(VI)→(I) can be carried out neat, in the absence of a solvent, or in a solvent.", "Suitable solvents are organic solvents which are inert under the reaction conditions.", "These include ketones, such as acetone and methyl ethyl ketone, acyclic and cyclic ethers, such as diethyl ether, 1,2-dimethoxyethane or tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide (DMSO).", "Another suitable solvent is water.", "It is also possible to use mixtures of the solvents mentioned above.", "Preference is given to acetonitrile, 1,2-dimethoxyethane and tetrahydrofuran.", "Suitable bases are the customary inorganic or organic bases.", "These include alkali metal hydroxides, such as, for example, sodium hydroxide or potassium hydroxide, alkali metal carbonates, such as sodium carbonate or potassium carbonate or sodium bicarbonate or potassium bicarbonate, potassium tert-butoxide, sodium hydride, amides, such as sodium amide, lithium bis(trimethylsilyl)amide or lithium diisopropylamide, organometallic compounds, such as butyllithium or phenyllithium, amines, such as triethylamine or pyridine, or else the sodium or potassium salt of the compound of the general formula (VI) in question itself.", "Preference is given to potassium tert-butoxide and potassium carbonate.", "Here, the base can be employed in a ratio of from 1 to 10 mol, preferably in a ratio of from 1 to 5 mol, in particular in a ratio of from 1 to 4 mol, of base per mole of the compound (VI).", "The reaction is generally carried out in a temperature range of from −78° C. to +120° C., preferably in the range from +20° C. to +100° C., in particular from +20° C. to +80° C. The reaction can be carried out under atmospheric, elevated or reduced pressure, for example in the range of from 0.5 to 5 bar.", "In general, the reaction is carried out at atmospheric pressure.", "The reaction is generally carried out using an equivalent amount or an excess of compound (VI), preferably in a ratio of from 1 to 50 mol of the compound (VI) per mole of the compound (V).", "The compounds of the general formula (VI) are commercially available, known to the person skilled in the art or can be prepared by methods known from the literature.", "Process [C]: (VII)→(I), where R4=R5=H, is carried out analogously to a method known from the literature [Alvarez-Insua et al., Journal of Heterocyclic Chemistry 7, 1305-1309 (1970)].", "The reaction can be carried out neat, in the absence of a solvent, or in a solvent.", "Suitable solvents are organic solvents which are inert under the reaction conditions.", "These include ketones, such as acetone and methyl ethyl ketone, acyclic and cyclic ethers, such as diethyl ether, 1,2-dimethoxyethane or tetrahydrofuran, esters, such as ethyl acetate or butyl acetate, hydrocarbons, such as benzene, xylene, toluene, hexane or cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chlorobenzene or dichloroethane, or other solvents, such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide (DMSO).", "It is also possible to use mixtures of the solvents mentioned above.", "The reaction is preferably carried out neat, in the absence of a solvent.", "Suitable bases are the customary inorganic or organic bases.", "These include alkali metal hydroxides, such as, for example, sodium hydroxide or potassium hydroxide, alkali metal carbonates, such as sodium carbonate or potassium carbonate or sodium bicarbonate or potassium bicarbonate, potassium tert-butoxide, sodium hydride, amides, such as sodium amide, lithium bis(trimethylsilyl)amide or lithium diisopropylamide, organometallic compounds, such as butyllithium or phenyllithium, amines, such as triethylamine or pyridine, or else the sodium or potassium salt of the compound of the general formula (VI) in question itself.", "Preference is given to the sodium or potassium salt of the compound of the general formula (VI) in question.", "Here, the base can be employed in a ratio of from 1 to 10 mol, preferably in a ratio of from 1 to 5 mol, in particular in a ratio of from 1 to 4 mol, of base per mole of the compound (VII).", "The reaction is generally carried out in a temperature range of from −78° C. to +180° C., preferably in the range from +20° C. to +160° C., in particular from +20° C. to +120° C. The reaction can be carried out under atmospheric, elevated or reduced pressure, for example in the range of from 0.5 to 5 bar.", "In general, the reaction is carried out at atmospheric pressure.", "The reaction is generally carried out using an equivalent amount or an excess of compound (VI), preferably in a ratio of from 1 to 50 mol of (VI) per mole of the compound (VII).", "The compounds of the general formula (VII) are commercially available, known to the person skilled in the art or can be prepared by methods known from the literature.", "Surprisingly, the compounds of the formula (I) have an unforeseeable pharmacological activity spectrum and are therefore suitable in particular for the prophylaxis and/or treatment of disorders.", "The compounds of the formula (I) are suitable for the prophylaxis and/or treatment of a number of disorders, such as, for example, in particular disorders of the cardiovascular system (cardiovascular disorders).", "In the context of the present invention, cardiovascular disorders are to be understood as meaning, in particular, for example the following disorders: coronary heart disease, hypertension (high blood pressure), restenosis, for example after balloon dilation of peripheral blood vessels, arteriosclerosis, tachycardia, arrhythmias, peripheral vascular disorders and cardiovascular disorders, stable and unstable angina pectoris and atrial fibrillation.", "The compounds of the formula (I) are furthermore also particularly suitable, for example, for reducing the size of the myocardial area affected by an infarct.", "The compounds of the formula (I) are furthermore particularly suitable, for example, for the prophylaxis and/or treatment of thromboembolic disorders and ischemias, such as myocardial infarction, stroke and transitory ischemic attacks.", "Further areas of indication for which the compounds of the formula (I) are suitable are, for example, in particular the prophylaxis and/or treatment of disorders of the urogenital system, such as, for example, an irritable bladder, erectile dysfunction and female sexual dysfunction and cancer, but additionally also the prophylaxis and/or treatment of inflammatory disorders, such as, for example, asthma and inflammatory dermatoses, of neuroinflammatory disorders of the central nervous system, such as, for example, disorders after stroke, Alzheimer's disease, and furthermore also of neurodegenerative disorders, such as Parkinson's disease, and also of pain.", "A further area of indication is, for example, in particular the prophylaxis and/or treatment of disorders of the respiratory tract, such as, for example, asthma, chronic bronchitis, pulmonary emphysema, bronchiectases, cystic fibrosis (mucoviscidosis) and pulmonary hypertension.", "The compounds of the formula (I) are furthermore also suitable, for example, in particular for the prophylaxis and/or treatment of liver fibrosis and liver cirrhosis.", "Finally, the compounds of the formula (I) are in particular also suitable, for example, for the prophylaxis and/or treatment of diabetes, in particular diabetes mellitus.", "The present invention also relates to the use of the substances of the formula (I) for preparing medicaments and pharmaceutical compositions for the prophylaxis and/or treatment of the clinical pictures mentioned above.", "The present invention furthermore relates to a method for the prophylaxis and/or treatment of the clinical pictures mentioned above using the substances of the formula (I).", "The pharmaceutical activity of the compounds of the formula (I) mentioned above can be explained by their activity as selective ligands on individual subtypes or a plurality of subtypes of the adenosine receptors, in particular as selective ligands on adenosine A1, adenosine A2a and/or adenosine A2b receptors, preferably as selective ligands on adenosine A1 and/or adenosine A2b receptors.", "In the context of the present invention, adenosine receptor ligands are referred to as being “selective” if, firstly, they are clearly active on one or more adenosine receptor subtypes and, secondly, the activity that can be observed on one or more other adenosine receptor subtypes is considerably weaker, if present at all, where, with respect to the test methods for selectivity of action, reference is made to the test methods described in Section A. II.", "One advantage of the compounds of the formula (I) according to the invention is that they are more selective than adenosine receptor ligands of the prior art.", "In particular, compounds of the formula (1) in which R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O—, —O—CH2—CH2—O— or —O—(CH2)3—O— generally act selectively on adenosine A1 receptors.", "In particular, compounds of the formula (I) in which one of the radicals R1, R2 or R3 represents —NH—C(O)—R7 and one of the radicals R4 or R5 represents benzyl or pyridylmethyl generally act selectively on adenosine A1 and adenosine A2b receptors.", "The receptor selectivity can be determined by biochemical measurement of the intracellular messenger cAMP in the transfected cells which specifically only express one subtype of the adenosine receptors.", "Here, what is observed is, in the case of A2a and A2b agonists (coupling preferably via Gs proteins) an increase of the intracellular cAMP concentration and in the case of A2a and A2b antagonists a decrease of the intracellular cAMP concentration, respectively, following prestimulation with adenosine or adenosine-like substances (see the publications B. Kull, G. Arslan, C. Nilsson, C. Owman, A. Lorenzen, U. Schwabe, B.", "B. Fredholm, “Differences in the order of potency for agonists but not antagonists at human and rat adenosine A2A receptors”, Biochem.", "Pharmacol., 57 (1999) pages 65-75; and S. P. Alexander, J. Cooper, J.", "Shine, S. J. Hill, “Characterization of the human brain putative A2B adenosine receptor expressed in Chinese hamster ovary (CHO.A2B4) cells”, Br.", "J.", "Pharmacol., 119 (1996) pages 1286-90, the respective content of which is expressly incorporated herein by way of reference).", "Correspondingly, A1 agonists (coupling preferably via Gi proteins) and A1 antagonists result in a decrease and increase, respectively, of the cAMP concentration.", "Thus, compounds of the formula (I) which bind selectively to adenosine A1 receptors are preferably suitable for myocard protection and for the prophylaxis and/or treatment of tachycardia, atrial arrhythmias, cardiac insufficiency, myocardial infarction, acute kidney failure, diabetes, pain, and for wound healing.", "Compounds of the formula (I) which bind selectively to adenosine A2a receptors are preferably suitable for the prophylaxis and/or treatment of thromboembolic disorders, of neurodegenerative disorders such as Parkinson's disease and for wound healing.", "Compounds of the formula (I) which bind selectively to adenosine A2b receptors are preferably suitable for the prophylaxis and/or therapy of liver fibrosis, of myocardial infarction, of neuroinflammatory disorders, of Alzheimer's disease, of urogenital incontinence and of disorders of the respiractory tract, such as, for example, asthma and chronic bronchitis.", "The present invention also provides medicaments and pharmaceutical preparations comprising at least one compound of the formula (I), preferably together with one or more pharmacologically acceptable auxiliaries or carriers, and their use for the abovementioned purposes.", "Suitable for administering the compounds of the formula (I) are all customary administration forms, i.e.", "oral, parenteral, inhalative, nasal, sublingual, rectal, local, such as, for example, in the case of implants or stents, or external, such as, for example, transdermal.", "In the case of parenteral administration, particular mention may be made of intravenous, intramuscular and subcutaneous administration, for example as a subcutaneous depot.", "Particular preference is given to oral administration.", "Here, the active compounds can be administered on their own or in the form of preparations.", "Suitable preparations for oral administration are inter alia tablets, capsules, pellets, sugar-coated tablets, pills, granules, solid and liquid aerosols, syrups, emulsions, suspensions and solutions.", "Here, the active compound has to be present in such a quantity that a therapeutic effect is obtained.", "In general, the active compound can be present in a concentration of from 0.1 to 100% by weight, in particular from 0.5 to 90% by weight, preferably from 5 to 80% by weight, i.e.", "the active compound should be present in quantities sufficient to achieve the dosage range mentioned.", "To this end, the active compounds can be converted in a manner known per se into the customary preparations.", "This is achieved using inert nontoxic pharmaceutically suitable carriers, auxiliaries, solvents, vehicles, emulsifiers and/or dispersants.", "Auxiliaries which may be mentioned are, for example: water, nontoxic organic solvents, such as, for example, paraffins, vegetable oils (for example sesame oil), alcohols (for example ethanol, glycerol), glycols (for example polyethylene glycol), solid carriers, such as natural or synthetic ground minerals (for example talc or silicates), sugars (for example lactose), emulsifiers, dispersants (for example polyvinylpyrrolidone) and glidants (for example magnesium sulfate).", "In the case of oral administration, tablets may, generally, also contain additives such as sodium citrate, together with adjuvants such as starch, gelatin and the like.", "Aqueous preparations for oral administration may furthermore be admixed with flavor enhancers or colorants.", "In general, it has been found to be advantageous to administer, in the case of parenteral administration, quantities of from about 0.1 to about 10 000 μg/kg, preferably from about 1 to about 1000 μg/kg, in particular from about 1 μg/kg to about 100 μg/kg, of body weight, to obtain effective results.", "In the case of oral administration, the quantity is from about 0.1 to about 10 mg/kg, preferably from about 0.5 to about 5 mg/kg, in particular from about 1 to about 4 mg/kg, of body weight.", "It may sometimes be required, depending on body weight, administration route, individual response to the active compound, the type of preparation and the time or interval at which administration takes place, to deviate from the quantities mentioned.", "The present invention is illustrated by the following examples, which do not restrict the invention in any way.", "A. Assessing Physiological Activity I. Detecting the Cardiovascular Effect Langendorff Heart of the Rat: After the thorax has been opened, the heart is removed from anesthetized rats and introduced into a conventional Langendorff apparatus.", "The coronary arteries are perfused at constant volume (10 ml/min), and the resulting perfusion pressure is recorded by way of an appropriate pressure sensor.", "In this set-up, a decrease in the perfusion pressure corresponds to a relaxation of the coronary arteries.", "At the same time, the pressure which the heart develops during each contraction is measured by way of a balloon, which has been introduced into the left ventricle, and a second pressure sensor.", "The frequency of the heart, which is beating in isolation, is calculated from the number of contractions per time unit.", "II.", "Assessing the Receptor Selectivity a) Adenosine A1, A2a, A2b and A3 Receptor Selectivity Cells of the CHO (Chinese Hamster Ovary) permanent cell line are transfected stably with the cDNA for the adenosine receptor subtypes A1, A2a, A2b and A3.The binding of the substances to the A2a or A2b receptor subtypes is determined by measuring the intracellular cAMP content in these cells using a conventional radioimmunological assay (cAMP RIA).", "When the substances act as agonists, the binding of the substances is expressed as an increase in the intracellular content of cAMP.", "The adenosine-analogous compound NECA (5-N-ethylcarboxamido-adenosine), which binds to all adenosine receptor subtypes with high affinity but not selectively and possesses an agonistic effect, is used as the reference compound in these experiments (Klotz, K. N., Hessling, J., Hegler, J., Owman, C., Kull, B., Fredholm, B.", "B., Lohse, M. J., Comparative pharmacology of human adenosine receptor subtypes—characterization of stably transfected receptors in CHO cells, Naunyn Schmiedebergs Arch Pharmacol, 357 (1998), 1-9).", "The adenosine receptors A1 and A3 are coupled to a Gi protein, i.e.", "stimulation of these receptors leads to inhibition of the adenylate cyclase and consequently to a lowering of the intracellular cAMP level.", "In order to identify A1/A3 receptor agonists, the adenylate cyclase is stimulated with forskolin.", "However, an additional stimulation of the A1/A3 receptors inhibits the adenylate cyclase, which means that A1/A3 receptor agonists can be detected by a comparatively low content of cAMP in the cell.", "In order to detect an antagonistic effect on adenosine receptors, the recombinant cells which are transfected with the corresponding receptor are prestimulated with NECA and the effect of the substances on reducing the intracellular content of cAMP occasioned by this prestimulation is investigated.", "XAC (xanthine amine congener), which binds to all adenosine receptor subtypes unselectively but with high affinity and possesses an antagonistic effect, is used as the reference compound in these experiments (Müller, C. E., Stein, B., Adenosine receptor antagonists: structures and potential therapeutic applications, Current Pharmaceutical Design, 2 (1996) 501-530).", "b) Adenosine A1, A2a, A2b Receptor Selectivity Cells of the CHO (Chinese Hamster Ovary) permanent cell line are transfected stably with the cDNA for the adenosine receptor subtypes A1, A2a and A2b.", "The adenosine A1 receptors are coupled to the adenylate cyclase by way of Gi proteins, while the adenosine A2a and A2b receptors are coupled by way of Gs proteins.", "In correspondence with this, the formation of cAMP in the cell is inhibited or stimulated, respectively.", "After that, expression of the luciferase is modulated by way of a cAMP-dependent promoter.", "The luciferase test is optimized, with the aim of high sensitivity and reproducibility, low variance and good suitability for implementation on a robot system, by varying several test parameters, such as cell density, duration of the growth phase and the test incubation, forskolin concentration and medium composition.", "The following test protocol is used for pharmacologically characterizing cells and for the robot-assisted substance test screening: The stock cultures are grown, at 37° C. and under 5% CO2, in DMEM/F12 medium containing 10% FCS (fetal calf serum) and in each case split 1:10 after 2-3 days.", "The test cultures are seeded in 384-well plates at the rate of from 1 000 to 3 000 cells per well and grown at 37° C. for approx.", "48 hours.", "The medium is then replaced with a physiological sodium chloride solution (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 20 mM HEPES, 1 mM MgCl2.6H2O, 5 mM NaHCO3, pH 7.4).", "The substances, which are dissolved in DMSO, are diluted 1:10 three times with this physiological sodium chloride solution and pipetted into the test cultures (maximum final concentration of DMSO in the test mixture: 0.5%).", "In this way, final substance concentrations of, for example, from 5 μM to 5 nM are obtained.", "10 minutes later, forskolin is added to the A1 cells and all the cultures are subsequently incubated at 37° C. for 4 hours.", "After that, 35 μl of a solution which is composed of 50% lysis reagent (30 mM disodium hydrogenphosphate, 10% glycerol, 3% TritonX100, 25 mM Tris HCl, 2 mM dithiothreitol (DTT), pH 7.8) and 50% luciferase substrate solution (2.5 mM ATP, 0.5 mM luciferin, 0.1 mM coenzyme A, 10 mM tricine, 1.35 mM MgSO4, 15 mM DTT, pH 7.8) are added to the test cultures, the plates are shaken for approx.", "1 minute and the luciferase activity is measured using a camera system.", "B.", "WORKING EXAMPLES Abbreviations Used: DMSO Dimethyl sulfoxide HPLC High pressure, high performance liquid chromatography NMR Nuclear magnetic resonance spectroscopy THF Tetrahydrofuran Example 1 2-Amino-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedicarbonitrile 344 mg (15 mmol) of sodium were dissolved in 20.7 ml of benzyl alcohol.", "660 mg (10 mmol) of malonitrile and 750 mg (5 mmol) of piperonal are then added, and the mixture is stirred at room temperature for about 16 h. 20 ml of water are added to the reaction mixture, and the mixture is neutralized using 1N hydrochloric acid.", "The mixture is extracted three times with in each case 50 ml of dichloromethane and the combined organic phases are dried with sodium sulfate and concentrated under reduced pressure.", "The concentration residue is purified by silica gel chromatography (mobile phase: dichloromethane).", "Yield: 872 mg (=40.1% of theory) Mass spectrum: molar mass required: 370, found [M+H]+=371 NMR spectrum: [1H-NMR, DMSO-d6] 5.45 [2H] s; 6.15 [2H] s; 7.0-7.2 [3H] m; 7.3-7.6 [5H] m; 7.8-8.2 [2H] s broad.", "Example 2 2-Amino-4-(1,3-benzodioxol-5-yl)-6-ethoxy-3,5-pyridinedicarbonitrile The compound is prepared analogously to example 1.Yield: 850 mg (=41.4% of theory) Mass spectrum: molar mass required: 308, found [M+H]+=309 Example 3 2-Amino-4-(1,3-benzodioxol-5-yl)-6-(2-hydroxyethoxy)-3,5-pyridinedicarbonitrile The compound is prepared analogously to example 1.Yield: 1100 mg (=50.9% of theory) Mass spectrum: molar mass required: 324, found [M+H]+=325 Example 4 2-Ethylamino-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedicarbonitrile Step 1: 2-Chloro-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedicarbonitrile 400 mg (1.08 mmol) of 2-amino-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedicarbonitrile (example 1) are dissolved in 10 ml of acetonitrile.", "759 mg (0.87 ml, 6.48 mmol) of isoamyl nitrite and 871 mg (6.48 mmol) of copper(I) chloride are then added, and the mixture is stirred at 40° C. for about 16 h. The reaction solution is then diluted with 1N hydrochloric acid and extracted three times with in each case 50 ml of dichloromethane.", "The combined organic phases are dried with sodium sulfate and concentrated under reduced pressure.", "The concentration residue is crystallized from dichloromethane/methanol.", "Yield: 214 mg (=50.9% of theory) Mass spectrum: molar mass required: 389, found [M+H]+=390 Step 2 2-Ethylamino-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedicarbonitrile 30 mg (0.08 mmol) of 2-chloro-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedicarbonitrile (example 4, step 1) and 10.4 mg (0.23 mmol) of ethylamine are shaken together in 0.12 ml of THF for about 16 h. Water is added to the reaction solution and the product that crystallizes out is filtered off with suction and dried under reduced pressure.", "Yield: 22 mg (=72.7% of theory) Mass spectrum: molar mass required: 398, found [M+H]+=399 Example 5 2-(2-Hydroxyethylamino)-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedi-carbonitrile 30 mg (0.08 mmol) of 2-chloro-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedicarbonitrile (example 4, step 1) and 14.1 mg (0.23 mmol) of 2-hydroxyethylamine are shaken together in 0.5 ml of THF for about 16 h. Water is added to the reaction solution and the product that crystallizes out is filtered off with suction and dried under reduced pressure.", "The crude product is purified by silica gel chromatography using the mobile phases dichloromethane and dichloromethane/methanol 50:1.Yield: 16 mg (=50% of theory) Mass spectrum: molar mass required: 414, found [M+H]+=415 NMR spectrum: [1H-NMR, DMSO-d6] 3.55 [4H] s; 4.8 [1H] s; 5.5 [2H] s; 6.15 [2H] s; 7.0-7.2 [3H] m; 7.3-7.6 [5H] m; 8 [1H] s. Example 6 2-Methylamino-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedicarbonitrile The compound is prepared analogously to example 4, step 2.Yield: 26 mg (=88.9% of theory) Mass spectrum: molar mass required: 384, found [M+H]+=385 Example 7 2-(2-Methoxyethylamino)-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedi-carbonitrile The compound is prepared analogously to example 4, step 2.Yield: 27 mg (=82.8% of theory) Mass spectrum: molar mass required: 428, found [M+H]+=429 Example 8 2-Cyclopropylamino-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedicarbonitrile The compound is prepared analogously to example 4, step 2.Yield: 24 mg (=76.9% of theory) Mass spectrum: molar mass required: 410, found [M+H]+=411 Example 9 2-Cyclopropylmethylamino-4-(1,3-benzodioxol-5-yl)-6-benzyloxy-3,5-pyridinedicarbonitrile The compound is prepared analogously to example 4, step 2.Yield: 24 mg (=73.5% of theory) Mass spectrum: molar mass required: 424, found [M+H]+=425 Example 10 2-(2-Hydroxyethylamino)-4-(1,3-benzodioxol-5-yl)-6-ethoxy-3,5-pyridinedicarbonitrile The starting material 2-chloro-4-(1,3-benzodioxol-5-yl)-6-ethoxy-3,5-pyridinedicarbonitrile is prepared analogously to example 4, step 1, the product 2-(2-hydroxylethylamino)-4-(1,3-benzodioxol-5-yl)-6-ethoxy-3,5-pyridinedicarbonitrile is prepared analogously to example 4, step 2.Yield: 22 mg (=39.9% of theory) Mass spectrum: molar mass required: 352, found [M+H]+=353 Example 11 2-Cyclopropylamino-4-(1,3-benzodioxol-5-yl)-6-(2-hydroxyethoxy)-3,5-pyridinedicarbonitrile The starting material 2-chloro-4-(1,3-benzodioxol-5-yl)-6-(2-hydroxyethoxy)-3,5-pyridinedicarbonitrile is prepared analogously to example 4, step 1, the product 2-cyclopropylamino-4-(1,3-benzodioxol-5-yl)-6-(2-hydroxyethoxy)-3,5-pyridinedicarbonitrile is prepared analogously to example 4, step 2.Yield: 51 mg (=80% of theory) Mass spectrum: molar mass required: 364, found [M+H]+=365 Example 12 2-(2-methoxyethylamino)-4-(1,3-benzodioxol-5-yl)-6-ethoxy-3,5-pyridinedicarbonitrile The starting material 2-chloro-4-(1,3-benzodioxol-5-yl)-6-ethoxy-3,5-pyridinedicarbonitrile is prepared analogously to example 4, step 1, the product 2-(2-methoxyethylamino)-4-(1,3-benzodioxol-5-yl)-6-ethoxy-3,5-pyridinedicarbonitrile is prepared analogously to example 4, step 2.Yield: 54 mg (=96.5% of theory) Mass spectrum: molar mass required: 366, found [M+H]+=367 Example 13 2-Amino-4-phenyl-6-(2-pyridinylmethoxy)-3,5-pyridinedicarbonitrile 102 mg (0.4 mmol) of 2-amino-6-chloro-4-phenyl-3,5-pyridinedicarbonitrile [Quintela et al., Heterocycles 38, 1299-1305 (1994)] and 54 mg (0.48 mmol) of potassium tert-butoxide are dissolved together in 1.696 g (=1.5 ml, 15.55 mmol) of 2-hydroxymethylpyridine, and the mixture is stirred at 60° C. for about 16 h. The product which crystallizes out is filtered off with suction, washed with water and dried under reduced pressure.", "Yield: 128 mg (=97.8% of theory) Mass spectrum: molar mass required: 327, found [M+H]+=328 NMR spectrum: [1H-NMR, DMSO-d6] 5.55 [2H] s; 7.35 [1H] m; 7.5 [6H] m; 7.85 [1H] m; 8.05 [2H] s broad; 8.6 [1H] m. Example 14 2-Amino-4-phenyl-6-(3-pyridinylmethoxy)-3,5-pyridinedicarbonitrile 102 mg (0.4 mmol) of 2-amino-6-chloro-4-phenyl-3,5-pyridinedicarbonitrile [Quintela et al., Heterocycles 38, 1299-1305 (1994)] and 54 mg (0.48 mmol) of potassium tert-butoxide are dissolved together in 1.696 g (=1.5 ml, 15.55 mmol) of 3-hydroxymethylpyridine, and the mixture is stirred at 60° C. for about 16 h. The resulting suspension is acidified with two drops of glacial acetic acid.", "The product which crystallizes out is filtered off with suction, washed with water and dried under reduced pressure.", "Yield: 130 mg (=99.7% of theory) Mass spectrum: molar mass required: 327, found [M+H]+=328 NMR spectrum: [1H-NMR, DMSO-d6] 5.5 [2H] s; 7.45 [6H] m; 7.95 [1H] m; 8.1 [2H] s broad; 8.55 [1H] m; 8.8 [1H] d. Example 15 2-Amino-4-phenyl-6-(4-pyridinylmethoxy)-3,5-pyridinedicarbonitrile 102 mg (0.4 mmol) of 2-amino-6-chloro-4-phenyl-3,5-pyridinedicarbonitrile [Quintela et al., Heterocycles 38, 1299-1305 (1994)] are, together with 54 mg (0.48 mmol) of potassium tert-butoxide and 131 mg (1.2 mmol) of 4-hydroxymethylpyridine, dissolved in 1.5 ml of DMSO, and the mixture is stirred at 60° C. for about 16 h. The reaction mixture is acidified with two drops of glacial acetic acid.", "The solution is purified by preparative HPLC on reversed-phase silica gel (gradient: water+0.1% formic acid/acetonitrile 90:1010:90 in 12 minutes).", "Yield: 66 mg (=50.5% of theory) Mass spectrum: molar mass required: 327, found [M+H]+=328 Example 16 N-{4-[2-Amino-6-(benzyloxy)-3,5-dicyano-4-pyridinyl]phenyl}acetamide 58 mg (0.15 mmol) of N-{4-[2-amino-3,5-dicyano-6-(phenylsulfanyl)-4-pyridinyl]phenyl}acetamide [prepared analogously to Kambe et al., Synthesis, 531-533 (1981)] are, together with 41 mg (0.3 mmol) of potassium carbonate, dissolved/suspended in 1.88 g (=1.8 ml, 17.4 mmol) of benzyl alcohol, and the mixture is stirred at 100° C. for 3.5 h. The benzyl alcohol is evaporated under reduced pressure and the evaporation residue is purified by preparative HPLC on reversed-phase silica gel (gradient: water/acetonitrile 90:1010:90 in 38 minutes).", "Yield: 38 mg (=67% of theory) Mass spectrum: molar mass required: 383, found [M+H]+=384 Example 17 2-Benzyloxy-6-(2-hydroxyethylamino)-4-phenyl-3,5-pyridinedicarbonitrile Step 1: 2-Chloro-4-phenyl-6-(2-hydroxyethylamino)-3,5-pyridinedicarbonitrile 1 g (3.65 mmol) of 2,6-dichloro-4-phenyl-3,5-pyridinedicarbonitrile [Quintela et al., Heterocycles 38, 1299-1305 (1994)] is, together with 0.33 g (5.47 mmol) of 2-aminoethanol, dissolved in 3 ml of THF, and the mixture is heated under reflux for 8 h. The reaction mixture is purified by preparative HPLC on reversed-phase silica gel (gradient: water+0.1% formic acid/acetonitrile 90:105:95 in 35 minutes).", "Yield: 977 mg (=89.7% of theory) Mass spectrum: molar mass required: 298, found [M+H]+=299 Step 2: 2-Benzyloxy-6-(2-hydroxyethylamino)-4-phenyl-3,5-pyridinedicarbonitrile 58 mg (0.2 mmol) of 2-chloro-4-phenyl-6-(2-hydroxyethylamino)-3,5-pyridinedicarbonitrile (example 17, step 1) are, together with 27 mg (0.24 mmol) of potassium tert-butoxide, dissolved in 0.96 g (=1 ml, 8.9 mmol) of benzyl alcohol, and the mixture is stirred at 40° C. for about 16 h. The reaction mixture is purified by preparative HPLC on reversed-phase silica gel (gradient: water+0.1% formic acid/acetonitrile 90:105:95 in 35 minutes).", "Yield: 62 mg (=83.3% of theory) Mass spectrum: molar mass required: 370, found [M+H]+=371 Example 18 N-{4-[2-Amino-6-(3-pyridylmethyloxy)-3,5-dicyano-4-pyridinyl]phenyl}-acetamide 115 mg (0.3 mmol) of N-{4-[2-amino-3,5-dicyano-6-(phenylsulfanyl)-4-pyridinyl]phenyl}acetamide [prepared analogously to Kambe et al., Synthesis, 531-533 (1981)] are, together with 50 mg (0.45 mmol) of potassium tert-butoxide, dissolved/suspended in 1.12 g (=1 ml, 10.3 mmol) of 3-pyridylmethyl alcohol, and the mixture is stirred at 60° C. for 2 h. The precipitate is filtered off with suction and suspended in a mixture of water and ethanol, and 0.3 ml of 5N acetic acid is added.", "After filtration, the product is purified by preparative HPLC on reversed-phase silica gel (gradient: water/acetonitrile 95:510:90 in 38 minutes).", "Yield: 24 mg (=20% of theory) Mass spectrum: molar mass required: 384, found [M+H]+=385 NMR spectrum: [1H-NMR, DMSO-d6] 2.1 [3H] s; 5.5 [2H] s; 7.45 [3H] m; 7.7 [2H] d; 7.95 [1H] m; 8.1 [2H] s broad; 8.6 [1H] m; 8.8 [1H] s; 10.2 [1H] s. Example 19 2-Amino-4-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[(2-methyl-1,3-thiazol-4-yl)-methoxy]-3,5-pyridinedicarbonitrile 100 mg (0.26 mmol) of 2-amino-4-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-(phenylsulfanyl)-3,5-pyridinedicarbonitrile [prepared analogously to Kambe et al., Synthesis, 531-533 (1981)] are, together with 44 mg (0.39 mmol) of potassium tert-butoxide and 334 mg (2.59 mmol) of (2-methyl-1,3-thiazol-4-yl)methyl alcohol, stirred in 5 ml of 1,2-dimethoxyethane at room temperature overnight.", "The reaction solution is concentrated under reduced pressure and the residue is then purified by preparative HPLC on reversed-phase silica gel.", "Yield: 41 mg (=39% of theory) Mass spectrum: molar mass required: 405, found [M+H]+=406 NMR spectrum: [1H-NMR, DMSO-d6] 2.65 (s, 3H); 4.3 (s, 4H); 5.45 (s, 2H); 7.0 (m, 3H); 7.7 (s, 1H); 8.0 (s broad; 2H).", "Example 20 2-Amino-4-(1,3-benzodioxol-5-yl)-6-(3-thienylmethoxy)-3,5-pyridinedicarbonitrile 100 mg (0.26 mmol) of 2-amino-4-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-(methylsulfanyl)-3,5-pyridinedicarbonitrile [prepared analogously to Dyachenko et al., Russian Journal of Chemistry, Vol.", "33, No.", "7, 1997, pages 1014-1017 or Vol.", "34, No.", "4, 1998, pages 557-563] are, together with 181 mg (1.6 mmol) of potassium tert-butoxide and 184 mg (1.6 mmol) of 3-hydroxymethylthiophene, stirred at 50° C. for 4 h. After cooling, the reaction mixture is diluted with dichloromethane and washed with water.", "The organic phase is dried with sodium sulfate and concentrated under reduced pressure.", "The residue is crystallized from diethyl ether.", "Yield: 57 mg (=47% of theory) Mass spectrum: molar mass required: 376, found [M+H]+=377 NMR spectrum: [1H-NMR, DMSO-d6] 5.45 (s, 2H); 6.15 (s, 2H); 7.0-7.15 (m, 3H); 7.25 (d, 1H); 7.55 (dd, 1H); 7.7 (d, 1H); 8.0 (s broad; 2H)." ] ]	Patent_10469556
[ [ "Vaccine", "The present invention relates to an isolated polypeptide useful for immunisation against self-antigens.", "In particular the invention relates to a self-protein that is capable of raising auto-antibodies when administered in vivo.", "The invention particularly relates to rendering human cytokines immunogenic in humans.", "The invention further relates to pharmaceutical compositions comprising such compounds and their use in medicine and to methods for their production." ], [ "1.An isolated protein which is at least 30% but less than 100% identical to a human protein wherein said isolated polypeptide (a) contains at least one mutation which is characteristic of an analogous non-human protein; (b) is capable of raising antibodies in a human, (c) is sufficiently structurally similar to the human protein that said antibodies bind to both the human protein and the isolated polypeptide and; wherein the isolated protein is not an antibody.", "2.A protein having B-cell epitopes from a self-antigen of a first mammalian species and a mutation that gives rise to a sequence of an analogous protein of a second mammalian species such that the protein is able to raise in the first species from which the B-cell epitopes derived, an immune response that recognises the natural protein from which the B-cell epitopes are derived.", "3.A protein having B-cell epitopes of a self-protein from a first mammalian species which are grafted, by substitution, into a frame work of an analogous protein from a second mammalian species such that the protein is able to raise in the species in which the B-cell epitopes are derived, an immune response that recognises the natural protein from which the B-cell epitopes are derived.", "4.A protein as claimed in claim 3, wherin said mutation comprises a conserved surface region introduced into the non-surface exposed region, said mutation giving rise to a sequence of an analogous protein such that the protein is able to raise an immune response to the self protein in the species from which the self-protein is derived.", "5.A protein as claimed in claim 2 wherein the immune response is a neutralising antibody response.", "6.A protein as claimed in claim 2 wherein the human protein, or B-cell epitope is derived from a cytokine.", "7.A cytokine as claimed in claim 6, which is a 4-helical cytokine.", "8.A cytokine as claimed in claim 7 which is IL-4 or IL-13.9.A mutated human—IL-13 having one or more of the following substitutions or a substitution involving a conservative substitution thereof: R → K at position 30 V → S at position 37 Y → F at position 63 A → V at position 65 E → D at position 68 E → Y at position 80 K → R at position 81 M → I at position 85 G → H at position 87 Q → H at position 113 V → I at position 115 D → K at position 117 10.A mutated human IL-13 as claimed in claim 9 having a plurality of substitutions as set forth in claim 9.11.A mutated human IL-13 as claimed in claim 9 having one or more of the following sequences LKELIEELSN FCVALDSL AIYRTQRILHG KIEVAHFITKLL or a variant of said sequence comprising one or more conservative substitutions.", "12.A mutated human IL-13 as shown in FIG.", "9.13.A polynucleotide encoding a protein of claim 2.14.A polynucleotide of claim 13 which is a DNA and is operably linked to a promoter.", "15.A vector comprising a polynucleotide of claim 13.16.A host transformed with a polynucleotide of claim 13.17.A pharmaceutical composition comprising the protein of claim 2 with a pharmaceutically acceptable carrier or excipient.", "18.A pharmaceutical composition as claimed in claim 17 additionally comprising an adjuvant.", "19.A pharmaceutical composition as claimed in claim 18 further comprising a protein as set forth in claim 2 and an immunostimulatory oligonucleotide.", "20.A pharmaceutical composition as claimed in claim 19 wherein the immunostimulatory oligonucleotide is selected from the group: OLIGO 1: TCC ATG ACG TTC CTG ACG TT (CpG 1826) (SEQ ID NO:1) OLIGO 2: TCT CCC AGC GTG CGC CAT (CpG 1758 (SEQ ID NO:2) OLIGO 3: ACC GAT GAC GTC GCC GGT GAC GGC (SEQ ID NO:3) ACC ACG OLIGO 4: TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO:4) (CpG 2006) OLIGO 5: TCC ATG ACG TTC CTG ATG CT (CpG 1668) (SEQ ID NO:5) 21.A protein as claimed in claim 2 for use in medicine.", "22.Use of a protein as claimed in claim 2 in the manufacture of a medicament for the treatment of IL-13 mediated diseases.", "23.Use as claimed in claim 22 for the treatment of asthma.", "24.A method for the treatment of prophylaxis of IL-13 mediated disease comprising the administration of a safe and effective amount of composition according to claim 17 to a patient in need thereof.", "25.A method for the preparation of a protein according to claim 2 which method comprises: (a) identification of one or more regions of a self, typically human, protein against which an antibody response is desired, (b) identification of the amino-acid sequence of the self protein, and (c) identification of the amino-acid sequence of an analogous protein construction by recombinant DNA techniques of a chimaeric molecule containing at least one target region identified in step (a), whose amino-acid sequence is taken from the sequence identified in step (b), and sufficient amino-acids from the sequence(s) identified in step (c) to enable the resulting protein to fold into a shape similar to that of the self protein such that the mutated protein can raise an immune response that recognises the self protein." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Asthma is a chronic lung disease, caused by inflammation of the lower airways and is characterised by recurrent breathing problems.", "Airways of patients are sensitive and swollen or inflamed to some degree all the time, even when there are no symptoms.", "Inflammation results in narrowing of the airways and reduces the flow of air in and out of the lungs, making breathing difficult and leading to wheezing, chest tightness and coughing.", "Asthma is triggered by super-sensitivity towards allergens (e.g.", "dust mites, pollens, moulds), irritants (e.g.", "smoke, fumes, strong odours), respiratory infections, exercise and dry weather.", "The triggers irritate the airways and the lining of the airways swell to become even more inflamed, mucus then clogs up the airways and the muscles around the airways tighten up until breathing becomes difficult and stressful and asthma symptoms appear.", "COPD is an umbrella term to describe diseases of the respiratory tract, which shows similar symptoms to asthma and is treated with the same drugs.", "COPD is characterised by a chronic, progressive and largely irreversible airflow obstruction.", "The contribution of the individual to the course of the disease is unknown, but smoking cigarettes is thought to cause 90% of the cases.", "Symptoms include coughing, chronic bronchitis, breathlessness and respiratory injections.", "Ultimately the disease will lead to severe disability and death.", "As a result of the various problems associated with the production, administration and tolerance of monoclonal antibodies there is an increased focus on methods of instructing the patient's own immune system to generate endogenous antibodies of the appropriate specificity by means of vaccination.", "However, mammals do not generally have high-titre antibodies against self-proteins present in serum, as the immune system contains homeostatic mechanisms to prevent their formation.", "The importance of these tolerance mechanisms is illustrated by diseases like myasthenia gravis, in which auto-antibodies directed to the nicotinic acetylcholine receptor of skeletal muscle cause weakness and fatigue (Drachman, 1994, N Engl J Med 330:1797-1810).", "There is therefore a need for a vaccine approach which is able to circumvent antibody tolerance mechanisms without inducing auto-antibody-mediated pathology.", "A number of techniques have been designed with the aim of breaking B cell tolerance without necessarily inducing unacceptable autoimmune toxicity.", "However, all have significant drawbacks.", "One technique involves chemically cross-linking either the self-protein (or peptides derived from it) to a highly immunogenic carrier protein, such as keyhole limpet haemocyanin (Antibodies: A laboratory manual” Harlow, E and Lane D. 1988.Cold Spring Harbor Press).", "This approach is a variant of the widely used hapten-carrier system for raising antibodies to poorly immunogenic targets, such as low-molecular weight chemical compounds.", "However, the process of chemical conjugation can destroy potentially valuable epitopes, and much of the evoked antibody response is directed at the carrier protein.", "Furthermore this approach is only applicable to protein vaccination, and is not compatible with nucleic acid immunogens.", "A variant on the carrier protein technique involves the construction of a gene encoding a fusion protein comprising both carrier protein (for example hepatitis B core protein) and self-protein (The core antigen of hepatitis B virus as a carrier for immunogenic peptides”, Biological Chemistry.", "380(3):277-83, 1999).", "The fusion gene may be administered directly as part of a nucleic acid vaccine.", "Alternatively, it may be expressed in a suitable host cell in vitro, the gene product purified and then delivered as a conventional vaccine, with or without an adjuvant.", "However, fusing a large carrier protein to the self-protein can constrain or distort the self-protein's conformation, reducing its efficiency in evoking antibodies cross-reactive with the native molecule.", "Also, like the traditional cross-linked carrier systems, much of the antibody response is directed to the carrier part of the fusion.", "Anti-carrier responses may limit the effectiveness of subsequent booster administrations of vaccine or increase the chance of allergic or anaphylactic reactions.", "A more refined approach has been described by Dalum and colleagues wherein a single class II MHC-restricted epitope is inserted into the target molecule.", "They demonstrated the use of this method to induce antibodies to ubiquitin (Dalum et al, 1996, J Immunol 157:4796-4804; Dalum et al, 1997, Mol Immunol 34:1113-1120) and the cytokine TNF (Dalum et al, 1999, Nature Biotech 17:666-669).", "As a result, all T cell help must arise either from this single epitope or from junctional sequences.", "While this approach may work well in subjects possessing the appropriate MHC class II haplotype for which the vaccine was designed, or indeed those fortunate enough to have class II molecules capable of binding junctional epitopes, in any normal outbred population, such as those typical of humans, there will be a significant portion of the population for whom the vaccine will not work.", "Additionally, since the inserted epitope is typically from a quite unrelated protein, such as ovalbumin or lysozyme, it is likely that the additional sequence will to some degree interfere with the folding of the target protein, preventing the adoption of a fully native conformation of the target protein.", "In contrast to all of the above, the present invention provides a multiplicity of potential T cell epitopes, yet retains the target molecule in a conformation close to the native form.", "These properties allow the vaccines of the present invention to be effective immunogens in complex outbred populations, such as those composed of human patients.", "These properties are achieved by rendering a mutation in a self-protein to produce a sequence at that point which can be found in an analogous protein.", "A number of recent papers have defined a critical role for the Th2 cytokine IL-13 in driving pathology in the ovalbumin model of allergenic asthma (Wills-Karp et al, 1998; Grunig et al, 1998).", "In this work, mice previously sensitised to ovalbumin were injected with a soluble IL-13 receptor which binds and neutralises IL-13.Airway hyper-responsiveness to acetylcholine challenge was completely ablated in the treated group.", "Histological analysis revealed that treated mice had reversed the goblet-cell metaplasia seen in controls.", "In complementary experiments, lung IL-13 levels were raised by over-expression in a transgenic mouse or by installation of protein into the trachea in wild-type mice.", "In both settings, airway hyper-responsiveness, eosinophil invasion and increased mucus production were seen (Zhu et al, 1999).", "These data show that IL-13 activity is both necessary and sufficient to produce several of the major clinical and pathological features of allergic asthma in a well-validated model.", "A vaccine capable of directing a neutralising response to IL-13 would therefore constitute a useful therapeutic for the treatment of allergic asthma in humans.", "It would also have application in the treatment of certain helminth infection-related disorders (Brombacher, 2000) and diseases where IL-13 production is implicated in fibrosis (Chiaramonte et al, 1999), such as chronic obstructive pulmonary disease.", "The present invention addresses this need.", "The concepts and principles of the invention are thus set forth with respect to IL-13, but can be applied to any mammalian self-protein having an analogous protein in a second species." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides an isolated polypeptide which is at least 30% but less than 100% identical to a human protein which polypeptide (a) contains at least one mutation which is characteristic of an analogous non human protein; and b) is capable of raising antibodies in humans and (c) is sufficiently structurally similar to the human protein that the antibodies bind to both the human protein and the polypeptide; and (d) wherein the polypeptide is not an antibody.", "Thus the invention provides in one embodiment; a protein having B cell epitopes from a mammalian self-antigen and a mutation that gives rise to a sequence of an analogous protein from a second mammalian species, such that the protein is able to raise in the species from which the B-cell epitopes are derived, an immune response that recognises the native protein from which the B-cell epitopes are derived.", "Preferably the sequence of the analogous protein is more than 5, more preferably greater than 8 contiguous amino acids.", "Thus the protein of the present invention contains a sequence that is identical to the analogous sequence for at least 5, preferably at least 8 consecutive amino acids.", "In an alternative embodiment a protein is provided having B cell epitopes of a self protein which are grafted by substitution, into a framework of an analogous protein from a second mammalian species such that the protein is able to raise in the species in which the B cell epitopes are derived an immune response that recognises the natural protein from which the B-cell epitopes are derived.", "It will be appreciated that the protein of the present invention are not an antibody.", "The immune response raised is preferably an antibody response, most preferably a neutralising antibody response.", "In general the mutation is introduced preferably into the non-surface exposed region of the molecule, such that surface exposed regions are conserved.", "Surface exposed regions are accessible to the immune-system and consequently often contain B-cell epitopes.", "Accordingly the present invention provides a protein comprising conserved surface exposed regions of a self protein, and a mutation introduced into the non-surface exposed region, said mutation giving rise to a sequence of an analogous protein such that the protein is able to raise an immune response to the self-protein arises in the species from which the self-protein is derived.", "The self protein is preferably a human protein, but can be a protein from any mammal in which it is desired to raise an auto immune response to.", "The immune response is preferably specific to the native protein and immunogen of the invention.", "That is having minimal cross-reactivity or neutralising capacity with respect to other self proteins.", "The self antigen is preferably a cytokine, more preferably a 4 helical cytokine, more preferably IL-4 or IL-13, most preferably IL-13.Thus in a preferred embodiment of the present invention there is provided a chimaeric protein comprising B cell epitopes from Human IL-13 presented in a murine IL-13 back bone.", "Such a construct is capable of raising a specific anti IL-13 antibody response in humans.", "Such a construct is shown in FIG.", "9 (seq: ID No 21 and 22).", "Similarly an IL-4 construct comprising human IL-surface regions and murine framework is presented in FIG.", "13 (Seq ID: No 25).", "The invention also provides: an expression vector which comprises a polynucleotide of the invention and which is capable of expressing a polypeptide of the invention; a host cell comprising an expression vector of the invention; a method of producing a polypeptide of the invention which method comprises maintaining a host cell of the invention under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide: a vaccine composition comprising a polypeptide or polynucleotide of the invention and a pharmaceutically acceptable carrier.", "In another aspect, the invention provides a method for the design and preparation of a polypeptide according to the invention which method comprises: 1.identification of one or more regions of a self, typically human, protein against which an antibody response is desired.", "2.identification of the amino-acid sequence of the self protein.", "3.identification of the amino-acid sequence of an analogous protein construction by recombinant DNA techniques of a chimaeric molecule containing at least one target region identified in step 1, whose amino-acid sequence is taken from the sequence identified in step 2, and sufficient amino-acids from the sequence(s) identified in step 3 to enable the resulting protein to fold into a shape similar to that the self protein such that the mutated protein can raise an immune response that recognises the self protein." ], [ "The present invention relates to an isolated polypeptide useful for immunisation against self-antigens.", "In particular the invention relates to a self-protein that is capable of raising auto-antibodies when administered in vivo.", "The invention particularly relates to rendering human cytokines immunogenic in humans.", "The invention further relates to pharmaceutical compositions comprising such compounds and their use in medicine and to methods for their production.", "BACKGROUND OF THE INVENTION Asthma is a chronic lung disease, caused by inflammation of the lower airways and is characterised by recurrent breathing problems.", "Airways of patients are sensitive and swollen or inflamed to some degree all the time, even when there are no symptoms.", "Inflammation results in narrowing of the airways and reduces the flow of air in and out of the lungs, making breathing difficult and leading to wheezing, chest tightness and coughing.", "Asthma is triggered by super-sensitivity towards allergens (e.g.", "dust mites, pollens, moulds), irritants (e.g.", "smoke, fumes, strong odours), respiratory infections, exercise and dry weather.", "The triggers irritate the airways and the lining of the airways swell to become even more inflamed, mucus then clogs up the airways and the muscles around the airways tighten up until breathing becomes difficult and stressful and asthma symptoms appear.", "COPD is an umbrella term to describe diseases of the respiratory tract, which shows similar symptoms to asthma and is treated with the same drugs.", "COPD is characterised by a chronic, progressive and largely irreversible airflow obstruction.", "The contribution of the individual to the course of the disease is unknown, but smoking cigarettes is thought to cause 90% of the cases.", "Symptoms include coughing, chronic bronchitis, breathlessness and respiratory injections.", "Ultimately the disease will lead to severe disability and death.", "As a result of the various problems associated with the production, administration and tolerance of monoclonal antibodies there is an increased focus on methods of instructing the patient's own immune system to generate endogenous antibodies of the appropriate specificity by means of vaccination.", "However, mammals do not generally have high-titre antibodies against self-proteins present in serum, as the immune system contains homeostatic mechanisms to prevent their formation.", "The importance of these tolerance mechanisms is illustrated by diseases like myasthenia gravis, in which auto-antibodies directed to the nicotinic acetylcholine receptor of skeletal muscle cause weakness and fatigue (Drachman, 1994, N Engl J Med 330:1797-1810).", "There is therefore a need for a vaccine approach which is able to circumvent antibody tolerance mechanisms without inducing auto-antibody-mediated pathology.", "A number of techniques have been designed with the aim of breaking B cell tolerance without necessarily inducing unacceptable autoimmune toxicity.", "However, all have significant drawbacks.", "One technique involves chemically cross-linking either the self-protein (or peptides derived from it) to a highly immunogenic carrier protein, such as keyhole limpet haemocyanin (Antibodies: A laboratory manual” Harlow, E and Lane D. 1988.Cold Spring Harbor Press).", "This approach is a variant of the widely used hapten-carrier system for raising antibodies to poorly immunogenic targets, such as low-molecular weight chemical compounds.", "However, the process of chemical conjugation can destroy potentially valuable epitopes, and much of the evoked antibody response is directed at the carrier protein.", "Furthermore this approach is only applicable to protein vaccination, and is not compatible with nucleic acid immunogens.", "A variant on the carrier protein technique involves the construction of a gene encoding a fusion protein comprising both carrier protein (for example hepatitis B core protein) and self-protein (The core antigen of hepatitis B virus as a carrier for immunogenic peptides”, Biological Chemistry.", "380(3):277-83, 1999).", "The fusion gene may be administered directly as part of a nucleic acid vaccine.", "Alternatively, it may be expressed in a suitable host cell in vitro, the gene product purified and then delivered as a conventional vaccine, with or without an adjuvant.", "However, fusing a large carrier protein to the self-protein can constrain or distort the self-protein's conformation, reducing its efficiency in evoking antibodies cross-reactive with the native molecule.", "Also, like the traditional cross-linked carrier systems, much of the antibody response is directed to the carrier part of the fusion.", "Anti-carrier responses may limit the effectiveness of subsequent booster administrations of vaccine or increase the chance of allergic or anaphylactic reactions.", "A more refined approach has been described by Dalum and colleagues wherein a single class II MHC-restricted epitope is inserted into the target molecule.", "They demonstrated the use of this method to induce antibodies to ubiquitin (Dalum et al, 1996, J Immunol 157:4796-4804; Dalum et al, 1997, Mol Immunol 34:1113-1120) and the cytokine TNF (Dalum et al, 1999, Nature Biotech 17:666-669).", "As a result, all T cell help must arise either from this single epitope or from junctional sequences.", "While this approach may work well in subjects possessing the appropriate MHC class II haplotype for which the vaccine was designed, or indeed those fortunate enough to have class II molecules capable of binding junctional epitopes, in any normal outbred population, such as those typical of humans, there will be a significant portion of the population for whom the vaccine will not work.", "Additionally, since the inserted epitope is typically from a quite unrelated protein, such as ovalbumin or lysozyme, it is likely that the additional sequence will to some degree interfere with the folding of the target protein, preventing the adoption of a fully native conformation of the target protein.", "In contrast to all of the above, the present invention provides a multiplicity of potential T cell epitopes, yet retains the target molecule in a conformation close to the native form.", "These properties allow the vaccines of the present invention to be effective immunogens in complex outbred populations, such as those composed of human patients.", "These properties are achieved by rendering a mutation in a self-protein to produce a sequence at that point which can be found in an analogous protein.", "A number of recent papers have defined a critical role for the Th2 cytokine IL-13 in driving pathology in the ovalbumin model of allergenic asthma (Wills-Karp et al, 1998; Grunig et al, 1998).", "In this work, mice previously sensitised to ovalbumin were injected with a soluble IL-13 receptor which binds and neutralises IL-13.Airway hyper-responsiveness to acetylcholine challenge was completely ablated in the treated group.", "Histological analysis revealed that treated mice had reversed the goblet-cell metaplasia seen in controls.", "In complementary experiments, lung IL-13 levels were raised by over-expression in a transgenic mouse or by installation of protein into the trachea in wild-type mice.", "In both settings, airway hyper-responsiveness, eosinophil invasion and increased mucus production were seen (Zhu et al, 1999).", "These data show that IL-13 activity is both necessary and sufficient to produce several of the major clinical and pathological features of allergic asthma in a well-validated model.", "A vaccine capable of directing a neutralising response to IL-13 would therefore constitute a useful therapeutic for the treatment of allergic asthma in humans.", "It would also have application in the treatment of certain helminth infection-related disorders (Brombacher, 2000) and diseases where IL-13 production is implicated in fibrosis (Chiaramonte et al, 1999), such as chronic obstructive pulmonary disease.", "The present invention addresses this need.", "The concepts and principles of the invention are thus set forth with respect to IL-13, but can be applied to any mammalian self-protein having an analogous protein in a second species.", "SUMMARY OF THE INVENTION The present invention provides an isolated polypeptide which is at least 30% but less than 100% identical to a human protein which polypeptide (a) contains at least one mutation which is characteristic of an analogous non human protein; and b) is capable of raising antibodies in humans and (c) is sufficiently structurally similar to the human protein that the antibodies bind to both the human protein and the polypeptide; and (d) wherein the polypeptide is not an antibody.", "Thus the invention provides in one embodiment; a protein having B cell epitopes from a mammalian self-antigen and a mutation that gives rise to a sequence of an analogous protein from a second mammalian species, such that the protein is able to raise in the species from which the B-cell epitopes are derived, an immune response that recognises the native protein from which the B-cell epitopes are derived.", "Preferably the sequence of the analogous protein is more than 5, more preferably greater than 8 contiguous amino acids.", "Thus the protein of the present invention contains a sequence that is identical to the analogous sequence for at least 5, preferably at least 8 consecutive amino acids.", "In an alternative embodiment a protein is provided having B cell epitopes of a self protein which are grafted by substitution, into a framework of an analogous protein from a second mammalian species such that the protein is able to raise in the species in which the B cell epitopes are derived an immune response that recognises the natural protein from which the B-cell epitopes are derived.", "It will be appreciated that the protein of the present invention are not an antibody.", "The immune response raised is preferably an antibody response, most preferably a neutralising antibody response.", "In general the mutation is introduced preferably into the non-surface exposed region of the molecule, such that surface exposed regions are conserved.", "Surface exposed regions are accessible to the immune-system and consequently often contain B-cell epitopes.", "Accordingly the present invention provides a protein comprising conserved surface exposed regions of a self protein, and a mutation introduced into the non-surface exposed region, said mutation giving rise to a sequence of an analogous protein such that the protein is able to raise an immune response to the self-protein arises in the species from which the self-protein is derived.", "The self protein is preferably a human protein, but can be a protein from any mammal in which it is desired to raise an auto immune response to.", "The immune response is preferably specific to the native protein and immunogen of the invention.", "That is having minimal cross-reactivity or neutralising capacity with respect to other self proteins.", "The self antigen is preferably a cytokine, more preferably a 4 helical cytokine, more preferably IL-4 or IL-13, most preferably IL-13.Thus in a preferred embodiment of the present invention there is provided a chimaeric protein comprising B cell epitopes from Human IL-13 presented in a murine IL-13 back bone.", "Such a construct is capable of raising a specific anti IL-13 antibody response in humans.", "Such a construct is shown in FIG.", "9 (seq: ID No 21 and 22).", "Similarly an IL-4 construct comprising human IL-surface regions and murine framework is presented in FIG.", "13 (Seq ID: No 25).", "The invention also provides: an expression vector which comprises a polynucleotide of the invention and which is capable of expressing a polypeptide of the invention; a host cell comprising an expression vector of the invention; a method of producing a polypeptide of the invention which method comprises maintaining a host cell of the invention under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide: a vaccine composition comprising a polypeptide or polynucleotide of the invention and a pharmaceutically acceptable carrier.", "In another aspect, the invention provides a method for the design and preparation of a polypeptide according to the invention which method comprises: 1.identification of one or more regions of a self, typically human, protein against which an antibody response is desired.", "2.identification of the amino-acid sequence of the self protein.", "3.identification of the amino-acid sequence of an analogous protein construction by recombinant DNA techniques of a chimaeric molecule containing at least one target region identified in step 1, whose amino-acid sequence is taken from the sequence identified in step 2, and sufficient amino-acids from the sequence(s) identified in step 3 to enable the resulting protein to fold into a shape similar to that the self protein such that the mutated protein can raise an immune response that recognises the self protein.", "DESCRIPTION OF FIGURES GST=glutathione S-transferase, rmIL-13=recombinant mouse IL-13, rhIL-13=recombinant human IL-13, cIL-13=chimaeric IL-13 FIG.", "1.Sequence of mouse chimaeric IL-13 vaccine construct.", "Underlined aminoacid symbols denote sequence human IL-13, unmodified symbols are from murine IL-13.FIG.", "2.Analysis of GST cIL-13 by 4-20% Tris-glycine SDS-PAGE gel (Novex), stained for total protein with Coomassie Blue.", "FIG.", "3.Western blot analysis of GST-cIL-13.FIG.", "4.ELISA analysis of cIL-13 and GST-cIL-13 interaction with anti-mIL-13 polyclonal antibody, anti-hIL-13 polyclonal antibody and anti-GST polyclonal antibody.", "FIG.", "5.ELISA analysis of the interaction of cIL-13 and GST-cIL-13 with the mIL-13 receptors, mIL-13Rα1 and mIL-13Rα2.FIG.", "6.Anti-phospho-STAT6 Western blot of A549 lysates.", "FIG.", "7.Antibody responses induced by immunisation with GST-cIL-13 (mouse F5) or cIL-13 (mouse E5).", "FIG.", "8.Anti-phospho-STAT6 Western blot analysis of A549 lysates.", "FIG.", "9 Chimaeric IL-13 vaccine for use in humans.", "Underlined aminoacid symbols denote sequence found in murine IL-13, unmodified symbols are from human IL-13.FIG.", "10.Anti-mouse IL-13 antibody profiles follow administration of cIL-13 in combination with various adjuvants.", "FIG.", "11.Serum neutralisation capacity of mice following administration of cIL-13.FIG.", "12.Alternative cIL-13 for use as a mouse immunogen.", "FIG.", "13.Chimaeric IL-4 for use in human anti IL-4 vaccine.", "DETAILED DESCRIPTION OF THE INVENTION Throughout this specification and the appended claims, unless the context requires otherwise, the words “comprise” and “include” or variations such as “comprising”, “comprises”, “including”, “includes” etc., are to be construed inclusively, that is, use of these words will imply the possible inclusion of integers or elements not specifically recited.", "As described herein, the present invention relates isolated polypeptides and isolated polynucleotides.", "In the context of this invention the term “isolated” is intended to convey that the polypeptide or polynucleotide is not in its native state, insofar as it has been purified at least to some extent or has been synthetically produced, for example by recombinant methods, or mechanical synthesis.", "The term “isolated” therefore includes the possibility of the polypeptides or polynucleotides being in combination with other biological or non-biological material, such as cells, suspensions of cells or cell fragments, proteins, peptides, expression vectors, organic or inorganic solvents, or other materials where appropriate, but excludes the situation where the polynucleotide is in a state as found in nature.", "An advantage of the invention is that the polypeptide of the invention contains regions of the self, eg human protein against which an antibody response is desired, in association with regions characteristic of an analogous protein which are sufficiently different to the human protein to provide excellent T cell help, but yet are optimised by evolution to fold into a shape highly similar to the human protein.", "This allows antibodies to be raised that recognise the self antigen.", "Typically, the immune response raised includes the raising of a neutralising antibody response.", "The human protein according to the invention may be a full length protein encoded by the human genome or a domain or sub-unit of a full length protein encoded by the human genome.", "Where it is desired to raise neutralising antibodies against a functional domain of the self antigen—or a receptor binding domain a chimaeric antigen involving only these regions may be prepared.", "Thus the exposed region of such a domain, or the B cell epitopes of such a domain are conserved and mutation of an analogues protein is introduced in the non-B cell epitope or surface exposed domains.", "The term ‘protein’ is intended to include, for example, shorter sequences of amino acid residues which may be referred to as peptides, such as neuropeptides.", "The human protein will typically be the subject of post-translational modification such as glycosylation, proteolytic cleavage, phosphorylation, and others well known to those skilled in the field.", "The human protein is preferably a cytokine, a hormone, a growth factor or an extracellular protein, more preferably a 4-helical cytokine, most preferably IL-13.Cytokines include, for example, IL1, IL2, IL3, IL-4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL20, IL21, IL25, TNF, TGF, GMCSF, MCSF and OSM.", "4-helical cytokines include IL2, IL3, IL-4, IL5, IL13, GMCSF and MCSF.", "Hormones include, for example, luteinising hormone (LH), follicle stimulating hormone (FSH), chorionic gonadotropin (CG), VGF, GHrelin, agouti, agouti related protein and neuropeptide Y.", "Growth factors include, for example, VEGF.", "Extracellular proteins include, for example, APP or B-amyloid.", "An analogous protein is one which is orthologous or paralogous to the self-protein, eg human protein, wherein an orthologous protein can be traced by descent to a common ancestor of the different organisms and is therefore likely to perform similar conserved functions in the different organisms.", "Thus an orthologous gene means genes which are so similar in sequence they have originated from a single ancestral gene and thus are an equivalent gene in a different species and have evolved from a common ancestor by specification.", "In particular in humans the orthologous protein is a structually equivalent molecule in a non human mammal.", "A paralogous protein is one which appears in more than one copy in a given organism by a duplication event (Venter, Science; 1336, vol 291; 2001), that is homologous sequence (sharing a common evolutionary ancestors) that have diversed by gene duplication.", "Preferably the analogous protein is an orthologue.", "An orthologous protein will typically have the same name as the human protein and will typically perform the same function, for example murine IL-13 is the orthologue to human IL-13.The analogous protein is typically mammalian or avian, for example, bovine, ovine, rodent, such as murine, porcine, simian, feline, canine or human.", "Preferably the analogous protein is murine.", "Thus in the context of the present invention, Murine IL-13 is an analogous (and orthologous) protein to human IL-13.Similiarly simian IL-4 is an analogous (and orthologous) protein to human IL-4.The polypeptide of the invention preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more mutations characteristic of an analogous protein.", "More preferably the polypeptide comprises at least three mutations.", "Each mutation may be characteristic of the same or different analogous proteins.", "Thus a first mutation might be characteristic of a murine analogue and a second mutation might be characteristic of a simian analogue.", "According to one feature, the polypeptide comprises at least three mutations, where each mutation is characteristic of a different analogue.", "Preferably, however, each mutation is characteristic of the same analogue.", "A mutation is a change in the amino acid sequence of the protein and includes, for example, deletions, insertions and substitutions.", "Preferably the mutation is a substitution.", "Preferably more than one amino acids are replaced in each non-surfaced exposed region.", "A mutation which is characteristic of an analogous protein is one which results in the sequence of the human protein being closer in identity to the sequence of the analogous protein after the mutation has been made to the human protein.", "For example when the human sequence is ProProArgVal and the murine analogue has the sequence ProProTyrVal, a mutation characteristic of the analogous protein is to substitute Arg for Tyr.", "Preferably the mutation is not made in residues which are surface residues in native folded active protein in aqueous solution under physiological conditions.", "These surface residues particular those forming loop structures are often B cell epitopes and it is preferred that all of these regions are conserved.", "The mutations thus introduced have the function of breaking the tolerance of the self-protein and being immunogenic in the species that the non-mutated protein is derived from.", "In an embodiment the polypeptides of the invention are at least 30% and less than 100% identical to a human protein, preferably over the whole length of the human protein.", "Preferably the polypeptides are at least 40%, for example at least 50% identical to the human protein.", "More preferably the polypeptides are at least 60%, for example, at least 70% identical to the human protein.", "Most preferably the polypeptides are at least 85% identical to the human protein, for example, about 90% identical.", "Such proteins are capable of raising an immune response in humans that recognise the human protein.", "For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).", "The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul (1993) J. Mol.", "Evol.", "36:290-300; Altschul et al (1990) J. Mol.", "Biol.", "215:403-10.Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).", "This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.", "T is referred to as the neighbourhood word score threshold (Altschul et al., 1990).", "These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them.", "The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.", "Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.", "The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.", "The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.", "Natl.", "Acad.", "Sci.", "USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands, when the program is being used on polynucleotides.", "The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc.", "Natl.", "Acad.", "Sci.", "USA 90: 5873-5787.One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.", "For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.The successful design of a polypeptide according to the present invention can be verified for example by demonstrating that, when expressed in an appropriate host cell, the polypeptide adopts a conformation sufficiently similar to that of the self protein that antibodies are generated which are cross-reactive with the native self protein.", "This may be shown using immunological techniques, such as binding of monoclonal or polyclonal antibodies in ELISA, or by physicochemical techniques such as circular dichroism, or by crystallographic techniques such as X-ray crystallography or by computer modelling, or by numerous other approaches well known to those skilled in the art.", "Further confirmation of a successful design can be obtained by administering the resulting polypeptide in a self-context in an appropriate vaccination regime, and observing that antibodies capable of binding the protein are induced.", "This binding may be assessed through use of ELISA techniques employing recombinant or purified native protein, or through bioassays examining the effect of the protein on a sensitive cell or tissue.", "A particularly favoured assessment is to observe a phenomenon causally related to activity of the protein in the intact host, and to determine whether the presence of antibodies induced by the methods of the invention modulate that phenomenon.", "Thus a protein of the present invention will be able to raise antibodies to the native antigen in the species from which the native protein is derived.", "The polypeptide of the invention may be further modified by mutation, for example substitution, insertion or deletion of amino-acids in order to add desirable properties (such as the addition of a sequence tag that facilitates purification or increase immunogenicity) or remove undesirable properties (such as an unwanted agonistic activity at a receptor) or trans-membrane domains.", "In particular the present invention specifically contemplates fusion partners that ease purification such as poly histidine tags or GST expression partners that enhance expression.", "In a preferred embodiment there is provided a human IL-13 having one or more of the following mutations or a conservative substitution thereof characteristic of mouse IL-13.The following numbering refers to IL-13 expressed with its signal sequence in E. coli.", "R → K at position 30 V → S at position 37 Y → F at position 63 A → V at position 65 E → D at position 68 E → Y at position 80 K → R at position 81 M → I at position 85 G → H at position 87 Q → H at position 113 V → I at position 115 D → K at position 117 More preferably the human IL-13 comprises at least two preferably at least 3, 4, 5, 6 or more of the above mutations or a conservative substitution thereof.", "It is preferred that all twelve mutations are present.", "A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.", "For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules.", "Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties.", "It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.", "In making such changes, the hydropathic index of amino acids may be considered.", "The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference).", "It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.", "Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).", "These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); ysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).", "It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e.", "still obtain a biological functionally equivalent protein.", "In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.", "It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.", "U.S. Pat.", "No.", "4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.", "As detailed in U.S. Pat.", "No.", "4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).", "It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.", "In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.", "As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.", "Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.", "These are preferred conservative substitutions.", "Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.", "For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.", "Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.", "In a preferred embodiment, the mutated IL-13 of the present invention comprises one or more of the following sequences or a variant thereof comprising a conservative substitution: LKELIEELSN; (SEQ ID No 1) FCVALDSL; (SEQ ID No 2) AIYRTQRILHG; (SEQ ID No 3) KIEVAHFITKLL; (SEQ ID No 4) The polypeptide of the invention is encoded by polynucleotides of the invention.", "A person skilled in the art will readily be able to determine the sequence of the polynucleotide which encodes the polypeptide by applying the genetic code.", "Once the required nucleic acid sequence has been determined, the polynucleotide with the desired sequence can be produced as described in the examples.", "A skilled person will readily be able to adapt any parameters necessary, such as primers and PCR conditions.", "It will also be understood by a person skilled in the art that, due to the degeneracy of the genetic code, there is potentially more than one polynucleotide which encodes a polypeptide of the invention.", "The polynucleotide of the invention is typically RNA, for example mRNA, or DNA, for example genomic DNA, cDNA or synthetic DNA.", "Preferably the polynucleotide is DNA.", "Particularly preferably it is cDNA.", "The present invention further provides an expression vector, which is a nucleic acid construct, comprising the polynucleotide of the invention.", "Additionally, the nucleic acid construct will comprise appropriate initiators, promoters, enhancers and other elements, such as for example, polyadenylation signals, which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression within a mammalian cell.", "The promoter may be a eukaryotic promoter for example a CD68 promoter, Gal1, Gal10, or NMT1 promoter, a prokaryotic promoter for example Tac, Trc, or Lac, or a viral promoter, for example the cytomegalovirus promoter, the SV40 promoter, the polyhedrin promoter, the P10 promoter, or the respiratory syncytial virus LTR promoter.", "Preferably the promoter is a viral promoter.", "Particularly preferred is when the promoter is the cytomegalovirus immediate early promoter, optionally comprising exon 1 from the HCMV IE gene.", "The transcriptional regulatory elements may comprise enhancers, for example the hepatitis B surface antigen 3′untranslated region, the CMV enhancer; introns, for example the CD68 intron, or the CMV intron A, or regulatory regions, for example the CMV 5′ untranslated region.", "The polynucleotide is preferably operably linked to the promoter on the nucleic acid construct such that when the construct is inserted into a mammalian cell, the polynucleotide is expressed to produce a encoded polypeptide.", "The nucleic acid construct backbone may be RNA or DNA, for example plasmid DNA, viral DNA, bacterial DNA, bacterial artificial chromosome DNA, yeast artificial chromosome DNA, synthetic DNA It is also possible for the nucleic acid construct to be artificial nucleic acid, for example phosphorothioate RNA or DNA.", "Preferably the construct is DNA.", "Particularly preferred is when it is plasmid DNA.", "The present invention further provides a host cell comprising an expression vector of the invention.", "Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, using for example a baculovirus expression system, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells.", "Particular examples of cells which may be modified by insertion of vectors encoding for a polypeptide according to the invention include mammalian HEK293T, CHO, HeLa, NS0 and COS cells.", "Preferably the cell line selected will be one which is not only stable, but also allows for mature glycosylation of a polypeptide.", "Expression may be achieved in transformed oocytes.", "A polypeptide of the invention may be expressed in cells of a transgenic non-human animal, preferably a mouse or expressed into the milk of larger mammals, such as goats, sheep and cows.", "A transgenic non-human animal expressing a polypeptide of the invention is included within the scope of the invention.", "A polypeptide of the invention may also be expressed in Xenopus laevis oocytes.", "The present invention also includes pharmaceutical or vaccine compositions, which comprise a therapeutically effective amount of nucleic acid construct or polypeptide of the invention, optionally in combination with a pharmaceutically acceptable carrier, preferably in combination with a pharmaceutically acceptable excipient such as phosphate buffered saline (PBS), saline, dextrose, water, glycerol, ethanol, liposomes or combinations thereof.", "The vaccine composition may alternatively comprise a therapeutically effective amount of a nucleic acid construct of the invention, formulated onto metal beads, preferably gold beads.", "The vaccine composition of the invention may also comprise an adjuvant, such as, for example, in an embodiment, imiquimod, tucaresol or alum.", "Protein adjuvant formulations are preferred as these induce high titre antibody responses.", "Preferably the adjuvant is administered at the same time as of the invention and in preferred embodiments are formulated together.", "Such adjuvant agents contemplated by the invention include, but this list is by no means exhaustive and does not preclude other agents: synthetic imidazoquinolines such as imiquimod [S-26308, R-837], (Harrison, et al.", "‘Reduction of recurrent HSV disease using imiquimod alone or combined with a glycoprotein vaccine’, Vaccine 19:1820-1826, (2001)); and resiquimod [S-28463, R-848] (Vasilakos, et al.", "‘Adjuvant activites of immune response modifier R-848: Comparison with CpG ODN’, Cellular immunology 204: 64-74 (2000).", "), Schiff bases of carbonyls and amines that are constitutively expressed on antigen presenting cell and T-cell surfaces, such as tucaresol (Rhodes, J. et al.", "‘Therapeutic potentiation of the immune system by costimulatory Schiff-base-forming drugs’, Nature 377: 71-75 (1995)), cytokine, chemokine and co-stimulatory molecules, Th1 inducers such as interferon gamma, IL-2, IL-12, IL-15 and IL-18, Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13 and other chemokine and co-stimulatory genes such as MCP-1, MIP-1 alpha, MIP-1 beta, RANTES, TCA-3, CD80, CD86 and CD40L, other immunostimulatory targeting ligands such as CTLA-4 and L-selectin, apoptosis stimulating proteins and peptides such as Fas, (49), synthetic lipid based adjuvants, such as vaxfectin, (Reyes et al., ‘Vaxfectin enhances antigen specific antibody titres and maintains Th1 type immune responses to plasmid DNA immunization’, Vaccine 19: 3778-3786) squalene, alpha-tocopherol, polysorbate 80, DOPC and cholesterol, endotoxin, [LPS], Beutler, B., ‘Endotoxin,’ Toll-like receptor 4, and the afferent limb of innate immunity’, Current Opinion in Microbiology 3: 23-30 (2000)); CpG oligo- and di-nucleotides, Sato, Y. et al., ‘Immunostimulatory DNA sequences necessary for effective intradermal gene immunization’, Science 273 (5273): 352-354 (1996).", "Hemmi, H. et al., ‘A Toll-like receptor recognizes bacterial DNA’, Nature 408: 740-745, (2000) and other potential ligands that trigger Toll receptors to produce Th1-inducing cytokines, such as synthetic Mycobacterial lipoproteins, Mycobacterial protein p19, peptidoglycan, teichoic acid and lipid A.", "Certain preferred adjuvants for eliciting a predominantly Th1-type response include, for example, a Lipid A derivative such as monophosphoryl lipid A, or preferably 3-de-O-acylated monophosphoryl lipid A. MPL® adjuvants are available from Corixa Corporation (Seattle, Wash.; see, for example, U.S. Pat.", "Nos.", "4,436,727; 4,877,611; 4,866,034 and 4,912,094).", "CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response.", "Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat.", "Nos.", "6,008,200 and 5,856,462.Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996.Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.", "); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.", "The present invention also provides methods of treating or preventing IL-13 mediated disease, any symptoms or diseases associated therewith, comprising administering an effective amount of a protein, a polynucleotide, a vector or a pharmaceutical composition according to the invention.", "Administration of a pharmaceutical composition may take the form of one or more individual doses, for example in a “prime-boost” therapeutic vaccination regime.", "In certain cases the “prime” vaccination may be via particle mediated DNA delivery of a polynucleotide according to the present invention, preferably incorporated into a plasmid-derived vector and the “boost” by administration of a recombinant viral vector comprising the same polynucleotide sequence, or boosting with the protein in adjuvant.", "Conversly the priming may be with the viral vector or with a protein formulation typically a protein formulated in adjuvant and the boost with a DNA vaccine of the present invention.", "For the treatment of self-antigen, for example IL-13, mediated disease it is preferred that the adjuvant is a preferable inducer of a TH-1 response.", "In particular, the adjuvant comprises an immunostimulatory CpG oligonucleotide, such as disclosed in (WO96102555).", "Typical immunostimulatory oligonucleotides will be between 8-100 bases in length and comprises the general formula X1 CpGX2 where X1 and X2 are nucleotide bases, and the C and G are unmethylated.", "The preferred oligonucleotides for use in adjuvants or vaccines of the present invention preferably contain two or more dinucleotide CpG motifs preferably separated by at least three, more preferably at least six or more nucleotides.", "The oligonucleotides of the present invention are typically deoxynucleotides.", "In a preferred embodiment the internucleotide in the oligonucleotide is phosphorodithioate, or more preferably a phosphorothioate bond, although phosphodiester and other internucleotide bonds are within the scope of the invention including oligonucleotides with mixed internucleotide linkages.", "e.g.", "mixed phosphorothioate/phophodiesters.", "Other internucleotide bonds which stabilise the oligonucleotide may be used.", "Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in U.S. Pat.", "No.", "5,666,153, U.S. Pat.", "No.", "5,278,302 and WO95/26204.Examples of preferred oligonucleotides have the following sequences.", "The sequences preferably contain phosphorothioate modified internucleotide linkages.", "OLIGO 1: TCC ATG ACG TTC CTG ACG TT (CpG 1826) (SEQ ID NO 5) OLIGO 2: TCT CCC AGC GTG CGC CAT (CpG 1758) (SEQ ID NO 6) OLIGO 3: ACC GAT GAC GTC GCC GGT GAC GGC (SEQ ID NO 7) ACC ACG OLIGO 4: TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO 8) (CpG 2006) OLIGO 5: TCC ATG ACG TTC CTG ATG CT (CpG 1668) (SEQ ID NO 9) Alternative CpG oligonucleotides may comprise the preferred sequences above in that they have inconsequential deletions or additions thereto.", "The CpG oligonucleotides utilised in the present invention may be synthesized by any method known in the art (eg EP 468520).", "Conveniently, such oligonucleotides may be synthesized utilising an automated synthesizer.", "An adjuvant formulation containing CpG oligonucleotide can be purchased from Qiagen under the trade name “ImmunEasy”.", "The compositions of the present invention may be used for both prophylaxis and therapy.", "The present invention provides a polypeptide or a polynucleotide according to the invention for use in medicine.", "The invention further provides the use of a polypeptide or a polynucleotide of the invention in the manufacture of a medicament for the treatment of allergies, respiratory ailments such as asthma and COPD, helminth-infection related disorders, fibrosis or cirrhosis of the liver.", "The present invention also provides a method of vaccinating which comprises administering an effective amount of a vaccine composition of the invention to a patient and provoking an immune response to the vaccine composition.", "The present invention also provides vaccine compositions as described herein for use in vaccination of a mammal against IL-13 mediated disorders such as allergies, respiratory ailments, helminth-infection related disorders, fibrosis and cirrhosis of the liver.", "Respiratory ailments include, for example, asthma, such as allergic asthma, and chronic obstructive pulmonary disease (COPD).", "Specifically, a vaccine composition capable of directing a neutralising response to IL-13 would therefore constitute a useful therapeutic for the treatment of asthma, particularly allergic asthma, in humans.", "It would also have application in the treatment of certain helminth infection-related disorders (Brombacher, 2000 Bioessays 22:646-656) and diseases where IL-13 production is implicated in fibrosis (Chiaramonte et al, 1999, J Clin Inv 104:777-785), such as chronic obstructive pulmonary disease (COPD) and cirrhosis of the liver.", "The vaccine compositions of the invention may be administered in a variety of manners for example via the mucosal, such as oral and nasal; pulmonary, intramuscular, subcutaneous or intradermal routes.", "Where the antigen is to be administered as a protein based vaccine, the vaccine will typically be formulated with an adjuvant and may be lyophilised and resuspended in water for injection prior to use.", "Such compositions may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.", "Typically such compositions will be administered intra muscularly, but other routes of administration are possible.", "One technique for intradermally administration involves particle bombardment (which is also known as ‘gene gun’ technology and is described in U.S. Pat.", "No.", "5,371,015).", "Proteins may be formulated with sugars to form small particles or DNA encoding the antigen may be coated on to inert particles (such as gold beads) and are accelerated at speeds sufficient to enable them to penetrate a surface of a recipient (e.g.", "skin), for example by means of discharge under high pressure from a projecting device.", "(Particles coated with nucleic acid vaccine constructs of the invention and protein sugar particles are within the scope of the present invention, as are devices loaded with such particles.)", "Other methods of administering the nucleic acid constructs or compositions containing said constructs directly to a recipient include ultrasound, electrical stimulation, electroporation and microseeding which is described in U.S. Pat.", "No.", "5,697,901.A nucleic acid construct of the present invention may also be administered by means of specialised delivery vectors useful in gene therapy.", "Gene therapy approaches are discussed for example by Verme et al, Nature 1997, 389:239-242.Both viral and non-viral systems can be used.", "Viral based systems include retroviral, lentiviral, adenoviral, adeno-associated viral, herpes viral and vaccinia-viral based systems.", "Non-viral based systems include direct administration of nucleic acids and liposome-based systems.", "For example, the vectors may be encapsulated by liposomes or within polylactide co-glycolide (PLG) particles.", "A nucleic acid construct of the present invention may also be administered by means of transformed host cells.", "Such cells include cells harvested from a subject.", "The nucleic acid vaccine construct can be introduced into such cells in vitro and the transformed cells can later be returned to the subject.", "The nucleic acid construct of the invention may integrate into nucleic acid already present in a cell by homologous recombination events.", "A transformed cell may, if desired, be grown up in vitro and one or more of the resultant cells may be used in the present invention.", "Cells can be provided at an appropriate site in a patient by known surgical or microsurgical techniques (e.g.", "grafting, micro-injection, etc.).", "Suitable cells include dendritic cells.", "The amount of vaccine composition which is delivered will vary significantly, depending upon the species and weight of mammal being immunised, the nature of the disease state being treated/protected against, the vaccination protocol adopted (i.e.", "single administration versus repeated doses), the route of administration and the potency and dose of the adjuvant compound chosen.", "Based upon these variables, a medical or veterinary practitioner will readily be able to determine the appropriate dosage level but it may be, for example, when the vaccine is a nucleic acid that the dose will be 0.5-51 g/kg of the nucleic acid constructs or composition containing them.", "In particular, the dose will vary depending on the route of administration.", "For example, when using intradermal administration on gold beads, the total dosage will preferably between 1 μg-10 ng, particularly preferably, the total dosage will be between 10 μg and 1 ng.", "When the nucleic acid construct is administered directly, the total dosage is generally higher, for example between 50 μg and 1 or more milligram.", "The above dosages are exemplary of the average case.", "In a protein vaccine, the amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees.", "Such amount will vary depending upon which specific immunogen is employed and how it is presented.", "Generally, it is expected that each dose will comprise 1-1000 μg of protein, preferably 1-500 μg, preferably 1-100 μg, most preferably 1 to 50 μg.", "An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in vaccinated subjects.", "Following an initial vaccination, subjects may receive one or several booster immunisation adequately spaced.", "Such a vaccine formulation may be either a priming or boosting vaccination regime; be administered systemically, for example via the transdermal, subcutaneous or intramuscular routes or applied to a mucosal surface via, for example, intra nasal or oral routes.", "There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.", "It is possible for the vaccine composition to be administered on a once off basis or to be administered repeatedly, for example, between 1 and 7 times, preferably between 1 and 4 times, at intervals between about 1 day and about 18 months, preferably one month.", "This may be optionally followed by dosing at regular intervals of between 1 and 12 months for a period up to the remainder of the patient's life.", "In an embodiment the patient will receive the antigen in different forms in a prime boost regime.", "Thus for example an antigen will be first administered as a DNA based vaccine and then subsequently administered as a protein adjuvant base formulation.", "Once again, however, this treatment regime will be significantly varied depending upon the size and species of animal concerned, the amount of nucleic acid vaccine and/or protein composition administered, the route of administration, the potency and dose of any adjuvant compounds used and other factors which would be apparent to a skilled veterinary or medical practitioner.", "The following example illustrates the theory of the invention in mice rather than in humans, so that the protein is murine with mutations characteristic of human protein, but the results can readily be extrapolated to treatment of humans where the protein will have B cell epitopes from Human with mutations characteristic of a mouse, or other analogous protein.", "Throughout the following examples of the invention, use is made of various widely known and practised techniques in molecular and cellular biology.", "Practical details of these may be found in a number of textbooks including Sambrook et al (1989, 2nd edition.", "Cold Spring Harbor Press: New York).", "Amino acid sequences or designations may be given in either the one letter code, or the three letter code.", "The prefix ‘h’ is used to denote a protein or gene of human origin, ‘m’, murine origin and ‘c’, a chimaeric construct.", "‘r’ is used to indicate a recombinant protein.", "EXAMPLES 1.Design of a Vaccine Against Murine IL-13 IL-13 belongs to the SCOP (Murzin et al, 1995, J Mol Biol 247:536-540) defined 4-helical cytokines fold family.", "Individual members of this fold superfamily are related structurally, but are difficult to align at the sequence level.", "The 3D structure of IL-13 has not yet been determined, but structures have been generated for a number of other 4-helical cytokines.", "Protein multiple sequence alignments were generated for IL-13 orthologues, and also for a number of other cytokines exhibiting this fold where the structure of at least one member had been determined (IL-4, GM-CSF, IL-5 and IL-2).", "Secondary structure predictions were performed for the IL-13 protein multiple sequence alignment using DSC (King and Sternberg, 1996, Prot Sci 5:2298-2310), SIMPA96 (Levin, 1997, Prot Eng 7:771-776) and Pred2ary (Chandonia and Karplus, 1995, Prot Sci 4:275-285).", "The individual cytokine protein multiple sequence alignments were aligned to each other, using both the sequence information and the structural information (from the known crystal structures and from the secondary structure prediction).", "Antigenic sites, specifically B-cell epitopes, were predicted for murine IL-13 using the Cameleon software (Oxford Molecular), and these were mapped onto the IL-4 structure (accession number 1 RCB in the Brookhaven database) using the protein multiple sequence alignment to give an idea of where they might be located structurally on IL-13.From this analysis, exposed regions which were potentially both antigenic and involved in receptor binding were selected.", "From this model, a chimaeric IL-13 sequence was designed in which the sequence of the predicted antigenic loops was taken from murine IL-13, and the sequence of the predicted structural (predominantly helical) regions was taken from human IL-13.The purpose of this design was to identify target epitopes from murine IL-13 against which neutralising antibodies might be raised, and to present them on a framework which was structurally similar to the native protein, but yet contained sufficient sequence variation to the native (murine) protein to ensure that one or more CD4 T helper epitopes would be present.", "The nucleic acid and protein sequences selected for this example of a chimaeric IL-13 vaccine are shown in FIG.", "1 (SEQ ID NO 19 and 20).", "The underlined sequences correspond to sequences found in the human orthologue.", "Twelve amino acids were substituted to achieve the sequence in FIG.", "1.It should be understood that the degeneracy of the genetic code allows many possible nucleic acid sequences to encode identical proteins.", "Furthermore, it will be appreciated that there are other possible chimaeric IL-13 vaccine designs within the scope of the invention, that have other orthologus mutations in non-exposed areas.", "1.2 Preparation of Chimaeric IL-13 Chimaeric IL-13 (cIL-13) DNA sequence was synthesised from a series of partially overlapping DNA oligonucleotides, with the sequences cIL-13-1 to cIL-13-6 shown in Table 1.These oligos were annealed, and cIL-13 DNA generated by a PCR with the cycle specification of 94° C. for 1 minute followed by 25 cycles of 94° C. for 30 seconds, 55° C. for 1 minute and 72° C. for 2 minutes.", "Followed by 72° C. for 7 minutes and cooling to 4° C. when finished.", "The reaction product comprised a band of the expected size, 361 base pairs, which was subcloned into the T/A cloning vector pCR2.1 (Invitrogen, Groningen, Netherlands) to generate pCR2.1-cIL-13.A BamH1 and Xho1 cIL-13 digested fragment from pCR2.1-cIL-13 was then subcloned into the BamH1 and Xho1 sites in pGEX4T3 (Amersham Pharmacia, Amersham, Bucks, UK) generating pGEX4T3-cIL-13/1.On sequencing the pGEX4T3-cIL-13/1 construct we discovered an extra 39 base pairs of DNA sequence (derived from the pCR2.1 vector) between the sequence for GST and cIL-13.To correct this, we repeated the PCR for cIL-13 using pGEX4T3-cIL-13/1 and primers cIL-13Fnew and cIL-13R.", "The PCR product obtained was then cloned back into pGEX4T3 using BamH1 and Xho1 restriction sites, to generate the expression vector pGEX4T3-cIL-13.The sequence of this construct was verified by dideoxy terminator sequencing.", "This vector encodes a genetic fusion protein consisting of glutathione-S-transferase and cIL-13 (GST-cIL-13).", "The two moieties of the protein are linked by a short spacer which contains the recognition site for thrombin.", "The fusion protein may be readily purified by glutathione sepharose affinity chromatography, and then used directly, or a preparation of free cIL-13 produced by cleavage with thrombin.", "TABLE 1 Oligonucleotides used to construct chimaeric IL-13.Oligo Sequence (5′-3′) cIL-13-1R TGTGATGTTGACCAGCTCCTCAATGAGCTCCCTAAG (SEQ ID NO 10) GGTCAGAGGGAGAGACACAGATCTTGGCACCGGCCC cIL-13-2F AGGAGCTGGTCAACATCACACAAGACCAGACTCCCC (SEQ ID NO 11) TGTGCAACGGCAGCATGGTATGGAGTG TGGACCTGGC cIL-13-3R GCAATTGGAGATGTTGGTCAGGGATTCCAGGGCTGC (SEQ ID NO 12) ACAGTACCCGCCAGCGGCCAGGTCCACACTCCATAC cIL-13-4F TGACCAACATCTCCAATTGCAATGCCATCGAGAAGA (SEQ ID NO 13) CCCAGAGGATGCTGGGCGGACTCTGTA ACCGCAAGGC cIL-13-5R AAACTGGGCCACCTCGATTTTGGTATCGGGAGGCTG (SEQ ID NO 14) GAGACCGTAGTGGGGGCCTTGCGGTTACAGAGTCC cIL-13-6F AAATCGAGGTGGCCCAGTTTGTAAAGGACCTGCTCA (SEQ ID NO 15) GCTACACAAAGCAACTGTTTCGCCACGGCCCCTTC cIL-13F CGCGGATTCGGGCCGGTGCCAAGATCTG (SEQ ID NO 16) cIL-13R CTCCGCTCGAGTCGACTTAGAAGGGGC (SEQ ID NO 17) CGTGGCGAAA cIL-13Fnew CGCGGATCCGGGCCGGTGCCAAGATCTG (SEQ ID NO 18) The pGEX4T3-cIL-13 expression vector was transformed into E. coli BLR strain (Novagen, supplied by Cambridge Bioscience, Cambridge, UK).", "Expression of GST-cIL-13 was induced by adding 0.5 mM IPTG to a culture in the logarithmic growth phase for 4 hrs at 37° C. The bacteria were then harvested by centrifugation and GST-cIL-13 purified from them by a method previously described for purification of a similar GST-human IL-13 fusion protein (McKenzie et al, 1993, Proc Natn Acad Sci 90:3735-3739).", "Characterisation of cIL-13 properties Samples of purified GST-cIL-13 were analysed by SDS-PAGE electrophoresis.", "FIG.", "2 shows that the purified preparation contains a protein of the expected size for GST-cIL-13.The lower band represents a small quantity of GST, arising due to partial cleavage of the fusion protein during preparation.", "To confirm that the purified protein was GST-cIL-13, samples were separated by SDS-PAGE, blotted onto PVDF membrane and then analysed for the presence of IL-13 and GST immunoreactivity by Western blotting.", "Since cIL-13 contains sequence arising from both human and murine IL-13, it was expected that it would be recognised by specific antisera directed at human IL-13 or mouse IL-13.Blots were blocked with 3% bovine serum albumin (BSA) in TBS (50 mM trizma hydrochloride, 138 mMsodium chloride, 2.7 mM potassium chloride, pH8.0) containing 0.05% Tween-20 (TBST) overnight at 4° C., incubated with primary antibody for 1 hour at room temperature (RT) with shaking then washed 4 times with TBST.", "Secondary antibody was added for 1 hour at RT with shaking, prior to washing 4 times and developing with SuperSignal Chemiluminescent Reagent (Pierce, Rockford, Ill., USA).", "FIG.", "3 (legend below) illustrates the results of this analysis, which indicate that the purified protein is recognised by antibodies to human IL-13, mouse IL-13 and GST, so confirming the expected structure.", "Lane Sample Primary Antibody 1 GST-cIL-13 Anti-mIL-13 2 rhIL-13 Anti-mIL-13 3 rmlL-13 Anti-mIL-13 4 Markers — 5 GST-cIL-13 Anti-hIL-13 6 rhIL-13 Anti-hIL-13 7 rmlL-13 Anti-hIL-13 8 Markers — 9 GST-cIL-13 Anti-GST 10 rhIL-13 Anti-GST 11 rmIL-13 Anti-GST 12 GST Anti-GST The primary antibodies used in this experiment were: anti-hIL-13, catalogue number AF-213-NA, R&D Systems, Abingdon, Oxford, UK, used at 1 μg/ml; anti-mIL-13, catalogue number AF-413-NA, R&D Systems, used at 1 μg/ml and anti-GST, catalogue number 27-4590D, Pharmacia, used at 1/200.The secondary antibodies used in this experiment were: HRP-conjugated anti-goat IgG, catalogue number A-5420, Sigma-Aldrich Company Ltd, Poole, Dorset, UK, used at 1/40,000.The protein samples were GST-cIL-13, prepared as described in Example 2, recombinant human IL-13 (rhIL-13), catalogue number CH1-013, Cambridge Bioscience, Cambridge, UK, recombinant mouse IL-13 (rmIL-13) catalogue number 413-ML-025, R&D Systems, and GST, prepared from E. coli transfected with empty pGEX4T3 vector as described (Sambrook et al, 1989, 2nd edition.", "Cold Spring Harbor Press: New York).", "1.3 Conformation of Chimaeric IL-13 To confirm that GST-cIL-13 adopts a similar conformation in solution to that of native IL-13, samples of GST-cIL-13 and cIL-13 (generated from GST-cIL-13 by thrombin cleavage) were analysed by ELISA.", "96-well Maxisorp plates (Life Technologies Ltd, Paisley, UK) were coated with cIL-13, GST-cIL-13, mIL-13, hIL-13 or gst in carbonate-bicarbonate buffer, overnight at 4° C. Plates were then blocked with 3% BSA/TBST for 1 hour at RT, washed 3 times in TBST, incubated with primary antibody for 1 hour at RT then washed 3 times in TBST.", "Secondary antibody was added for 1 hour, washed 3 times in TBST, then developed with 0-phenylenediamine dihydrochloride peroxidase substrate (OPD, Sigma Aldrich) for 30 minutes.", "The primary and secondary antibodies used in this experiment were as described above.", "As shown in FIG.", "4, GST-cIL-3 and cIL-13 were specifically recognised by antibodies to human IL-13 and mouse IL-13.These data confirm that the chimaerisation process has not grossly altered the protein confirmation.", "1.4 Binding of Chimaeric IL-13 to Receptors ELISAs were set up to determine whether cIL-13 could bind to either of the known mouse IL-13 receptors (mIL-13R1 or mIL-13R2).", "96-well Maxisorp plates were coated with anti-human IgG (catalogue number 1-3382, Sigma Aldrich) in carbonate-bicarbonate buffer overnight at 4° C. Plates were then blocked with 3% BSA/TBST for 1 hour at RT, washed 3 times in TBST, and incubated with mIL-13R1-Fc or mIL-13R2-Fc (catalogue numbers 491-1R-200 and 539-1R-100 respectively, R+D Systems) for 1 hour at RT.", "After washing, plates were incubated with dilutions of mIL-13 or cIL-13 or GST-cIL-13 for 1 hour at RT, washed again and incubated with biotinylated anti-mIL-13 (catalogue number BAF413, R+D Systems).", "Following further washing and incubation with streptavidin conjugated horse-radish peroxidase, the plates were developed with 0-phenylenediamine dihydrochloride peroxidase substrate for 30 minutes.", "As shown in FIG.", "5, cIL-13 and GST-cIL-13 are both able to bind to either of the mIL-13 receptors.", "Again, these data confirm that the chimaerisation process has not grossly altered the protein confirmation.", "1.5 Bioactivity of Chimaeric IL-13 The bioactivity of GST-cIL-13 was assessed by the ability of this protein to phosphorylate STAT6 in the human lung fibroblast cell line A549.These cells express the human type-2 IL-4 receptor that is responsive to both IL-4 and IL-13.Stimulation of these cells with hIL-4, hIL-13 or mIL-13 induces phosphorylation of the signalling protein STAT6.5×105 A549 cells were plated into 60 mm tissue culture dishes (Life Technologies) in RPMI (Life Technologies) and grown to 70% confluence.", "Cells were then incubated with between 2 and 150 ng/ml cytokine or purified cIL-13 for 15 mins at 37° C. Because the presence of a GST fusion partner may alter the bioactivity of cytokines, the chimaeric IL-13 was assayed as both GST-cIL-13 fusion protein, and free cIL-13 liberated from the fusion by thrombin cleavage.", "By way of control, rmIL-13 and GST were also tested.", "Cell lysates were then prepared and analysed by Western blot for the presence of phospho-STAT6 using rabbit anti-phospho-STAT6 polyclonal antibody (NEB, Hitchin, Herts, UK.", "Catalogue number 9361 S).", "Blots were blocked overnight in 5% BSA/TBST (BSA must be A-7906 from Sigma as primary antibody is phospho-specific, 0.1% Tween-20), primary antibody was added at 1/1000 for 1 hour at RT then washed 3 times with TBST.", "Anti-rabbit HRP conjugated secondary antibody (A-4914, Sigma Aldrich) was added at 1/5000 for 1 hour at RT then washed 4 times with TBST prior to developing with the HRP chemiluminescent substrate ECL Reagent (Amersham Pharmacia).", "The results of this experiment are shown in FIG.", "6.Each lane was loaded with the following protein: Lane Lysates of A549 cells treated with .", ".", ".", "1 50 ng/ml rmIL-13 (R&D Systems) 2 10 ng/ml rmIL-13 (R&D Systems) 3 2 ng/ml rmIL-13 (R&D Systems) 4 50 ng/ml cIL-13 5 10 ng/ml cIL-13 6 2 ng/ml cIL-13 7 150 ng/ml GST-cIL-13 8 30 ng/ml GST-cIL-13 9 6 ng/ml GST-cIL-13 10 No treatment 11 1 μg/ml GST 12 0.25 μg/ml GST 13 Molecular weight markers Recombinant protein reagents were as described in FIG.", "3.Treatment of A549 cells with 50 or 10 ng/ml (but not 2 ng/ml) rmIL-13 induced the phosphorylation of STAT6, indicating bioactivity.", "Treatment of A549 cells with 50 ng/ml (but not 10 or 2 ng/ml) cIL-13 induced the phosphorylation of STAT6, indicating bioactivity.", "Similarly, 150 ng/ml GST-cIL-13 (which is approximately equivalent in molar terms to 50 ng/ml cIL-13) is bioactive, whereas 30 and 6 ng/ml are not.", "CIL-13 is therefore an agonist at this receptor, but under these experimental conditions is approximately 5 fold less bioactive than mIL-13.1.6 Immunisation with cIL-13 cIL-13 and GST-cIL-13 were then used as immunogens to induce the formation of auto-antibodies against mouse IL-13 in Balb/c mice.", "Female mice aged 6-8 weeks were given one subcutaneous (sc) injection of approximately 30 μg protein in complete Freunds adjuvant (CFA) at the base of the tail.", "This was followed by three booster immunisations at the same site, each consisting of approximately 10 μg protein in incomplete Freunds adjuvant [IFA] for boosts.", "Each treatment group contained 5 animals, and they were immunised according to the protocol in Table 2.TABLE 2 Group Immunisation A Saline control in CFA/IFA s/c B 30/10 μg GST in CFA/IFA s/c C Non immunised naïve mice D 30/10 μg GST-hIL-13 in CFA/IFA s/c E 30/10 μg cIL-13 in CFA/IFA s/c F 30/10 μg GST-cIL-13 in CFA/IFA s/c Day Treatment −12 Pre-bleed 0 Primary immunisation 14 1st Boost Immunisation 27 Tail bleed 42 Tail bleed 49 2nd Boost Immunisation 70 Tail bleed 97 Tail bleed 99 3rd Boost Immunisation 113 Tail bleed 140 Tail bleed Serum samples were obtained by venepuncture of the tail vein at the timepoints specified in Table 2.After clarification by centrifugation, the samples were assayed by ELISA for the presence of specific IgG responses to mouse IL-13, human IL-13 and GST.", "None of the animals in groups A-D possessed anti-mouse IL-13 antibodies at any time point.", "All of the animals in groups B, D and F made a strong IgG response to GST (group E animals also made strong antibody responses to GST, because there was GST remaining in the cIL-13 sample used to immunise these mice).", "Anti-mouse IL-13 antibody responses were induced in five out of five animals in group F and four out of five animals in group E. FIG.", "7 (a and b) shows the serological analysis for one of these animals in group F and one of these animals from group E 7b (gst—cIL-13 immunised and cIL-13 immunised respectively).", "The results indicate that immunisation with GST-cIL-13 or cIL-13 was able to break tolerance to mIL-13, generating mouse anti-mIL-13 antibodies.", "Sera from two mice (F1d70 and F5d97) that had strong anti-mIL-13 IgG responses, were tested for the capacity to neutralise the bioactivity of rmIL-13 in the A549/phospho-STAT6 assay.", "20 ng/ml or 10 ng/ml rmIL-13 (R&D Systems) were incubated with 1% sera in serum free RPMI tissue culture media for 15 minutes at room temperature prior to a 15 minute incubation at 37° C. with A549 cells.", "Cell lysates were prepared and analysed by Western blot for the presence of phospho-STAT6 as previously described above.", "As a negative control, anti-hIL-13 serum was obtained from a Balb/c mouse immunised with GST-hIL-13 and shown by ELISA to have a strong anti-hIL-13 IgG response, but no anti-mIL-13 antibodies.", "As a positive control, normal mouse serum was spiked with a neutralising anti-mIL-13 antibody (R&D Systems, catalogue number AF-413-NA) to give a final concentration of 1 μg/ml.", "The results of this experiment are shown in FIG.", "8, in which the following was tested: Lane Cytokine Antibody 1 20 ng/ml rmIL-13 Normal mouse serum 2 10 ng/ml rmIL-13 Normal mouse serum 3 0 ng/ml rmlL-13 Normal mouse serum 4 20 ng/ml rmIL-13 Serum sample F1d70 5 10 ng/ml rmlL-13 Serum sample F1d70 6 0 ng/ml rmIL-13 Serum sample F1d70 7 20 ng/ml rmIL-13 Anti-hIL-13 mouse serum 8 10 ng/ml rmIL-13 Anti-hIL-13 mouse serum 9 0 ng/ml rmIL-13 Anti-hIL-13 mouse serum 10 Molecular weight markers — 11 0 ng/ml rmIL-13 Normal mouse serum + anti-mIL-13 12 20 ng/ml rmIL-13 Serum sample F5d97 13 10 ng/ml rmIL-13 Serum sample F5d97 14 0 ng/ml rmIL-13 Serum sample F5d97 15 20 ng/ml rmIL-13 Normal mouse serum + anti-mIL-13 16 10 ng/ml rmIL-13 Normal mouse serum + anti-mIL-13 Immunisation with a chimaeric IL-13 immunogen of the invention induces the production of auto-antibodies against mouse IL-13, capable of neutralising the biological activity of the mouse IL-13 (lanes 4, 5, 12, 13), in a fashion comparable to exogenously added anti-murine IL-13 antibody (lanes 15, 16).", "This activity is not present in normal mouse serum (lanes 1, 2), nor in serum from animals immunised with GST-hIL-13 (lanes 7, 8).", "These data provide a basis for treating mammals with an IL-13 dependent pathology by vaccinating them with cIL-13, and so inducing an endogenous neutralising antibody activity.", "1.7 Alternative Constructs 1.7.1 6 his Tagged cIL-13 Design.", "GST-cIL-13 is bacterially produced protein is insoluble and requires solubilisation and refolding in vitro.", "Size exclusion chromatography indicates that the refolding process generates several differentially folded forms, which suggest that a proportion of the immune response is being directed against forms that may be generating irrelevant antibodies that do not bind native mouse IL-13.Therefore this candidate may not be generating the most potent neutralising anti-mouse IL-13 antibody responses possible.", "For this reason 6 his-cIL-13 has been cloned into a mammalian expression vector, mammalian expressed 6 his-cIL-13 is soluble and does not require refolding in vitro.", "1.7.2 FIG.", "12 (SEQ ID NO 23 and 24) shows a vaccine antigen where different analogous mutations are made.", "Protein sequence numbering according to a scheme where the glycine residue in the sequence “GPVPR” is residue 1.Single underlined sequences correspond to the predicted helical regions from the revised structural model.", "Double underlined bold residues indicate points at which mutations are incorporated into the mouse sequence: 11 mouse Leu changed to Val (rat) 21 mouse Ser changed to Thr (non-orthologous) 63 mouse Tyr changed to Phe (non-orthologous) 71 mouse Gly changed to Ala (dog/pig/cow) 100 mouse Ser changed to Thr (dog) 104 mouse Gln changed to Asn (non-orthologous) 108 mouse His changed to Arg (non-orthologous) 1.8 Application to Human Therapy FIG.", "9 shows one possible vaccine antigen according to the invention directed at the production of anti-human IL-13 antibodies in humans.", "This will be useful for the treatment of diseases characterised by excessive or inappropriate IL-13, for example asthma.", "The sequence corresponding to mouse IL-13 are underlined.", "The construct contains twelve amino-acid substitutes that are analogous to murine IL-13.These are: R → K at position 30 V → S at position 37 Y → F at position 63 A → V at position 65 E → D at position 68 E → Y at position 80 K → R at position 81 M → I at position 85 G → H at position 87 Q → H at position 113 V → I at position 115 D → K at position 117 FIG.", "13 (SEQ ID NO 25) shows one possible vaccine for human use based on Chimaeric IL-4.It is an Example of a chimearic human IL-4 vaccine protein.", "Underlined amino-acid residues comprise the alpha-helical structural regions and are derived from mouse IL-4 with the inclusion of amino acid 21 into the first helix.", "Plain symbols indicate amino-acid residues derived from human IL-4.Positions of the alpha-helical regions are taken from Zuegg, J et al (2001) Immunol and Cell Biol 79:332-339.Example 2 Immune Response to gst-cIL-13 is Specific for Mouse IL-13 and does not Cross React with Mouse IL-4 As mouse IL-13 is structurally similar to mouse IL-4, sera from a GST-cIL-13 immunised mouse (that had been shown to contain high titre anti-mouse IL-13 autoantibodies) was analysed for cross-reactivity to mouse IL-4 using an anti-mouse IL-4 ELISA and an in vitro mIL-4 neutralisation bioassay.", "2.1 Anti-Mouse IL-4 ELISA.", "96-well Maxisorp plates were coated with anti-mouse IL-4 monoclonal antibody (Cat.", "No.", "MAB404, R+D Systems) in carbonate-bicarbonate buffer overnight at 4° C. Plates were then blocked with 3% BSA/TBST for 1 hour at RT, washed 3 times in TBST, and incubated with mouse IL-4 (Cat.", "No.", "404-ML-005, R+D Systems) for 1 hour at RT.", "After washing, plates were incubated with mouse sera for 1 hour at RT, washed again and incubated with HRP conjugated anti-mouse IgG polyclonal antibody (Cat.", "No.", "A-9309, SIGMA).", "Following further washing, the plates were developed with 0-phenylenediamine dihydrochloride peroxidase substrate for 30 minutes.", "The level of anti-mouse IL-4 antibodies in the serum was expressed as an endpoint titre.", "The endpoint titre is defined as that dilution of serum that is equivalent to twice the ELISA background reading.", "Anti-mouse IL-4 Anti-mouse IL-13 Mouse antibody endpoint titre antibody endpoint titre C2 1/900 1/80000 (serum sample taken at day 125, post 4 x GST- cIL-13 vaccine doses) A very low level of mouse IL-4 cross-reactivity was detected in this serum sample.", "In contrast, a much higher anti-mouse IL-13 antibody endpoint titre was previously determined in this serum sample, using an anti-mouse IL-13 antibody ELISA.", "The level of mouse IL-4 cross-reactivity determined by this ELISA, would not be expected to have mouse IL-4 neutralising effects in vivo.", "This serum sample was assessed for mouse IL-4 neutralisation capacity in an in vitro mouse IL-4 bioassay.", "2.2 In Vitro mous IL-4 Neutralization Bioassay.", "Mouse IL-4 stimulates the proliferation of CTLL cells in vitro.", "An assay was therefore developed in these cells, to assess the mouse IL-4 neutralisation capacity of serum from this GST-cIL-13 vaccinated mouse.", "To measure the ability of mouse serum to neutralise the bioactivity of recombinant mouse IL-4 on mouse CTLL cells (Cat.", "No.", "87031904, ECACC), 3 ng/ml recombinant mouse IL-4 was incubated with various concentrations of sera for 1 hour at 37° C. in a 96-well tissue culture plate (Invitrogen).", "Following this pre-incubation period, CTLL cells were added.", "The assay mixture, containing various serum dilutions, recombinant mouse IL-4 and CTLL cells, was incubated at 37° C. for 70 hours in a humidified CO2 incubator.", "MTT substrate (Cat.", "No.", "G4000, Promega) was added during the final 4 hours of incubation, after which the reaction was stopped with an acid solution to solubilise the metabolised blue formazan product.", "The absorbance of the solution in each well was read in a 96-well plate reader at 570 nm wavelength.", "Note that this assay is only able to measure mouse IL-4 neutralisation capacity in serum dilutions greater than or equivalent to 1/100.Serum dilutions less than 1/100 induce non-specific proliferative effects in CTLL cells.", "The capacity of the serum to neutralise mouse IL-4 bioactivity was expressed as, that dilution of serum required to neutralise the bioactivity of a defined amount of mouse IL-4 by 50% (=ND50).", "The more dilute serum sample required, the more potent the neutralisation capacity.", "The highest concentration of mouse C2 serum tested was a 1/100 dilution.", "This did not neutralise the bioactivity of 3 ng/ml mouse IL-4 by 50%, therefore the ND50 is expressed as < 1/100 dilution.", "Mouse IL-4 Mous IL-13 neutralisation capacity neutralisation capacity Mous (ND50) (ND50) C2 <1/100 1/5300 (serum sample taken at day 125, post 4 × GST- cIL-13 vaccine doses) No mouse IL-4 neutralisation capacity was detected in this serum sample at the dilutions of serum tested.", "In contrast (when assessed for mouse IL-13 neutralisation capacity), this serum sample potently neutralised mouse IL-13 bioactivity.", "These data demonstrate that although a very low level of mouse IL-4 cross-reactivity can measured in the serum by an anti-mouse IL-4 antibody ELISA, there is no associated mouse IL-4 neutralisation capacity.", "2.3 New Mouse IL-13 Neutralisation Bioassay to Assess the Mouse IL-13 Neutralisation Capacity of Mouse Serum Samples.", "Previous GST-cIL-13 bioactivity and mouse IL-13 neutralisation capacity data were generated using a STAT-6 phosphorylation readout in A549 cells.", "This assay was cumbersome and not easily amenable for the generation of quantitative data.", "Mouse IL-13 stimulates the proliferation of TF-1 cells in vitro.", "An assay was therefore developed in these cells to assess the mouse IL-13 neutralisation capacity of serum from GST-cIL-13 vaccinated mice.", "2.4 In Vitro Mouse IL-13 Neutralisation Bioassay.", "To measure the ability of mouse serum to neutralise the bioactivity of recombinant mouse IL-13 on human TF-1 cells (obtained in-house), 5 ng/ml recombinant mouse IL-13 was incubated with various concentrations of sera for 1 hour at 37° C. in a 96-well tissue culture plate (Invitrogen).", "Following this pre-incubation period, TF-1 cells were added.", "The assay mixture, containing various serum dilutions, recombinant mouse IL-13 and TF-1 cells, was incubated at 37° C. for 70 hours in a humidified CO2 incubator.", "MTT substrate (Cat.", "No.", "G4000, Promega) was added during the final 4 hours of incubation, after which the reaction was stopped with an acid solution to solubilise the metabolised blue formazan product.", "The absorbance of the solution in each well was read in a 96-well plate reader at 570 nm wavelength.", "Note that this assay is only able to measure mouse IL-13 neutralisation capacity in serum dilutions greater than or equivalent to 1/100.Serum dilutions less than 1/100 induce non-specific proliferative effects in TF-1 cells.", "The capacity of the serum to neutralise mouse IL-13 bioactivity was expressed as, that dilution of serum required to neutralise the bioactivity of a defined amount of mouse IL-13 by 50% (=ND50).", "The more dilute serum sample required, the more potent the neutralisation capacity.", "The mouse IL-13 neutralisation capacity of serum from GST-cIL-13 immunised mice was measured by the above method.", "Potent IL-13 neutralising responses were generated, as indicated below.", "Mouse (Serum samples taken at day 125, post 4 × Mouse IL-13 GST-cIL-13 vaccine neutralisation capacity doses) (ND50) C1 1/1250 C2 1/5230 C3 1/523 C4 1/417 C5 1/1670 2.5 Determination of the Level of Mouse IL-13 Neutralisation Required for Efficacy in the ‘Ovalbumin Challenge’ Mouse Asthma Model.", "In order to benchmark the required potency of an IL-13 autovaccine for treatment of asthma, mice were treated with various doses of rabbit anti-mouse IL-13 polyclonal antibody (administered passively by intra-peritoneal injection) during ovalbumin challenge, in the ‘ovalbumin challenge’ mouse asthma model.", "Model parameters such as airway hyper-responsiveness (AHR), goblet cell metaplasia (GCM) and lung inflammatory cell content were measured at the end of this experiment.", "Efficacy in this model was correlated to the levels of mouse IL-13 neutralisation achieved in mouse serum.", "The mouse IL-13 neutralisation bioassay was used to determine the level of mouse IL-13 neutralisation in serum samples.", "Treatment group (Dose of passively Mouse IL-13 administered rabbit anti- neutralisation capacity mouse IL-13 antibody) (ND50) Highest dose 1/4100 High dose 1/2670 Mid dose 1/476 Lowest dose 1/207 Treatment groups given the highest three doses of antibody all performed similarly.", "All of these three groups showed efficacy equivalent to (for AHR) or better than (for GCM) the gold standard treatment (dexamethasone, administered by the intraperitoneal route at 3×1.5 mg/kg) used in this model.", "The ‘lowest dose’ of antibody administered, showed efficacy somewhere between that of dexamethasone and the ‘no treatment’ positive control groups.", "Therefore the level of IL-13 neutralisation achieved in the ‘mid dose’ treatment group, represents the required potency threshold for an IL-13 autovaccine in this animal model.", "The potency threshold is defined as the lowest level of IL-13 neutralisation in mouse serum, required to show 100% efficacy in the asthma model (=ED100).", "1×ED100 is therefore equivalent to an ND50 of 1/476.Significance of Defined Potency Threshold.", "The level of IL-13 neutralisation required for efficacy in the ‘ovalbumin challenge’ mouse asthma model has been defined above.", "The levels of IL-13 neutralisation induced by GST-cIL-13 in mice C1-3 and C5, are in excess of the potency threshold required for efficacy in the asthma model.", "These results are illustrated in FIG.", "11.Therefore the GST-cIL-13 vaccine would be expected to show efficacy in the mouse asthma model.", "Example 3 Immunogenicity Profile of GST-cIL-13 in Combination with Various Adjuvants 3.1 Immunisation Protocol.", "GST-cIL-13 was used as an immunogen to induce the formation of auto-antibodies against mouse IL-13 in Balb/c mice.", "Female mice aged 6-8 weeks were given one injection of approximately 100 μg protein in adjuvant.", "This was followed by four booster immunisations each consisting of 50 μg protein in adjuvant (See below for immunogen+adjuvant formulations).", "Each treatment group contained 5 animals, immunised according to the protocol in the table below.", "Serum samples were obtained by venepuncture of the tail vein at the timepoints specified.", "After clarification by centrifugation, the samples were assayed by ELISA for the presence of specific IgG responses to mouse IL-13.Group Immunisation A GST-cIL-13 in AS03 i/m B GST-cIL-13 in Alum i/p C GST-cIL-13 in ‘ImmunEasy’ i/m D GST-cIL-13 in CFA/IFA s/c E GST-cIL-13 in PBS s/c F No immunisations Day Treatment −7 Pre-bleed 0 Primary immunisation 21 1st boost immunisation 35 Tail bleed 49 2nd boost immunisation 63 Tail bleed 77 3rd boost immunisation 92 Tail bleed 106 4th boost immunisation 125 Tail bleed 3.2 Immunogen+Adjuvant Formulation.", "Preparation of emulsion Adjuvant AS03: Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS.", "To provide 100 ml two-fold concentrate emulsion 5 g of DL alpha tocopherol and 5 ml of squalene are vortexed to mix thoroughly.", "90 ml of PBS/Tween solution is added and mixed thoroughly.", "The resulting emulsion is then passed through a syringe and finally microfluidised by using an M110S microfluidics machine.", "The resulting oil droplets have a size of approximately 180 nm.", "Mix adjuvant 1:1 with protein solution, vortex briefly (10 seconds at middle speed) and incubate for 10 minutes at room temperature on an orbital shaker.", "Vortex briefly before injection and administer 100 ul total suspension per mouse by the intramuscular route at 2 separate sites (ie.", "2×50 ul per mouse, one injection in each quadriceps muscle).", "Prepare fresh before each immunisation.", "Alum Supplied by SIGMA (Cat.", "No.", "A-1577).", "Prepare a 2 mg/ml suspension of alum in PBS.", "Mix adjuvant 1:1 with protein solution, vortex briefly and incubate shaking gently for 10 minutes at room temperature.", "Vortex briefly before injection and administer 100 ul total suspension per mouse i/p.", "Prepare fresh before each immunisation.", "CpG—ImmunEasy Supplied by Qiagen (Cat.No.", "303101).", "Mix the stock pot of adjuvant by gentle vortexing, then mix adjuvant 1:1 with protein by gently pipetting up and down 5 times.", "Incubate at room temperature for 15 minutes.", "Gently pipette the mix up and down 5 times and administer 100 ul suspension per mouse by the intramuscular route at 2 separate sites (ie.", "2×50 ul per mouse, one injection in each quadriceps muscle).", "Prepare fresh before each immunisation.", "CFA/IFA Supplied by SIGMA (Cat.", "Nos.", "F-5881, F-5506).", "Formulate 1:1 with pre-mixed CFA for primary or IFA for boosts.", "Whirlimix sample to ensure an even white suspension with the CFA/IFA.", "Store on ice for at least 30 mins prior to use and whirlimix thoroughly prior to dosing.", "3.3 Anti-Mouse IL-13 Antibody Responses.", "Anti-mouse IL-13 antibody responses were monitored in the serum samples using an anti-mouse IL-13 antibody detection ELISA.", "96-well Maxisorp plates were coated with anti-mouse IL-13 monoclonal antibody (Cat.", "No.", "MAB, R+D Systems) in carbonate-bicarbonate buffer overnight at 4° C. Plates were then blocked with 3% BSA/TBST for 1 hour at RT, washed 3 times in TBST, and incubated with mouse IL-13 (Cat.", "No.", "413-ML-025, R+D Systems) for 1 hour at RT.", "After washing, plates were incubated with mouse sera for 1 hour at RT, washed again and incubated with HRP conjugated anti-mouse IgG polyclonal antibody (SIGMA, Cat.", "No.", "A-9309).", "Following further washing the plates were developed with O-phenylenediamine dihydrochloride peroxidase substrate for 30 minutes.", "The level of anti-mouse IL-13 antibodies in the serum was expressed as an endpoint titre.", "The endpoint titre is defined as that dilution of serum that is equivalent to twice the ELISA background reading.", "Anti-mouse IL-13 antibody endpoint titre Mouse AS03 Alum CpG CFN/FA 1 1/875 1/7250 1/67500 1/6750 2 1/9250 1/800 1/80000 1/975 3 1/160 1/9000 1/54000 1/6000 4 1/9000 1/6500 1/62500 1/16000 5 1/3600 1/10000 1/77500 1/31000 FIG.", "10 illustrates the anti-mouse IL-13 antibody profiles in the various treatment groups at day 125, for serum samples diluted at 1/100.All five mice immunised with GST-cIL-13 in combination with CpG adjuvant raised strong anti-mouse IL-13 auto-antibody responses.", "This is in contrast to the other adjuvants, where responses were less consistent throughout each group, some mice raising very weak responses indeed.", "These results indicate that CpG adjuvant is much more effective at raising consistent high titre anti-mouse IL-13 auto-antibody responses compared to the other adjuvants tested.", "These serum samples were analysed for IL-13 neutralising ability in an in vitro IL-13 neutralisation bioassay.", "3.4 IL-13 Neutralisation Capacity.", "To measure the ability of mouse serum to neutralise the bioactivity of recombinant mouse IL-13 on human TF-1 cells (ATCC Cat.", "No.", "CRL-2003), 5 ng/ml recombinant mouse IL-13 was incubated with various concentrations of sera for 1 hour at 37° C. in a 96-well tissue culture plate (Gibco BRL).", "Following this pre-incubation period, TF-1 cells were added.", "The assay mixture, containing various serum dilutions, recombinant mouse IL-13 and TF-1 cells, was incubated at 37° C. for 70 hours in a humidified CO2 incubator.", "MTT substrate (Cat.", "No.", "G4000, Promega) was added during the final 4 hours of incubation, after which the reaction was stopped with an acid solution to solubilise the metabolised blue formazan product.", "The absorptance of the solution in each well was read in a 96-well plate reader at 570 nm wavelength.", "Note that this assay is only able to measure mouse IL-13 neutralisation capacity in serum dilutions greater than or equivalent to 1/100.Serum dilutions less than 1/100 induce non-specific proliferative effects in TF-1 cells.", "The capacity of the serum to neutralise mouse IL-13 bioactivity was expressed as, that dilution of serum required to neutralise the bioactivity of 5 ng/ml mouse IL-13 by 50% (=ND50).", "The more dilute serum sample required, the more potent the neutralisation capacity.", "The highest concentration of mouse D5 serum tested was a 1/100 dilution.", "This did not neutralise the bioactivity of 5 ng/ml mouse IL-13 by 50%, therefore the ND50 is expressed as < 1/100 dilution.", "Mouse Mouse IL-13 (Serum samples taken neutralisation capacity at day 125) (ND50) C1 1/1250 C2 1/5230 C3 1/523 C4 1/417 C5 1/1670 D5 <1/100 Day 125 serum samples from all five mice immunised with GST-cIL-13 in combination with CpG adjuvant, were able to potently neutralise the bioactivity of mouse IL-13 in an in vitro bioassay.", "In contrast, the day 125 serum sample from mouse D5 (immunised with GST-cIL-13 in CFA/IFA) was unable to neutralise the bioactivity of mouse IL-13 at all dilutions tested.", "These results indicate that CpG adjuvant is much more effective at raising neutralising anti-mouse IL-13 auto-antibody responses compared to the other adjuvants tested." ] ]	Patent_10469561
[ [ "Sprue apparatus", "A sprue apparatus for use in a molding apparatus for connecting the melt duct of a molding machine nozzle with a runner system of a molding apparatus.", "The sprue apparatus includes a plurality of thermal regulators that regulate a plurality of thermal zones that segment the length of the sprue apparatus for the purpose of localized temperature control in support of a molding process.", "The plurality of zones may be thermally regulated such as to enable a substantially leak-free junction between the machine nozzle and the molding apparatus.", "The sprue apparatus may include an isolating coupler that substantially isolates a heated sprue bushing from carriage force.", "The invention has been found particularly useful when injecting metal alloys such as magnesium based alloys when in the thixotropic state." ], [ "1) A sprue apparatus (51) configured to be received in a molding apparatus (26) for connecting a melt duct (89) of a molding machine nozzle (48) with a runner system of the molding apparatus (26), the sprue apparatus (51) comprising: a sprue bushing (52) that is configured to cooperate with an isolating coupler (53); the sprue bushing (52) comprising: a tubular body having a first and a second end; a nozzle connection interface (94) configured on an inner surface of the tubular body, at the first end thereof, for connecting with a complementary connection interface on the machine nozzle (48); an outer surface of the tubular body being configured to receive a plurality of thermal regulators; a first isolating coupler connection interface (72) configured on the outer surface of the tubular body, at the first end thereof, substantially adjacent the nozzle connection interface (94); a second isolating coupler connection interface (74) configured on the outer surface of the tubular body, at the second end thereof; and a melt duct (89) configured through the tubular body, on the inner surface thereof, from the first to the second end; and the isolating coupler (53) comprising: a tubular body having a first and a second end; a first sprue bushing connection interface (76) configured on an inner surface of the tubular body, at the first end thereof, for receiving the first isolating coupler connection interface (72) on the sprue bushing (52); a second sprue bushing connection interface (78) configured on an inner surface of the tubular body, in proximity to the second end thereof, for receiving the second isolating coupler connection interface (74) on the sprue bushing (52); a mold connection interface (93) configured at the second end of the tubular body for connecting with a complementary connection interface on the molding apparatus (26); the first isolating coupler connection interface (72) and the first sprue bushing connection interface (76) are configured to cooperate to substantially isolate the sprue bushing (52) from an applied carriage force from the machine nozzle (48).", "2) The sprue apparatus (51) according to claim 1 wherein the nozzle connection interface (94) is configured as a cylindrical spigot interface.", "3) The sprue apparatus (51) according to claim 2 wherein at least one of the plurality of zones is a thermally regulated sealing zone that maintains a temperature at the nozzle connection interface (94) below a liquidus temperature of a molding material that provides a substantially leak-free connection with the machine nozzle (48).", "4) The sprue apparatus (51) according to claims 1 or 2 wherein the mold connection interface (93) is configured as a cylindrical spigot interface.", "5) The sprue apparatus (51),according to claim 4 wherein at least one of the plurality of zones is a thermally regulated sealing zone that maintains a temperature at the mold connection interface (93) below a liquidus temperature of a molding material that provides a substantially leak-free connection with the molding apparatus (26).", "6) The sprue apparatus (51) according to claim 4 wherein the isolating coupler (53) includes a melt duct extension configured along the inner surface of the tubular member at the second end thereof that interconnects the melt duct (89) of the sprue bushing (52) with the runner system of the molding apparatus (26).", "7) The sprue apparatus (51) according to claim 6 wherein the connection between the second isolating coupler connection interface (74) of sprue bushing (52) and the second sprue bushing connection interface (78) of isolating coupler (53) is a spigot junction.", "8) The sprue apparatus (51) according to claim 7 wherein at least one of the plurality of zones is a thermally regulated sealing zone that maintains a temperature at the mold connection interface (93) below a liquidus temperature of a molding material that provides a substantially leak-free connection with the molding apparatus (26).", "9) The sprue apparatus (51) according to claim 7 wherein the isolating coupler (53) further comprises a front housing (54) connected with a cooling insert (56), the cooling insert (56) providing the function of a thermal regulator adjacent the nozzle connection interface (94).", "10) The sprue apparatus (51) according to claim 9 wherein the cooling insert (56) includes a cooling channel (142) for circulating cooling fluid.", "11) The sprue apparatus (51) according to claim 10 wherein the cooling insert (56) includes a thermocouple in mount (128) located adjacent an interface area between the cooling insert (56) and sprue bushing (52).", "12) The sprue apparatus (51) according to claim 9 wherein the front housing (54) provides a thermal conduit for heat conduction between the cooled molding apparatus (26) and the second end of sprue bushing (52) within a second sealing zone, the thermal conduit providing the function of a thermal regulator adjacent the connection between the second isolating coupler connection interface (74) of sprue bushing (52) and the second sprue bushing connection interface (78) of isolating coupler (53).", "13) The sprue apparatus (51) according to claim 12 wherein the front housing (54) includes an bore (90) that extends through the front housing (54) from a first end to a second end thereof and provides a pocket surrounding the sprue bushing along a substantial portion of its length.", "14) The sprue apparatus (51) according to claim 13 wherein the bore (90) further includes a cylindrical surface in proximity to the second end of the front housing (54) that provides the second sprue bushing connection interface 78, and an outward taper immediate the second end that provides the melt duct extension (89).", "15) The sprue apparatus (51) according to claim 14 wherein the front housing (54) includes an outer surface that is configured to provide the mold connection interface (93).", "16) The sprue apparatus (51) according to claim 1 wherein the nozzle connection interface (94) is provided by an inner surface of a recessed cylindrical bore (87) at a first end of the sprue bushing (52).", "17) The sprue apparatus (51) according to claim 16 wherein the first isolating coupler connecting interface (74) is provided by a surface on the outer diameter of a cylindrical flange (86) located at the first end of sprue bushing (52).", "18) The sprue apparatus (51) according to claim 17 further including a heat choke (124) formed as a groove on an inside face of flange (86).", "19) The sprue apparatus (51) according to claim 18 wherein melt duct (89) further includes an inwardly contracting tapered portion adjacent the bore (87), a stepped transition (170) between a main melt portion and an outwardly expanding tapered portion immediate the second end of the sprue bushing (52).", "20) The sprue apparatus (51) according to claim 19 wherein sprue bushing (52) further includes an extended circular spigot ring portion (88) at its second end with an outer surface that provides the second isolating coupler connection interface (74.)", "21) The sprue apparatus (51) according to claim 20 wherein sprue bushing (52) includes heaters (96a, 96b, 96c and 96d) located along the longitudinal axis of sprue bushing (52) and may be selectively controlled, the heaters providing the function of a thermal regulator for selective heating of at least one conditioning zone between sealing zones.", "22) The sprue apparatus (51) according to claim 21 wherein the sprue bushing (52) further includes thermocouples in mounts (122a, 122b and 122c) located along its length for housing thermocouples that provide, in use, temperature feedback of the thermal conditions along the length of the sprue bushing (52) to at least one controller that controls thermal settings of the thermal regulators.", "23) The sprue apparatus (51) according to claim 22 wherein the sprue bushing (52) includes a narrowed portion in proximity to its second end to enable rapid temperature changes in the molding material.", "24) The sprue apparatus (51) according to claim 1 further including the isolating coupler.", "25) A method of controlling the temperature along a sprue apparatus 51 in accordance with claim 1 that connects the melt duct of a machine nozzle (48) with the runner system of a molding apparatus (26), the method including the steps of: configuring a plurality of thermal zones (z1, Z2, Z3, Z4) that segment the sprue apparatus (51) along its length; the plurality of zones including a nozzle sealing zone (Z1) that encompasses a nozzle connection interface (94) and a melt duct portion (89a) at a first end of the sprue apparatus (51); configuring one or more thermal regulators (54, 56, 96) for regulating the temperature in the plurality of thermal zones; operating a controller for driving at least a subset of the thermal regulators, based upon temperature feedback from their respective thermal zones and a desired temperature setting; said controller driving said thermal regulator in said nozzle sealing zone (Z1) to maintain a temperature at the mold connection interface (94) below a liquidus temperature of a molding material that provides a substantially leak-free connection with a machine nozzle (48).", "26) The method of controlling the temperature along a sprue apparatus (51) according to claim 25, further including the step of configuring at least one of the plurality of thermal zones as a conditioning zone (Z2 and Z3) located adjacent the nozzle sealing zone (Z1) wherein the molding material within the encompassed melt duct portion (89b and 89c) is maintained at any desired processing temperature.", "27) The method of controlling the temperature along a sprue apparatus (51) according to claim 26, further including the step of configuring one of the plurality of thermal zones as a cycling zone (Z4) located at the second end of the sprue apparatus (51) for the controlled formation of a localized plug of solidified molding material in an encompassed melt duct portion (89d).", "28) The method of controlling the temperature along a sprue apparatus (51) according to claim 27, wherein the sprue apparatus (51) comprises a sprue bushing (52) housed within 40 an isolating coupler (53) that itself includes a front housing (54) connected with a cooling insert (56), the cooling insert (56Y providing the function of a thermal regulator for selective cooling of the nozzle sealing zone (Z1).", "29) The method of controlling the temperature along a sprue apparatus (51) according to claim 28, wherein sprue bushing (52) includes heaters (96a, 96b, 96c and 96d) located along the longitudinal axis of sprue bushing (52) and may be selectively controlled, the heaters providing the function of a thermal regulator for selective heating of the least one conditioning zone (Z2 and Z3).", "30) The method of controlling the temperature along a sprue apparatus (51) according to claim 29, wherein the isolating coupler (53) provides a thermal conduit for heat conduction between the cooled molding apparatus (26) and the second end of sprue bushing (52) within a second sealing zone contained in the cycling zone (Z4), the thermal conduit providing the function of a thermal regulator." ], [ "<SOH> BACKGROUND OF TEE INVENTION <EOH>Sprue bushings for molding apparatus are well known in the art.", "For example, the book entitled “Understanding Injection Molding Technology” by Herbert Rees, copyright 1994, ISBN 1-56990-130-9, describes Hot Sprues on page 61.Essentially, sprue bushings provide a connection between the machine nozzle and the runner system of a mold for injecting at least partially molten molding material into the cavity of a mold.", "The at least partially molten material, sometimes called the melt, travels from the machine nozzle into a duct located within the sprue bushing and into a cavity of a mold.", "A carriage force is typically directed longitudinally through the sprue bushing for sealing the connection between the machine nozzle and the sprue bushing during the molding process.", "Generally, there are two.", "categories of sprues, cold and hot.", "Cold sprues are not heated.", "Any arrested flow of molten material in a cold sprue will solidify within a portion of the duct in the sprue bushing.", "The solidified material must be removed from the sprue bushing before a subsequent injection cycle.", "The solidified material is wasteful and increases the cost of the part as a result of the scrap material.", "Hot sprues, generally, are electrically heated.", "The heat may be applied either internally or externally to the sprue.", "Generally, the hot sprue keeps the material molten within the duct of the sprue bushing through a single heat zone.", "U.S. Pat.", "No.", "5,884,687 issued on Mar.", "23, 1999 to Hotset teaches a hot sprue with a heated chamber for a die casting machine.", "A feed sleeve includes a central passage for receiving a melt of material.", "A heater providing a single heat zone surrounds the feed sleeve.", "One end of the feed sleeve engages a supply of liquid metal and a carriage force is directed through a portion of the sprue bushing.", "A plug of solid material forms near a gate and is pushed out during the molding process through the application of injection pressure.", "U.S. Pat.", "No.", "6,095,789 issued on Aug. 1, 2000 to Polyshot Corporation teaches an adjustable hot sprue bushing.", "Resistive heaters are shown surrounding the body of the bushing.", "The number of turns of wire is increased at the distant ends of the bushing to provide more heat energy at the distant ends to compensate for the high heat transfer, or heating losses, at the distant ends.", "This is an attempt to provide a constant temperature along the entire length of the bushing in a single uniform thermal zone.", "PCT application WO 01/19552 to Hotflo Die Casting teaches a sprue tip insert in combination with a separate transition channel.", "The temperature along the entire length of the sprue appears to be controlled as a single uniform thermal zone.", "The material in the entire length of the sprue is at a temperature high enough to ensure flow.", "A separate mating die includes the transition channel downstream from the sprue.", "The transition channel is controlled independently of the sprue to freeze the material in the transition channel.", "U.S. Pat.", "No.", "6,357,511 issued on Mar.", "19, 2002 to the assignee of the present invention teaches a spigot junction that provides an improved connection interface between melt channel components of an injection molding machine, and in particular between a machine nozzle and an otherwise typical sprue bushing for thixotropic molding of a metallic material.", "The spigot junction includes an annular cylindrical portion of a first component received in a cylindrical bore of a second component.", "The fit of a spigot junction is characterized as having a close diametric fit between an outer surface of the annular cylindrical portion and a corresponding inner surface of the cylindrical bore, the close fit may include a small annular gap to support an initial melt seepage, and a longitudinal engagement of sufficient length to permit limited relative axial movement without a loss of sealing.", "The spigot junction provides a seal against melt leakage by virtue of the fit, that may be augmented by a seal of solidified molding material seepage that forms in the small gap.", "European patent publication 0 444 748 to Boekel et al., published on Sep. 4, 1991, describes a mold sprue bushing that includes a number of thermal control zones configured therealong.", "Japanese patent publication 2002-059456 to Atsuki et al.", "describes a machine nozzle for use with a metal molding system that includes a structure for controlling the formation of a cold plug therein.", "There are a number of problems with known sprue apparatus that result from poor thermal regulation along the length of the sprue bushing, with only a single thermal zone dedicated to maintaining the conditions of the molding material flowing through the sprue bushing.", "For example, with the single thermal control zone dedicated to the thermal regulation of molding material in support of the molding process, it is not possible to independently thermally regulate the junction between mating melt channel components; as is required with a spigot junction, to ensure a reliable seal against molding material leakage.", "The leakage of molding material is of particular concern when processing light-alloys, such as magnesium in a thixomolding process, due to the possibility of rapid and uncontrolled oxidation at elevated processing temperatures.", "Further, it would be desirable to provide localized temperature control along the length of the sprue apparatus to counter problems such as undesirable molding temperature variances, control sprue plug formation, or provide general processing flexibility.", "Another problem relates to the susceptibility of known sprue apparatus to permanent deformation due to longitudinally applied carriage force, required for the purpose of maintaining a seal between the machine nozzle and the sprue apparatus, especially when the sprue apparatus is weakened at the high operating temperatures required for thixomolding magnesium.", "In particular, the sprue apparatus, in use, is constrained along its length between the machine nozzle and the molding apparatus and is therefore compressed under the applied carriage force directed though the machine nozzle.", "The sprue apparatus is susceptible to permanent deformation from the compression due to its slender construction; the slender construction of the sprue apparatus provides a short heat conduction path, and therefore a fast thermal response, between heaters provided along the length of the sprue apparatus and the molding material within its melt duct.", "Yet another problem relates to undesirable process fluctuations that result from the formation and ejection of sprue plugs of inconsistent length, the variations in sprue plugs may be attributed to inadequate thermal regulation and melt channel configuration." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In a first aspect of the present invention there is provided a sprue apparatus that connects a melt duct of a molding machine nozzle with a runner system of a molding apparatus.", "The sprue apparatus is configured to be received in the molding apparatus and includes a nozzle connection interface at a first end configured to form a junction with a complementary connection interface on the machine nozzle, a melt duct that extends through the sprue apparatus from the first end to a second end, and a mold connection interface at the second end configured to form a junction with a complementary connection interface on the molding apparatus for connection of the melt duct with the mold runner system.", "The sprue apparatus further includes a plurality of thermal regulators, disposed along the sprue apparatus, that thermally regulate a plurality of thermal zones that segment the length of the sprue apparatus for the purpose of localized temperature control of molding material within encompassed melt duct portions.", "The sprue apparatus may be an assembly of components with a junction between mating components.", "Further, any junction may be thermally regulated such as to enable a substantially leak-free connection between the machine nozzle and the runner system of the molding apparatus.", "The connection interface of the sprue apparatus may be configured to complete a spigot junction as described in U.S. Pat.", "No.", "6,357,511.In another aspect of the present invention there is provided a method of controlling the temperature along a sprue apparatus that connects the melt duct of a machine nozzle with the runner system of a molding apparatus, including the steps of: i) configuring a plurality of thermal zones that segment the sprue apparatus along its length; and ii) configuring one or more thermal regulators for regulating the temperature in at least a subset of the plurality of thermal zones; and iii) operating one or more controllers for driving at least a subset of the thermal regulators based upon temperature feedback from their respective thermal zones in accordance with a molding process.", "Preferably, the method of controlling the temperature along a sprue apparatus further includes the step of configuring one of the plurality of thermal zones as a nozzle sealing zone that encompasses a machine nozzle connection interface and a melt duct portion at a first end of the sprue apparatus, wherein the temperature at the nozzle connection interface is maintained below the melting point of the molding material while simultaneously maintaining the molding material within the melt duct portion at any desired processing temperature.", "The method may further include the step of configuring one of the plurality of thermal zones as a conditioning zone, located adjacent the nozzle sealing zone, wherein the molding material within an encompassed melt duct portion is maintained at any desired processing temperature.", "The method may further include the step of configuring one of the plurality of thermal zones as a cycling zone, located at the second end of the sprue apparatus, for the controlled formation of a localized plug of solidified molding material in an encompassed melt duct portion.", "An advantage of embodiments of the sprue apparatus of the present invention is the thermal regulation and control of a plurality of distinct thermal zones along a sprue apparatus for maintaining the molding material in the melt duct at a desired temperature and physical state to support a molding process.", "The plurality of thermal zones may also provide thermal regulation of spigot junctions for ensuring a reliable sealing connection between the machine nozzle and the molding apparatus.", "Another advantage of embodiments of the sprue apparatus of the present invention is a durable configuration, even at the high operating temperatures required for the thixotropic molding of magnesium.", "In particular, vulnerable components of the sprue apparatus may be substantially isolated from applied carriage force.", "Another advantage of embodiments of the sprue apparatus of the present invention is the provision of a cycling zone that controls the formation of a molding material plug used for flow control.", "The cycling zone is configured and controlled such that the size of the injected plug is of a consistent and minimal size and hence there is less process variation from shot to shot.", "The invention has been found particularly useful when molding with metal alloys such as with magnesium-based alloys in a thixotropic state in an injection molding system, although it will be understood that the concept is widely applicable in any molding system in which a molding material is plastecized or at least partially melted prior to delivery to a mold." ], [ "TECHNICAL FIELD This invention broadly relates to a sprue apparatus for use in a molding apparatus.", "More specifically, the invention relates to a sprue apparatus for use in an injection molding or die-casting machine, and is particularly, but not exclusively, applicable to injection molding a metal material into a cavity of a mold when the material is in a thixotropic state.", "BACKGROUND OF TEE INVENTION Sprue bushings for molding apparatus are well known in the art.", "For example, the book entitled “Understanding Injection Molding Technology” by Herbert Rees, copyright 1994, ISBN 1-56990-130-9, describes Hot Sprues on page 61.Essentially, sprue bushings provide a connection between the machine nozzle and the runner system of a mold for injecting at least partially molten molding material into the cavity of a mold.", "The at least partially molten material, sometimes called the melt, travels from the machine nozzle into a duct located within the sprue bushing and into a cavity of a mold.", "A carriage force is typically directed longitudinally through the sprue bushing for sealing the connection between the machine nozzle and the sprue bushing during the molding process.", "Generally, there are two.", "categories of sprues, cold and hot.", "Cold sprues are not heated.", "Any arrested flow of molten material in a cold sprue will solidify within a portion of the duct in the sprue bushing.", "The solidified material must be removed from the sprue bushing before a subsequent injection cycle.", "The solidified material is wasteful and increases the cost of the part as a result of the scrap material.", "Hot sprues, generally, are electrically heated.", "The heat may be applied either internally or externally to the sprue.", "Generally, the hot sprue keeps the material molten within the duct of the sprue bushing through a single heat zone.", "U.S. Pat.", "No.", "5,884,687 issued on Mar.", "23, 1999 to Hotset teaches a hot sprue with a heated chamber for a die casting machine.", "A feed sleeve includes a central passage for receiving a melt of material.", "A heater providing a single heat zone surrounds the feed sleeve.", "One end of the feed sleeve engages a supply of liquid metal and a carriage force is directed through a portion of the sprue bushing.", "A plug of solid material forms near a gate and is pushed out during the molding process through the application of injection pressure.", "U.S. Pat.", "No.", "6,095,789 issued on Aug. 1, 2000 to Polyshot Corporation teaches an adjustable hot sprue bushing.", "Resistive heaters are shown surrounding the body of the bushing.", "The number of turns of wire is increased at the distant ends of the bushing to provide more heat energy at the distant ends to compensate for the high heat transfer, or heating losses, at the distant ends.", "This is an attempt to provide a constant temperature along the entire length of the bushing in a single uniform thermal zone.", "PCT application WO 01/19552 to Hotflo Die Casting teaches a sprue tip insert in combination with a separate transition channel.", "The temperature along the entire length of the sprue appears to be controlled as a single uniform thermal zone.", "The material in the entire length of the sprue is at a temperature high enough to ensure flow.", "A separate mating die includes the transition channel downstream from the sprue.", "The transition channel is controlled independently of the sprue to freeze the material in the transition channel.", "U.S. Pat.", "No.", "6,357,511 issued on Mar.", "19, 2002 to the assignee of the present invention teaches a spigot junction that provides an improved connection interface between melt channel components of an injection molding machine, and in particular between a machine nozzle and an otherwise typical sprue bushing for thixotropic molding of a metallic material.", "The spigot junction includes an annular cylindrical portion of a first component received in a cylindrical bore of a second component.", "The fit of a spigot junction is characterized as having a close diametric fit between an outer surface of the annular cylindrical portion and a corresponding inner surface of the cylindrical bore, the close fit may include a small annular gap to support an initial melt seepage, and a longitudinal engagement of sufficient length to permit limited relative axial movement without a loss of sealing.", "The spigot junction provides a seal against melt leakage by virtue of the fit, that may be augmented by a seal of solidified molding material seepage that forms in the small gap.", "European patent publication 0 444 748 to Boekel et al., published on Sep. 4, 1991, describes a mold sprue bushing that includes a number of thermal control zones configured therealong.", "Japanese patent publication 2002-059456 to Atsuki et al.", "describes a machine nozzle for use with a metal molding system that includes a structure for controlling the formation of a cold plug therein.", "There are a number of problems with known sprue apparatus that result from poor thermal regulation along the length of the sprue bushing, with only a single thermal zone dedicated to maintaining the conditions of the molding material flowing through the sprue bushing.", "For example, with the single thermal control zone dedicated to the thermal regulation of molding material in support of the molding process, it is not possible to independently thermally regulate the junction between mating melt channel components; as is required with a spigot junction, to ensure a reliable seal against molding material leakage.", "The leakage of molding material is of particular concern when processing light-alloys, such as magnesium in a thixomolding process, due to the possibility of rapid and uncontrolled oxidation at elevated processing temperatures.", "Further, it would be desirable to provide localized temperature control along the length of the sprue apparatus to counter problems such as undesirable molding temperature variances, control sprue plug formation, or provide general processing flexibility.", "Another problem relates to the susceptibility of known sprue apparatus to permanent deformation due to longitudinally applied carriage force, required for the purpose of maintaining a seal between the machine nozzle and the sprue apparatus, especially when the sprue apparatus is weakened at the high operating temperatures required for thixomolding magnesium.", "In particular, the sprue apparatus, in use, is constrained along its length between the machine nozzle and the molding apparatus and is therefore compressed under the applied carriage force directed though the machine nozzle.", "The sprue apparatus is susceptible to permanent deformation from the compression due to its slender construction; the slender construction of the sprue apparatus provides a short heat conduction path, and therefore a fast thermal response, between heaters provided along the length of the sprue apparatus and the molding material within its melt duct.", "Yet another problem relates to undesirable process fluctuations that result from the formation and ejection of sprue plugs of inconsistent length, the variations in sprue plugs may be attributed to inadequate thermal regulation and melt channel configuration.", "SUMMARY OF THE INVENTION In a first aspect of the present invention there is provided a sprue apparatus that connects a melt duct of a molding machine nozzle with a runner system of a molding apparatus.", "The sprue apparatus is configured to be received in the molding apparatus and includes a nozzle connection interface at a first end configured to form a junction with a complementary connection interface on the machine nozzle, a melt duct that extends through the sprue apparatus from the first end to a second end, and a mold connection interface at the second end configured to form a junction with a complementary connection interface on the molding apparatus for connection of the melt duct with the mold runner system.", "The sprue apparatus further includes a plurality of thermal regulators, disposed along the sprue apparatus, that thermally regulate a plurality of thermal zones that segment the length of the sprue apparatus for the purpose of localized temperature control of molding material within encompassed melt duct portions.", "The sprue apparatus may be an assembly of components with a junction between mating components.", "Further, any junction may be thermally regulated such as to enable a substantially leak-free connection between the machine nozzle and the runner system of the molding apparatus.", "The connection interface of the sprue apparatus may be configured to complete a spigot junction as described in U.S. Pat.", "No.", "6,357,511.In another aspect of the present invention there is provided a method of controlling the temperature along a sprue apparatus that connects the melt duct of a machine nozzle with the runner system of a molding apparatus, including the steps of: i) configuring a plurality of thermal zones that segment the sprue apparatus along its length; and ii) configuring one or more thermal regulators for regulating the temperature in at least a subset of the plurality of thermal zones; and iii) operating one or more controllers for driving at least a subset of the thermal regulators based upon temperature feedback from their respective thermal zones in accordance with a molding process.", "Preferably, the method of controlling the temperature along a sprue apparatus further includes the step of configuring one of the plurality of thermal zones as a nozzle sealing zone that encompasses a machine nozzle connection interface and a melt duct portion at a first end of the sprue apparatus, wherein the temperature at the nozzle connection interface is maintained below the melting point of the molding material while simultaneously maintaining the molding material within the melt duct portion at any desired processing temperature.", "The method may further include the step of configuring one of the plurality of thermal zones as a conditioning zone, located adjacent the nozzle sealing zone, wherein the molding material within an encompassed melt duct portion is maintained at any desired processing temperature.", "The method may further include the step of configuring one of the plurality of thermal zones as a cycling zone, located at the second end of the sprue apparatus, for the controlled formation of a localized plug of solidified molding material in an encompassed melt duct portion.", "An advantage of embodiments of the sprue apparatus of the present invention is the thermal regulation and control of a plurality of distinct thermal zones along a sprue apparatus for maintaining the molding material in the melt duct at a desired temperature and physical state to support a molding process.", "The plurality of thermal zones may also provide thermal regulation of spigot junctions for ensuring a reliable sealing connection between the machine nozzle and the molding apparatus.", "Another advantage of embodiments of the sprue apparatus of the present invention is a durable configuration, even at the high operating temperatures required for the thixotropic molding of magnesium.", "In particular, vulnerable components of the sprue apparatus may be substantially isolated from applied carriage force.", "Another advantage of embodiments of the sprue apparatus of the present invention is the provision of a cycling zone that controls the formation of a molding material plug used for flow control.", "The cycling zone is configured and controlled such that the size of the injected plug is of a consistent and minimal size and hence there is less process variation from shot to shot.", "The invention has been found particularly useful when molding with metal alloys such as with magnesium-based alloys in a thixotropic state in an injection molding system, although it will be understood that the concept is widely applicable in any molding system in which a molding material is plastecized or at least partially melted prior to delivery to a mold.", "BRIEF DESCRIPTION OF THE DRAWINGS Exemplary embodiments of the present invention will now be described with reference to the accompanying drawings in which: FIG.", "1 is a partially cut-away side diagrammatic view of a molding system including a clamp unit and an injection unit, that can support the present invention; FIG.", "2A is a more detailed cross-sectional view of a sprue apparatus according to a preferred embodiment of the present invention, the sprue apparatus shown in-situ within the molding system of FIG.", "1; FIG.", "2B is another, more detailed cross-sectional view of the sprue apparatus according to a preferred embodiment of FIG.", "2A; FIG.", "3 is an exploded view illustrating the component parts of the sprue apparatus according to a preferred embodiment of FIG.", "2B; FIGS.", "4A and 4B are perspective views of a front housing of the sprue apparatus of FIG.", "3; FIG.", "4C is an end view of the front housing of FIG.", "4A; FIG.", "4D is a cross-sectional side view of the front housing taken along line 4D-4D of FIG.", "4C; FIG.", "4E is a view of the other end of the front housing of FIG.", "4A.", "; FIG.", "5A is a perspective view of an alternative embodiment of the front housing of FIG.", "3; FIG.", "5B is a sectional view of the housing of FIG.", "5A; FIG.", "6 is a cross-sectional view of a sprue bushing taken along line 6-6 of FIG.", "3; FIG.", "7 is a perspective view of the cooling insert of the sprue apparatus of FIG.", "3; FIG.", "8 is an end view of the cooling insert of FIG.", "7 when facing the injection unit; FIG.", "9 is another end view of the cooling insert of FIG.", "7 when facing the mold; FIG.", "10 is a cross-sectional view of the cooling insert taken along line 10-10 of FIG.", "9; FIG.", "11 is a bottom side view of the cooling insert of FIG.", "7; FIG.", "12 is a cross-sectional view of the insert taken along line 12-12 from FIG.", "11; and FIG.", "13 is a cross-sectional view of the insert taken along line 13-13 from FIG.", "11.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S) An embodiment of the invention is described in the context of (and in situ within) a typical injection molding system 10, as shown in FIG.", "1.The injection molding system 10 includes an injection unit 14 and a clamp unit 12.The injection unit 14 processes molding material for injection into a mold.", "The injection unit 14 includes a frame 32 that typically supports a housing for electrical components for machine, control and operation (not shown), as well as a housing for a power pack (not shown).", "A carriage (not shown) supports a barrel assembly 34 that includes a barrel 42.The carriage is movable relative to the frame 32 by actuation of a pair of carriage cylinders (not shown).", "A screw 40 is located within a bore of the barrel 42.In operation, the screw 40 is rotated and usually axially translated within the barrel 42 by a screw drive 36 in a manner that is well known in the art.", "The screw drive 36 may be a combination of an electric motor to rotate the screw 40, and hydraulic components to translate the screw.", "40 for injection.", "Persons skilled in the art appreciate that a completely, hydraulic drive system or a completely electric motor drive system could be applied to the injection unit 14.Further, a single stage reciprocating screw injection unit is illustrated, however, persons skilled in the art will appreciate a two-stage injection unit may be used.", "The clamp unit 12 opens, closes, and applies a closing force to a mold.", "The clamp unit 12 includes a stationary platen 16 and a moving platen 20 mounted over a frame 18, and a clamp drive (not shown) for stroking the moving platen 20 relative to the stationary platen 16.The stationary platen 16 and the drive are typically interconnected by four tie bars 38, only two of which are shown.", "A first mold half 24 is attached to the moving platen 20 and a second mold half 26 is attached to the stationary platen 16.Persons skilled in the art appreciate that the clamp unit 12 can be driven by a hydraulic drive unit, a completely electric motor clamp drive or a combination of electric motor and hydraulic components.", "In the context of an exemplary thixotropic molding system, magnesium chips 178 or other appropriate material are fed into a hopper 180 and metered through feed throat 132 of barrel 42.The screw 40 is rotated to convey molding material from the feed throat 132 along the barrel 42, past a check valve 46 at the end of screw 40, and into an accumulation zone 82 at the head of the barrel and in front of the nozzle 48.As the screw conveys material into the accumulation zone 82 and nozzle 48, the screw 40 moves back within the barrel 42 to accumulate a shot of material.", "When sufficient material has been conveyed into the accumulation zone 82, a shot of material is injected into the mold 24, 26 through a sprue apparatus.", "To accomplish this injection, a hydraulic actuator in screw drive 36 forces screw 40 forward towards the mold and thereby injects the material from the accumulator zone 82 into the mold through nozzle 48.Check valve 46 prevents material from flowing back into barrel 42 while the screw 40 is being moved forward.", "Heaters 44 are placed along the barrel 42 and nozzle 48 (see FIG.", "2A) to achieve and maintain a desired processing temperature and physical state of the molding material.", "The sprue apparatus of the present invention is exemplified with reference to the embodiment of FIG.", "2A & 2B.", "The sprue apparatus 51 provides a connection between a melt duct of machine nozzle 48 of the injection unit 14 and a runner system (not shown) of the second mold half 26.The sprue apparatus 51 is configured to be received in the second mold half 26 and includes a nozzle connection interface 94 at a first end configured to form a junction with a complementary connection interface of the machine nozzle 48.A melt duct 89 extends through the sprue apparatus 51 from the first end to a second end.", "A mold connection interface 93 at the second end is configured to form a junction with a complementary connection interface on the second mold half 26 for connection of melt duct 89 with the mold runner system.", "The sprue apparatus 51 further includes a plurality of thermal regulators, disposed along the sprue apparatus 51, that thermally regulate a plurality of thermal zones that segment the length of the sprue apparatus 51 for the purpose of localized temperature control of molding material within encompassed melt duct portions.", "The sprue apparatus may be an assembly of components with a junction between mating components.", "Further, any junction may be thermally regulated such as to enable a substantially leak-free connection between the machine nozzle 48 and the runner system of the second mold half 26.The connection interface 49 of the machine nozzle 48 is provided by a longitudinal surface of a cylindrical spigot tip extension 92.The junction between the nozzle connection interface 94 and the connection interface 49 of the machine nozzle 48 includes a small gap, into which molding material is allowed to seep and solidify to seal,the gap, as is typical of a spigot junction.", "Thermal regulation maintains the temperature at the junction below the freezing point of the molding material.", "With this arrangement, the sprue apparatus,51 can expand and contract without losing sealing contact with the nozzle 48 or the second mold half 26.Preferably, the sprue apparatus further comprises a sprue bushing 52 housed within an isolating coupler 53.The sprue bushing 52 includes the nozzle connection interface 94 at its first end, a first isolating coupler connection interface 72 in proximity to the nozzle connection interface 94.The melt duct 89 extends through the sprue bushing 52 from the first end to a second end.", "A second isolating coupler connection interface 74, at the second end.", "The first and second isolating coupler connection interfaces 72 and 74 are configured to form a junction with complementary first and second sprue bushing connection interfaces 76 and 78 provided on the isolating coupler 53.The isolating coupler 53 is configured to be at least partially received in the second mold half 26, and is further configured to interconnect the melt duct 89 of the sprue bushing 52 with the runner system of the second mold half 26.The isolating coupler 53 preferably connects with the sprue bushing 52 in such a way as to distribute a longitudinally applied carriage force, acting through the first end of the sprue bushing 52, through to the second mold half 26 whereby a substantial portion of the sprue bushing 52 is isolated from the carriage force.", "In particular, isolating coupler 53 longitudinally constrains the first end of sprue bushing 52 while allowing its remaining portion unrestricted longitudinal movement.", "The isolating coupler further assists in the thermal regulation of at least one of the plurality of thermal zones that segment the length of the sprue apparatus 51 through the provision of one or more thermal regulator to raise or lower a temperature of the one or more of the plurality of thermal zones.", "Preferably, the isolating coupler 53 is an assembly that comprises a front housing 54 connected to a cooling insert 56.The front housing 54 fits into the second mold half 26, with the cooling insert 56 held in position by locating ring 84.Bolts 130 (see FIG.", "3) extend through retaining ring 58 and into threaded holes 136 to retain sprue bushing 52 within the isolating coupler 53.An extending inner lip (on retaining ring 58) holds sprue bushing 52 within the isolating coupler whenever the nozzle 48 comes out of engagement with the sprue bushing 52.Preferably, the front housing 54 provides a thermal conduit, providing the function of a thermal regulator, for heat conduction between the cooled second mold half 26 and the second end of sprue bushing 52, thereby controlling the temperature at the junction between the second isolating coupler connection interface 74 and the second sprue bushing connection interface 78 in proximity to the second end of the front housing.", "Preferably, the junction is a spigot junction wherein the thermally regulation provides for the formation of a seal of solidified molding material as previously explained.", "The cooling insert 56 also provides the function of a thermal regulator.", "Cooling tube 66 with connector 70, having a bushing 168, provide cooling fluid, preferably oil, to cooling insert 56 to enable selective cooling of the sprue bushing 52, as will be more fully explained hereinafter.", "Thermal regulators, such as heaters 96a, 96b, 96c and 96d, are also located along the longitudinal axis of sprue bushing 52, and are shaped to ensure and maintain thermal contact.", "The heaters can be selectively controlled to adjust the temperature of the molding material; as needed.", "Heater 96d may surround a narrowed portion of sprue bushing 52.In the current example, the narrowed portion of sprue bushing 52 provides a shorter heat conduction path, and therefore a fast thermal response, between heater 96d and the molding material within melt duct portion 89d.", "It should be noted that the number of heaters and the location of the heaters can be changed.", "Also, the major portion of the sprue bushing 52 could be of a single diameter throughout its length so that all the heaters would have the same inner circumference.", "Thermocouples located along the length of sprue bushing 52 provide temperature feedback, regarding the thermal zones, to at least one controller (not shown) that controls thermal, settings of at least a subset of the thermal regulators.", "A platen locating ring 30 cooperates with mold locating ring 84 to position the second mold half 26 in correct alignment with the injection unit 34 in a manner well known in the art.", "Nozzle 48 is attached to barrel head 50 by bolts 60.Barrelhead.", "50 is attached to barrel 42 by bolts 188.Referring now to FIG.", "3, the sprue apparatus 51 of a preferred embodiment is shown as an exploded view of component parts.", "Front housing 54 includes an access slot 100 allowing routing of electrical leads 95 of heaters 96b, 96c and 96d and thermocouples.", "Bolts 68 extend through enlarged collar 102 of front housing 54 to join housing 54 to cooling insert 56.Cooling insert 56 includes a slot 104 for receiving the electrical leads for the heater 96a and a second slot 106, (see FIG.", "7) for receiving the cooling lines 66.Heater elements 96b, 96c and 96d fit within housing 54 while heating element 96a fits within cooling insert 56.In the embodiment shown in FIG.", "3, four heater elements are illustrated by way of example only, and it will therefore be appreciated that the number and location of the heater elements can be varied as desired or as necessary.", "Further, the type of heater used is entirely optional, and may include without restriction, any combination of resistive, inductive; inducto-resistive, and may further include thin or thick film heaters.", "As described hereinbefore, sprue bushing 52 fits inside the heating elements and is held within and between the front housing 54 and cooling insert 56 by the retaining ring 58 that is secured to the cooling insert 56 by bolts 130.Locating ring 84 abuts against shoulder 108 of cooling insert 56 and is bolted to mold backing plate 64 (see FIG.", "2A) by bolts 62.The second mold half 26 may include an insulative plate 110 (of FIG.", "2A) between backing plate 64 and locating ring 84 to provide thermal isolation between stationary platen 16 and second mold half 26.The front housing 54 is shown in more detail in FIGS.", "4A, 4B, 4C, 4D and 4E.", "As shown therein, the front housing includes an outer surface that is configured to be received in the second mold half 26.A portion of the front housing 54 located adjacent a second end provides a mold connection interface 93 for ensuring a sealed connection with the second mold half 26.Bore 90 extends through the front housing from the first end to the second end.", "The bore 90 provides a pocket surrounding a substantial length of the sprue bushing 52 between the first and second isolating coupler connection interface 72 and 74.A clearance space in the pocket between the sprue bushing 52 and the isolating coupler 53 provides an insulative airspace for the sprue bushing from the relatively cold second mold half 26 and space for the routing of heater and thermocouple leads 95.The bore 90 further includes a cylindrical surface in proximity to the second end of the front housing 54 that provides the second sprue bushing connection interface 78 for receiving the complementary second isolating coupler connection interface of the sprue bushing 52.An outward taper immediately adjacent the second end extends the melt duct 89, the outward taper having a function of allowing formation of a plug of molding material, as will be explained later.", "A channel 114 in the interior wall of the front housing 54 provides a space for the wiring clamps used in conjunction with the heaters 96b, 96c and 96d.", "Apertures 118 in conjunction with an alignment dowel provide means for properly orientating housing 54 with respect to cooling insert 56.Shoulder 112 is provided to mate with a corresponding ridge 120 (see FIG.", "9) on cooling insert 56 to provide close coupling between the housing 54 and cooling insert 56.Bolt holes 116 receive bolts 68 for coupling housing 54 to cooling insert 56.FIGS.", "5A and 5B illustrate another embodiment of the front housing 154.In this embodiment, housing 154 is a substantially uniform annular cylinder 184 through a major portion of its length with the end adjacent the mold 26 of reduced diameter so as to fit within second mold half 26.Cut-outs 182 are provided on each side of the annular cylinder 184 to provide access to bolt holes 186 to enable housing 154 to be attached to the cooling insert 56.FIG.", "6 shows the sprue bushing 52 of FIG.", "3 in more detail.", "Located at a first end of the sprue bushing 52 is a recessed cylindrical bore 87, an inner surface of which provides a nozzle connection interface 94.Also located at the first end of sprue bushing 52 is cylindrical flange 86, the first isolating coupler connecting interface 72 provided on an outer diameter of the flange 86.A shoulder 138 on an inside face of the cylindrical flange 86 provides a mating surface for cooling insert 56.A heat choke 124 is formed as a groove on the inside face of flange 86.The heat choke 124 provides a degree of thermal isolation between the portion of the sprue bushing 52 in contact with the cooling insert 56 and the remainder of the sprue bushing 52, thereby reducing conduction heat transfer from the melt duct 89 to the cooling insert 56.Melt duct 89 extends through the sprue bushing 52 from the first end to a second end, and may include a inwardly contracting tapered portion adjacent the bore 87 that gradually reduces the diameter of the melt duct 89 to match the nozzle melt duct to the sprue bushing melt duct.", "The melt duct may also include a stepped transition 170 between a main melt portion and an outwardly expanding tapered portion immediately adjacent the second end of the sprue bushing 52.The stepped transition 170 functions, under applied injection pressure, to shear a plug of consistent length from one injection cycle to the next, while the outwardly expanding tapered melt duct portion 89d further assists in the formation and ready discharge of the sprue plug at the beginning of each injection cycle with the application of injection pressure.", "The ability to eject a plug of a consistent length permits the appropriate sizing of a sprue catcher (not shown) in the second mold half 26 and the configuration of the molding apparatus runner system to optimize the melt flow in support of a stable molding process.", "Located at the second end of the sprue bushing 52 is an extended circular spigot ring portion 88 with an outer surface that provides the second isolating coupler connection interface 74.Thermocouple mount points, such as 122a, 122b and 122c, are located along the length of the sprue bushing 52 and provide temperature feedback in specific control zones as will be more fully explained hereinafter.", "FIG.", "7 is an enlarged perspective view of the cooling insert 56.A thermocouple mount point 128 is located at the base of slot 106 as a channel within the sidewall of the cooling insert accessible through slot 106 at the base of the cooling insert.", "Placement of the thermocouple mount point 128 is best shown in FIG.", "10.A longitudinally oriented slot 104 through the sidewall of the cooling insert permits electrical access to heater 96a (not shown).", "Longitudinally oriented channel 114b extends through an inner bore in cooling insert 56 and provides a space for screw clamps (not shown) that retain heater 96a around sprue bushing 52 in a manner well understood in the art.", "FIG.", "8 shows the end view of cooling insert 56 that faces locating ring 84 and retaining ring 58.Bolts 130 (see FIG.", "3) extend through retaining ring 58 into threaded holes 136 in cooling insert 56 to retain sprue bushing 52.With reference to FIGS.", "2B and 6, the shoulder 138 on an inner face of flange 86 of sprue bushing 52 (see FIG.", "6) engages surface 140 on an inner face of recessed bore 91 formed in the cooling insert 56, and is held by retaining ring 58.The surface of the inside diameter of cylindrical bore 91 provides the first sprue bushing connection interface 76 for receiving the first isolating coupler interface of the sprue bushing 52.FIG.", "9 shows the end view of cooling insert 56 that faces front housing 54.Bolts 68 extend through slots 116 in front housing 54 (see FIG.", "3) and into threaded slots 134 in cooling insert 56 thereby to retain front housing 54 in attachment with cooling insert 56.The shoulder 112 (see FIG.", "4A) of front housing 54 receives ring portion 120 of cooling insert 56 to ensure close coupling of the cooling insert 56 and housing 54.The remaining slots 172 are formed during creation of a cooling channel and contain plugs to close off the cooling channel as will become apparent from the subsequent description.", "FIG.", "10 is a cross-sectional view of the cooling insert 56 taken along the sectional lines 10-10 indicated in FIG.", "9.The cooling channel circulates cooling fluid, preferably oil, through the cooling insert 56.The thermocouple mount point 128 is located adjacent the interface area between the cooling insert 56 and sprue bushing 52 (see FIG.", "2B) as the temperature at this point is critical to the proper operation of the injection molding process as will be described later.", "An aperture 174 receives a dowel 176 (see FIG.", "3) that engages with a corresponding aperture (not shown) in sprue bushing 52 to assure alignment of the sprue bushing 52 with the cooling insert 56.FIGS.", "11, 12 and 13 show a particularly suitable arrangement of cooling channels for the cooling insert 56.The cooling insert 56 has cooling channels located on two separate planes that are connected by vertical channel connectors.", "Channels 144, 146 and 148 are shown in the sectional view in FIG.", "13 and channels 150 and 152 are shown in the sectional view in FIG.", "12.Vertical channel connectors 154, 156, 158, 160, 162 and 164 interconnect the channels 144, 146 and 148 to channels 150 and 152 and cooling tubes 66.The cooling tubes and channels are interconnected in the following manner.", "One cooling tube 66 is connected to vertical channel connector 164.Vertical channel connector 164 is connected to channel 144.Coolant flows through channel 144 to vertical channel connector 162.Vertical channel connector 162 carries coolant to channel 150.The coolant flows through channel 150 to vertical channel connector 160.Vertical channel connector 160 carries the coolant to channel 146.The coolant flows through channel 146 to vertical channel connector 158.Vertical channel connector 158 carries the coolant to channel 152.The coolant flows through channel 152 to vertical channel connector 156.Vertical channel connector 156 carries the coolant to channel 148.The coolant flows through channel 148 to vertical channel connector 154 from where it is transported through the other cooling tube 66.In this manner, the cooling fluid flows longitudinally through and around the circumference of the cooling insert 56 to provide very efficient and effective cooling of the cooling insert 56 and attached front housing 54.The thermal regulator of the isolating coupler could alternatively include a tubular coil for circulating a fluid where the tubular coil, is disposed about the nozzle connection interface.", "Another alternative might provide a wrap disposed about the nozzle connection interface for circulating a fluid.", "The thermal regulator could include a hollow jacket disposed about the nozzle connection interface for circulating a fluid or a plurality of fins for convection heat transfer could be provided.", "The present invention is not limited to a specific means for regulating the temperature in the thermal regulator.", "For the better understanding of the invention, the preferred operation of the injection molding unit and, in particular, the sprue apparatus 51 will be described with particular reference to FIG.", "1 & 2A operating a thixotropic molding process for a magnesium alloy.", "As described hereinbefore, molding material is fed through feed throat 132 where it is received by screw 40.Screw 40 shears the molding material in the barrel 42 while it is also heated to achieve, in the case of thixotropic molding of light-metal alloys, a thixotropic state.", "The thixotropic material is fed past the check valve 46 into the accumulation zone 82 as previously described.", "The thixotropic melt material is maintained in a thixotropic state by heaters 44 in the nozzle 48 and barrel 42 and the heaters 96a, 96b, 96c, and 96d on the sprue bushing 52.When sufficient material has been conveyed into the accumulation zone 82, carriage force is applied and the screw is driven forward by the screw drive unit 36 to inject a shot of material into the mold 24, 26 through a sprue apparatus 51.Upon injection a small plug at the end of the sprue bushing 52 is driven into a mold plug catcher (not shown) in a manner that is well understood by those skilled in the art of injection molding.", "The plug was formed during a previous injection cycle by permitting the melt located in the melt duct near the ring portion 88 to solidify and block the duct to prevent further egress of the melt material.", "The carriage forte counteracts the separating force that result from injection and thereby maintains the injection nozzle 48 in a sealed connection with the sprue apparatus 51.In the embodiments, the carriage force acts through the machine nozzle 48 to a portion of sprue bushing 52 constrained between a shoulder of nozzle 48 and cooling insert 56, and then through cooling insert 56 to front housing 54 and into the second mold half 26.This arrangement isolates a portion of the sprue bushing 52, adjacent the portion confined between nozzle 48 and cooling insert 56, from the carriage force.", "Furthermore, since sprue bushing 52 can move laterally within the bore 90, any pressure transmitted into the bushing 52 would be relieved.", "The force isolated portion of the sprue bushing 52 can therefore have a relatively slender construction with associated improvements in thermal response characteristics without regard to the effects of applied carriage force, and is of particular significance at the high operating temperatures typical for the thixotropic molding of magnesium.", "The sprue apparatus 51 of FIG.", "2B includes the delimitation of the plurality of thermal zones for the exemplary process.", "The present invention is not limited to specific number or configuration of the plurality of thermal zones that may be required in support of alternate processes.", "The plurality of thermal zones includes a nozzle sealing zone, Z1, located at the first end of the sprue apparatus 51, that encompasses the nozzle connection interface 94 and a melt duct portion 89a.", "The temperature in the nozzle sealing zone Z1 is thermally regulated to maintain a desired sealing temperature at the nozzle connection interface 94, and hence the junction between the sprue bushing 52 and the machine injection nozzle 48, while simultaneously maintaining the molding material within the melt duct portion 89a at any desired processing temperature in support of the molding process.", "In particular, the thermal regulation of the spigot junction that forms the junction between the sprue bushing 52 and the machine nozzle 48 requires that the temperature at the connection interface 94 be maintained low enough at to solidify any molding material that may seep into the junction, thereby to form a seal.", "Therefore, to accomplish the thermal regulation requirements in the nozzle sealing zone, Z1, an equilibrium conduction heat flow is established between an adjacent sprue bushing conditioning zone, Z2, itself thermally regulated by thermal regulator heaters 96b and 96c, and the thermal regulator of cooling insert 56.The establishment of the equilibrium heat flow is assisted by providing the sprue bushing 52 with a heat choke (124 of FIG.", "6) between the first isolating coupler connection interface and the nozzle connection interface such that heat flow originating from the adjacent conditioning zone, Z2, is preferentially directed into the melt duct portion 89a and heat flow from the nozzle junction is preferentially directed to the cooling insert 56.The cooling insert 56 includes a flow circuit for circulating a flow of coolant that is temperature conditioned by a thermolator with temperature feedback from thermocouples embedded in mounting point 128 in cooling insert 56, and in mounting point 122c in the sprue bushing flange 86.As previously mentioned, the plurality of thermal zones includes conditioning zone, Z2, located along a central portion of the sprue apparatus 51 adjacent the nozzle sealing zone, Z1, wherein the molding material within the encompassed melt duct portion 89b is maintained at a desired processing temperature in support of the molding process.", "Thermal regulation in the zone is provided by thermal regulator/heaters 96a, 96b, and 96c based on feedback from a thermocouple installed in mount point 122b (see FIG.", "6).", "The plurality of thermal zones includes another conditioning zone, Z3, located along the reduced diameter portion of the sprue apparatus 51 that is adjacent conditioning zone, Z2, wherein the molding material within the encompassed melt duct portion 89c is again maintained at a desired processing temperature in support of the molding process.", "As previously mentioned, the shorter heat conduction path, provided by the reduced diameter portion, provides for a relatively fast thermal response for the temperature adjustment of the molding material within melt duct portion 89d by thermal regulator/heater 96d based on feedback from a thermocouple installed in mount point 122a (see FIG.", "6).", "The fast thermal response compensates for the frequent temperature variations in an adjacent cycling zone, Z4.The plurality of thermal zones also includes a cycling zone, Z4, located at the second end of the sprue apparatus 51, for the controlled formation of a localized plug of solidified molding material in an encompassed melt duct portion 89d.", "The plug may be used to prevent the egress of molding material during various intervals of a molding process cycle and may obviate the need for a mechanical melt shut-off.", "The thermal regulation of the cycling zone is provided as a conduction heat flow between the adjacent sprue bushing conditioning zone, Z3, itself thermally regulated by thermal regulator heaters 96d, and the thermal regulator/thermal conduit of the front housing 54.The thermal conduit of the front housing is not actively controlled, but again provides a heat conduction path between the cooled second mold half 26 and the sprue bushing.", "Alternatively, the cycling zone Z4 may be thermally regulated to at least partially re-melt the molding material, and may be assisted by yet another thermal regulator.", "The plurality of thermal zones may also include a second sealing zone (not shown) that incorporates the junction between the second isolating coupler connection interface 74 and the second sprue bushing connection interface 78 in proximity to the second end of the front housing 54.In the present embodiment the second sealing zone is embedded in cycling zone Z4.The junction is a spigot junction that is thermally regulated, in use, to cause solidification of any molding material that may seep into the junction, thereby forming a seal.", "Thus, what has been described is a novel sprue apparatus that is useful in a molding apparatus that requires thermal management and control of a plurality of distinct thermal zones.", "The invention has been found particularly useful when injecting metal alloys, such as magnesium based alloys, when in the thixotropic state.", "All U.S. and foreign patent documents, and articles, discussed above are hereby incorporated by reference into the Detailed Description of the Preferred Embodiment.", "The individual components shown in outline or designated by blocks, in the attached Drawings are all well-known in the injection molding arts, and their specific construction and operation are not critical to the operation or best mode for carrying out the invention." ] ]	Patent_10469590
[ [ "Polymeric bioresorbable composites containing an amorphous calcium phosphate polymer ceramic for bone repair and replacement", "A bioresorbable composite of a non-crystalline calcium phosphate ceramic synthesized within an encapsulating microspheres of bioresorbable polymeric material for use in bone repair and replacement is provided.", "Also provides are methods for producing these composites as well as porous, 3-dimensional scaffold produced by sintering together microspheres of this bioresorbable composite." ], [ "1.A bioresorbable composite comprising a non-crystalline calcium phosphate ceramic synthesized within an encapsulating microsphere of bioresorbable polymeric material.", "2.A method for producing the bioresorbable composite of claim 1 comprising synthesizing the calcium phosphate ceramic within the encapsulating microspheres of the bioresorbable polymeric material.", "3.A porous, 3-dimensional scaffold produced by sintering together microspheres of the bioresorbable composite of claim 1." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>With demographics shifting towards an older population and with more people living more active lifestyles, the number of orthopaedic injuries and disorders continues to rise.", "In the United States alone, there were more than 6 million fractures each year from 1992 to 1994 (Praemer et al.", "American Academy of Orthopaedic Surgeons 1999 182).", "In 1995, there were 216,000 total knee replacements, 134,000 total hip replacements, and close to 100,000 bone grafting procedures performed.", "Traditionally, autografts and allografts have been used by orthopaedic surgeons to repair fractures and other bone defects.", "However, limitations including donor-site morbidity, risk of disease transfer, potential immunogenicity, and insufficient supply has led investigators to search for alternative bone repair materials.", "Since the main mineral component of bone is a complex calcium phosphate system called apatite, hydroxyapatite and other materials within the calcium phosphate family have been and continue to be extensively investigated (DeMaeyer et al.", "J. Biomed.", "Mater.", "Res.", "2000 52:95-106; Keller, L. and Dollase, W. A. J. Biomed.", "Mater.", "Res.", "2000 49:244-249; Zeng et al.", "Biomaterials 1999 20:443-451; Ma et al.", "J. Biomed.", "Mater.", "Res.", "2001 54:284-293; and Duracan, C. and Brown, P. W. J. Biomed.", "Mater.", "Res.", "2000 51:726-734).", "Further, calcium phosphate ceramics have been reported to be osteoconductive and to directly bond to bone (Jarcho, M. Clin.", "Orthop.", "Rel.", "Res.", "1981 157:259-278; Kitsugi et al.", "Clin.", "Orthop.", "Rel.", "Res.", "1988 234:280-290).", "In addition, calcium phosphate ceramics are believed to serve as precursors to bone apatite formation in vivo.", "Accordingly, the good bone compatibility of calcium phosphate ceramics is indicative of their suitability for repair or replacement of damaged or diseased bone.", "However, the brittleness of these materials limits their widespread use in orthopaedics, particularly in load-bearing applications.", "Accordingly, various attempts has been made to overcome this limitation.", "One example has been to prepare composities of these ceramics with bioresorbable polymeric materials such as collagen and polymers of lactic acid and glycolic acid (TenHuisen et al.", "J. Biomed.", "Mater.", "Res.", "1995 29:803-810; Yasunaga et al.", "J. Biomed.", "Mater.", "Res.", "1999 47:412-419; Zhang et al.", "J. Biomed.", "Mater.", "Res.", "1999 45:285-293; Devin et al.", "J. Biomater.", "Sci.", "Polyner Edn.", "1996 7:661-669; Boeree et al.", "Biomaterials 1993 14:793-796).", "In general, these composites are made porous in order to create a 3-dimensional scaffold that allows the ingrowth of new bone and the eventual replacement of the scaffold with new skeletal tissue (Zhang et al.", "J. Biomed.", "Mater.", "Res.", "1999 45:285-293; Devin et al.", "J. Biomater.", "Sci.", "Polymer.", "End.", "1996 7:661-669).", "In these composites, the ceramic typically comprises a calcium phosphate compound with moderate to high crystallinity (TenHuisen et al.", "J. Biomed.", "Mater.", "Res.", "1995 29:803-810; Yasunaga et al.", "J. Biomed.", "Mater.", "Res.", "1999 47:412-419; Zhang et al.", "J. Biomed.", "Mater.", "Res.", "1999 45:285-293; Devin et al.", "J. Biomater.", "Sci.", "Polymer.", "Edn.", "1999 7:661-669; Boeree et al.", "Biomaterials 1993 14:793-796).", "In contrast, bone apatite is poorly crystalline and non-stoichiometric due to the presence of other ions such as magnesium and carbonate ions (Posner, A.S. and Betts, F. Acc.", "Chem.", "Res.", "1975 8:274-281; Bigi et al.", "Calcif.", "Tissue Int.", "1992 50:439-444).", "Further, crystalline forms of hydroxyapatite have been shown to resorb at a slower rate than that of new bone formation.", "In fact, the rate of new bone formation coincides more closely with the resorption rate of poorly crystalline or amorphous calcium phosphate ceramics (Frayssinet et al.", "Biomaterials 1993 14:423-429; Klein et al.", "J. Biomed.", "Mater.", "Res.", "1983 17:769-784; Knaack et al.", "J. Biomed.", "Mater.", "Res.", "(Appl.", "Biomater.)", "1998 43:399-409).", "In the present invention, crystalline hydroxyapatite is replaced with a poorly crystalline or amorphous calcium phosphate ceramic believed to resorb concurrently with new bone growth.", "Also, the degradation of the amorphous calcium phosphate forms alkali products that serve to buffer the acidic degradation product of either lactic or glycolic acid in a composite of the two materials." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>An object of the present invention is to provide a bioresorbable composite for use in bone repair and replacement which comprises a non-crystalline or amorphous calcium phosphate ceramic synthesized within encapsulating microspheres of a bioresorbable polymeric material.", "Another object of the present invention is to provide a method for producing a bioresorbable composite which comprises a bioresorbable polymeric material and a non-crystalline or amorphous calcium phosphate ceramic for use in bone repair and replacement wherein the method comprises synthesizing the calcium phosphate ceramic within encapsulating microspheres of the bioresorbable polymeric material.", "Yet another object of the present invention is to provide porous, 3-dimensional scaffolds with uniform composition throughout the scaffold, wherein said scaffolds are produced by sintering together microspheres of a non-crystalline or amorphous calcium phosphate ceramic synthesized within encapsulating microspheres of a bioresorbable polymeric material." ], [ "INTRODUCTION This work was supported in part by the National Science Foundation (Grant No.", "NSG-BES9817872) and the U.S. Government may have certain rights in this invention.", "BACKGROUND OF THE INVENTION With demographics shifting towards an older population and with more people living more active lifestyles, the number of orthopaedic injuries and disorders continues to rise.", "In the United States alone, there were more than 6 million fractures each year from 1992 to 1994 (Praemer et al.", "American Academy of Orthopaedic Surgeons 1999 182).", "In 1995, there were 216,000 total knee replacements, 134,000 total hip replacements, and close to 100,000 bone grafting procedures performed.", "Traditionally, autografts and allografts have been used by orthopaedic surgeons to repair fractures and other bone defects.", "However, limitations including donor-site morbidity, risk of disease transfer, potential immunogenicity, and insufficient supply has led investigators to search for alternative bone repair materials.", "Since the main mineral component of bone is a complex calcium phosphate system called apatite, hydroxyapatite and other materials within the calcium phosphate family have been and continue to be extensively investigated (DeMaeyer et al.", "J. Biomed.", "Mater.", "Res.", "2000 52:95-106; Keller, L. and Dollase, W. A. J. Biomed.", "Mater.", "Res.", "2000 49:244-249; Zeng et al.", "Biomaterials 1999 20:443-451; Ma et al.", "J. Biomed.", "Mater.", "Res.", "2001 54:284-293; and Duracan, C. and Brown, P. W. J. Biomed.", "Mater.", "Res.", "2000 51:726-734).", "Further, calcium phosphate ceramics have been reported to be osteoconductive and to directly bond to bone (Jarcho, M. Clin.", "Orthop.", "Rel.", "Res.", "1981 157:259-278; Kitsugi et al.", "Clin.", "Orthop.", "Rel.", "Res.", "1988 234:280-290).", "In addition, calcium phosphate ceramics are believed to serve as precursors to bone apatite formation in vivo.", "Accordingly, the good bone compatibility of calcium phosphate ceramics is indicative of their suitability for repair or replacement of damaged or diseased bone.", "However, the brittleness of these materials limits their widespread use in orthopaedics, particularly in load-bearing applications.", "Accordingly, various attempts has been made to overcome this limitation.", "One example has been to prepare composities of these ceramics with bioresorbable polymeric materials such as collagen and polymers of lactic acid and glycolic acid (TenHuisen et al.", "J. Biomed.", "Mater.", "Res.", "1995 29:803-810; Yasunaga et al.", "J. Biomed.", "Mater.", "Res.", "1999 47:412-419; Zhang et al.", "J. Biomed.", "Mater.", "Res.", "1999 45:285-293; Devin et al.", "J. Biomater.", "Sci.", "Polyner Edn.", "1996 7:661-669; Boeree et al.", "Biomaterials 1993 14:793-796).", "In general, these composites are made porous in order to create a 3-dimensional scaffold that allows the ingrowth of new bone and the eventual replacement of the scaffold with new skeletal tissue (Zhang et al.", "J. Biomed.", "Mater.", "Res.", "1999 45:285-293; Devin et al.", "J. Biomater.", "Sci.", "Polymer.", "End.", "1996 7:661-669).", "In these composites, the ceramic typically comprises a calcium phosphate compound with moderate to high crystallinity (TenHuisen et al.", "J. Biomed.", "Mater.", "Res.", "1995 29:803-810; Yasunaga et al.", "J. Biomed.", "Mater.", "Res.", "1999 47:412-419; Zhang et al.", "J. Biomed.", "Mater.", "Res.", "1999 45:285-293; Devin et al.", "J. Biomater.", "Sci.", "Polymer.", "Edn.", "1999 7:661-669; Boeree et al.", "Biomaterials 1993 14:793-796).", "In contrast, bone apatite is poorly crystalline and non-stoichiometric due to the presence of other ions such as magnesium and carbonate ions (Posner, A.S. and Betts, F. Acc.", "Chem.", "Res.", "1975 8:274-281; Bigi et al.", "Calcif.", "Tissue Int.", "1992 50:439-444).", "Further, crystalline forms of hydroxyapatite have been shown to resorb at a slower rate than that of new bone formation.", "In fact, the rate of new bone formation coincides more closely with the resorption rate of poorly crystalline or amorphous calcium phosphate ceramics (Frayssinet et al.", "Biomaterials 1993 14:423-429; Klein et al.", "J. Biomed.", "Mater.", "Res.", "1983 17:769-784; Knaack et al.", "J. Biomed.", "Mater.", "Res.", "(Appl.", "Biomater.)", "1998 43:399-409).", "In the present invention, crystalline hydroxyapatite is replaced with a poorly crystalline or amorphous calcium phosphate ceramic believed to resorb concurrently with new bone growth.", "Also, the degradation of the amorphous calcium phosphate forms alkali products that serve to buffer the acidic degradation product of either lactic or glycolic acid in a composite of the two materials.", "SUMMARY OF THE INVENTION An object of the present invention is to provide a bioresorbable composite for use in bone repair and replacement which comprises a non-crystalline or amorphous calcium phosphate ceramic synthesized within encapsulating microspheres of a bioresorbable polymeric material.", "Another object of the present invention is to provide a method for producing a bioresorbable composite which comprises a bioresorbable polymeric material and a non-crystalline or amorphous calcium phosphate ceramic for use in bone repair and replacement wherein the method comprises synthesizing the calcium phosphate ceramic within encapsulating microspheres of the bioresorbable polymeric material.", "Yet another object of the present invention is to provide porous, 3-dimensional scaffolds with uniform composition throughout the scaffold, wherein said scaffolds are produced by sintering together microspheres of a non-crystalline or amorphous calcium phosphate ceramic synthesized within encapsulating microspheres of a bioresorbable polymeric material.", "DETAILED DESCRIPTION OF THE INVENTION In recent years, bioresorbable, porous, 3-dimensional scaffolds have been studied as matrices for the regeneration of tissues such as skin, cartilage and bone.", "The rationale for this design is that the interconnected pores allow the ingrowth of new tissue, which eventually replaces the degrading scaffold.", "In orthopaedic applications, the scaffolds are typically made of resorbable polymers, ceramics or composites of each.", "The present invention relates to bioresorbable composites which can be sintered into 3-dimensional scaffolds for bone repair and replacement.", "These bioresorbable composites comprise a non-cyrstalline or amorphous calcium phosphate ceramic synthesized within an encapsulating microsphere of a bioresorbable polymeric material.", "Calcium phosphate ceramics useful in tissuse scaffolds are well known in the art.", "Examples of calcium phosphate ceramics which can be used in the present invention include, but are not limited to, hydroxyapatite, monocalcium phosphate, tricalcium phosphate, and tetracalcium phosphate.", "Various bioresorbable, biocompatible polymers have also been created for use in medical applications.", "One of the most common polymers used as a biomaterial has been the polyester copolymer poly(lactic acid-glycolic acid) referred to herein as PLAGA.", "PLAGA is highly biocompatible, degrades into harmless nomoner units and has a wide range of mechanical properties making this copolymer and its homopolymer derivatives, PLA and PGA, useful in skeletal repair and regeneration (Coombes, A.D. and Heckman, J.D.", "Biomaterials 1992 13:217-224; Mikos et al.", "Polymer 1994 35:1068-1077; Robinson et al.", "Otolaryngol.", "Head and Neck Surg.", "1995 112:707-713; Thomson et al.", "J. Biomater.", "Sci.", "Polymer Edn.", "1995 7:23- 38; Devin et al.", "J. Biomater.", "Sci.", "Polylmer Edn.", "1996 7:661-669).", "In a preferred embodiment of the present invention, the bioresorbable polymeric material comprises PLAGA (50:50).", "However, as will be understood by those of skill in the art upon this disclosure, other polymeric materials including, but not limited to, poly(anhydrides), poly(phosphazenes), poly(orthoesters), poly(caprolactones), polyhydroxybutyrates, polyanhydroideco-imides, polypropylene fumarates, polydiaxonanes, and poly(urethanes) can also be used to form the encapsulating microspheres.", "In the composites of the present invention, the calcium phosphate ceramic is synthesized within the encapsulating microspheres of the bioresorbable polymeric material.", "Thus, in the present invention, the microspheres serve as microreactors for the synthesis of the calcium phosphate ceramic.", "By carrying out the synthesis within the confined space of the microsphere's interior, crystal growth is impeded due to the constraints imposed by the small internal space of the microsphere.", "As a result, the formation of crystalline calcium phosphates is prevented or at least minimized.", "Accordingly, the calcium phosphate ceramic of the present invention more closely mimics the low crystallinity of bone apatite and thus provides a better bone substitute than a highly crystalline calcium phosphate material.", "In addition, amorphous or low crystalline calcium phosphates have a faster resorption rate than their crystalline counter parts, which allows them to be more readily replaced by new bone.", "The degradation of an amorphous calcium phosphate also forms alkali products that serve to buffer the acidic degradation products of the bioresorbable polymeric material of the microsphere.", "Inclusion of the bioresorbable polymeric material, however, reduces the brittleness of the ceramic component and serves to bind the microspheres together during sintering of the microspheres of this composite into tissue scaffolds.", "Additionally, because each individual microsphere is in itself a composite, the resulting scaffold has uniform composition throughout.", "The reaction between calcium nitrate tetrahydrate and ammonium hydrogen phosphate in basic aqueous solution has been shown to produce an amorphous calcium phosphate, which becomes crystalline over time (Eanes et al.", "Nature 1965 208:365-367).", "In the present invention, a similar reaction was carried out within microspheres made from the bioresorbable polymer, PLAGA.", "Microspheres were prepared using a water-in-oil emulsion containing PLAGA, calcium nitrate tetrahydrate, and ammonium hydrogen phosphate.", "The wate-in-oil emulsion was prepared by adding a basic aqueous solution of the nitrate and phosphate components to a solution of PLAGA in methylene chloride followed by rapid mixing.", "The differnet components of the emulsion were cooled prior to mixing in order to prevent the premature reaction of calcium nitrate with the phosphate reagent which would result in precipitation of a calcium phosphate compound at this stage of the process.", "Once the emulsion was made, it was added to a solution of poly (vinyl alcohol) containing an excess of calcium nitrate tetrahydrate which was being stirred with a mechanical stirrer.", "Excess calcium nitrate was added to minimize the diffusion of calcium ions from inside the mocropheres to the surrounding solution, thereby increasing the chances of obtaining a calcium phosphate with a high Ca/P molar ratio similar to that of bone apatite, which has a Ca/P ratio of approximately 1.6 (Bigi et al.", "Calcif.", "Tissue Int.", "1992 50:439-444).", "The resulting microparticles were collected after a 30-hour reaction period.", "Thses collected microparticles consisted of the composite microspheres as well as unencapsulated calcium phosphate particles.", "To separate the micorphseres from the unencapsulated ceramic particles, the micorparticle mixture was added to a hexane-water mixture, which was then shaken and allowed to stand.", "This separation method is based on the preference of the unencapsulated calcium phosphate particles for the aqueous layer and the preference of the microspheres for the organic layer due to the presence of the more hydrophobic PLAGA in the microspheres.", "The hexane layer was then collected and the microspheres isolated by filtration.", "The ceramic content of the microspheres as determined by gravimetric analysis, was approximately 28%.", "This can be increased, however, if different mechanical properties are desired.", "For example, ceramic content can be increased to increase compressive modulus and other mechanical properties.", "The synthesized calcium phosphate was characterized via elemental analysis to determine its Ca/P molar ratio.", "Tetracalcium phosphate was one of the products expected, the other being hydroxyapatite.", "At pH 10, which was the pH of the reaction mixture, both of these compounds are the least soluble of the various calcium phosphates and are therefore more likely to form and precipitate at this pH (Chow et al.", "Mat.", "Res.", "Soc.", "Symp.", "Proc.", "1991 179:3-24).", "Table 1 shows the % calcium (Ca), % phosphate (P), and Ca/P ratio of the synthesized ceramic together with the calculated values for commercially available hydroxyapatite and tetracalcium phosphate.", "TABLE 1 Ca and P analysis of synthesized calcium phosphate and calculated values for known calcium phosphate ceramics Elemental Analysis % Ca % P Ca/P molar ratio Synthesized calcium 36.30 13.84 2.02 Tetracalcium 43.72 16.94 2.00 Hydroxyapatite 39.84 18.53 1.67 As shown, the Ca/P ratio closely matches that of tetracalcium phosphate.", "The synthesized material, as well as the commercially available hydroxyapatite, were analyzed via infrared (IR).", "The IR spectra for both compounds were very similar except that the spectra of the synthesized calcium phosphate had peaks around 1400-1700 cm−1 indicating the presence of carbonate.", "These peaks are similar to the ones observed for a carbonated apatite that was obtained after implanting brushite for 2 weeks in the femoral metaphysis of a rabbit (Constantz et al.", "J. Biomed.", "Mater.", "Res.", "(Appl.", "Biometer.)", "1998 43:451-461).", "Since bone apatite has also been reported to contain substantial amounts of carbonate (Posner, A.S. and Betts, F. Acc.", "Chem.", "Res.", "1975: 8:273-281), the synthesized calcim phosphate of the present invention more closely resembles the composition of bone apatite than does tetracalcium phosphate or hydroxyapatite.", "To detemine the crystallinity of the ceramic that was synthesized, X-ray powder diffraction (XRD) was performed.", "From the XRD spectra it could be seen that the hydroxyapatite was highly crystalline while the synthesized calcium phosphate material appeared non-crystalline.", "Further, the non-crystalline pattern was similar to those seen in other amorphous calcium phosphate XRD spectra (Eanes et al.", "Nature 1965 208:365-367).", "This non-crystalline pattern evidences the fact that by restricting the synthesis of the calcium phosphate to the small space within the microsphere's interior, crystalline formation was prevented.", "As discussed, bone apatite is a poorly crystalline calcium phosphate ceramic.", "Further, amorphous or poorly crystalline calcium phosphates are more resorbable than their crystalline counterparts.", "Thus, these results are indicative of the synthesized calcium phosphate ceramic of the present invention being a suitable material for bone repair when encapsulated within a bioresorbable polymeric matherial such as PLAGA microsphere to offset its brittleness.", "The present invention also relates to 3-dimensional scaffolds produced by sintering together micropheres of a bioresorbable polymeric material and a non-crystalline calcium phosphate ceramic synthesized within the microspheres.", "Various conditions for sintering the microspheres can be used.", "Further, such conditions can be routinely determined by those of skill in the art upon reading this disclosure and based upon the melting temperature of the bioresorbable polymeric material.", "For example, composite microspheres comprising PLAGA were sintered at 150° C. for 1 hour in order to fuse the microspheres together.", "Fusion of the microspheres was possible because this sintering temperature was above the melting temperature of PLAGA.", "The resulting 3-dimensional scaffold was porous and did not crumble upon handling unlike some sintered, porous, calcium phosphate ceramics.", "Image analysis of a cross-section of this scaffold by scanning electron microscopy showed fused microspheres and deep pores.", "In addition, some of the microspheres split open probably due to the slight pressure provided by the piston of the cylindrical mold assembly.", "As a result, the porosity of the scaffold was increased further, which also increased the surface area for cell attachment.", "Furthermore, the sintering process transformed the surface of the microspheres into a rougher one, characterized by the preponderance of micron-sized pores.", "Decades ago, the minimum pore size for effective bone ingrowth into a porous scaffold was established to be approximately 100 μ (Klawitter, J. J. and Hulbert, S. F. J. Biomed.", "Mater.", "Res.", "Symp.", "1971 2:161; Gauthier et al.", "Biomaterial 1998 19(1-3):133; and Daculsi, G. and Passuit, N. Biomaterials 1990 11:86).", "Mercury intrusion porosimetry on the scaffold indicated that a majority of the pores have diameters of at least 100 μ, thus meeting the minimum requirement for bone ingrowth.", "Porosimetry results also showed that the scaffold had a porosity of approximately 75%.", "Thus, as demonstrated herein, by restricting the reaction of a calcium salt with a phosphate compound to the small confine of a polymeric microsphere's interior, a non-crystalline, carbonated calcium phosphate ceramic useful in combination with a bioresorbable polymeric material as a composite for bone repair and replacement was obtained.", "Further, sintering the composite microspheres together produced a highly porous, 3-dimensional scaffold with a rough surface.", "In all, the combination of high-porosity and a calcium phosphate component that is non-crystalline and carbonated renders the 3-dimensional commposite scaffolds produced from the composites particularly suitable for bone repair and/or replacement applications.", "The following nonlimiting examples are provided to further illustrate the present invention.", "EXAMPLES EXAMPLE 1 Materials Poly(lactide-co-glycolide) or PLAGA (50:50) with a molecular weight of 50,000 was obtained from American Cyanamid.", "Calcium nitrate tetrahydrate, ammonium hydrogen phosphate, and concentrated ammonium hydroxide were obtained from Aldrich and used as received.", "Commercial hydroxyapatite used for comparison was obtained from Stryker Howmedica (Allendale, NJ).", "Methylene chloride was obtained from Fisher Scientific (Camden, NJ) and used as received.", "EXAMPLE 2 Preparation of Composite Microspheres PLAGA (1.50 grams, 0.0115 mol) was dissolved in 9 mL of methylene chloride.", "Calcium nitrate tetrahydrate (3.54 grams, 0.0150 moles) was dissolved in 1.5 mL of water previously adjusted to pH 10 with ammonium hydroxide.", "In a separate vial, ammonium hydrogen phosphate (0.99 grams, 0.0075 mol) was dissolved in 2.25 mL of water, pH 10.The polymer solution and the calcium nitrate solution were Kept at −70° C. prior to use, while the phosphate solution was maintained at room temperature.", "The cooled nitrate solution was added quickly to the cooled polymer solution followed immediately by the addition of the phosphate solution, and vortex-mixed at high speed for 20 seconds to create an emulsion.", "The emulsion was then poured in a slow steady stream into 600 mL of a 1% polyvinyl alcohol (PVA) solution (pH 10) containing 30 grams of calcium nitrate tetrahydrate which was being stirred at a speed of 250 rpm during the addition and was raised to 500 rpm after the addition.", "The reaction was allowed to proceed for 30 hours, during which ammonium hydroxide was added at regular intervals in order to maintain a pH of 10.Methylene chloride was allowed to evaporate slowly during the reaction period.", "After 30 hours, the hollow microspheres that floated to the top of the reaction mixture were removed using a pipette.", "The microparticles that settled to the bottom of the reaction vessel were collected by suction filtration and washed several times with water, pH 10.The residue was air-dried for 24 hours.", "The air-dried residue was added to a separatory funnel containing 100 mL of a 50:50 mixture of water and hexane.", "The whole mixture was shaken and allowed to stand for a few minutes.", "Most of the encapsulated calcium phosphate particles settled into the lower aqueous layer while most of the microsphere stayed in the upper organic layer.", "The aqueous layer was removed and more water was added and the separation process was repeated.", "This step was repeated one more time and the microspheres in the organic layer were collected by vacuum filtration, air-dried and then lyophilized for 24 hours.", "EXAMPLE 3 Percent Ceramic Content A known weight of microspheres was placed in methylene chloride to dissolve away the PLAGA component.", "The mixture was filtered and the residue placed in methylene chloride to remove more PLAGA.", "The extraction step was repeated one more time.", "The residue was collected, lyophilized and weighed.", "This experiment was performed in triplicate.", "EXAMPLE 4 Characterization of Synthesized Calcium Phosphate The calcium phosphate that was synthesized within the microsphere was analyzed for calcium and phosphorus.", "Elemental analysis was obtained from Quantitative Technologies, Inc. (Whitehouse, NJ).", "IR analysis was carried out using a Nicolet Magna IR 560 (Madison, WI).", "Powder X-ray diffraction was performed using a Siemens D500 diffractometer using Ni-filtered CuK radiation with a 20 range from 2-60° C. EXAMPLE 5 Preparation and Characterization of 3-dimensional Scaffold The composite microspheres (100-250 μ in diameter) were placed in a 5 -mm stainless steel cylinder mold and pressed lightly with the corresponding sized piston.", "The whole assembly was placed in an oven at 150° C. for 1 hour in order to sinter the microspheres.", "A cross-section of the resulting 3-dimensional scaffold was viewed by scanning electron microscopy (SEM).", "Samples were gold-coated using a Denton Desk 1 sputter coater in an argon-purged chamber evacuated in 500 mTorr.", "The images were viewed using a Amray 1830 D4 scanning electron microscope (20 kV) with a tungsten gun and a diffusion pump.", "The porosity and the pore-size distribution of the scaffold was dermined using a Micromeritics Autopore III Mercury Intrusion Porosimeter (Norcross, GA) by infusing mercury into the samples with a pressure of 40 psi.", "Pore size and porosity was determined as a function of pressure and corresponding mercury intrusion volume.", "As the mercury is forced into the pores of the sample, both the pressure necessary to move the mercury and the volume of mercury moved are recorded.", "From these data, both porosity and pore size are computed." ] ]	Patent_10469617
[ [ "Purification materials and method of filtering using the same", "The invention relates to a purification material (1) comprising filtration particulate matter aggregated with a first binder and further processed with a second binder to generate a porous fluid filtration material or a non-pourous coating, a filtering device comprising a housing (11) and the purification material (1), and a method of filtering and/or purifying a fluid including water or other solutions containing chemical and microbiological contaminants, such as fluids containing heavy metals, pesticides, by products of oxidation chemicals and including cysts, bacteria and/or viruses, where the fluid is passed through ot made to contact a surface of the purification material (1)." ], [ "1.A purification material for fluids, wherein the material comprises insoluble filtration particles aggregated with a first binder and combined into the form of a porous block, porous sheet, porous coating or a non-porous coating using a second binder.", "2.The purification material of claim 1, wherein the material is in the form of a porous block.", "3.The purification material of claim 2, wherein the porous block is rigid.", "4.The purification material of claim 2, wherein the porous block is flexible.", "5.The purification material of claim 1, wherein the material is in the form of a porous sheet.", "6.The purification material of claim 5, wherein the porous sheet is rigid.", "7.The purification material of claim 5, wherein the porous sheet is flexible.", "8.The purification material of claim 1, wherein the material is in the form of a porous coating.", "9.The purification material of claim 8, wherein the porous coating is contained on a substrate which is rigid.", "10.The purification material of claim 8, wherein the porous coating is contained on a substrate which is flexible.", "11.The purification material of claim 1, wherein the material is in the form of a non-porous coating.", "12.The purification material of claim 11, wherein the non-porous coating is contained on a substrate which is rigid.", "13.The purification material of claim 11, wherein the non-porous coating is contained on a substrate which is flexible.", "14.The purification material of claim 1, wherein at least a portion of said insoluble filter material is carbon in a form selected from particles, fibers, or a combination thereof.", "15.The purification material of claim 1, wherein at least a portion of said insoluble filtration material is derived from minerals containing apatites, phosphates, silicates, hydroxides, oxides or combinations thereof.", "16.The purification material of claim 1, wherein the first binder is a polymer material.", "17.The purification material of claim 16, wherein the first binder is a polymer material containing positive charges, negative charges, hydrogen bonding sites, or a combination thereof.", "18.The purification material of claim 17, wherein the first binder is a polyelectrolyte material derived from natural polymers or modified natural polymers.", "19.The purification material of claim 18, wherein the first binder is a polyelectrolyte material known as cationic starch.", "20.The purification material of claim 17, wherein the first binder is a polyelectrolyte material selected from the group consisting of polyamines, polyamides, polyalcohols, polysaacharides, polyacrylamides, polyacrylates, humic acids, proteins, poly(DADMAC), Poly-DADM, polyamine-poly(DADMAC) blends, polyquartenary amines, inorganic-polyamine blends, and inorganic Poly(DADMAC) blends, cationic starch, cationic polymethylmethacrylates, copolymers of vinylimidazolium methochloride and vinylpyrrolidone, quarternized vinylpyrrolidone/dimethyl-aminoethyl-methacrylate copolymer, and polyethyleneimine.", "21.The purification material of claim 1, wherein the first binder is selected from the group consisting of metal hydroxides and oxides.", "22.The purification material of claim 21, wherein the first binder is selected from the group containing aluminum, calcium, magnesium, iron, polyaluminum sulfates, polyaluminum chlorides, polyorganozirconates, polyorganoaluminates, polysiloxanes, polysilanes, polysilazanes, polycarbosilanes, polyborosilanes, zirconium dimethacrulate, zirconium tetramethacrylate, zirconium 2-ethylhexanoate, aluminum butoxides, aluminum diisopropoxide ethylacetoacetate, tetramethyldisiloxanes and derivatives thereof, tristrimethylsilylphosphate, and tristrimethylsiloxyboron.", "23.The purification material of claim 21, wherein the first binder is a metal oxide or hydroxide derived from aluminum, calcium, magnesium, or iron.", "24.The purification material of claim 1, wherein the second binder is a polymer material.", "25.The purification material of claim 24, wherein the binder is a polymer melting between about 50° C. and about 500° C. 26.The purification material of claim 24, wherein the polymer is stable under sterilization conditions.", "27.The purification material of claim 24, wherein said binder is selected from the group consisting of thermoplastics, polyethylene glycols or a derivative thereof, polyvinyl alcohols, polyvinylacetate, and polylactic acids.", "28.The purification material of claim 24, wherein the polymer material is a thermoplastic is selected from the group consisting of nylon, polyethylene, polyvinylchloride, fluorocarbon resins, polystyrene, polypropylene, cellulosic resins, and acrylic resins.", "29.The purification material of claim 24, wherein the polymer material comprises a naturally occurring polymer.", "30.The purification material of claim 29, wherein the naturally occurring polymer is selected from the group consisting of natural and synthetically modified celluloses, collagens, and organic acids.", "31.The composite purification material of claim 29, wherein the naturally occurring polymer is selected from the group consisting of natural and synthetically modified celluloses, collagens, and organic acids.", "32.The composite purification material of claim 29, wherein the naturally occurring polymer is a biodegradable polymer selected from the group consisting of a polyethyleneglycol, a polylactic acid, a polyvinylalcohol, a co-polylactideglycolide, cellulose, alginic acids, carrageenans isolated from seaweeds, polysaccharides, pectins, xanthans, starch, and combinations thereof.", "33.The purification material of claim 24, wherein the polymer material comprises an electrically conductive polymer.", "34.The purification material of claim 24, wherein the polymer material comprises a biodegradable polymer.", "35.The purification material of claim 34, wherein the biodegradable polymer is a polyethyleneglycol, a polylactic acid, a polyvinylalcohol, or a co-polylactideglycolide.", "36.The purification material of claim 24, wherein said binder is selected from the group consisting of gelling or absorbent materials.", "37.The purification material of claim 36, wherein said binder is selected from the group consisting of superabsorbents.", "38.The composite purification material of claim 37, wherein said superabsorbent comprises a material selected from polyacrylic acids, polyacrylamides, poly-alcohols, polyamines, polyethylene oxides, cellulose, chitins, gelatins.", "starch, polyvinyl alcohols and polyacrylic acid, polyacrylonitrile, carboxymethyl cellulose, alginic acids, carrageenans isolated from seaweeds, polysaccharides, pectins, xanthans, poly-(diallyldimethylammonium chloride), poly-vinylpyridine, poly-vinylbenzyltrimethylammonium salts, polyvinylacetates, polylactic acids or a combination thereof.", "39.The composite purification material of claim 37, wherein the superabsorbent material comprises an ionically charged surface.", "40.The composite purification material of claim 37, wherein the superabsorbent material comprises a biodegradable polymer.", "41.The purification material of claim 37, wherein said binder is selected from the group consisting polylactic acids, polyacrylamides or combinations of the polymers thereof.", "42.The composite purification material of claim 37, wherein the absorbent material comprises a clay or aluminosilicate material.", "43.The composite purification material of claim 42, wherein the absorbent material comprises is bentonite.", "44.The composite purification material of claim 24, wherein the superabsorbent comprises a material selected from the group consisting of resins obtained by polymerizing acrylic acid and resins obtained by polymerizing acrylamide.", "45.The composite purification material of claim 24, wherein the polymer material comprises a naturally occurring polymer, cellulose, alginic acids, carrageenans isolated from seaweeds, polysaccharides, pectins, xanthans, starch, or combinations thereof.", "46.The composite purification material of claim 24, wherein the superabsorbent material comprises an ionically charged surface ranging from 1-100% of the material surface.", "47.The purification material of claim 6, wherein the purification material is in the form of a sheet and is disposed on a woven web.", "48.The purification material of claim 6, wherein the purification material is in the form of a sheet and is disposed on a nonwoven web.", "49.The purification material of claim 7, wherein the purification material is in the form of a sheet and is disposed on a woven web.", "50.The purification material of claim 7, wherein the purification material is in the form of a sheet and is disposed on a nonwoven web.", "51.The purification material of claim 8, wherein the purification material is in the form of a coated substrate and the substrate is rigid.", "52.The purification material of claim 51, wherein the substrate is a metal.", "53.The purification material of claim 52, wherein the substrate is a coinage metal.", "54.The purification material of claim 52, wherein the substrate contains iron.", "55.The purification material of claim 51, wherein the substrate is a polymer.", "56.The purification material of claim 24, wherein the polymer is derived from synthetic sources.", "57.The purification material of claim 56, wherein the synthetic source produces a woven substrate.", "58.The purification material of claim 24, wherein the synthetic source produces a non-woven substrate.", "59.The purification material of claim 24, wherein the polymer is derived from natural sources.", "60.The purification material of claim 59, wherein the natural source produces a woven substrate.", "61.The purification material of claim 59, wherein the natural source produces a non-woven substrate.", "62.The purification material of claim 8, wherein the purification material is in the form of a coated substrate and the substrate is flexible.", "63.The purification material of claim 62, wherein the polymer is derived from synthetic sources.", "64.The purification material of claim 63, wherein the synthetic source produces a woven substrate.", "65.The purification material of claim 63, wherein the synthetic source produces a non-woven substrate.", "66.The purification material of claim 62, wherein the polymer is derived from natural sources.", "67.The purification material of claim 66, wherein the natural source produces a woven substrate.", "68.The purification material of claim 66, wherein the natural source produces a non-woven substrate.", "69.The purification material of claim 51, wherein the substrate conducts electricity.", "70.The purification material of claim 62, wherein the substrate conducts electricity.", "71.The purification material of claim 1, wherein the binders are present in an amount ranging from about 1 wt % and about 99.9 wt % of the total weight of the purification material.", "72.The purification material of claim 1, further comprising one or more additional non-carbon adsorptive materials.", "73.The purification material of claim 72, wherein said additional adsorptive material comprises a calcium or magnesium containing phosphate or a calcium or magnesium containing silicate.", "74.The purification material of claim 72, wherein said adsorptive material comprises apatite obtained from bone char.", "75.The purification material of claim 72, wherein said adsorptive material comprises an aluminum containing silicate, oxide, or hydroxide.", "76.The purification material of claim 72, wherein said adsorptive material comprises a magnesium containing hydroxide, oxide, or silicate.", "77.The purification material of claim 72, wherein said additional adsorbent material and said insoluble filtration particles are present in approximately equal amounts, further wherein the insoluble filtration particles comprise granulated activated carbon.", "78.The purification material of claim 77, wherein said additional adsorbent material and said granulated activated charcoal are each present in amounts of about 42.5 wt %, and said binders are present in an amount of about 15 wt %, based upon the total weight of said purification material.", "79.The purification material of claim 72, wherein said additional adsorptive material comprises an ion-binding material selected from the group consisting of synthetic ion exchange resins, zeolites, and phosphate minerals.", "80.The purification material of claim 79, wherein the phosphate minerals are members of the phosphate class of minerals.", "81.The purification material of claim 79, wherein the phosphate minerals are members of the aluminosilicate group of minerals.", "82.The purification material of claim 79, wherein the synthetic ion exchange resins are functionalized styrenes, vinylchlorides, divinyl benzenes, methacrylates, acrylates, and mixtures, copolymers, and blends thereof.", "83.The purification material of claim 79 wherein the natural or synthetic zeolites are silicate containing minerals known as clinoptilolite.", "84.The purification material of claim 1, further comprising one or more materials that undergo an chemical oxidation or a chemical reduction in the presence of water or aqueous fluid.", "85.A device for filtering microbiological contaminants from water or aqueous fluid, comprising: a housing; and a porous block of the purification material of claim 1.86.The device according to claim 85, wherein the housing comprises an inlet, an outlet, and a contacting chamber therebetween, and wherein said porous block is disposed within the contacting chamber, such that fluid can flow into the housing from the inlet passes through the porous block and then can flow out of the housing through the outlet.", "87.A method for filtering a fluid to remove any microorganisms therefrom, comprising causing the fluid to flow through the purification material of claim 1, thereby obtaining filtered fluid.", "88.The method of claim 87, wherein said fluid is water.", "89.The method of claim 89, wherein the filtered water is potable.", "90.The method of claim 87, wherein said fluid is an aqueous solution.", "91.The method of claim 90, wherein said aqueous solution is blood.", "92.The method of claim 90, wherein said aqueous solution is a fermentation broth.", "93.The method of claim 90, wherein said aqueous solution is a recycled stream in a chemical or biological process.", "94.The method of claim 90, wherein the aqueous solution is a recycled stream in a cell culturing process.", "95.The method of claim 90, wherein the aqueous solution has been used in a surgical procedure.", "96.The method of claim 87, wherein the fluid comprises breathable air.", "97.The method of claim 87, wherein the fluid comprises a purge gas.", "98.The method of claim 97, wherein the purge gas is selected from the group consisting of O2, CO2, N2, or Ar.", "99.The method of claim 87, wherein the fluid is an anesthetic gas.", "100.The method of claim 99, wherein the anesthetic gas comprises nitrous oxide.", "101.The method of claim 87, further comprising regenerating said purification material by sterilization.", "102.The method of claim 101, wherein said sterilization comprises exposing the purification material to elevated temperature, pressure, radiation levels, or chemical oxidants or reductants, or a combination thereof.", "103.The method of claim 101, wherein said sterilization comprises autoclaving.", "104.The method of claim 101, wherein said sterilization comprises electrochemical treatment.", "105.The method of claim 101, wherein said sterilization comprises a combination of chemical oxidation and autoclaving.", "106.The method of claim 87, wherein said fluid is a gaseous mixture.", "107.The method of claim 106, wherein the filtered gas is air.", "108.The method of claim 87, wherein said fluid is a chemically unreactive gas.", "109.The method of claim 87, wherein said gas is oxygen, carbon dioxide, nitrogen, argon, or nitrogen oxides.", "110.The method of claim 87, wherein said gas is used to pressurize a chamber.", "111.The method of claim 87, wherein said gas is used to sparge or purge an aqueous solution for the purpose of increasing the concentration of the sparging gas in the solution.", "112.The method of claim 87, wherein said gas is used to sparge or purge an aqueous solution for the purpose of decreasing the concentration of the gases initially present in the solution.", "113.The method of claim 87, wherein said gas is used to remove particulate material from surfaces.", "114.An immobilization and contacting medium for microorganisms, comprising magnesium containing mineral and a binder therefor, the medium in the form of a rigid, porous block or a sheet.", "115.The immobilization and contacting medium of claim 114, further comprising one or more microorganisms disposed within the pores thereof.", "116.The regeneration of the material of claim 1 through the use of solutions comprising salt, acid, or caustic.", "117.A method for filtering a fluid to remove any microorganisms therefrom, comprising causing the fluid to flow over the purification material of claim 11, thereby obtaining filtered fluid." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Purification or filtration of water or other aqueous solutions is necessary for many applications, from the provision of safe or potable drinking water to biotechnology applications including fermentation processing and separation of components from biological fluids.", "Similarly, the removal of microbial organisms from breathable air in hospitals and clean rooms, where ultrapurified air is required, and in environments where the air will be recirculated, such as aircraft or spacecraft, is also an important application for filtration media.", "In recent years, the need for air filtration and purification in the home has become more recognized, and the competing concerns of energy efficiency and indoor air quality have lead to numerous air filtration products, such as HEPA filters and the like, that purport to remove small particles, allergens, and even microorganisms from the air.", "There are many well-known methods currently used for water purification, such as distillation, ion-exchange, chemical adsorption, filtering or retention, which is the physical occlusion of particulates.", "Particle filtration may be completed through the use of membranes or layers of granular materials, however in each case the pore size of the material and the space between the granular materials controls the particle size retained.", "Additional purification media include materials that undergo chemical reactions, which alter the state or identity of chemical species in the fluid to be purified.", "In most cases a combination of techniques are required in order to completely purify fluids, such as water.", "Combinations of technologies may be implemented by combining functions in a single device or using several devices in series where each performs a distinct function.", "Examples of this practice include the use of mixed resins that remove both negative and positively charged chemical species as well as species without charge.", "Many of these fluid purification techniques and practices are costly, energy inefficient and/or require significant technical know-how and sophistication.", "Traditional means of reducing these complications require extensive processing or specially designed apparatus.", "Unfortunately, development of low cost techniques do not adequately address the removal of harmful chemical and biological contaminates, such as, bacteria and viruses.", "For example, simple point-of-use purification devices, such as filters attached to in-house water supply conduits or portable units for campers and hikers, cannot sufficiently remove bacteria and viruses unless relatively costly membrane technology or strong chemical oxidizers, such as halogens or reactive oxygen species, are utilized.", "The Environmental Protection Agency (EPA) has set forth minimum standards for acceptance of a device proposed for use as a microbiological water purifier.", "Common coliforms, represented by the bacteria E. coli and Klebsiella terrigena , must show a minimum 6-log reduction, 99.9999% of organisms removed, from an influent concentration of 1×10 7 /100 ml.", "Common viruses, represented by poliovirus 1 (LSc) and rotavirus (Wa or SA-11), which show resistance to many treatment processes, must show a minimum 4 log reduction, 99.99% of organisms removed, from an influent concentration of 1×10 7 /L.", "Cysts, such as those represented by Giardia muris or Giardia lamblia , are widespread, disease-inducing, and resistant to chemical disinfection.", "Devices that claim cyst removal must show a minimum 3 log reduction, 99.9% of cysts removed, from an influent concentration of 1×10 6 /L or 1×10 7 /L, respectively.", "The EPA has accepted the use of other particles in the appropriate size range as a means of testing devices that claim this function.", "Materials that are highly efficient at removing and immobilizing microbial organisms have numerous applications, but a particular area of application is in the biotechnology and fermentation industries.", "Not only would such materials be useful in the processing of fermentation broth for recycling or reuse, they also would have utility as microbial immobilization materials for the microbes of interest to the fermentation process.", "It is well known to use granular, particulate, or fibers of natural or synthetic materials for fluid treatment.", "These materials are commonly used singularly and in mixtures.", "In some cases a material which immobilizes the individual particles or fibers together, referred to as a binder, is used.", "Techniques for generating porous blocks of carbon using a polymer binder is described in prior art by companies such as KX Industries, Amway Corporation, and Cuno.", "Natural materials used in filter applications include carbonaceous materials such as activated carbon and minerals such as apatites, oxides, hydroxides, phosphates, and silicates and combinations thereof.", "Synthetic materials used in filter applications include hydrocarbon polymers, and mineral species such as apatites, oxides, hydroxides, phosphates, and silicates and combinations thereof.", "The particle size of the filtration material used in filtration devices controls many of the technical specifications and successful application of a filtration device.", "Particle sizes commonly used include those in a range between 80 and 325 mesh.", "Grinding and milling of both natural and synthetic materials can be required to generate particles in this size range.", "Although particle sizes outside this range can be used they present practical problems.", "As example, small particles such as those smaller than 325 mesh are difficult to retain in the filtration device while particles larger than 80 mesh lack the needed surface area for many applications.", "The grinding and milling of natural and synthetic materials often produces particles of varying size and distribution.", "Particles size distributions and mixtures of distributions are modified by sieving, particle collection, and recombination.", "Particle sizes that are too small for use in filtration devices often go to waste or must be left for other applications.", "It is also common for synthetic materials to be synthesized in particle sizes that are too small for use in many filtration devices.", "The use of soluble treatment chemicals for increasing particulate matter size in water and waste water treatment is well known.", "It is commonly understood that inorganic and organic materials can be used to flocculate, coagulate, and aggregate small particulate material found in a water stream.", "The resulting larger particles are now able to be filtered or to be removed from the water system through standard sedimentation and clarification methods.", "It is also well understood that many particulates in the water stream carry positive or negative charges and that this characteristic may be used to aggregate the small particles into larger species.", "Accordingly, there remains a need in the art of fluid filtration for an uncomplicated, safe, inexpensive fluid purification and filtration method and device incorporating insoluble small filtration particles (<325 mesh) and soluble water treatment chemicals.", "It is the intention of this invention and art to generate filtration particulate material and filtration devices through the use of multiple different chemical binders.", "Furthermore it is the intention of this invention and method to permit the simultaneous use of activated carbon, silicates, oxides, metal hydroxides, and phosphates in the forms which are readily available and commonly found or synthesized by a variety of different methods.", "There is also a need in the art for a method and device that can address the EPA requirements for designation as chemical and microbiological water purifiers, such that the device is more than suitable for consumer and industry point-of-use and point-of-entry applications." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>To this end, the present inventors have discovered that a significant problem in the known use of small particulate inorganic and organic materials, called fines, incorporated into filter devices is that the particles are difficult to contain thus requiring very small pore size containers which increases the back pressure of devices.", "Small particles also tend to plug devices which leads to short product lifetimes.", "Loss of particulate material decreases or inhibits product performance, can cause illness, and presents a general annoyance for devices users.", "Finally, as with all particulate containing devices, grinding of particles as a result of particle movement generates even smaller particles.", "Additionally, the present inventors have discovered that there also exists a significant problem in the known binding methods used to generate filter devices.", "As the size of the filtration particles decreases an increase in the amount of polymer binder is required in order to retain and immobilize the particles.", "The increase in binder levels required often generates filtration blocks with small pores which increases device backpressure.", "The invention disclosed provides a means for using small filtration particulate matter (<325 mesh), material fines, generated from the processing of natural or synthetic materials or from the synthesis of materials for the generation of porous blocks suitable for fluid filtration.", "The invention in general involves the use of multiple organic and/or inorganic binders to first increase the particulate matter size, and subsequently to generate porous blocks or sheets or coatings.", "Non-flow through coated surfaces which function by contact filtration are also considered an important material that may be used in the invention.", "The method of the invention involves two steps.", "The first step involves taking small particles or fines in the size range between 10 nanometers and 200 microns and aggregating or agglomerating them into larger particles through the use of positively charged, negatively charged, and/or uncharged organic or inorganic polymers, and/or compounds such as oxides, hydroxides, phosphates, and silicates.", "Examples of suitable binders include, polyelectrolytes such as polyamines, polyalcohols, polysaacharides, polyacrylates, polyacrylamides and derivitized natural and synthetic polymers, oxides of magnesium and calcium, and hydroxides of calcium, magnesium, aluminum, and iron.", "Additionally, precipitation of hydroxide and phosphate compounds may be used.", "This first processing step may also include mechanical steps, such as mixing, spraying, dripping or fluidic processing.", "This step may also include heat treatment, including digestion, calcining, sintering, and firing.", "The chemical and physical processing of this step may be repeated until the particles are of appropriate size, which is usually greater than 325 mesh.", "It should be understood that during the first step of the invention, the binder may be partially or fully removed by the various processing methods.", "The second step in the invention involves taking the particulate material generated in step 1 , particles of significantly greater size, and combining them with a second binder, of different type, which immobilizes the particles into a porous block.", "This second step may utilize standard techniques such as extrusion, molding, and pressure.", "The invention and method provides an efficient means of using small particles which are difficult to implement in the generation of filtration devices.", "There are numerous advantageous to the method of this invention.", "First, the initial starting particles have very large surface areas which increases the filtration efficiency of the filtration device when they are included as components of larger particles which are now retained in the device.", "Second, the invention provides the ability to simultaneously use a mixture or agglomeration of different filtration particle types.", "As an example, carbon, apatite, silicate, metal oxide, hydroxide, and/or sulfur containing particles may be agglomerated to generate a mixed composition particle.", "Third, the method provides a means of producing insoluble water treatment polymer materials from previously soluble polymeric compounds and retaining them in the filtration device.", "As an example, high molecular weight charged water treatment polymers have a plethora of active binding sites.", "By using some of the active binding sites to bind particulate material the polymer is rendered insoluble.", "Since only some of the many active binding sites are used for particle binding there still remains many active binding sites which are now available for participating in the fluid stream filtration, for the removal of chemical and biological contaminants.", "Fourth, the method allows the use of materials that are hazardous in larger sizes such as magnesium containing silicates in asbestos form to be used in safer smaller particulate sizes.", "Fifth, the method provides a means for utilizing nanometer size particles of metals and metal oxides that are of interest in fluid catalysis and chemical stream processing.", "It should be understood that the present invention may also be used for generating particles in a size range greater than 100 mesh.", "Unfortunately, the effectiveness of filters generated with larger materials with or without a binder is compromised by channeling and by-pass effects caused by the pressure of fluid, in particular, water and aqueous solutions, flowing through the filter media as well as particle erosion and aggregation.", "Because many chemicals, viruses and bacteria are removed by intimate contact with the adsorption material, even relatively small channels or pathways in the granular material formed over time by water pressure, water flow, particle erosion, or particle aggregation are easily sufficient to allow passage of the undesirable chemical and microbiological contaminants through the filter.", "For example, taking water as an exemplary fluid and using the material of the invention as a filtration medium for microbial organisms, calculations based on a virus influent concentration of 1×10 6 /L show that where a 4-log reduction is to be expected, only a 3.7 log reduction actually occurs if only 0.01% of the water bypasses treatment by passing through channels formed in the filter media during filtration.", "If 0.1% of the water passes through untreated, then only a 3 log reduction occurs.", "If 1% passes through untreated, only a 2 log reduction occurs, and if 10% passes untreated, only a 1 log reduction occurs.", "Where a 6-log reduction is expected, the detrimental results of channeling are even more dramatic, with only a 4-log reduction actually occurring when 0.01% of the water bypasses treatment.", "This invention solves this problem by providing a method and device for removing contaminants, including chemicals, bacteria and viruses, where very small particulate filtration materials and device adsorptive filter media are immobilized with multiple chemical binders material to form a porous filter material that eliminates the possibility of channeling and active material by-pass.", "This invention is, in general, a device and method for the purification and filtration of aqueous fluids, in particular water (such as drinking water or swimming or bathing water), or other aqueous solutions (such as fermentation broths and solutions used in cell culture), or gases and mixtures of gases, such as breathable air, found in clean rooms, hospitals, diving equipment homes, aircraft, or spacecraft, and gases used to sparge, purge, or remove particulate matter from surfaces.", "The use of the device and method of the invention results in the removal of an extremely high percentage of microbiological contaminants, including bacteria and viruses and components thereof as well as chemical contaminants such as heavy metals, pesticides and by products of chemical treatment processes.", "In particular, the use of the device and method of the invention results in purification of water to a level that addresses the EPA standards for chemical or microbiological water purification.", "In one embodiment, the invention relates to a purification material for fluids that contains particulate carbon that is in the form of a porous block as the result of employing multiple binders.", "Typically, at least a portion of this carbon is activated and from natural sources.", "In another embodiment, the invention relates to a purification material for fluids that contains particulate apatite minerals that is in the form of a porous block as the result of the presence of the multiple binders.", "Apatites are commonly mined, prepared from natural sources (bone char), or synthesized from calcium and phosphorus containing compounds.", "In another embodiment, the invention relates to a purification material for fluids that contains particulate oxide and hydroxide minerals that are in the form of a porous block as the result of the presence of the multiple binders.", "Aluminum, iron, and magnesium oxides are commonly mined and purified from natural sources (alumina/bauxite, chlorides), or synthesized from aluminum, magnesium containing minerals, or generated from synthetic sources such as the mixing of aluminum, iron, and magnesium containing compounds.", "In another embodiment, the invention relates to a purification material for fluids that contains particulate silicate minerals that are in the form of a porous block as the result of the presence of the multiple binders.", "Aluminum, calcium, iron, magnesium, sodium, and potassium containing silicates are commonly mined and purified from natural sources, or synthesized from aluminum, calcium, iron, magnesium, sodium, or potassium containing compounds.", "In another embodiment, the invention relates to a purification material for fluids that contains particulate metals important in catalysis that are in the form of a porous block as the result of the presence of the multiple binders.", "Platinum group metals, such as platinum, rhodium, and palladium, as well as coinage metals, such as gold, silver, copper, and nickel, as well as heavy metals, such as cadmium and chromium, are commonly mined and purified from natural sources, or reclaimed from spent electronic components.", "In another embodiment, the invention relates to a purification material for fluids that contains a mixture of the particulate filtration materials described in the individual embodiments of this invention that are in the form of a porous block as the result of the presence of the multiple binders.", "The mixtures included in this embodiment can vary dramatically with individual components included varying from less than 1% through greater than 99%.", "In yet another embodiment, the invention relates to a purification material for fluids that contains a mixture of particulate filtration materials generated by combining particulate material generated by a method consistent with the first step of this invention with materials that have been generated through traditional methods, such as grinding and or milling.", "The mixture of particles is then processed into the form of a porous block as the result of the presence of the binders and method consistent with the second step of the method.", "The mixtures of particles generated in the different processes included in this embodiment can vary dramatically with individual components included varying from less than 1% through greater than 99%.", "Also typically, the binders used are inorganic or organic compounds including polymeric or oligomeric materials that are capable of maintaining the particulate material in a particulate form (first binder) and in block structure form (second binder).", "This allows the purification material to be extruded, molded or pressed into any desired shape, e.g., a shape suitable for inclusion into the housing of a filtration device, which provides for fluid inflow and outflow, and which filtration device has one or more chambers for contact of the fluid with the purification material.", "Such a device forms another embodiment of the invention.", "In addition to maintaining the filtration particles immobilized in a unitary block, the polymeric binders also provide desirable physical characteristics to the filter material, e.g., rendering it rigid or flexible, depending upon the type and amount of polymeric binders used.", "In another embodiment, the invention relates to a purification material for fluids that is in the form of a self-supporting sheet or membrane containing the particulate filtration immobilized with the binders.", "In another embodiment, the invention relates to a purification material for fluids that is in the form of a porous coating supported by a porous substrate containing the particulate filtration immobilized with the binders.", "In another embodiment, the invention relates to a purification material for fluids that is in the form of a nonporous coating supported by a porous or nonporous substrate containing the particulate filtration immobilized with the binders.", "Here the fluid is filtered by surface contact.", "The invention also relates to methods of filtering fluids, such as water, aqueous solutions, and gases, to remove a large proportion of one or more types of chemicals, microorganisms contained therein, by contacting the fluid with the purification material of the invention.", "In a particular aspect of this embodiment of the invention, this contacting occurs within the device described above, with the unfiltered fluid flowing through an inlet, contacting the purification material in one or more chambers, and the filtered fluid flowing out of the chamber through an outlet.", "The purification material of the invention can be used to purify drinking water, to purify water used for potable and/or recreational purposes, such as in swimming pools, hot tubs, and spas, to purify process water, e.g.", "water used in cooling towers, to purify aqueous solutions, including, but not limited to, blood, fermentation broths, and cell culture solutions (e.g., for solution recycling in fermentation or other cell culture processes) and aqueous fluids used in surgical procedures for recycle or reuse, and to purify gases and mixtures of gases such as breathable air, for example, air used to ventilate hospital or industrial clean rooms, air used in diving equipment, or air that is recycled, e.g., in airplanes or spacecraft, and gases used to sparge, purge or remove volatile or particulate matter from surfaces, containers, or vessels.", "The purification material of the invention has the additional advantage of making use of readily available filtration materials and more especially small particulate materials, including those obtained from natural and synthetic sources, while still maintaining high purification efficiency.", "In yet another embodiment of the invention, the materials of the invention, namely small filtration particulate matter and optionally other adsorptive materials with a multiple binder matrix and which is formed into a block or sheet or coating, can be used as an immobilization medium for microorganisms used in biotechnology applications such as fermentation processes and cell culture.", "In this embodiment, biological process fluids, such as nutrient broths, substrate solutions, and the like, are passed through or over the immobilization material of the invention in a manner that allows the fluids to come into contact with the microorganisms immobilized therein and thereon, and effluent removed from the material and further processed as needed." ], [ "FIELD OF THE INVENTION This invention relates generally to the field of solution and fluid filters or purification devices, primarily to aqueous solution filters and water purification, devices for gases and water and other aqueous liquids, which remove contaminants from the gas or aqueous liquid solution passed through them.", "In its more particular aspects, the invention relates to the field of such devices that remove chemical and microbiological contaminants, including pesticides, byproducts of chemical treatment processes, cysts, bacteria and viruses and their components, from water or aqueous solutions.", "BACKGROUND OF THE INVENTION Purification or filtration of water or other aqueous solutions is necessary for many applications, from the provision of safe or potable drinking water to biotechnology applications including fermentation processing and separation of components from biological fluids.", "Similarly, the removal of microbial organisms from breathable air in hospitals and clean rooms, where ultrapurified air is required, and in environments where the air will be recirculated, such as aircraft or spacecraft, is also an important application for filtration media.", "In recent years, the need for air filtration and purification in the home has become more recognized, and the competing concerns of energy efficiency and indoor air quality have lead to numerous air filtration products, such as HEPA filters and the like, that purport to remove small particles, allergens, and even microorganisms from the air.", "There are many well-known methods currently used for water purification, such as distillation, ion-exchange, chemical adsorption, filtering or retention, which is the physical occlusion of particulates.", "Particle filtration may be completed through the use of membranes or layers of granular materials, however in each case the pore size of the material and the space between the granular materials controls the particle size retained.", "Additional purification media include materials that undergo chemical reactions, which alter the state or identity of chemical species in the fluid to be purified.", "In most cases a combination of techniques are required in order to completely purify fluids, such as water.", "Combinations of technologies may be implemented by combining functions in a single device or using several devices in series where each performs a distinct function.", "Examples of this practice include the use of mixed resins that remove both negative and positively charged chemical species as well as species without charge.", "Many of these fluid purification techniques and practices are costly, energy inefficient and/or require significant technical know-how and sophistication.", "Traditional means of reducing these complications require extensive processing or specially designed apparatus.", "Unfortunately, development of low cost techniques do not adequately address the removal of harmful chemical and biological contaminates, such as, bacteria and viruses.", "For example, simple point-of-use purification devices, such as filters attached to in-house water supply conduits or portable units for campers and hikers, cannot sufficiently remove bacteria and viruses unless relatively costly membrane technology or strong chemical oxidizers, such as halogens or reactive oxygen species, are utilized.", "The Environmental Protection Agency (EPA) has set forth minimum standards for acceptance of a device proposed for use as a microbiological water purifier.", "Common coliforms, represented by the bacteria E. coli and Klebsiella terrigena, must show a minimum 6-log reduction, 99.9999% of organisms removed, from an influent concentration of 1×107/100 ml.", "Common viruses, represented by poliovirus 1 (LSc) and rotavirus (Wa or SA-11), which show resistance to many treatment processes, must show a minimum 4 log reduction, 99.99% of organisms removed, from an influent concentration of 1×107/L.", "Cysts, such as those represented by Giardia muris or Giardia lamblia, are widespread, disease-inducing, and resistant to chemical disinfection.", "Devices that claim cyst removal must show a minimum 3 log reduction, 99.9% of cysts removed, from an influent concentration of 1×106/L or 1×107/L, respectively.", "The EPA has accepted the use of other particles in the appropriate size range as a means of testing devices that claim this function.", "Materials that are highly efficient at removing and immobilizing microbial organisms have numerous applications, but a particular area of application is in the biotechnology and fermentation industries.", "Not only would such materials be useful in the processing of fermentation broth for recycling or reuse, they also would have utility as microbial immobilization materials for the microbes of interest to the fermentation process.", "It is well known to use granular, particulate, or fibers of natural or synthetic materials for fluid treatment.", "These materials are commonly used singularly and in mixtures.", "In some cases a material which immobilizes the individual particles or fibers together, referred to as a binder, is used.", "Techniques for generating porous blocks of carbon using a polymer binder is described in prior art by companies such as KX Industries, Amway Corporation, and Cuno.", "Natural materials used in filter applications include carbonaceous materials such as activated carbon and minerals such as apatites, oxides, hydroxides, phosphates, and silicates and combinations thereof.", "Synthetic materials used in filter applications include hydrocarbon polymers, and mineral species such as apatites, oxides, hydroxides, phosphates, and silicates and combinations thereof.", "The particle size of the filtration material used in filtration devices controls many of the technical specifications and successful application of a filtration device.", "Particle sizes commonly used include those in a range between 80 and 325 mesh.", "Grinding and milling of both natural and synthetic materials can be required to generate particles in this size range.", "Although particle sizes outside this range can be used they present practical problems.", "As example, small particles such as those smaller than 325 mesh are difficult to retain in the filtration device while particles larger than 80 mesh lack the needed surface area for many applications.", "The grinding and milling of natural and synthetic materials often produces particles of varying size and distribution.", "Particles size distributions and mixtures of distributions are modified by sieving, particle collection, and recombination.", "Particle sizes that are too small for use in filtration devices often go to waste or must be left for other applications.", "It is also common for synthetic materials to be synthesized in particle sizes that are too small for use in many filtration devices.", "The use of soluble treatment chemicals for increasing particulate matter size in water and waste water treatment is well known.", "It is commonly understood that inorganic and organic materials can be used to flocculate, coagulate, and aggregate small particulate material found in a water stream.", "The resulting larger particles are now able to be filtered or to be removed from the water system through standard sedimentation and clarification methods.", "It is also well understood that many particulates in the water stream carry positive or negative charges and that this characteristic may be used to aggregate the small particles into larger species.", "Accordingly, there remains a need in the art of fluid filtration for an uncomplicated, safe, inexpensive fluid purification and filtration method and device incorporating insoluble small filtration particles (<325 mesh) and soluble water treatment chemicals.", "It is the intention of this invention and art to generate filtration particulate material and filtration devices through the use of multiple different chemical binders.", "Furthermore it is the intention of this invention and method to permit the simultaneous use of activated carbon, silicates, oxides, metal hydroxides, and phosphates in the forms which are readily available and commonly found or synthesized by a variety of different methods.", "There is also a need in the art for a method and device that can address the EPA requirements for designation as chemical and microbiological water purifiers, such that the device is more than suitable for consumer and industry point-of-use and point-of-entry applications.", "SUMMARY OF THE INVENTION To this end, the present inventors have discovered that a significant problem in the known use of small particulate inorganic and organic materials, called fines, incorporated into filter devices is that the particles are difficult to contain thus requiring very small pore size containers which increases the back pressure of devices.", "Small particles also tend to plug devices which leads to short product lifetimes.", "Loss of particulate material decreases or inhibits product performance, can cause illness, and presents a general annoyance for devices users.", "Finally, as with all particulate containing devices, grinding of particles as a result of particle movement generates even smaller particles.", "Additionally, the present inventors have discovered that there also exists a significant problem in the known binding methods used to generate filter devices.", "As the size of the filtration particles decreases an increase in the amount of polymer binder is required in order to retain and immobilize the particles.", "The increase in binder levels required often generates filtration blocks with small pores which increases device backpressure.", "The invention disclosed provides a means for using small filtration particulate matter (<325 mesh), material fines, generated from the processing of natural or synthetic materials or from the synthesis of materials for the generation of porous blocks suitable for fluid filtration.", "The invention in general involves the use of multiple organic and/or inorganic binders to first increase the particulate matter size, and subsequently to generate porous blocks or sheets or coatings.", "Non-flow through coated surfaces which function by contact filtration are also considered an important material that may be used in the invention.", "The method of the invention involves two steps.", "The first step involves taking small particles or fines in the size range between 10 nanometers and 200 microns and aggregating or agglomerating them into larger particles through the use of positively charged, negatively charged, and/or uncharged organic or inorganic polymers, and/or compounds such as oxides, hydroxides, phosphates, and silicates.", "Examples of suitable binders include, polyelectrolytes such as polyamines, polyalcohols, polysaacharides, polyacrylates, polyacrylamides and derivitized natural and synthetic polymers, oxides of magnesium and calcium, and hydroxides of calcium, magnesium, aluminum, and iron.", "Additionally, precipitation of hydroxide and phosphate compounds may be used.", "This first processing step may also include mechanical steps, such as mixing, spraying, dripping or fluidic processing.", "This step may also include heat treatment, including digestion, calcining, sintering, and firing.", "The chemical and physical processing of this step may be repeated until the particles are of appropriate size, which is usually greater than 325 mesh.", "It should be understood that during the first step of the invention, the binder may be partially or fully removed by the various processing methods.", "The second step in the invention involves taking the particulate material generated in step 1, particles of significantly greater size, and combining them with a second binder, of different type, which immobilizes the particles into a porous block.", "This second step may utilize standard techniques such as extrusion, molding, and pressure.", "The invention and method provides an efficient means of using small particles which are difficult to implement in the generation of filtration devices.", "There are numerous advantageous to the method of this invention.", "First, the initial starting particles have very large surface areas which increases the filtration efficiency of the filtration device when they are included as components of larger particles which are now retained in the device.", "Second, the invention provides the ability to simultaneously use a mixture or agglomeration of different filtration particle types.", "As an example, carbon, apatite, silicate, metal oxide, hydroxide, and/or sulfur containing particles may be agglomerated to generate a mixed composition particle.", "Third, the method provides a means of producing insoluble water treatment polymer materials from previously soluble polymeric compounds and retaining them in the filtration device.", "As an example, high molecular weight charged water treatment polymers have a plethora of active binding sites.", "By using some of the active binding sites to bind particulate material the polymer is rendered insoluble.", "Since only some of the many active binding sites are used for particle binding there still remains many active binding sites which are now available for participating in the fluid stream filtration, for the removal of chemical and biological contaminants.", "Fourth, the method allows the use of materials that are hazardous in larger sizes such as magnesium containing silicates in asbestos form to be used in safer smaller particulate sizes.", "Fifth, the method provides a means for utilizing nanometer size particles of metals and metal oxides that are of interest in fluid catalysis and chemical stream processing.", "It should be understood that the present invention may also be used for generating particles in a size range greater than 100 mesh.", "Unfortunately, the effectiveness of filters generated with larger materials with or without a binder is compromised by channeling and by-pass effects caused by the pressure of fluid, in particular, water and aqueous solutions, flowing through the filter media as well as particle erosion and aggregation.", "Because many chemicals, viruses and bacteria are removed by intimate contact with the adsorption material, even relatively small channels or pathways in the granular material formed over time by water pressure, water flow, particle erosion, or particle aggregation are easily sufficient to allow passage of the undesirable chemical and microbiological contaminants through the filter.", "For example, taking water as an exemplary fluid and using the material of the invention as a filtration medium for microbial organisms, calculations based on a virus influent concentration of 1×106/L show that where a 4-log reduction is to be expected, only a 3.7 log reduction actually occurs if only 0.01% of the water bypasses treatment by passing through channels formed in the filter media during filtration.", "If 0.1% of the water passes through untreated, then only a 3 log reduction occurs.", "If 1% passes through untreated, only a 2 log reduction occurs, and if 10% passes untreated, only a 1 log reduction occurs.", "Where a 6-log reduction is expected, the detrimental results of channeling are even more dramatic, with only a 4-log reduction actually occurring when 0.01% of the water bypasses treatment.", "This invention solves this problem by providing a method and device for removing contaminants, including chemicals, bacteria and viruses, where very small particulate filtration materials and device adsorptive filter media are immobilized with multiple chemical binders material to form a porous filter material that eliminates the possibility of channeling and active material by-pass.", "This invention is, in general, a device and method for the purification and filtration of aqueous fluids, in particular water (such as drinking water or swimming or bathing water), or other aqueous solutions (such as fermentation broths and solutions used in cell culture), or gases and mixtures of gases, such as breathable air, found in clean rooms, hospitals, diving equipment homes, aircraft, or spacecraft, and gases used to sparge, purge, or remove particulate matter from surfaces.", "The use of the device and method of the invention results in the removal of an extremely high percentage of microbiological contaminants, including bacteria and viruses and components thereof as well as chemical contaminants such as heavy metals, pesticides and by products of chemical treatment processes.", "In particular, the use of the device and method of the invention results in purification of water to a level that addresses the EPA standards for chemical or microbiological water purification.", "In one embodiment, the invention relates to a purification material for fluids that contains particulate carbon that is in the form of a porous block as the result of employing multiple binders.", "Typically, at least a portion of this carbon is activated and from natural sources.", "In another embodiment, the invention relates to a purification material for fluids that contains particulate apatite minerals that is in the form of a porous block as the result of the presence of the multiple binders.", "Apatites are commonly mined, prepared from natural sources (bone char), or synthesized from calcium and phosphorus containing compounds.", "In another embodiment, the invention relates to a purification material for fluids that contains particulate oxide and hydroxide minerals that are in the form of a porous block as the result of the presence of the multiple binders.", "Aluminum, iron, and magnesium oxides are commonly mined and purified from natural sources (alumina/bauxite, chlorides), or synthesized from aluminum, magnesium containing minerals, or generated from synthetic sources such as the mixing of aluminum, iron, and magnesium containing compounds.", "In another embodiment, the invention relates to a purification material for fluids that contains particulate silicate minerals that are in the form of a porous block as the result of the presence of the multiple binders.", "Aluminum, calcium, iron, magnesium, sodium, and potassium containing silicates are commonly mined and purified from natural sources, or synthesized from aluminum, calcium, iron, magnesium, sodium, or potassium containing compounds.", "In another embodiment, the invention relates to a purification material for fluids that contains particulate metals important in catalysis that are in the form of a porous block as the result of the presence of the multiple binders.", "Platinum group metals, such as platinum, rhodium, and palladium, as well as coinage metals, such as gold, silver, copper, and nickel, as well as heavy metals, such as cadmium and chromium, are commonly mined and purified from natural sources, or reclaimed from spent electronic components.", "In another embodiment, the invention relates to a purification material for fluids that contains a mixture of the particulate filtration materials described in the individual embodiments of this invention that are in the form of a porous block as the result of the presence of the multiple binders.", "The mixtures included in this embodiment can vary dramatically with individual components included varying from less than 1% through greater than 99%.", "In yet another embodiment, the invention relates to a purification material for fluids that contains a mixture of particulate filtration materials generated by combining particulate material generated by a method consistent with the first step of this invention with materials that have been generated through traditional methods, such as grinding and or milling.", "The mixture of particles is then processed into the form of a porous block as the result of the presence of the binders and method consistent with the second step of the method.", "The mixtures of particles generated in the different processes included in this embodiment can vary dramatically with individual components included varying from less than 1% through greater than 99%.", "Also typically, the binders used are inorganic or organic compounds including polymeric or oligomeric materials that are capable of maintaining the particulate material in a particulate form (first binder) and in block structure form (second binder).", "This allows the purification material to be extruded, molded or pressed into any desired shape, e.g., a shape suitable for inclusion into the housing of a filtration device, which provides for fluid inflow and outflow, and which filtration device has one or more chambers for contact of the fluid with the purification material.", "Such a device forms another embodiment of the invention.", "In addition to maintaining the filtration particles immobilized in a unitary block, the polymeric binders also provide desirable physical characteristics to the filter material, e.g., rendering it rigid or flexible, depending upon the type and amount of polymeric binders used.", "In another embodiment, the invention relates to a purification material for fluids that is in the form of a self-supporting sheet or membrane containing the particulate filtration immobilized with the binders.", "In another embodiment, the invention relates to a purification material for fluids that is in the form of a porous coating supported by a porous substrate containing the particulate filtration immobilized with the binders.", "In another embodiment, the invention relates to a purification material for fluids that is in the form of a nonporous coating supported by a porous or nonporous substrate containing the particulate filtration immobilized with the binders.", "Here the fluid is filtered by surface contact.", "The invention also relates to methods of filtering fluids, such as water, aqueous solutions, and gases, to remove a large proportion of one or more types of chemicals, microorganisms contained therein, by contacting the fluid with the purification material of the invention.", "In a particular aspect of this embodiment of the invention, this contacting occurs within the device described above, with the unfiltered fluid flowing through an inlet, contacting the purification material in one or more chambers, and the filtered fluid flowing out of the chamber through an outlet.", "The purification material of the invention can be used to purify drinking water, to purify water used for potable and/or recreational purposes, such as in swimming pools, hot tubs, and spas, to purify process water, e.g.", "water used in cooling towers, to purify aqueous solutions, including, but not limited to, blood, fermentation broths, and cell culture solutions (e.g., for solution recycling in fermentation or other cell culture processes) and aqueous fluids used in surgical procedures for recycle or reuse, and to purify gases and mixtures of gases such as breathable air, for example, air used to ventilate hospital or industrial clean rooms, air used in diving equipment, or air that is recycled, e.g., in airplanes or spacecraft, and gases used to sparge, purge or remove volatile or particulate matter from surfaces, containers, or vessels.", "The purification material of the invention has the additional advantage of making use of readily available filtration materials and more especially small particulate materials, including those obtained from natural and synthetic sources, while still maintaining high purification efficiency.", "In yet another embodiment of the invention, the materials of the invention, namely small filtration particulate matter and optionally other adsorptive materials with a multiple binder matrix and which is formed into a block or sheet or coating, can be used as an immobilization medium for microorganisms used in biotechnology applications such as fermentation processes and cell culture.", "In this embodiment, biological process fluids, such as nutrient broths, substrate solutions, and the like, are passed through or over the immobilization material of the invention in a manner that allows the fluids to come into contact with the microorganisms immobilized therein and thereon, and effluent removed from the material and further processed as needed.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a cross-sectional view illustrating a particular embodiment of the invention, namely a water filter housing containing a block filter incorporating synthetic apatite minerals and granulated activated charcoal (GAC) in a multiple binder matrix according to the invention.", "FIGS.", "2a and 2b are schematic views of a particular embodiment of the invention, namely a filter material containing synthetic apatite minerals and granulated activated charcoal (GAC) and a multiple binder matrix in the form of a membrane or sheet.", "DETAILED DESCRIPTION OF THE INVENTION As indicated above, one embodiment of the invention relates to a purification material in the form of a block filter containing granulated carbon, such as activated carbon, and synthetic or natural apatite (hydroxy-calcium-phosphate) with a first binder to increase filtration particle size and a second binder which generates a porous block.", "The first binder is typically an organic polymer material, such as a polyamine, polyacrylamide, polyvinylalcohol, polyacrylic, or polyelectrolyte derived from natural or synthetic polymers or an inorganic material such as a metal oxide or hydroxide or a polysilane.", "The second binder employed for the purpose of generating the porous block is typically a thermoplastic such as polyethylene or material that generates immobilization pressure through fluid absorption.", "In a particular aspect of this embodiment, the invention relates to a rigid porous block filter that contains a mixture of granulated carbon and apatite derivatives, or granulated activated charcoal (GAC) or bone char or other adsorptive filter media using a first binder material which is an inorganic, and a second binder, such as a thermoplastic material, such that the mineral materials and derivatives and GAC are fixed within the binder matricies, and that channeling from flow during water treatment cannot occur.", "The final purification material of the invention can be produced by extrusion, molding including injection molding, or by fluid absorption/compression methods.", "Fibrillation may also be used to prepare fibrils of the mixture of first binder and mineral and carbon particulates that can then be formed into a sheet, film, or block.", "It may be produced in any shape or size and may be rigid or flexible.", "The pore size of the filter block influences flow rates of the fluid through the filter, and is a function of the size of the granular particles generated in the first step and incorporated into the filter block in the second step.", "As used herein, the term “block” does not denote any particular geometrical shape, but rather that the material is not a sheet or membrane, or coating.", "Nonlimiting examples of “blocks” as this term is intended to be used include tubes, annular rings, as well as more conventional geometrical solids.", "Material formed into flexible blocks is particularly suitable for use in pipes or tubes that serve as the fluid filter medium.", "One of the desirable features of the purification material of the invention is that it may be formed into any desired shape, and thus provides ease of handling and use.", "For example, the purification material may be formed into a monolith or block that fits into conventional housings for filtration media or it can be shaped to provide purification as part of a portable or personal filtration system.", "Alternatively, the material may be formed into several different pieces, through which water flows in series or in parallel.", "Sheets or membranes of the purification material may also be formed.", "The rigidity of the purification material, whether in block form or in sheet/membrane/coating form, may be altered through inclusion of flexible polymers in the binder material.", "While not wishing to be bound by any theory, it is believed that the purification material of the invention achieves its unusually high efficiency in removing chemicals and microorganisms from fluids partly as the result of the immobilization of the filtration particles in the binders, and the necessity for fluid flowing through the purification material to follow an extended and tortuous path therethrough, instead of forming channels through the purification material as occurs in prior particulate-containing purification materials.", "This path ensures that the fluid contacts a larger proportion of the surface area of the filtration particles, and it prevents sustained laminar flow of the fluid through the filtration material.", "This latter effect is believed to help prevent laminae of fluid containing chemicals and microorganisms from avoiding sustained contact with the filtration particles in the filter.", "After the purification material has been in service for a period of time, additional filtration by occlusion will occur as adsorbed material accumulates in the pores of the purification material.", "Those familiar with the art of fluid filtration will understand that the pore size and physical dimensions of the purification material may be manipulated for different applications and that variations in these variables will alter flow rates, back-pressure, and the level of chemical and microbiological contaminant removal.", "Likewise those knowledgeable in the art will recognize that variations in the percentages of each component of the purification material will provide some variability in utility.", "For example, increasing the percentage of filtration particulate material in the purification material will result in a material having an increased number of interaction sites for chemical and biological species, while increasing the percentage of a binder with no active fluid treatment sites will result in a purification material having material and mechanical properties closer to that of the binder material and with reduced interaction sites.", "In one particular embodiment of the invention, the mineral material used is in the form of apatite, and the hydroxyapatite and GAC material are present in approximately equal amounts, with the percentage of both the first and second binder materials kept to a minimum.", "However, the mineral adsorbents used in the invention may be obtained from other natural or synthetic/industrial sources and mixtures of the different derivatives can provide differences in the properties of the purification material.", "For example, adding fluoride to the filter block will result in a decreased reduction of fluoride in the effluent water if water is used as the fluid.", "This can be useful in, e.g.", "purifying fluorinated water in such a way as to maintain desirable fluorine levels therein.", "Fluoride in the filter material may be obtained either by inclusion of fluoride containing apatite, inclusion of fluoride salts and compounds, or by pre-conditioning the purification material by passing fluoride-containing solutions therethrough.", "Likewise, as the number of binding sites is increased through the use of different crystal and material structures and orientation of different crystal faces, the binding of metal ions, radioactive isotopes, and microorganisms can also be increased.", "Commonly, exposure to increased temperatures allows conversion between crystalline and amorphous forms.", "Furthermore choosing a first, and or second binder material with active fluid treatment sites such as those which occur with charged and uncharged binders facilitates the “tailoring” of the filtration material for specific applications.", "Those experienced in the art will also understand that different crystal or amorphous lattices are possible for mineral filtration particles, and for other adsorbent materials used in the invention, and that these variations will yield differences in properties of the resulting purification material, as certain crystal structures improve and inhibit interactions with chemicals, microorganisms and other biological materials.", "These differences in properties result from differences in interactions between the chemicals and microorganisms and other biological materials and the different positive, negative ions, and neutral species that are included in the crystal structure.", "In another embodiment of the invention, the purification material is constructed to withstand sterilization.", "Sterilization processes include thermal processes, such as steam sterilization or other processes wherein the purification material is exposed to elevated temperatures or pressures or both, resistive heating, radiation sterilization wherein the purification material is exposed to elevated radiation levels, including processes using ultraviolet, infrared, microwave, and ionizing radiation, and chemical sterilization, wherein the purification material is exposed to elevated levels of oxidants or reductants or other chemical species, and which is performed with chemicals, such as halogens, reactive oxygen species, formaldehyde, surfactants, metals and gases such as ethylene oxide, methyl bromide, beta-propiolactone, and propylene oxide.", "Additionally, sterilization may be accomplished with electrochemical methods by direct oxidation or reduction with microbiological components or indirectly through the electrochemical generation of oxidative or reductive chemical species.", "Combinations of these processes may also used.", "It should also be understood that sterilization processes may be used on a continuous or batch basis while the purification material is in use.", "In general, the invention comprises a device and a method for the filtration and purification of a fluid, in particular an aqueous solution or water, to remove organic and inorganic elements and compounds present in the water as particulate material.", "In particular, the device and method can be used to remove microbiological contaminants, including cysts, bacteria and viruses and components thereof, as well as chemical species, such as pesticides and byproducts of chemical treatment processes, from water or other fluids or gasses destined for consumption or other use by humans or other animals.", "The method and device of the invention are particularly useful in these applications where the reduction in concentration of chemical and microbiological contaminants made possible by the invention addresses the EPA standards for microbiological and chemical water purification devices, and also significantly exceeds the effectiveness of other known filtration and purification devices incorporating granulated adsorption media that contain filtration particulate matter in the absence of binder materials.", "In a particular embodiment of the invention, the purification material is a porous block formed by granulated or particulate apatite, oxide, or silicate material, which is defined herein to include hydroxyapatite, alumina, and iron and/or magnesium containing silicates and other optional adsorptive granular materials, described in more detail below, such as granulated activated charcoal (GAC), retained within a multiple polymer binder matrix.", "In the method corresponding to this particular embodiment, the microbiological contaminants are removed from the water when the water is forced through the porous block by water pressure on the influent side, or by a vacuum on the effluent side, of the filter block.", "In an embodiment of the invention where the purification material is composed of a mixture of apatite and/or an adsorptive granular filter media, for example GAC, such components can be dispersed in a random manner throughout the block.", "The purification material can also be formed with spatially distinct gradients or separated layers.", "For example, apatite and alumina and GAC granules may be immobilized in separate layers using a solid second binder matrix, for instance, a polymer thermoplastic such as polyethylene or the like, so that movement of the mineral particulate and GAC particles is precluded and detrimental channeling effects during fluid transport through the block are prevented.", "If the components reside in separate locations, the fluid flow is sequential through these locations.", "In a particular example of this embodiment, at least a portion of the apatite present originates from synthetic mixtures thereof.", "An example of a suitable material is that designated as tricalcium phosphate as sold by Murlin Chemical in PA and/or Brimac Carbon Services, UK, and carbon as provided by KX Industries in CT.", "The carbon material may be ground to a desirable particle size, e.g., 80×325 mesh.", "A typical analysis of these materials shows greater than 90% purity.", "The element binding characteristics of these materials have been reported and such elements include chlorine, fluorine, aluminum, cadmium, lead, mercury (organic and inorganic), copper, zinc, iron, nickel, strontium, arsenic, chromium, manganese, and certain radionuclides.", "The organic molecule binding capabilities have been reported for complex organic molecules, color-forming compounds, compounds that add taste to fluids, compounds that add odors to fluids, and trihalomethane precursors.", "In this embodiment, the mineral species (apatite, oxide, hydroxide, silicate, etc.)", "and the GAC are mixed in approximately equal amounts with the minimal amount of first binder material required to generate particles in the 80×325 mesh size and the minimum amount of second binder necessary to generate a monolithic purification material.", "However, the amounts of mineral particulate matter, GAC, and binder are substantially variable, and materials having different concentrations of these materials may be utilized in a similar fashion without the need for any undue experimentation by those of skill in the art.", "In general, however, when GAC, or bone char is used as the additional adsorbent material, its concentration in the mixture is generally less than 50% by weight, based upon the weight of the composition before any drying or compacting.", "Additionally, adsorbents other than GAC may be substituted completely for, or mixed with, the GAC in a multicomponent mixture.", "Examples of these adsorbents include various ion-binding materials, such as synthetic ion exchange resins, zeolites (synthetic or naturally occurring), diatomaceous earth, metal hydroxides and oxides, in particular those containing the metals such as aluminum, calcium, magnesium, and iron and one or more other phosphate-containing materials, such as minerals of the phosphate class, in particular, minerals of the aluminosilicate group.", "In particular, minerals of the aluminosilicate group that contain magnesium, calcium, iron, sodium and or potassium, and mixtures thereof, are particularly suitable for the invention.", "These materials may be calcined, sintered, and/or purified by any of the well known mineral processing methods.", "Additionally, polymeric materials for ion-binding, including derivatised resins of styrene and divinylbenzene, and methacrylate, may be used.", "The derivatives include functionalized polymers having anion binding sites based on quaternary amines, primary and secondary amines, aminopropyl, diethylaminoethyl, and diethylaminopropyl substituents.", "Derivatives including cation binding sites include polymers functionalized with sulfonic acid, benzenesulfonic acid, propylsulfonic acid, phosphonic acid, and/or carboxylic acid moieties.", "Natural or synthetic zeolites may also be used or included as ion-binding materials, including, e.g., naturally occurring aluminosilicates such as clinoptilolite.", "Suitable materials for the first binder which is used to flocculate, coagulate and/or aggregate, small particulate matter into large particulate matter may include any material capable of aggregating the particulate materials together and maintaining this aggregation under the conditions of use.", "They are generally included in amounts ranging from about 1 wt % to about 99.9 wt %, more particularly from about 15 wt % to about 50 wt %, based upon the total weight of the purification material.", "If a polymeric binder material is used it may be charged positively, negatively, or uncharged and may originate from synthetic sources or natural sources.", "Suitable binders include polyamides, polyalcohols, polysaacharides, polyacrylamides, polyacrylates, humic acids, and proteins.", "Binders may also include materials such as metal hydroxides and oxides including those containing aluminum, calcium, magnesium, and iron and including polyaluminum sulfates and polyaluminum chlorides.", "Binders appropriate for this first step can include polorganozirconates, polyorganoaluminates, polysiloxanes, polysilanes, polysilazanes, polycarbosilanes, polyborosilanes, zirconium dimethacrulate, zirconium tetramethacrylate, zirconium 2-ethylhexanoate, aluminum butoxides, aluminum diisopropoxide ethylacetoacetate, tetramethyldisiloxanes and derivatives thereof, tristrimethylsilylphosphate, and tristrimethylsiloxyboron.", "Exempliary first step binders also include polyelectrolytes carrying positive or negative charged chemical functionalities, or a combination thereof.", "These may include but are not limited to polyamines such as poly(DADMAC), Poly-DADM, Polyamine-Poly(DADMAC) blends, polyquartenary amines, inorganic-polyamine blends, and inorganic Poly(DADMAC) blends.", "Additionally, cationic starch, and cationic polymethylmethacrylates may be used.", "Also, copolymers of vinylimidazolium methochloride and vinylpyrrolidone, quarternized vinylpyrrolidone/dimethyl-aminoethyl-methacrylate copolymer, and polyethyleneimine.", "Companies manufacturing suitable materials include Vinings Industries, Cytec, BASF Corporation, Ontario Specialty Coatings Corp., International Specialty Products, and EKA Chemicals.", "The primary requirement for the first binder is that it yields the appropriate particle size under the conditions which the second binder is employed for porous solid generation.", "Those skilled in the art will understand that the first binder may be employed through a number of physical processing methods including, but not limited to, dripping, spraying, solid state reaction, and solution state reaction.", "It should be understood by those experienced in the art that step processing methods may remove portions of the first binder while maintaining appropriate particle size and that processing with the second binder may remove or displace some of the first binder.", "It should also be understood by those experienced in the art of water treatment that the interactions between insoluble treatment particles and the first binder may be classified as that of flocculation, coagulation, aggregation and combinations thereof.", "Additionally, it should be understood that different binders will react to varying degrees with water treatment oxidizers such as chlorine.", "It should also be understood that regulatory agencies such as the US FDA, US EPA, CEN, DWI, and KIWA have regulatory requirements for the concentration of these materials that may be present in food stuffs and potable waters.", "Suitable materials for the second and other binders which are used to generate the monolithic porous block include any polymeric material capable of aggregating the particulate materials together and maintaining this aggregation under the conditions of use.", "They are generally included in amounts ranging from about 1 wt % to about 99.9 w %, more particularly from about 15 wt % to about 50 wt %, based upon the total weight of the purification material.", "Suitable polymeric materials include both naturally occurring and synthetic polymers, as well as synthetic modifications of naturally occurring polymers.", "The polymeric binder materials generally include one or more thermoset, thermoplastic, elastomer, or a combination thereof, depending upon the desired mechanical properties of the resulting purification material.", "In general, polymers melting between about 50° C. and about 500° C., more particularly, between about 75° C. and about 350° C., even more particularly between about 80° C. and about 200° C., are suitable polymeric binders for the invention.", "For instance, polyolefins melting in the range from about 85° C. to about 180° C., polyamides melting in the range from about 200° C. to about 300° C., and fluorinated polymers melting in the range from about 300° C. to about 400° C., can be particularly mentioned as suitable.", "Examples of types of polymers suitable for use as binders in the invention include, but are not limited to, thermoplastics, polyethylene glycols or derivatives thereof, polyvinyl alcohols, polyvinylacetates, and polylactic acids.", "Suitable thermoplastics include, but are not limited to, nylons and other polyamides, polyethylenes, including LDPE, LLDPE, HDPE, and polyethylene copolymers with other polyolefins, polyvinylchlorides (both plasticized and unplasticized), fluorocarbon resins, such as polytetrafluoroethylene, polystyrenes, polypropylenes, cellulosic resins, such as cellulose acetate butyrates, acrylic resins, such as polyacrylates and polymethylmethacrylates, thermoplastic blends or grafts such as acrylonitrile-butadiene-styrenes or acrylonitrile-styrenes, polycarbonates, polyvinylacetates, ethylene vinyl acetates, polyvinyl alcohols, polyoxymethylene, polyformaldehyde, polyacetals, polyesters, such as polyethylene terephthalate, polyether ether ketone, and phenol-formaldehyde resins, such as resols and novolacs.", "Those of skill in the art will recognize that other thermoplastic polymers can be used in the invention in an analogous manner.", "Suitable thermoset polymers for use as, or inclusion in, the binder used in the invention include, but are not limited to, polyurethanes, silicones, fluorosilicones, phenolic resins, melamine resins, melamine formaldehyde, and urea formaldehyde.", "Suitable elasomers for use as or inclusion in, the binder used in the invention include but are not limited to natural and/or synthetic rubbers, like styrene-butadiene rubbers, neoprenes, nitrile rubber, butyl rubber, silicones, polyurethanes, alkylated chlorosulfonated polyethylene, polyolefins, chlorosulfonated polyethylenes, perfluoroelastomers, polychloroprene (neoprene), ethylene-propylene-diene terpolymers, chlorinated polyethylene, VITON (fluoroelastomer), and ZALAK® (Dupont-Dow elastomer).", "Those of skill in the art will realize that some of the thermoplastics listed above can also be thermosets, depending upon the degree of crosslinking, and that some of each may be elastomers, depending upon their mechanical properties, and that the particular categorization used above is for ease of understanding and should not be regarded as limiting or controlling.", "Naturally occurring and synthetically modified naturally occurring polymers suitable for use in the invention include, but are not limited to, natural and synthetically modified celluloses, such as cotton, collagens, and organic acids.", "Biodegradable polymers suitable for use in the invention include, but are not limited to, polyethylene glycols, polylactic acids, polyvinylalcohols, co-polylactideglycolides, and the like.", "In the specific embodiment of a filter material that may be sterilized the mineral filtration material originating from natural or synthetic sources and GAC or bone char material are present in approximately equal amounts, with the percentage of binders kept to a minimum.", "The binders used must be stable to the temperature, pressure, electrochemical, radiative, and chemical conditions presented in the sterilization process, and should be otherwise compatible with the sterilization method.", "Examples of first binders can include acrylamides, acrylates, and any stable polyelectrolytes.", "Examples of second binders suitable for sterilization methods involving exposure to high temperatures (such as steam sterilization or autoclaving) include cellulose nitrate, polyethersulfone, nylon, polypropylene, polytetrafluoroethylene (teflon), and mixed cellulose esters.", "Purification materials prepared with these binders can be autoclaved when the binder polymers are prepared according to known standards.", "Desirably, the purification material is stable to both steam sterilization or autoclaving and chemical sterilization or contact with oxidative or reductive chemical species, as this combination of sterilizing steps is particularly suitable for efficient and effective regeneration of the purification material.", "Additionally, sterilization and regenerating of devices incorporating the mineral materials may be conducted by passing solutions of apatite, alum, acid, and/or caustic through the filter.", "In the embodiment of the invention wherein sterilization is at least in part conducted through the electrochemical generation of oxidative or reductive chemical species, the electrical potential necessary to generate said species can be attained by using the purification material itself as one of the electrodes.", "For example, the purification material, which contains polymeric binder, can be rendered conductive through the inclusion of a sufficiently high level of conductive particles, such as GAC, carbon black, or metallic particles to render a normally insulative polymeric material conductive.", "Alternatively, if the desired level of carbon or other particles is not sufficiently high to render an otherwise insulative polymer conductive, an intrinsically conductive polymer may be used as or blended into the binder.", "Examples of suitable intrinsically conductive polymers include doped polyanilines, polythiophenes, and other known intrinsically conductive polymers.", "These materials can be incorporated into the binder in sufficient amount to provide a resistance of less than about 1 kΩ, more particularly less than about 300 Ω.", "The purification material of the present invention need not be in the form of a block, but may also be formed into a self-supporting sheet or film.", "This sheet or film may, in a particular embodiment, be disposed on a woven or nonwoven web of, e.g., a polymer.", "The polymer used to form the woven or nonwoven web may be any thermoplastic or thermosetting resin typically used to form fabrics.", "Polyolefins, such as polypropylene and polyethylene, are particularly suitable in this regard.", "The purification material of the present invention need not be in the form of a block, or sheet but may also be formed into a coating which may be disposed on a porous or nonporous supporting substrate.", "This coating may, in a particular embodiment, be disposed on a woven or nonwoven web of, e.g., a polymer.", "The polymer used to form the woven or nonwoven web may be any thermoplastic or thermosetting resin typically used to form fabrics.", "Polyolefins, such as polypropylene and polyethylene, are particularly suitable in this regard.", "The coating may be disposed on a metal surface such as a sheet, fiber, or wire.", "Suitable metals include steel, iron containing alloys, precious metals, silver, copper, and gold, and aluminum, and coinage metals.", "The coating may be disposed on a metal surface such as a mesh or screen.", "Suitable meshes include those made from steel, iron containing alloys, precious metals, silver, copper, and gold, and aluminum, and coinage metals.", "The efficiency of the purification material and the method for using it to reduce chemical and microbiological contaminants and the flow rate of the fluid through the material, are a function of the pore size within the block and the influent fluid pressure.", "At constant fluid pressure, flow rate is a function of pore size, and the pore size within the block can be regulated by controlling the size of the mineral particulate and GAC granules.", "For example, a large granule size provides a less dense, more open purification material which results in a faster flow rate, and small granule size provides a more dense, less open purification material which results in a slower flow rate.", "A block 17 formed with relatively large filtration particle granules will have less surface area and interaction sites than a block formed with smaller granules.", "Accordingly, the purification material of large granules must be of thicker dimension to achieve equal removal of microbiological contaminants.", "Because these factors are controllable within the manufacturing process, the purification materials can be customized by altering pore size, block volume, block outer surface area, and geometric shape to meet different application criteria.", "Average pore size in a particular embodiment is kept to below several microns, and more particularly to below about one micron, to preclude passage of cysts.", "It should be noted that the pore size described herein does not refer to the pores within the enlarged particles or in the particles comprising the enlarged particles or other adsorbent particles themselves, but rather to the pores formed within the purification material when the particles are aggregated together by the binder.", "The method of making the material of the invention, in its most general aspect, involves combining the particulate filtration material (and optional additional particulate adsorbent material(s) with the first binder material under conditions of pressure and temperature that allow interaction between the materials and flocculation, coagulation, agglomeration, or a combination thereof, and a second binder material under conditions of pressure and temperature that allow at least a portion of the binder to be present in liquid form and that allow for compaction of the particulate, and then solidifying the binder around and/or between the particles.", "The precise nature of the production process will depend to a certain extent upon the nature of the binder materials.", "For example, if the first binder material is supplied in the form of a liquid solution, suspension, or emulsion (e.g., in a volatile solvent), it may be contacted with the particles by dipping or spraying, and the wet particles compressed in a mold.", "The mold may be optionally heated to evaporate any necessary solvent or binder.", "The resulting molded material is then dried to form a purification material which can then be milled to the desired particle size desired for interaction with the second binder.", "The wet material may also be extruded or dewatered and then processed to the desired particle size.", "In similar fashion, if the first or second binder material is supplied in the form of a liquid solution, suspension, or emulsion (e.g., in a volatile solvent), it may be contacted with the particles generated in the first binding process by dipping or spraying, and the wet particles compressed in a mold.", "The mold may be optionally heated to evaporate any necessary solvent.", "The resulting molded material is then dried to form the purification material of the invention.", "If, on the other hand, the first or second binders are a polymer resin, it will typically be mixed in pellet form with the particles of the adsorbent material, and the resulting mixture heated and extruded or molded into the desired shape.", "Examples of suitable particulate/binder extrusion processes and equipment are disclosed in U.S. Pat.", "Nos.", "5,189,092; 5,249,948; and 5,331,037.Other extrusion equipment and processes may also be used.", "Moreover, the mixture may be heated and injection molded, without the need for any extrusion.", "Additionally, the binder, a thermoset, may be generated through a crosslinking process that incorporates initiation by chemical processes, electrochemical processes, irradiation and through physical parameters of temperature and pressure variations.", "With reference to the drawings, the invention and a mode of practicing it will now be described with regard to one particular embodiment, which significantly exceeds the EPA requirements for microbiological filters.", "FIG.", "1 illustrates a typical specific embodiment of a filtration apparatus containing the purification material of the invention, which incorporates a rigid porous block filter.", "A removable housing 11 is mated with a cap 12, the cap 12 having an inflow orifice 13 and an outflow orifice 14.A water supply conduit 15 is joined to the inflow orifice 13 to deliver non-treated water into the device, and a water discharge conduit 16 is joined to the outflow orifice 14 to conduct treated water from the device.", "Water passes into the housing 11.The pressure of the water flow forces it through the porous block filter member 17, which as shown is formed in the shape of hollow cylinder with an axial bore 18.The treated water then passes into the axial bore 18 which connects to the outflow orifice 14.FIG.", "2 is provided as a representative illustration of one possible configuration.", "It is to be understood that other configurations where water is caused to pass through a porous filter block (which may have different geometrical shapes and/or different flow properties) are contemplated to be within the scope of the invention.", "The block 17 may be formed by any of a number of known methods, such as by extrusion, compression, molding, sintering or other techniques.", "FIGS.", "2a and 2b shows two embodiments where the purification material of the invention is used in the form of a sheet or film.", "FIG.", "2a shows purification material 1 used in connection with normal flow-through filtration, indicated by arrow 2, which represents the fluid being filtered by passage through the sheet or film 1.FIG.", "2b shows purification material 1 used in connection with crossflow filtration.", "Fluid flowing across the filter is indicated by double-headed arrow 3, while fluid flowing through the purification material 1 is indicated by arrow 2.The cross flow fluid indicated by arrow 3 sweeps across the surface of the purification material 1, decreasing the level of particulate matter deposited thereon.", "EXAMPLE 1 A cylindrical filter block 17 of the shape shown in FIG.", "2 may be prepared with a material composition of approximately 42.5% apatite obtained from Murlin Chemical in PA in the form of hydroxycalciumphosphate and approximately 42.5% GAC obtained from KX Industries.", "Approximately 10% inorganic binder, selected from one or more of the inorganics described above, as a first binder is used to increase the particle size of the hydroxycalciumphosphate and 15% (polyethylene) thermoplastic binder material selected from one or more of the thermoplastics described above is used as a second binder to generate the porous block of all components.", "The material may be extruded at a temperature that provides a uniform mixture of mineral adsorbent, GAC, aggregating binder and thermoplastic binder.", "The cylindrical or toroidally shaped block 17 is approximately 9.8 inches in length, with an outer diameter of approximately 2.5 inches and an inner diameter (the bore 18) of approximately 1.25 inches.", "This shape filter fits into a standard water filtration housing used in the home and industrial settings.", "The filter material has a resistance of about 300 Ω.", "EXAMPLE 2 The filter prepared in Example 1 may be challenged by exposing it to tap water that is filtered with activated carbon and then seeded with 2.3×108 colony forming units per liter of K. terrigena bacteria and 1.0×107 units per liter of MS2 virus.", "The seeded water is passed through the filter block 17 at a flow rate of approximately 2 liters/minute for 3 minutes, followed by collection of a 500 ml effluent sample.", "Bacteria may be assayed on m-Endo agar plates by membrane filtration procedure, while the MS2 virus may be assayed by standard methods.", "EXAMPLE 3 The composite prepared in Example 1 may be used to reduce a water soluble chlorine species such as hypochlorous acid in an oxidized state to a chlorine species in a reduced state (choride).", "Chlorine levels of approximately 2.0 mg/L were reduced to below the detection limits of standard test strip based assays.", "As described above, the material of the invention is extremely useful in the area of water purification, particularly the area of drinking water purification.", "Because of the extremely high efficiency with which the material of the present invention removes chemicals and microorganisms from water, it meets and exceeds the EPA guidelines for materials used as microbiological and chemical water purifiers.", "In addition to functioning as a purifier for drinking water, the material of the invention can also be used to purify water used for recreational purposes, such as water used in swimming pools, hot tubs, and spas.", "As the result of the ability of the material of the invention to efficiently remove and immobilize microorganisms and other cells from aqueous solutions, it has numerous applications in the pharmaceutical and medical fields.", "For example, the material of the invention can be used to fractionate blood by separating blood components, e.g., plasma, from blood cells, and to remove microorganisms from other physiological fluids.", "The material can also be used in hospital or industrial areas requiring highly purified air having extremely low content of microorganisms, e.g., in intensive care wards, operating theaters, and clean rooms used for the therapy of immunosuppressed patients, or in industrial clean rooms used for manufacturing electronic and semiconductor equipment.", "The material of the invention has multiple uses in fermentation applications and cell culture, where it can be used to remove microorganisms from aqueous fluids, such as fermentation broths or process fluids, allowing these fluids to be used more efficiently and recycled, e.g., without cross-contamination of microbial strains.", "In addition, because the material is so efficient at removing microorganisms and at retaining them once removed, it can be used as an immobilization medium for enzymatic and other processing requiring the use of microorganisms.", "A seeding solution containing the desired microorganisms is first forced through the material of the invention, and then substrate solutions, e.g., containing proteins or other materials serving as enzymatic substrates, are passed through the seeded material.", "As these substrate solutions pass through the material, the substrates dissolved or suspended therein come into contact with the immobilized microorganisms, and more importantly, with the enzymes produced by those microorganisms, which can then catalyze reaction of the substrate molecules.", "The reaction products may then be eluted from the material by washing with another aqueous solution.", "The material of the invention has numerous other industrial uses, e.g., filtering water used in cooling systems.", "Cooling water often passes through towers, ponds, or other process equipment where microorganisms can come into contact with the fluid, obtain nutrients and propagate.", "Microbial growth in the water is often sufficiently robust that the process equipment becomes clogged or damaged and requires extensive chemical treatment.", "By removing microorganisms before they are able to propagate substantially, the present invention helps to reduce the health hazard associated with the cooling fluids and the cost and dangers associated with chemical treatment programs.", "Similarly, breathable air is often recycled in transportation systems, either to reduce costs (as with commercial airliners) or because a limited supply is available (as with submarines and spacecraft).", "Efficient removal of microorganisms permits this air to be recycled more safely.", "In addition, the material of the invention can be used to increase indoor air quality in homes or offices in conjunction with the air circulation and conditioning systems already in use therein.", "The purification material of the invention can also be used to purify other types of gases, such as anesthetic gases used in surgery or dentistry (e.g., nitrous oxide), gases used in the carbonated beverage industry (e.g., carbon dioxide), gases used to purge process equipment (e.g., nitrogen, carbon dioxide, argon), and/or to remove particles from surfaces, etc.", "In each of these applications, the method of using the material of the invention is relatively simple and should be apparent to those of skill in the filtration art.", "The fluid or gas to be filtered is simply conducted to one side of a block or sheet of material of the invention, typically disposed in some form of housing, and forced through the material as the result of a pressure drop across the purification material.", "Purified, filtered fluid or gas is then conducted away from the “clean” side of the filter and further processed or used.", "The invention having been thus described by reference to certain of its specific embodiments, it will be apparent to those of skill in the art that many variations and modifications of these embodiments may be made within the spirit of the invention, which are intended to come within the scope of the appended claims and equivalents thereto." ] ]	Patent_10469653
[ [ "Texturing method and apparatus", "A method and apparatus is disclosed for conveying and applying texturing instructions within a very speed, or bandwidth environment, or where storage capacity is limited; the invention and of particular use in image displaying mobile telephones (16), but also applies to image data storage (184, 186, 188, 190, 192) and use on the Internet.", "Image texturing is defined by a texture string (198) defining a seed number (200) for pseudo random generation of z axis displacement of pixels from an initial surface, a roughness value (202) for the surface, warp function values (204, 206), distortion function values (208, 210), texturing cell style and size data (212, 214) colour information (216, 218, 220, 222, 224, 226) and indication of where the texture is to be used (228).", "Cells (20) tile and wrap." ], [ "1-27 (cancelled) 28: A method of generating a surface texture in an image, said method comprising the steps of: providing a texture field of pixels; allocating a random value for each of said pixels in the field; moving said pixels; dividing said texture field into wrappable cells; and allocating a color to each of said pixels.", "29.A method, according to claim 28, wherein said step of allocating a random value for each of said pixels comprises controlling a roughness of said random value.", "30.A method, according to any one of the preceding claims, wherein said step of moving said pixels comprise one of warping said pixels and distorting said pixels 31.A method, according to claim 28 or claim 29, wherein the step of dividing said texture field into wrappable cells comprises at least one of the step of selecting a shape of said cells and the step of selecting a size of said cells.", "32.A method, according to claim 30, wherein the step of dividing said texture field into wrappable cells comprises at least one of the step of selecting a shape of said cells and the step of selecting a size of said cells.", "33.A method, according to claim 28 or claim 29, wherein said step of allocating a color to each of said pixels comprises at least one of the step of taking account of a position of the pixel, the step of taking account of a movement of the pixel and, the step of providing a selected range of colors and of taking allocation from within the selected range of colors.", "34.A method, according to claim 30, wherein said step of allocating a color to each of said pixels comprises at least one of the step of taking account of a position of the pixel, the step of taking account of a movement of the pixel and, the step of providing a selected range of colors and of taking allocation from within the selected range of colors.", "35.A method, according to claim 31, wherein said step of allocating a color to each of said pixels comprises at least one of the step of taking account of a position of the pixel, the step of taking account of a movement of the pixel and, the step of providing a selected range of colors and of taking allocation from within the selected range of colors.", "36.A method, according to claim 32, wherein said step of allocating a color to each of said pixels comprises at least one of the step of taking account of a position of the pixel, the step of taking account of a movement of the pixel and, the step of providing a selected range of colors and of taking allocation from within the selected range of colors.", "37.A method, according to claim 28 or claim 29, comprising at least one of the step of using a computer program to generate the surface texture and the step of using a texture data string to generate the surface texture.", "38.A method, according to claim 30, comprising the step of generating the surface texture by using a computer program which utilizes a texture data string.", "39.A method, according to claim 31, comprising the step of generating the surface texture by using a computer program which utilizes a texture data string.", "40.A method, according to claim 32, comprising the step of generating the surface texture by using a computer program which utilizes a texture data string.", "41.A method, according to claim 33, comprising the step of generating the surface texture by using a computer program which utilizes a texture data string.", "42.A method, according to claim 34, comprising the step of generating the surface texture by using a computer program which utilizes a texture data string.", "43.An apparatus for generating a surface texture in an image, said apparatus comprising: means for generating a texture field of pixels; means for allocating a random value for each pixel in the field; means for moving the pixels; means for dividing said texture field into wrappable cells; and means for allocating a colour to each pixel.", "44.An apparatus, according to claim 43, wherein said means for allocating a random value to each pixel includes means for controlling a roughness of said random values.", "45.An apparatus, according to claim 43 or claim 44, wherein said means for moving said pixels comprises at least one of means for warping said pixels and means for distorting said pixels.", "46.An apparatus, according to claim 43 or claim 44, wherein said means for dividing said texture field into wrappable cells comprises at least one of means for selecting a shape of said cells and means for selecting a size of said cells.", "47.An apparatus, according to claim 45, wherein said means for dividing said texture field into wrappable cells comprises at least one of means for selecting a shape of said cells and means for selecting a size of said cells.", "48.An apparatus, according to claim 43 or claim 44, wherein said means for allocating a color to each pixel comprises at least one of means for responding to a position of the pixel, means for responding to a movement of the pixel and, means for providing a selected range of colors and for allocating a color from within said selected range of colors.", "49.An apparatus, according to claim 45, wherein said means for allocating a color to each pixel comprises at least one of means responsive to a position of the pixel, means for responding to a movement of the pixel and, means for providing a selected range of colors and for allocating a color from within said selected range of colors.", "50.An apparatus, according to claim 46, wherein said means for allocating a colour to each pixel comprises at least one of means responsive to a position of the pixel, means for responding to a movement of the pixel and, means for providing a selected range of colors and for allocating a color from within said selected range of colors.", "51.An apparatus, according to claim 47, wherein said means for allocating a colour to each pixel comprises at least one of means responsive to a position of the pixel, means for responding to a movement of the pixel and, means for providing a selected range of colors and for allocating a color from within said selected range of colors.", "52.An apparatus, according to claim 43 or claim 44 comprising a computer program which utilizes a texture data string for generating the surface texture.", "53.An apparatus, according to claim 45 comprising a computer program which utilizes a texture data string for generating the surface texture.", "54.An apparatus, according to claim 46 comprising a computer program which utilizes a texture data string for generating the surface texture.", "55.An apparatus, according to claim 47 comprising a computer program which utilizes a texture data string for generating the surface texture.", "56.An apparatus, according to claim 48 comprising a computer program which utilizes a texture data string for generating the surface texture.", "57.An apparatus, according to claim 49 comprising a computer program which utilizes a texture data string for generating the surface texture.", "58.An apparatus, according to claim 50 comprising a computer program which utilizes a texture data string for generating the surface texture.", "59.An apparatus, according to claim 51 comprising a computer program which utilizes a texture data string for generating the surface texture.", "60.An apparatus, according to claim 52 comprising a computer program which utilizes a texture data string for generating the surface texture.", "61.A computer program comprising an instruction set for generating a surface texture in an image by employing a texture field of pixels; allocating a random value for each of said pixels in the field; moving said pixels; dividing said texture field into wrappable cells; and allocating a color to each of said pixels.", "62.A memory device, bearing a copy of a computer program, said computer program comprising an instruction set for generating a surface texture in an image by the steps of employing a texture field of pixels; allocating a random value for each of said pixels in the field; moving said pixels; dividing said texture field into wrappable cells; and allocating a color to each of said pixels.", "63.A computer device programmed to generating a surface texture in an image by performing the steps of employing a texture field of pixels; allocating a random value for each of said pixels in the field; moving said pixels; dividing said texture field into wrappable cells; and allocating a color to each of said pixels.", "64.A texture string message for generating a surface texture in an image by the steps of: employing a texture field of pixels; allocating a random value for each of said pixels in the field; moving said pixels; dividing said texture field into wrappable cells; and allocating a color to each of said pixels, wherein a texture data string is used to generate the surface texture 65.A computer programmed to generate a surface texture in an image by using a texture data string, by the steps of: providing a texture field of pixels; allocating a random value for each of said pixels in the field; moving said pixels; dividing said texture field into wrappable cells; and allocating a color to each of said pixels." ], [ "The present invention relates to the surface texturing of graphic images for display on a screen.", "More particularly, the present invention relates to compact methods for applying surface textures to graphic images.", "A key feature of most three-dimensional computer models, for viewing in two dimensions on a screen, is the selection of suitable texture maps.", "Texture maps are used to provide details of various surface properties, such as colour, opacity, roughness, shading, transparency, and so on.", "Often, texture maps are created from photographs or scanned images.", "This can result in very large file sizes.", "New applications of texture maps, such as in images transmitted to be viewed on mobile telephone displays, require a very small bandwidth.", "There is simply not enough time to send large files.", "The present invention seeks to provide a method and apparatus apt for use with mobile telephones.", "It is a problem of image storage on discs that the images take up very large amounts of space.", "The texturing information forms a large part of the required storage.", "The present invention seeks to provide an improved method and apparatus for image storage on discs, allowing storage of more images on the same disc.", "In a limited band width or capacity situation, the present invention seeks to provide a means whereby more surface texture can be transmitted in a fixed time, or over a fixed bandwidth, or stored in a fixed space.", "Users of the World Wide Web are often, with good reason, impatient.", "A user will wait only a certain amount of time before abandoning a web page and going elsewhere.", "By allowing the rapid transmission of texture data, the present invention seeks to provide a means whereby web pages are more rapidly made available.", "In general terms, the present invention applies to any application which relies on the general appearance of an image rather than the specifics of an image itself, and which relies on limited storage or bandwidth.", "The present invention is particularly useful for display of background images and spot graphical effects on web pages, generation of a wide range of paper texture effects for paint programmes, display of pleasing images on storage limited devices such as palm top computers, and efficient storage and transmission of very large high resolution textures for printing.", "According to a first aspect, the present invention consists in a method of generating a surface texture in an image, comprising the steps of: employing a texture of pixels; allocating a random value for each pixel in the field; moving the pixels; dividing the texture into wrappable cells; and allocating a colour to each pixel.", "The first aspect further provides that the step of allocating a random value to each pixel includes controlling the roughness of the random values.", "The first aspect further provides that the step of moving the pixels includes warping the pixels.", "The first aspect further provides that the step of moving the pixels includes distorting the pixels.", "The first aspect, further provides that the step dividing the texture into wrappable cells includes the step of selecting the shape of the cells.", "The first aspect, further provides that the step dividing the texture into wrappable cells includes the step of selecting the size of the cells.", "The first aspect of the invention, further, provides that the step of allocating a colour to each pixel includes taking account of the position of the pixel.", "The first aspect of the invention, further, provides that the step of allocating a colour to each pixel includes taking account of the movement of the pixel.", "The first aspect of the invention, further, provides that the step of allocating a colour to each pixel includes taking allocating from within a selected range of colours.", "The first aspect of the invention further provides for use of a computer program to generate the surface texture.", "The first aspect of the invention further provides for use of a texture data string to generate the surface texture.", "According to a second aspect, the present invention consists in an apparatus, operating according to the method of the first aspect.", "According to a third aspect, the present invention consists in a computer program, operating according to the first aspect, According to a fourth aspect, the present invention consists in a memory device, bearing a copy of the computer program.", "According to a fifth aspect, the present invention consists in a texture string message.", "According to a sixth aspect, the present invention consists in a computer device, containing a program to cause it to operate according to the first aspect.", "According to a seventh aspect, the present invention consists in a computer, operative to generate the texture data string.", "The invention is further explained, by way of an example, by the following description, read in conjunction with the appended drawings in which: FIG.", "1 shows a schematic view of the general situation wherein the preferred embodiment of the invention is employed.", "FIG.", "2A-FIG.", "2E show examples of different textures, having different styles of cell, used in the present invention.", "FIG.", "3A and FIG.", "3B show the wrapping feature of the textures of FIG.", "1, in this instance showing tessellating cells.", "FIG.", "4A and FIG.", "4B illustrate the wrapping of textures of FIG.", "1 using matching cells.", "FIG.", "5 shows an object, to be covered with a surface texture, in relation to the cells of a texture.", "FIG.", "6 shows individual pixels within a texture.", "FIG.", "7 shows individual pixels prior to a randomising process.", "FIGS.", "8A to 8D are project views illustrating the randomising process and FIG.", "8E is a flow chart of the randomising process.", "FIGS.", "9A to FIG.", "9F show various degrees of roughness which can be achieved using the present invention.", "FIG.", "10A shows a linear interpolation method employed in a warping process.", "FIG.", "10B shows a smooth interpolation method used in a warping process.", "FIGS.", "11A to 11E show various examples of the effects that can be achieved with warping.", "FIG.", "12 is a flow chart illustrating the manner in which warping is achieved.", "FIG.", "13 is a flow chart showing how distortion is achieved.", "FIG.", "14A to FIG.", "14F illustrate the effect of various degrees of warping.", "FIG.", "15 shows how pixels are allocated to a cell.", "FIG.", "16 shows how weighting functions are applied to pixel properties for use in colour selection.", "FIG.", "17 illustrates, in general terms, how the weighting functions of FIG.", "16 are actually applied in the formulae thereon.", "FIG.", "18 is a diagram illustrating the meaning of some of the pixel properties used in relation to FIG.", "17.FIG.", "19 is a diagram illustrating the meaning of the term “radial” in FIG.", "17.FIG.", "20 is a diagram illustrating the meaning of the terms “edgewards”, “angle”, “cell plasma”, “pixel plasma” and “pixel plasma 2”, shown in FIG.", "17.FIG.", "21 shows a diagram illustrating how the colour values derived according to the flow chart of FIG.", "16, are applied to select the colour of a pixel in the final texture.", "FIG.", "22A to FIG.", "22L illustrate different textured effects which can be achieved using the present invention.", "FIG.", "23 is a flow chart illustrating how the image generation source of FIG.", "1 provides the receiving apparatus with a copy of the texture programme should the receiving apparatus not already possess the texture programme.", "FIG.", "24 shows the manner in which the texture information is conveyed to the apparatus which is to generate and display a texture.", "FIG.", "25 is a flow chart showing how the apparatus, which is to display the textured image, achieves that effect.", "FIG.", "26 is a schematic diagram showing various ways in which the texturing information and method of the present invention may be transmitted and/or stored.", "FIG.", "27 is an example of a texture string, a sequence of binary digits, which can be sent to a receiving apparatus so that a texture can be generated therein and displayed.", "Attention is drawn to FIG.", "1 which shows one of the environments wherein the present invention can be applied.", "An image generation source 10, here shown as a computer, sends image date to the telephone network 12, which, in turn, sends image data to the mobile telephone network 14 and thence to a mobile telephone 16 which displays the image 18 represented by the data from the image generation source 10.It is the object of the present invention to provide the image 18 which can have a wide range of surface textures for a very small amount of image data.", "FIGS.", "2A to 2E show the starting point of the surface texture generation process.", "A texture 20 is provided.", "The texture 20, in this instance, is square.", "In general, this is a rectangle the same height and width as the final texture.", "The texture 20 is divided into cells 22.The cells 22 form a regular pattern in the texture 20 and have the property of “wrapping”.", "That is to say that when the top edge 24 is, figuratively speaking, wrapped around to join the bottom edge 26, the pattern of cells 22 is continuous.", "Equally, when the right edge 28 is wrapped around to the left edge 30 the pattern of cells 22 is again continuous.", "Different kinds of cell 22 are possible.", "FIG.", "2A shows a pattern of cells called “Weave”.", "FIG.", "2B shows a pattern called “Brick”.", "FIG.", "2C shows a pattern called “Rectangular”.", "FIG.", "2D shows a “Hexagonal” cell 22 in the texture 20.FIG.", "2E shows a cell 22 pattern “Rings”.", "Attention is drawn to 3A showing the wrapping feature in more detail.", "FIG.", "3A shows the Hexagonal cell 22 texture 20 and, immediately below, a tessellation of five textures 20 in FIG.", "3B showing how the Hexagonal cells 22 form a continuous pattern at every edge 24, 26, 28, 30 of the texture.", "In this case, the Hexagonal cells 22 form a complete tessellation.", "FIGS.", "4A and 4B show the same process as FIGS.", "3A and 3B, this time using the “Rings” cells 22 otherwise shown in FIG.", "2E.", "In this case the Rings simply match at the edges 24, 26, 28, 30 and do not form, in the classic sense, a tessellation.", "FIGS.", "4A and 4B show that the essence, of the “wrapping” process wrapability depends on there being a match across the edges 24, 26, 28, 30.The surface, to be textured, is covered with a texture 20.One of the various types of texture 20 is selected.", "The cell 22 types , being simple, can either be generated by a mathematical algorithm or simply stored and retrieved with little use of memory.", "One of the cell 22 types is selected.", "FIG.", "5 illustrates one possible way how cells 22 can cover an object.", "An object 32, generally represented in this example as a heptagon, is covered in an array of cells 22 of a single texture 20 to at least its edge.", "The cells 22 will be visible, in the completed image, only to the edge of the object 32, (cropped at the edge of the object 32).", "There must, however, be a degree of overlapping so that this condition can be fulfilled.", "In practice, it is assumed that the texture repeats infinitely many times across the whole plane.", "Alternatively, the texture 20 can be “wrapped” around the object or “projected onto” the object, in a manner known in the art.", "The object 32 is either a two dimensional surface, or a two dimensional projection of a three dimensional surface, being part of an image generated by, for example, but not restricted to, United Kingdom Patent Application Number 9926131.5“Image Enhancement” filed 5 Nov. 1999 by the same applicants.", "Any other object 32 generated by any other means can be the subject of the present invention.", "All that is required is that the object 32 has a surface capable of accepting a texture.", "FIG.", "6 shows a texture 20.The texture 20 comprises a plurality of spaced pixels 34 (here shown more widely spaced and fewer in number than in reality) each having its own coordinates within the texture 20.As an example, shown in FIG.", "6 is the individual mth pixel, 34m, has its X and Y coordinates X(m), Y(m).", "The texturing process, the subject of the present invention, is effectively a mapping from the pixel coordinates X(m) Y(m) to a new positional and colour value.", "In FIG.", "7 is a projected view of a section of the individual is shown imposed, where the X Y plane is the plane of the pixel 34, and is located on the plane surface at Z=0.Also shown is a lower bounding plane 38, located at the value Z=−0.5 and an upper bounding plane 40, located at the value Z=+0.5.The first action in the texturing operation is to assign a number to each of the pixels 34.The assigned number is illustrated, in a figurative way in FIG.", "8, as a Z value on the axes 36.Each pixel 34 is shown displaced along the Z axis by its assigned value.", "FIGS.", "8A to 8D show successive stages in the assignment of Z values to a pixel 34, and FIG.", "8E shows a flowchart of the assignment process.", "Attention is drawn to FIGS.", "8A to 8D.", "A value is assigned to each pixel 34 using a pseudo random methos which generates a set of values known as a plasma field.", "A seed number is selected, and a roughness value.", "The seed number is applied to a pseudo-random number generation process, which supplies pseudo-random values when required.", "Initially, a pseudo-random value between −0.5 and +0.5 is assigned to each corner pixel 34′ in the plasma field, and an initial step value of ½ is assigned.", "Three pseudo-random values, P,Q and R, between −0.5 and +0.5 are selected such that their sum is 0.The midpoint pixel 34″ along the top edge is selected and assigned a value which is the average of the two values of the adjacent corner pixels 34′, added to the previously selected pseudo-random value P multiplied by the current step value.", "The midpoint pixel 34′″ along the left edge is selected and assigned a value which is the average of the two values of the adjacent corner pixels, added to the previously selected pseudo-random value Q multiplied by the current step value.", "The centre pixel 34″″′, is assigned a value which is the average of the four corner pixels, added to the previously selected pseudo-random value R multiplied by the current step value.", "The field is then broken into four smaller squares, each half the size the step value is halved, and the process is then repeated with each smaller square starting with the selection of new pseudo-random values P′,Q′and R′, until all pixels 34 in the field have been assigned a value.", "The assigned roughness is used to modify the initial step, where the corner pixels 34′ are given their initial pseudo-random values.", "If the roughness is non-zero, then this step is applies to the smaller squares at a number of subsequent stages also, that number being determined by the roughness value.", "Thus the user is able to select a second feature for the texture.", "Attention is drawn to FIG.", "8E which shows a flow chart of the Z value assignment process.", "From and entry 11 a set up operation 13 places the seed number in the pseudo-random number generator, initialises the pseudo-random number generator and sets the roughness value.", "Thereafter, a corner test 15 checks to see if the corner pixels 34′ have come into contact with each other.", "This signals the end of the process.", "If they have, the assignment operation exits to a return 17, if it has not, a roughness test 19 checks to see if roughness is to be applied.", "If it is, a roughness operation 21 assigns random values to the corner pixels 34′.", "Both the roughness test 19 and the roughness operation 21 pass control to an edge centre operation 23 which assigns values to the edge centre pixels 34″, 34′″ thereafter a centre pixel operation 25 assigns a value to the centre pixel 34″″.", "A division operation 27 then divides the field into four equal regions.", "A repeat operation 29 repeats the process for each smaller region until the corner test 15 detects that the entire field has been covered, each pixel 34 having been assigned a Z value.", "FIGS.", "9A to FIG.", "9F illustrate the effects of roughness on a final image.", "In FIG.", "9A, the roughness value is set to zero and the image is substantially smooth.", "In FIG.", "9B the roughness value is set to 1 and the image becomes a little finer in detail.", "FIGS.", "9C, 9D, 9E and 9F have respective roughness values of 2, 3, 4, 5, and 6.The effect on the final image is clear from the figures.", "FIG.", "9A shows a surface which might, for example, appear on a piece of satin fabric, whereas FIG.", "9F resembles sandpaper.", "Just by varying the roughness value on the same set of pixels 34 on a texture 20, with the same pseudo random number generator seed number, all these different effects can be achieved.", "The next action in the texture forming operation is “warping”.", "Warping is the large-scale deformation of the texture.", "It can be used to create controlled curves, zig-zags and ripples in the final image.", "For each axis X, Y in the texture, a warping function is defined by the user.", "For every X coordinate the Y warping returns an off-set to be applied to the Y coordinate.", "Similarly for every Y coordinate, the X warping function returns an off-set to be applied to the X coordinate.", "The warping functions are defined by a list of pairs of values a, b representing points on a curve.", "FIG.", "10A shows a linear interpolation used in warping.", "The points 42, selected by the texture designer, of the warping curve, are shown, as separate locations on a control icon 44.In this example, a smooth option has not been selected.", "Consequently, the interpolation between the points 42 is linear.", "A display 46 shows the resulting warping function comprising straight line segments.", "FIG.", "10B shows the same set of points 42 as is shown in FIG.", "10A, but where the smooth option 48 has been selected.", "The curve, on the display 46, now smoothly interpolates between the points 42, with no sharp angles or straight lines.", "For preference, the smoothing function used in FIG.", "10B is a Hermite spline curve.", "This is just one of many smoothing functions which can be used in the invention.", "For example a Bezier function, or a Bezier spline, can equally be used.", "Those, skilled in the art, will be aware of numerous other functions which can fulfill this purpose.", "FIGS.", "11A to 11E show examples of the effects of various warping functions.", "FIG.", "11A shows the initial unwarped cell, laid out as a chequerboard for clarity of illustration of effect.", "FIG.", "11B shows linear warping in the X axis.", "FIG.", "11C shows smooth warping in the X axis.", "FIG.", "11D shows smooth warping in the Y axis.", "FIG.", "11E shows simultaneous smooth warping in the X axis and the Y axis.", "The texture designer, is thus able to add a further effect to the texture.", "To warp the texture, the designer selects points 42 and a linear or smooth option to achieve the desired effect.", "The next stage in texture design, if required, enables the texture designer to apply distortion.", "A variable amount of pseudo-random distortion can be applied to a texture.", "This can be used to change a regular cell pattern, such as a simple square grid, into something that looks more “organic”, perhaps similar to the scales on a crocodile skin.", "FIG.", "12 is a flow chart showing the manner in which a texture 20 is warped.", "Entry 50 is to a first operation 52 where the warp curve points 42 (from FIG.", "9 and FIG.", "10) are entered by the texture designer and the preferred manner of interpolation is selected.", "Thereafter, a second operation 54 performs the interpolation to produce the curves (as seen on the displays 46 of FIGS.", "9 and 10).", "A third operation 56 then selects the first pixel 34 in the texture 20.Next, a fourth operation 58 finds the y warping factor.", "To do this, it goes to the curve generated in the second operation 54 at the X value of the pixel 34 which is being processed.", "The “warp(y)” value, used in a later operation, is the value of the curve at the X value of the pixel 34.A fifth operation 60 then finds the x warping factor.", "To do this, reference is made to the interpolated curve generated in the second operation 54.The Y value of the pixel 34 being processed is selected, and the value of the X coordinate, on the curve, is taken.", "This is the x warping factor “warp(x)”.", "A sixth operation 62 then calculates the warped x position of the pixel 34 w(x) by adding the X coordinate of the pixel 34 to the product of the X coordinate of the pixel 34 and the y warping factor warp(y).", "A seventh operation 64 then calculates the warped y position of the pixel 34 w(y) by adding the Y coordinate of the pixel 34 being processed to the product of the Y coordinate of the pixel being processed and the x warping factor warp(x).", "Thereafter, a first test 66 checks to see if the last pixel 34 in the texture 20 has been processed.", "If it has, the routine goes to an exit 68.If it has not, control goes to an eighth operation 70 which selects the next pixel 34 in the texture 20 and passes control to the fourth operation 58.In this way, each pixel 34 in the cell 22 is processed in turn until all of the pixels 34 have been processed.", "FIG.", "13 is a flow chart of the process whereby distortion is added to a texture 20.From an entry 72, a ninth operation 74 selects the first pixel 34 in the texture 20.The coordinates of a pixel 34 that the distortion process selects are not the initial coordinates of a pixel 34, X, Y which form the starting point of the process of FIG.", "12, but, rather, the warped X and Y coordinates from the warping process of FIG.", "12, namely W(x), W(y).", "If there had been no warping, the coordinates of the pixel 34 would have remained as X, Y.", "These would then be the values of W(x), W(y) had no warping taken place.", "Thereafter a tenth operation 76 goes to the initial plasma field and reads the Z value (see FIG.", "8) at a pixel 34, displaced from the warped coordinate W(x), W(y), in the Y direction by one quarter of the Z value (see FIG.", "8) assigned to the pixel 34 being processed.", "This produces an X distortion factor f(x).", "An eleventh operation 78 then reads the Z value of a pixel 34, displaced from the pixel 34 being processed, in the Y direction by one half of the Z value assigned to the pixel being processed (see FIG.", "8).", "A twelfth operation 80 then selects a width distortion factor D(w).", "A thirteenth operation 82 similarly, selects a height distortion factor D(h).", "Both the distortion factors D(w), D(h), are provided by the texture designer as another input variable to the final texture.", "Thereafter a fourteenth operation 84 calculates the distorted X value d(x) by summing the warped X coordinate W(x) with the product of the X distortion factor f(x) found in the tenth operation 76, and the width distortion factor D(w), selected in the twelfth operation 80.A fifteenth operation 86 then calculates the distorted Y value d(y) by summing the warped Y coordinate W(y) of the pixel 34 being processed with the product of the Y displacement factor f(y), found in the eleventh operation 78, and the height distortion factor D(h), selected in the thirteenth operation 82.A second test 88 then checks to see if the last pixel 34 in the texture 20 has been processed.", "If all the pixels 34 in the texture 20 have been processed the distortion process passes to exit 90.If the last pixel 34 has not been processed, a sixteenth operation 92 selects the next pixel 34 to be processed and passes control to the tenth operation 76.In this way, all of the pixels 34 in the texture 20 are processed, one by one, until the whole texture 20 is complete.", "FIGS.", "14A to 14F show examples of different degrees of distortion, on a simple square cell 22 pattern for clarity of visible effect.", "FIG.", "14A shows the texture 20 with no warping or distortion.", "FIG.", "14B shows the grid of FIG.", "14A with slight distortion.", "FIG.", "14C shows the grid of FIG.", "14A with a little bit more distortion than FIG.", "14B.", "FIG.", "14D shows the grid of FIG.", "14A with a fair degree of distortion.", "FIG.", "14E shows the grid of FIG.", "14A with a great deal of distortion.", "FIG.", "14F shows the grid of FIG.", "14A with the maximum distortion.", "The regular grid pattern of time distortion has reached the levels of FIG.", "14F achieved an appearance which is apparently, to the eye, chaotic rather than regular.", "Such a pattern as shown in FIG.", "14F emulates the surface of materials like marble.", "FIG.", "15 is a flow chart of the next activity in the preparation of a texture.", "Having the performed the distortion activity described in FIG.", "13, it now becomes necessary to find out, so that further processing can take place, where all of the pixels 34 have moved to.", "From entry 94, a seventeenth operation 96 selects the first pixel 34 according to the distorted outputs D(x), D(y) from the operation shown in FIG.", "13.An eighteenth operation 98 then calculates which cell 22 the pixel 34 under scrutiny actually occupies now that it has been moved around.", "The cell 22 shape and size is chosen by the texture designer.", "The analysis is made on the distorted coordinates d(x), d(y) where the pixels 34 have been moved by any warping and distortion which may have been applied.", "If no warping or distortion has been applied, of course, the pixel 34 remains in its original position X,Y.", "The designer of the texturing can choose various types of cell, shown in FIG.", "2.The cell 22 width and height are specified by the designer, and are usually constrained to provide tileability) such that a whole number of cells 22 will fit across and down the image.", "Using the boundaries of the selected cell 22 type and size, the pixel 34 is allocated to a specific cell 22.The position of the pixel 34 within the cell 22 is also used to determine the Xoffset 134 and Yoffset 136 values as shown in FIG.", "18.A third test 100 checks to see if the last pixel 34 has been processed.", "If it has, the activity proceeds to exit 102.If it has not, a nineteenth operation 104 selects the next pixel 34 and returns control to the eighteenth operation 98.In this way, all the pixels 34 are processed and allocated to a cell 22.Attention is drawn to FIG.", "16 showing the first stage of colouring the texture 20.From an entry 106 a twentieth operation 108 selects a first weighting function “aweigh” and a second weighting function “bweigh”.", "These are sets of coefficients for multiplication with properties of a “pixel vector”.", "These will be used, as described hereafter in relation to FIG.", "17, to assist in the calculation of a colour value for each pixel 34.A twenty-first operation 110 then selects the first pixel 34 in the texture.", "A twenty-second operation 112 then calculates a first colour value A according to the equation: A=0.5+sum(aweigh{i}*value{i})/sum(abs(aweigh{i})) “Abs” is the absolute value of aweigh{i}.", "Value{i} is explained in FIG.", "17.The sum is taken over the pixel vector for the pixel being processed.", "Thereafter a twenty-third operation 114 calculates a second colour value B according to the equation shown in the box of the twenty-third operation 114.B=0.5+sum(bweigh{i}·value{i}/sum(abs(bweigh{i})) A fourth test 116 checks to see if the last pixel 34 has been processed.", "If it has not, a twenty-fourth operation 118 selects the next pixels 34 in the texture 20 and returns control to the twenty-second operation 112 so that all the pixel 34 can be processed in turn.", "If the fourth test 116 finds that the last pixel 34 has been processed, the process then goes to exit 122.In this way, each pixel 34 in the texture 20 is allocated an A value and a B value.", "Attention is now drawn to FIG.", "17 which shows, in tabulated form, the elements in the equations shown in the twenty-second operation 112 and the twenty-fourth operation 114 of FIG.", "16.While only the equation in the twenty-second operation 112 is explained, it is to be understood that the equation in the twenty-third operation 114 behaves in just the same way, except that a different weighting table bweigh is used.", "A first column 124 shows the elements that make up the vector {i} which is used to characterise each pixel 34 in the texture 20.A second column 126 shows the aweigh weighting function where each of the elements a1-a16 corresponds to one of the 16 separate elements in the first column 124.A third column 128 shows the result of multiplying the first column 124 with the second column 124.Each element x,y etc is multiplied by its respective coefficient a1, a2 etc in the second column to produce the corresponding result in the third column 128.Finally for each pixel 34, the terms appearing in the equations in the twenty-second operation 112 and the twenty-third operation 114 are created.", "Value(i) is the value of each of the elements in the first column 124.Aweigh{i} is the value of each of the elements in the third column 128.The particular coefficients a1-a16 in the second column 126 can be chosen by the user when creating a texture, or can be fixed values.", "Certainly, when reconstructing an image, they must be the same values used for its creation.", "The aweigh weighting function has different coefficients a1-a16 than appear in the corresponding positions in the bweigh weighting function.", "For example, sum(aweigh{i}·value{i})=[(x·a1)·(x)+(y·a2)·(y)+ .", ".", ".", "+(pixelrand·a16)·(pixelrand)] Attention is drawn to FIG.", "18 explaining some of the elements shown in the first column 124 of FIG.", "17.An origin 130 is shown on a texture 20 at one of its corners.", "A pixel starting point 132, in one of the cells 221 has its X offset and Y offset measured from the edge of the cell 22′, as indicated by first and second arrows 134, 136.The X offset and Y offset are thus the coordinates of the pixel starting point 132 within its particular cell 22′.", "A warped position 138 has coordinates w(x), w(y) measured from the origin 130 of the texture 20.The warped position 138 is where the starting point 132 ends up after the warping function, shown in FIG.", "10, has been applied.", "Equally a warped and distorted position 140, where the pixel 34 arrives having been distorted away from the warped position 138, has coordinates d(x) and d(y), also measured from the origin 130 of the texture 20.Attention is drawn to FIG.", "19 explaining the term radial in the first column 124 of FIG.", "17.A line is drawn from the cell 22 centre 142, through the position of the pixel 34 to intersect an ellipse 144 which bounds the cell 22.This ellipse has the same ratio of width to height as the cell that it bounds.", "For a square cell, the ellipse has equal width and height and is therefore a circle.", "The line is used as a scale, with the value 0 being given at the cell centre 142 and the value 1 being assigned at the point 146 of intersection with the ellipse 144.The value “radial” is then the proportional point on the line occupied by the coordinates of the pixel 34.In the example shown, it can be seen that “radial” has a value of about 0.25.“The value of radial” is always less than 1.FIG.", "20 illustrates the term “edgewards”; also shown in the first column 124 of FIG.", "17.Instead of the line from the centre 142 of the cell 22 through the pixel 34 being extended to the ellipse 144, the line is terminated at a cell edge intersection point 148.Once again, the line to the edge of the cell 22 is used as a scale and the centre of the cell 142 is assigned the value 0 and the cell edge intersection point 148 is assigned the value 1.The proportional position of the pixel 34 along the line gives the value “edgewards”.", "In the example shown, the value of “edgewards” is around 0.5.The value of “edgewards” is always less than 1.Also shown in FIG.", "20 is the term “angle” which is simply the angle, between the line that was used to create “edgewards” and “radial” and the X axis.", "Likewise, the term “cellplasma” is the Z value (see FIG.", "8) of the plasma field at the centre of the cell 142.“Pixelplasma” is the value of the pixel 34 on the plasma field (the Z value, as shown in FIG.", "8) at the point d(x), d(y) indicated by the numeral 150 in FIG.", "20.Similarly, “pixelplasma2” is the value of the plasma field at the point d(x),((z/2)-d(y)), where z is the z value derived at d(x), d(y).", "The term “cellrand”, used in the first column 124 of FIG.", "17, is a pseudo random value, derived from the cell coordinates.", "The corner, of the cell 22, from which xoffset and yoffset are measured, gives the coordinates within the texture 20 of the cell 22.The cell coordinate is used as the seed for a pseudo random number generator.", "The result of the pseudo random generated number is “cellrand”.", "The term “pixelrand” is a pseudo random value derived from the coordinates of the pixel 34 (x,y) and, again, is a pseudo random number in which the pixel 34 coordinates (x,y) are the seed.", "Each of the values in the first column 124 of FIG.", "17 is scaled so that it lies between 0 and 1.Thus, the equations used in the twenty-second operation 112 and the twenty-third operation 114 always yield a value between 0 and 1.The A Value and the B value represent colour coordinates by which the individual pixels are coloured.", "The A and B values can further be processed by employing a warping function, similar to that shown in FIG.", "10.Attention is drawn to FIG.", "21 where the A colour value and the B colour value are used to determine the colour of each pixel 34.The texture designer selects four colours C1, C2, C3 and C4 to represent the corners of a unit square 52.The colours C1 to C4 are specified, in this instance, by a 24 binary digit number which identifies their colour.", "Which colour C1 to C4 goes on which corner of the selection square 152 is entirely up to the texture designer.", "The texture designer can specify any colour they to occupy any of the corners.", "The pixel colour selection square 152 can be imagined as being filled with a field of all of the different mixed hues and saturations available from the corner colours C1 to C4.One of the sides of the selection square is calibrated as a scale 0 to 1 and this is the axis of selection for the A colour selection value.", "Likewise, an adjacent side of the square is also calibrated 0 to 1 and this is used as a selection axis for the B colour value.", "Whatever the value of A and B for a particular pixel 34, the corresponding colour is selected from the selection square 152 at the coordinates A,B and applied to that pixel 34 in the completed texture.", "In use, the texture designer creates a texture by adjusting all of the variables herein before mentioned, until a satisfactory image of a texture is found.", "This is then stretched to fit the object 32 (FIG.", "7)in the manner of stretching a rubber sheet to fit.", "For example, on a sphere, the texture 20 may be wrapped around to envelop the surface.", "Equally, the texture 20 can be projected in the manner of a slide projector, onto the surface to be textured.", "It can be placed and cropped.", "Many other methods of mapping a texture onto a surface will be known to one skilled in the art.", "FIGS.", "22A to 22L show different textures which can be achieved.", "Since the drawings are in black and white it is impossible to represent the rich variation in colour.", "That will have to be imagined.", "FIG.", "22A has a close resemblance to brickwork.", "FIG.", "22B is a fabric.", "FIG.", "22C is a pink granite.", "FIG.", "22D is a blue marble.", "FIG.", "22E is galvanised steel.", "FIG.", "22F is snake skin.", "FIG.", "22G is leopard skin.", "FIG.", "22H is a representation of a dawn sky.", "FIG.", "22I is wickerwork.", "FIG.", "22J is pine grain.", "FIG.", "22K is a cork tile.", "FIG.", "22L is a linoleum tile.", "It can be seen that, using the variables available and the techniques employed, may different textures can be achieved.", "FIG.", "23 is a flow chart of the activity of the image generation source 10 shown in FIG.", "1.The image generation source 10 can be a computer, a URL, an ISP or any other device from which a representation of an image may be sent.", "From an entry 154 a twenty-sixth operation 156 has the image generation source 10 interrogate the mobile telephone 16, or any other device which is to receive the image to determine whether or not the texture programme is stored in that device.", "If a fifth test 158, on receiving a response from the device which is to receive the image, detects that the programme is present, the operation goes to exit 160.If the fifth test 158 detects that the texture programme is not present in the device to receive the image, a twenty-seventh operation 162 has the image generation source 10 send the texture programme to the device 16 so that the device 16 can interpret a texture.", "Once the texture programme is in the device, the operation, shown in FIG.", "23, proceeds to exit 160.FIG.", "24 shows the activity of the image generation source 10 when sending an image to a device 18.From an entry 164 a twenty-eighth operation 166 first sends the image to the mobile phone, computer or other device which is to show the image.", "Thereafter a twenty-ninth operation 168 has the image generation source 10 send a texture string to the mobile phone 18 or computer, or other device, that is to generate the image.", "As will be explained hereafter, the texture string is a simple concatenation, in known order, of all of the selectable variables which determine a texture.", "Once the texture string has been sent to the mobile phone or computer, this operation proceeds to exit 170.Attention is drawn to FIG.", "25 showing the activity of the cell phone 18, computer or other device which is to regenerate an image.", "From an entry 172 a thirtieth operation 174 either retrieves the 3-D object which is to be textured from a memory, or receives the 3-D object from an outside source such as the image generation source 10.A thirty-first operation 176 then either retrieves from memory, or receives from an outside string which defines the texture to be applied to the three dimensional object.", "A thirty-second operation 178 then, having received or retrieved the texture string containing the concatenated variables which define the required texture, generates that texture for application to the object.", "A thirty-third operation 180 then applies the surface texture to the object by projection, wrapping, or any other technique known in the art.", "Thereafter, the thirty-third operation 180 exits 182.Attention is drawn to FIG.", "26, showing the various ways in which a texture can be employed.", "The image generation source 10 can store or retrieve the texture programme and texture strings, for use in providing surface texture on objects, in a disc data store 184 such as hard disc.", "The programmes and texture strings can likewise be stored and retrieved on removable media 186 such as a floppy disc, pre-recorded or re-writeable compact discs 188, fixed or removable magnetic tapes 190, or in a memory 192 which can be a RAM, ROM, electrically alterable ROM, or any other electronic or physical device which can store a volatile or non-volatile record.", "Likewise, the image generation source 10 sends messages 194, via a telecommunications network or internet service or telephone system as earlier described, to and from the remote devices such as the mobile phone 18 or a remote computer terminal 196.The messages from the image generation source 10 include texture strings and, on occasions, the texture programme.", "FIG.", "27 illustrates the texture string, a serial concatenation of binary words or binary digits, sent by the image generation source 10 to the device 18, 194, or stored and retrieved from a memory 184, 186, 188, 190, 192 for the reconstruction of a texture.", "Although the elements are here given a specific order, it is to be understood that a different order can be allocated within the invention, and some elements omitted and new elements added.", "A first element 200 conveys the seed for the pseudo random number generator which generates the z values for each pixel 34 as illustrated in FIG.", "8.A second element 202 contains the roughness value discussed in relation to FIG.", "9.A third element 204 contains the coordinates of the warp function points illustrated in FIGS.", "9 and 10.A fourth element 206 conveys the warp mould, selecting either a smooth or linear interpolation as discussed in connection with FIGS.", "9 and 10.A fifth element 208 contains the width distortion factor (Dw) described in relation to FIG.", "13.A sixth element 210 contains the height distortion factor (Dh) also described in relation to FIG.", "13.A seventh element 212 contains data to select the cell style, as illustrated in FIG.", "2A to FIG.", "2E.", "An eighth element 214 contains information to determine the cell size.", "A ninth element 216 contains either an indication of the first weighting function aweigh, or the values of another weighting function for use in place of aweigh, as described in relation to FIG.", "16.A tenth element 218 conveys either an indication of the second weighting function bweigh, or the values of a second weighting function to be used in its place.", "This is also described in relation to FIG.", "16.An eleventh element 220 contains C1, the colour to be used in a first corner of the selection square 152, illustrated in FIG.", "21.A twelfth element 222 contains an indication of the second colour C2 to be used on a second corner of the selection square 152.A thirteenth element 224 contains indication of a third colour C3 to be used on a third corner of the selection square 152.A fourteenth element 226 contains an indication of the fourth colour C4 to be used on the fourth corner of the selection square 152.Finally, and optionally a fifteenth element 228 contains an indication as to which object the texture, defined in the previous elements, is to be applied.", "The texture string 198 may be sent as a serial data stream over a radio wave or down a telephone line, using a carrier, or not using a carrier as is appropriate.", "It may also be sent and stored as a series of parallel words.", "This small amount of data is sufficient to generate complex textures and offers advantage in speed, bandwidth and storage.", "It also has the advantage that the texture program is small and fast, making it suitable for use in low capacity devices such as mobile telephones and palm top computers." ] ]	Patent_10469656
[ [ "Cell visual characteristic-modifying sequences", "The present invention relates generally to peptides, polypeptides or proteins having one or more amino acids or one or more amino acid sequences which exhibit color-facilitating properties, either on their own or following interaction with one or more other amino acids and to nucleic acid molecules encoding same.", "Such peptides, polypeptides and proteins are referred to herein as “color-facilitating molecules” or “CFMs”.", "The present invention further provides genetic constructs for use in genetically modifying eukaryotic or prokaryotic cells and more particularly eukaryotic tissue so as to alter their visual characteristics or capacity for exhibiting same to a human eye in the absence of excitation by an extraneous non-white light or particle emission.", "The present invention, therefore, extends to eukaryotic or prokaryotic cells and more particularly eukaryotic tissue, which are genetically modified to produce CFMs and which thereby exhibit altered visual characteristics in the absence of excitation by an extraneous non-white light or particle emission.", "In one particular embodiment, the CFMs are used to alter the visual characteristics of plants and even more particularly flower color.", "In another embodiment, the present invention provides gels or coatings or similar biomaterials in the form of a biomatrix comprising the CFMs such as for use as a UV sink, in a sun screen, in cosmetics, as an expression marker or other reporter molecule or for use as a photon trap to increase light intensity." ], [ "1.An isolated nucleic acid molecule comprising a nucleotide sequence encoding a color-facilitating molecule (CFM) which, in a cell, alone or together with one or more other molecules imparts an altered visual characteristic to said cell when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission, wherein the CFM is derived from Anemonia majano, Anemonia sulcata, Clavularia sp.", "Zoanthus sp.", "Discosoma sp (e. g. Discosoma striata), Aequorea sp (e. g. Aequorea victoria).", "Anthozoa sp.", "Cassiopea sp.", "(e. g. Cassiopea xamachana), Millepora sp.", "Acropora sp (e. g. Acropora aspera and Acropora nobilis).", "Montipora sp.", "Poritesmurrayensis, Pocillopora damiconnis, Pavona descussaca, Acanthastrea sp.", "Platygyra sp or Caulastrea sp, and wherein the CFM comprises an amino acid sequence in its N-terminal end selected from the group consisting of SVIAK (SEQ ID NO: 5).", "(M)SVIAT (SEQ ID NO: 6).", "SGIAT (SEQ ID NO: 7).", "SVIVT (SEQ ID NO: 8) or SVSAT (SEQ ID NO :9), SVIAK QMTY KVNM SGT (SEQ ID NO: 16), and SVIAK QMTY KVYM SDT (SEQ ID NO: 17).", "2.-3.", "(canceled) 4.The isolated nucleic acid molecule of claim 1 wherein the CFM comprises an amino acid sequence selected from the list comprising SVIAT QMTY KVYM SGT (SEQ ID NO:.10), SVIAT QMTY KVYM PGT (SEQ ID NO: 11), SVIAT QVTY KVYM SGT (SEQ_ID NO: 12), SGIAT QMTY KVYM SGT (SEQ ID NO:13), SVIVT QMTY KVYM SGT (SEQ_ID NO: 14), SVSAT QMTY KVYM SGT (SEQ ID NO: 15), SVIAK QMTY KVNM SGT (SEQ ID NO: 16), SVIAK QMTY KVYM SDT (SEQ ID NO: 17) and SVIAK QMTYXIX2YX3 SGT (SEQ_ID NO: 18), wherein XI, Xz and X3 may be any amino acid provided that XI is not K; Xz is not V; X3 is not M. 5.The isolated nucleic acid molecule of claim 4 wherein the CFM comprises an amino acid sequence selected from the list comprising SEQ ID NOs: 20,22, 24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68 70,72, 74, 76, 78, 80,82,84,86,88,90,92,94,96,98, 100,102,104,106,108,110,112, 114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148, 150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,190,192, 194,196,198,200 and 202 provided that, where the said amino acid sequence comprises the sequence SVIAK QMTYXIX2yX3SGT, Xi is not lysine,X2 is not valine, and X3 is not methionine or an amino acid sequence having at least 60% similarity to any one or more of the above referenced sequences.", "6.The isolated nucleic acid molecule of claim 5 comprising a nucleotide sequence encoding a color-facilitating molecule (CFM), wherein the nucleotide sequence is selected from the list comprising SEQ ID NOs.", "19,21,23,25,27,29,31,33,35,37,39, 41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89, 91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125, 127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161, 163,165,167,169,171,173,175,177,179,189,191,193,195,197,199 and 201 or a nucleotide sequence having at least 60% similarity to one or more of the above referenced sequences or a nucleotide'sequence capable of hybridizing to one of the above referenced sequences or a complementary form thereof under low stringency conditions.", "7.The isolated nucleic acid molecule of claim 4 wherein the cell is a prokaryotic cell.", "8.The isolated nucleic acid molecule of claim 4 wherein the cell is a eukaryotic cell.", "9.The isolated nucleic acid molecule of claim 8 wherein the eukaryotic cell is a mammalian animal cell.", "10.The isolated nucleic acid molecule of claim 8 wherein the eukaryotic cell is a non-mammalian animal cell.", "11.The isolated nucleic acid molecule of claim 10 wherein the eukaryotic cell is a plant cell.", "12.The isolated nucleic acid molecule of claim 11 wherein the plant cell is part of a plant callus or a whole plant.", "13.The isolated nucleic acid molecule of claim 12 wherein the whole plant is an ornamental or flowering plant or a part thereof.", "14.The isolated nucleic acid molecule of claim 13 wherein the plant part is a flower, root, leaf, stem, seed, fruit or fiber.", "15.The isolated nucleic acid molecule of claim 13 wherein the plant is selected froth a rose, carnation, lisianthus, petunia, lily, tulip, pansy, gerbera or chrysanthemum.", "16.The isolated nucleic acid molecule of claim 4 wherein the CFM is a GFP or derivative or homolog thereof.", "17.The isolated nucleic acid molecule of claim 16 wherein the homolog of GFP is a non-fluorescent GFP.", "18.An isolated color-facilitating molecule (CM) comprising a polypeptide which, in a cell, alone or together with one or more other molecules imparts an altered visual characteristic to said cell when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission, wherein the CFM is derived from Anemonia majano, Aneznonia sulcata, Clavularia sp, Zoanthus sp.", "Diycosoma sp (e. g.Discosoma striata).", "Aequorea sp (e.g.", "Aequorea victoria).", "Anthozoa sp.", "Cassiopea sp.", "(e. g. Cassiopea xamachana).", "Milleppora sp.", "Acropora sp (e. g. Acropora aspera and Acropora nobilis), Montipora sp.", "Poritesmurrayensis, Pocillopora damicorrrcis, Pavona descussaca, Acanthastrea sp, Platygyra sp or Caulastrea sp, and wherein the CFM comprises an amino acid sequence in its N-terminal end selected from SVIAK (SEQ ID NO: 5), (M)SVIAT (SEQ ID NO: 6), SGIAT (SEQ ID NO: 7), SVIVT (SEQ ID NO: 8) or SVSAT (SEQ ID NO: 9), SVIAK QMTY KVNM SGT (SEQ ID NO: 16), and SVIAK QMTY KVYM SDT (SEQ ID NO: 17).", "19.-20.", "(canceled) 21.The isolated CFM of claim 18 wherein the CFM comprises an amino acid sequence selected from the list comprising SVIAT QMTY KVYM SGT (SEQ ID NO 10),SVIAT QMTY KVYM PGT (SEQ ID NO:11), SVIAT QVTY KVYM SGT (SEQ ID NO: 12), SGIAT QMTY KVYM SGT (SEQ ID NO:13), SVIVT QMTY KVYM SGT (SEQ ID NO: 14), SVSAT QMTY KVYM SGT (SEQ ID NO: 15), SVIAK QMTY KVNM SGT (SEQ ID NO: 16), SVIAK QMTY KVYM SDT (SEQ_ID NO: 17) and SVIAK QMTYXIX2yX3 SGT (SEQ ID NO: 18) wherein_Xi, X2 and X3 may be any amino acid provided that Xi is not K;X2 is not V; X3 is not M. 22.The isolated CFM of claim 21 wherein the CFM comprises a polypeptide having an amino acid sequence selected from the list comprising SEQ ID NOs: 20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68 70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114, 116, 118,120,122, 124, 126,128,130,132 134,136,138,140,142,144,146,148,150, 152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,190,192,194, 196,198,200 and 202 provided that where the said amino acid sequence comprises the sequence SVIAK QMTYX, X2YX3 SGT, Xi is not lysine, X2 is not valine, and X3 is not methionine or an amino acid sequence having at least 60% similarity to any one or more of the above referenced sequences.", "23.The isolated CFM of claim 18 wherein the cell is a prokaryotic cell.", "24.The isolated CFM of claim 18 wherein the cell is a eukaryotic cell.", "25.The isolated CFM of claim 24 wherein the eukaryotic cell is a mammalian animal cell.", "26.The isolated CFM of claim 24 wherein the eukaryotic cell is a nonmammalian animal cell.", "27.The isolated CFM of claim 26 wherein the non-mammalian animal cell is a plant cell.", "28.The isolated CFM of claim 27 wherein the plant cell is part of a plant callus or a whole plant.", "29.The isolated CFM of claim 28 wherein the whole plant is an ornamental or flowering plant or a part thereof.", "30.The isolated CFM of claim 29 wherein the plant part is a flower, root, leaf, stem, seed, fruit or fiber.", "31.The isolated CFM of claim 29 wherein the plant is selected from a rose, carnation, lisianthus, petunia, lily, tulip, pansy, gerbera or chrysanthemum.", "32.The isolated CFM of claim 21 wherein the CFM is a GFP or derivative or homolog thereof.", "33.The isolated CFM of claim 32 wherein the homolog of GFP is a nonfluorescent GFP.", "34.An isolated cell wherein said cell or a parent cell is genetically modified to enable the production of a color-facilitating molecule (CFM) of claims 18 or 21 which alone or together with one or more other molecules imparts an altered visual characteristic to said cell when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "35.-38.", "(canceled) 39.The isolated cell of claim 34 wherein the cell is a prokaryotic cell.", "40.The isolated cell of claim 34 wherein the cell is a eukaryotic cell.", "41.-42.", "(canceled) 43.The isolated cell of claim 40 wherein the eukaryotic cell is a plant cell.", "44.The isolated plant cell of claim 43 wherein the cell is part of a plant callus or a whole plant.", "45.-76.", "(canceled) 77.An isolated antibody specific for a CFM, of any one of claims 18 or 21.78-114.", "(canceled)" ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.", "All-protein chromophores (pigments) have been isolated from the phylum Cnidaria (also known as Coelenterata).", "This phylum contains four classes: Scyphozoa, Cubozoa, Anthozoa and Hydrozoa.", "The first all-protein chromophore to be isolated, Green Fluorescent Protein (GFP), was cloned and sequenced from cDNA of the Hydrozoan Aequorea Victoria , commonly called jellyfish.", "Similar all-protein chromophores have been isolated from Anthozoans.", "Matz et al.", "( Nature Biotechnol.", "17: 969-973, 1999), used degenerative primers based on Aequorea victoria GFP nucleotide sequence to PCR amplify EDNA isolated from four of the five orders of Anthozoa: Stolonifera, Actiniaria, Zoanthidea, and Corallimorpharia.", "Lukyanov et al.", "( Journal of Biological Chemistry 275: 25879-25882, 2000) used the same methodology to isolate a non-fluorescent all-protein chromophore from Actiniaria.", "However, the methodology used was unable to isolate all-protein chromophores from the fifth order, Scleractinia.", "The Scleractinia are corals that form architecture for coral reefs.", "They are otherwise known as “hue” or “reef-building” corals.", "International Patent Publication No.", "WO 00/46233 and Dove et al.", "( Coral Reefs 19: 197-204, 2000) both relate to isolation of an all-protein chromophore derived from Scleractinia pigment protein from coral tissue (PPCT).", "All-protein chromophores isolated to date display a range of spectral properties which effect apparent color in specific environments.", "Color may be determined by absorption and/or fluorescence properties of the molecules as well as qualities of incident light.", "Spectral properties include absorption, excitation and emission energies, molar extinction coefficients, quantum yields and maturation parameters.", "In many cases, a simple amino acid substitution can have a dramatic effect on the polypeptide spectral parameters (e.g.", "Tsien, Ann.", "Rev.", "Biochem.", "67: 509, 1998; Lukyanov et al., 2000, supra).", "However, useful modifications of a particular molecule are limited, as directed and random mutagenesis of specific all-protein chromophores has failed to produce desired spectral features (Tsien, 1998, supra).", "The result is that all-protein chromophores isolated from different sources are finding specific application niches.", "One all-protein chromophores, primarily used as molecular marker, is GFP.", "This protein, when excited with either UV or blue light (maximally at 396 nm or 475 nm) emits green fluorescence (maximally at 500 nm) [Heim et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 91: 12501-12504, 1994].", "GFP mutants that are altered in their maximal excitation and emission characteristics have been generated by random mutagenesis (Crameri et al., Nature Biotechnology 14: 315-319, 1996).", "Other GFP mutants have been generated that have increased solubility and fluorescence (Davis and Vierstra, Soluble derivatives of green fluorescent protein (GFP) for use in Arabidopsis thaliana .", "Weeds of the World, The International Electronic Arabidopsis Newsletter ISSN 1358-6912, (Ed.", "Mary Anderson) vol 3ii, 1996).", "The fluorescence of GFP and its mutants has been exploited for non-invasive analysis and monitoring of biological samples in plants and other organisms for research purposes (Haseloff et al., Proc.", "Natl.", "Acad.", "Sci USA 94: 2122-2127, 1997; Hu and Cheng, FEBS Letters 369: 331-334, 1995; Wang and Hazelrigg, Nature 369: 400403, 1994).", "The use of these fluorescent GFPs, mutants and homologs as fluorescent marker pigments visible upon excitation by light of specific wavelengths is well documented (e.g.", "U.S. Pat.", "Nos.", "6,027,881 and 5,958,713; Japanese Patent No.", "11266883; International Patent Publication No.", "WO97/11094; U.S. Pat.", "No.", "5,625,048; International Patent Application No.", "PCT/US99/29472 and International Patent Publication No.", "PCT/AU00/00056).", "In contrast to other fluorescent proteins, the fluorescence of GOP is due to amino acid interaction within the molecule, generally after folding.", "A contiguous fluorophote-defining amino acid sequence of Ser-Tyr-Gly is modified upon folding to produce an extended aromatic system which imparts the characteristic green fluorescence to the mature protein (Cody et al., Biochemistry 32: 1212-1218, 1993; Ormö et al., Science 273: 1392-1395, 1996; Yang et al., Nature Biotechnol.", "14: 1246-1251, 1996).", "As stated above, GFP like molecules have been identified for nonbioluminscent Anthozoa species (Matz et al., 1999, supra) which provides evidence that GFP-like proteins are not necessarily components of bioluminescent systems but may just determine fluorescent coloration in animals (Lukyanov et al., 2000, supra).", "Other weakly fluorescent GFP homologs have been identified from Acropora formosa and Acropora digitifera (Dove et al., Biol.", "Bull.", "189: 288-297, 1995; Hoegh-Guldberg and Dove, 2000, supra; Salih et al., Nature 408: 850-853, 2000).", "All-protein chromophores are now finding application as molecular markers for monitoring polypeptide expression and localization in the fields of biochemistry, molecular and cell biology.", "The present invention now describes novel all-protein chromophores (or CFMs) as well as novel and useful applications of same.", "For example, the flower industry strives to develop new and different varieties of flowering plants, in particular through the manipulation of flower color.", "While classical breeding techniques have been used with some success to produce a wide range of colors for most of the commercial varieties of flowers, this approach has been limited by the constraints of a particular species gene pool.", "For this reason, it is rare for a single species to have a full spectrum of colored varieties.", "The development of blue varieties of major cut flower species such as rose, chrysanthemum, tulip, lily, carnation and gerbera, for example, has proved difficult and would offer a significant opportunity in both the cut flower and ornamental markets.", "Flower color is predominantly due to three types of pigment: flavonoids, carotenoids and betalains.", "Of the three, the flavonoids are the most common and contribute to a range of colors from yellow to red to blue.", "The flavonoid molecules which make the major contribution to flower color are the anthocyanins which are glycosylated derivatives of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin and are localized in the vacuole.", "Carotenoids are natural pigments that confer yellow, orange and red colors to flowers and fruit.", "In plants, these pigments are localized in chromoplasts in flowers, leaves, fruit and roots.", "Novel colors in ornamental plant and flowering plant species may be generated by modifying the anthocyanin pathway to produce novel anthocyanins and aurones (Davies et al., Plant Journal 13: 259-266, 1998) and to alter ratios of anthocyanins to co-pigments (Holton et al., Plant Journal 4: 1003-1010, 1993).", "Alternatively, the carotenoid biosynthetic pathway can be modified to produce novel flower colors Mann et al., Nature Biotech.", "18: 888-892, 2000).", "The levels of anthocyanin production can also be increased by the expression of heterologous anthocyanin pathway gene regulatory factors (e.g.", "see Borevitz et al., Plant Cell 12: 2383-2393, 2000).", "These approaches have been used with some, albeit limited, success and alternative novel approaches are constantly being sought.", "In work leading up to the present invention, the inventors sought, inter alia, to identify novel color-facilitating molecules (CFMs) and to use same to modify the visual characteristics of eukaryotic or prokaryotic organisms by introducing into eukaryotic or prokaryotic cells, genetic material encoding CFMs which impart a color visible to a human eye in the absence of excitation by extraneous non-white light or particle emission.", "In a preferred embodiment, the CFMs are proteins such as GFPs or their relatives, such as non-fluorescent GFP-homologs.", "The use of CFMs to modulate the color of plants or plant parts such as flowers and seeds, represents a new approach to developing plant varieties having altered color characteristics.", "Other uses contemplated herein for the CFMs include their use as expression markers or as general reporter molecules, as a photon trap, UV sink and in sun screen or cosmetic or may be embedded in a gel matrix and be used to convert less visible light to wavelengths which are more visible.", "All such compositions are encompassed by the term “biomatrix”." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.", "Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).", "The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1, <400>2, etc.", "A sequence listing is provided after the claims.", "The present invention provides peptides, polypeptides and proteins having one or more amino acid sequences which exhibit color-facilitating properties, either on their own or following interaction with one or more amino acids as well as nucleic acid molecules encoding same.", "Preferably, the peptides, polypeptides and proteins or their nucleic acid molecules are derived from one or more Anemonia majano, Anemonia sulcata, Clavularia sp, Zoanthus sp, Discosoma sp (e.g.", "Discosoma striata ), Aequorea sp (e.g.", "Aequorea victoria ), Anthozoa sp, Cassiopea sp, (e.g.", "Cassiopea xamachana ), Millepora sp, Acropora sp (e.g.", "Acropora aspera and Acropora nobilis ), Montipora sp, Porites murrayensis, Pocillopora damicormis, Pavona descussaca, Acanthastrea sp, Platygyra sp or Caulastrea sp.", "These peptides, polypeptides and proteins are referred to as “color-facilitating molecules” (CFMs) and may be in isolated form, be produced within or on a cell or may form part of a biomatrix.", "Accordingly, in one aspect of the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a color-facilitating molecule (CFM) which, in a cell, alone or together with one or more other molecules imparts an altered visual characteristic to said cell when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "The present invention also provides an isolated CFM comprising a polypeptide which, in a cell, alone or together with one or more other molecules imparts an altered visual characteristic to said cell when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "The preferred CFM comprises the amino-terminal end of the polypeptide set forth in SEQ ID NOs: 5, 6, 7, 8 or 9.Particularly preferred CFMs comprise amino acid sequences selected from SEQ ID NOs:10, 11, 12, 13, 14,15, 16, 17 or 18.Even more preferably, the CFM is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 189, 191, 193, 195, 197, 199 and 201 or a nucleotide sequence capable of hybridizing to one of the above sequences or a complementary form thereof under low stringency conditions or a nucleotide sequence having at least about 60% similarity to any one of the above sequences.", "Amino acid sequences corresponding to the above nucleotide sequences correpond to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 190, 192, 194, 196, 198, 200 and 202 as well as an amino acid sequence having at least about 60% similarity to any one of the above sequences.", "The CFM may be in isolated form or part of a biomatrix wherein the biomatrix includes a cell, solid support, gel or bioinstrument.", "The CFMs are particularly useful in generating eukaryotic or prokaryotic cells exhibiting altered visual characteristics as well as biomatrices in the form of sun screen, UV traps, photon traps and illuminescent intensifiers.", "In a particularly preferred embodiment, the present invention provides transgenic plants and parts thereof including flowers, roots, leaves, stems, fruit and fibers exhibiting an altered visual characteristic." ], [ "FIELD OF THE INVENTION The present invention relates generally to peptides, polypeptides or proteins having one or more amino acids or one or more amino acid sequences which exhibit color-facilitating properties, either on their own or following interaction with one or more other amino acids and to nucleic acid molecules encoding same.", "Such peptides, polypeptides and proteins are referred to herein as “color-facilitating molecules” or “CFMs”.", "The present invention further provides genetic constructs for use in genetically modifying eukaryotic or prokaryotic cells and more particularly eukaryotic tissue so as to alter their visual characteristics or capacity for exhibiting same to a human eye in the absence of excitation by an extraneous non-white light or particle emission.", "The present invention, therefore, extends to eukaryotic or prokaryotic cells and more particularly eukaryotic tissue, which are genetically modified to produce CFMs and which thereby exhibit altered visual characteristics in the absence of excitation by an extraneous non-white light or particle emission.", "In one particular embodiment, the CFMs are used to alter the visual characteristics of plants and even more particularly flower color.", "In another embodiment, the present invention provides gels or coatings or similar biomaterials in the form of a biomatrix comprising the CFMs such as for use as a UV sink, in a sun screen, in cosmetics, as an expression marker or other reporter molecule or for use as a photon trap to increases light intensity.", "BACKGROUND OF THE INVENTION Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.", "All-protein chromophores (pigments) have been isolated from the phylum Cnidaria (also known as Coelenterata).", "This phylum contains four classes: Scyphozoa, Cubozoa, Anthozoa and Hydrozoa.", "The first all-protein chromophore to be isolated, Green Fluorescent Protein (GFP), was cloned and sequenced from cDNA of the Hydrozoan Aequorea Victoria, commonly called jellyfish.", "Similar all-protein chromophores have been isolated from Anthozoans.", "Matz et al.", "(Nature Biotechnol.", "17: 969-973, 1999), used degenerative primers based on Aequorea victoria GFP nucleotide sequence to PCR amplify EDNA isolated from four of the five orders of Anthozoa: Stolonifera, Actiniaria, Zoanthidea, and Corallimorpharia.", "Lukyanov et al.", "(Journal of Biological Chemistry 275: 25879-25882, 2000) used the same methodology to isolate a non-fluorescent all-protein chromophore from Actiniaria.", "However, the methodology used was unable to isolate all-protein chromophores from the fifth order, Scleractinia.", "The Scleractinia are corals that form architecture for coral reefs.", "They are otherwise known as “hue” or “reef-building” corals.", "International Patent Publication No.", "WO 00/46233 and Dove et al.", "(Coral Reefs 19: 197-204, 2000) both relate to isolation of an all-protein chromophore derived from Scleractinia pigment protein from coral tissue (PPCT).", "All-protein chromophores isolated to date display a range of spectral properties which effect apparent color in specific environments.", "Color may be determined by absorption and/or fluorescence properties of the molecules as well as qualities of incident light.", "Spectral properties include absorption, excitation and emission energies, molar extinction coefficients, quantum yields and maturation parameters.", "In many cases, a simple amino acid substitution can have a dramatic effect on the polypeptide spectral parameters (e.g.", "Tsien, Ann.", "Rev.", "Biochem.", "67: 509, 1998; Lukyanov et al., 2000, supra).", "However, useful modifications of a particular molecule are limited, as directed and random mutagenesis of specific all-protein chromophores has failed to produce desired spectral features (Tsien, 1998, supra).", "The result is that all-protein chromophores isolated from different sources are finding specific application niches.", "One all-protein chromophores, primarily used as molecular marker, is GFP.", "This protein, when excited with either UV or blue light (maximally at 396 nm or 475 nm) emits green fluorescence (maximally at 500 nm) [Heim et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 91: 12501-12504, 1994].", "GFP mutants that are altered in their maximal excitation and emission characteristics have been generated by random mutagenesis (Crameri et al., Nature Biotechnology 14: 315-319, 1996).", "Other GFP mutants have been generated that have increased solubility and fluorescence (Davis and Vierstra, Soluble derivatives of green fluorescent protein (GFP) for use in Arabidopsis thaliana.", "Weeds of the World, The International Electronic Arabidopsis Newsletter ISSN 1358-6912, (Ed.", "Mary Anderson) vol 3ii, 1996).", "The fluorescence of GFP and its mutants has been exploited for non-invasive analysis and monitoring of biological samples in plants and other organisms for research purposes (Haseloff et al., Proc.", "Natl.", "Acad.", "Sci USA 94: 2122-2127, 1997; Hu and Cheng, FEBS Letters 369: 331-334, 1995; Wang and Hazelrigg, Nature 369: 400403, 1994).", "The use of these fluorescent GFPs, mutants and homologs as fluorescent marker pigments visible upon excitation by light of specific wavelengths is well documented (e.g.", "U.S. Pat.", "Nos.", "6,027,881 and 5,958,713; Japanese Patent No.", "11266883; International Patent Publication No.", "WO97/11094; U.S. Pat.", "No.", "5,625,048; International Patent Application No.", "PCT/US99/29472 and International Patent Publication No.", "PCT/AU00/00056).", "In contrast to other fluorescent proteins, the fluorescence of GOP is due to amino acid interaction within the molecule, generally after folding.", "A contiguous fluorophote-defining amino acid sequence of Ser-Tyr-Gly is modified upon folding to produce an extended aromatic system which imparts the characteristic green fluorescence to the mature protein (Cody et al., Biochemistry 32: 1212-1218, 1993; Ormö et al., Science 273: 1392-1395, 1996; Yang et al., Nature Biotechnol.", "14: 1246-1251, 1996).", "As stated above, GFP like molecules have been identified for nonbioluminscent Anthozoa species (Matz et al., 1999, supra) which provides evidence that GFP-like proteins are not necessarily components of bioluminescent systems but may just determine fluorescent coloration in animals (Lukyanov et al., 2000, supra).", "Other weakly fluorescent GFP homologs have been identified from Acropora formosa and Acropora digitifera (Dove et al., Biol.", "Bull.", "189: 288-297, 1995; Hoegh-Guldberg and Dove, 2000, supra; Salih et al., Nature 408: 850-853, 2000).", "All-protein chromophores are now finding application as molecular markers for monitoring polypeptide expression and localization in the fields of biochemistry, molecular and cell biology.", "The present invention now describes novel all-protein chromophores (or CFMs) as well as novel and useful applications of same.", "For example, the flower industry strives to develop new and different varieties of flowering plants, in particular through the manipulation of flower color.", "While classical breeding techniques have been used with some success to produce a wide range of colors for most of the commercial varieties of flowers, this approach has been limited by the constraints of a particular species gene pool.", "For this reason, it is rare for a single species to have a full spectrum of colored varieties.", "The development of blue varieties of major cut flower species such as rose, chrysanthemum, tulip, lily, carnation and gerbera, for example, has proved difficult and would offer a significant opportunity in both the cut flower and ornamental markets.", "Flower color is predominantly due to three types of pigment: flavonoids, carotenoids and betalains.", "Of the three, the flavonoids are the most common and contribute to a range of colors from yellow to red to blue.", "The flavonoid molecules which make the major contribution to flower color are the anthocyanins which are glycosylated derivatives of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin and are localized in the vacuole.", "Carotenoids are natural pigments that confer yellow, orange and red colors to flowers and fruit.", "In plants, these pigments are localized in chromoplasts in flowers, leaves, fruit and roots.", "Novel colors in ornamental plant and flowering plant species may be generated by modifying the anthocyanin pathway to produce novel anthocyanins and aurones (Davies et al., Plant Journal 13: 259-266, 1998) and to alter ratios of anthocyanins to co-pigments (Holton et al., Plant Journal 4: 1003-1010, 1993).", "Alternatively, the carotenoid biosynthetic pathway can be modified to produce novel flower colors Mann et al., Nature Biotech.", "18: 888-892, 2000).", "The levels of anthocyanin production can also be increased by the expression of heterologous anthocyanin pathway gene regulatory factors (e.g.", "see Borevitz et al., Plant Cell 12: 2383-2393, 2000).", "These approaches have been used with some, albeit limited, success and alternative novel approaches are constantly being sought.", "In work leading up to the present invention, the inventors sought, inter alia, to identify novel color-facilitating molecules (CFMs) and to use same to modify the visual characteristics of eukaryotic or prokaryotic organisms by introducing into eukaryotic or prokaryotic cells, genetic material encoding CFMs which impart a color visible to a human eye in the absence of excitation by extraneous non-white light or particle emission.", "In a preferred embodiment, the CFMs are proteins such as GFPs or their relatives, such as non-fluorescent GFP-homologs.", "The use of CFMs to modulate the color of plants or plant parts such as flowers and seeds, represents a new approach to developing plant varieties having altered color characteristics.", "Other uses contemplated herein for the CFMs include their use as expression markers or as general reporter molecules, as a photon trap, UV sink and in sun screen or cosmetic or may be embedded in a gel matrix and be used to convert less visible light to wavelengths which are more visible.", "All such compositions are encompassed by the term “biomatrix”.", "SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.", "Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).", "The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1, <400>2, etc.", "A sequence listing is provided after the claims.", "The present invention provides peptides, polypeptides and proteins having one or more amino acid sequences which exhibit color-facilitating properties, either on their own or following interaction with one or more amino acids as well as nucleic acid molecules encoding same.", "Preferably, the peptides, polypeptides and proteins or their nucleic acid molecules are derived from one or more Anemonia majano, Anemonia sulcata, Clavularia sp, Zoanthus sp, Discosoma sp (e.g.", "Discosoma striata), Aequorea sp (e.g.", "Aequorea victoria), Anthozoa sp, Cassiopea sp, (e.g.", "Cassiopea xamachana), Millepora sp, Acropora sp (e.g.", "Acropora aspera and Acropora nobilis), Montipora sp, Porites murrayensis, Pocillopora damicormis, Pavona descussaca, Acanthastrea sp, Platygyra sp or Caulastrea sp.", "These peptides, polypeptides and proteins are referred to as “color-facilitating molecules” (CFMs) and may be in isolated form, be produced within or on a cell or may form part of a biomatrix.", "Accordingly, in one aspect of the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a color-facilitating molecule (CFM) which, in a cell, alone or together with one or more other molecules imparts an altered visual characteristic to said cell when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "The present invention also provides an isolated CFM comprising a polypeptide which, in a cell, alone or together with one or more other molecules imparts an altered visual characteristic to said cell when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "The preferred CFM comprises the amino-terminal end of the polypeptide set forth in SEQ ID NOs: 5, 6, 7, 8 or 9.Particularly preferred CFMs comprise amino acid sequences selected from SEQ ID NOs:10, 11, 12, 13, 14,15, 16, 17 or 18.Even more preferably, the CFM is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 189, 191, 193, 195, 197, 199 and 201 or a nucleotide sequence capable of hybridizing to one of the above sequences or a complementary form thereof under low stringency conditions or a nucleotide sequence having at least about 60% similarity to any one of the above sequences.", "Amino acid sequences corresponding to the above nucleotide sequences correpond to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 190, 192, 194, 196, 198, 200 and 202 as well as an amino acid sequence having at least about 60% similarity to any one of the above sequences.", "The CFM may be in isolated form or part of a biomatrix wherein the biomatrix includes a cell, solid support, gel or bioinstrument.", "The CFMs are particularly useful in generating eukaryotic or prokaryotic cells exhibiting altered visual characteristics as well as biomatrices in the form of sun screen, UV traps, photon traps and illuminescent intensifiers.", "In a particularly preferred embodiment, the present invention provides transgenic plants and parts thereof including flowers, roots, leaves, stems, fruit and fibers exhibiting an altered visual characteristic.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 shows a representation of multiple alignment of encoded amino acid sequences having SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 70, 72, 74, 76, 78, 80, 82, 84 and 86, representing polypeptides comprising an N-terminal SVIAK (SEQ ID NO:5) sequence.", "FIG.", "2 shows corresponding nucleotide sequence alignments of nucleic acid molecules, having SEQ ID NOs:19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85, encoding the polypeptides shown in FIG.", "1.FIG.", "3 shows a representation of multiple alignment of encoded amino acid sequences having SEQ ID NOs:88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 and 168, for polypeptides comprising an N-terminal (M)SVIAT (SEQ ID NO:6), SGIAT (SEQ ID NO:7), SVIVT (SEQ ID NO:8) and SVSAT (SEQ ID NO:9) sequences.", "FIG.", "4 shows corresponding nucleotide sequence alignments of nucleic acid molecules, having SEQ ID NOs:87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 and 167, encoding the polypeptides shown in FIGS.", "3A-3D.", "FIG.", "5 shows a representation of an alignment of amino acid sequences having SEQ ID NOs:170, 172, 174, 176, 178 and 180, for polypeptides comprising an N-terminal SVIAK sequence (SEQ ID NO:5) and a stop codon corresponding to amino acid residue 14.FIG.", "6 shows corresponding nucleotide sequence alignments for nucleic acid molecules, having SEQ ID NOs:169, 171, 173, 175, 177 and 179, encoding the polypeptides shown in FIG.", "5.FIG.", "7 is a nucleotide sequence alignment of SEQ ID NO:19 and SEQ ID NO:169, being nucleic acid sequences encoding polypeptides without and with a stop codon corresponding to amino acid residue 14, respectively.", "FIG.", "8 shows a representation of multiple alignment of amino acid sequences for polypeptides comprising an N-terminal SVIAK sequence (SEQ ID NO:5), including SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 70, 72, 74, 76, 78, 80, 82, 84 and 86, as well as sequences Aapat-1 (SEQ ID NO:181) and Aapat-2 (SEQ ID NO:182) which are disclosed in International Patent Publication No.", "WO 00/46233.FIG.", "9 shows amino acid sequence alignments of pigment polypeptides from coral tissue, grouped according to their N-terminal 5-amino acid sequence.", "The name and SEQ ID NO for each peptide is indicated, as well as the “Type” to which each has been assigned based on the identity of the 29 amino acids which are located within 5 Angstroms of the “QYG” fluorophore.", "These 29 individual, non-contiguous amino acid residues are also indicated, as are the individual non-contiguous variable amino acids residues throughout the polypeptides shown.", "FIG.", "10 is a diagrammatic representation of a generic bacterial expression vector based on pQE-30 (Qiagen), into which is inserted an ˜0.7 kb cDNA; depending on the source of the cDNA clone, each plasmid is designated as follows: pCGP2915-A10 clone from Acropora sp.", "; pCGP2916-A11 clone from Acropora sp.", "; pCGP2917-A12 clone from Acropora sp.", "; pCGP2918-A8 clone from Acropora sp.", "(SEQ ID NO:189); pCGP2920-D10 clone from Discosoma sp.", "(SEQ ID NO:191); pCGP2922-T3 clone from Tubastrea sp.", "(SEQ ID NO:195); pCGP2924-S3 clone from Sinularia sp.", "(SEQ ID NO:193); pCGP2919-D1 clone from Discosoma sp.", "(SEQ ID NO:197); pCGP2921-T1 clone from Tubastrea sp.", "(SEQ ID NO:201); pCGP2923-S1 clone from Sinularia sp.", "(SEQ ID NO:199).", "Abbreviations are as follows: bla=β-lactamase gene; ColElori=plasmid origin of replication.", "The locations of restriction endonuclease recognition sites for PstI, HindIII and BamHI are also marked.", "Refer to Example 3 for further details.", "FIG.", "11 is a graphical representation of examples of absorption scans of five “Type 1” (refer to text in Example 2 and Tables 6 and 7 for further detail) colored proteins showing extinction coefficients (ελmax) based on the method of Whitaker and Granum, 1980 (Anal.", "Biochem.", "109:156-159) for calculating protein concentration.", "x-axis=relative absorption; y-axis=wavelength (nm); (a) Rtms5.pep (SEQ ID NO:166), where ε592=111,000 M−1 cm−1; (b) LGasv-C.pep (SEQ ID NO:44) where ε591=53,000 M−1 cm−1; (c) Ce61-7sv.pep (SEQ ID NO:38) where ε591.5=104,000 M−1 cm−1; (d) PPd57-2ms.pep (SEQ ID NO:140) where ε593=67,000 M−1 cm−1; (e) Mims-C.pep (SEQ ID NO:126) where ε589=48,000 M−1 cm−1.FIG.", "12 a graphical representation of examples of absorption scans of three “Type 2” (A) and two “Type 12” (B) (refer to text in Example 2 and Tables 6 and 7 for further detail) colored proteins, showing extinction coefficients (ελmax) based on the method of Whitaker and Granum (Anal.", "Biochem.", "109: 156-159, 1980) for calculating protein concentration.", "x-axis=relative absorption; y-axis=wavelength (mn); (A) (a) PMms-B.pep (SEQ ID NO:130) where ε579.5=39,000 M−1 cm−1; (b) LGAsv-D.pep (SEQ ID NO:46) where ε579=72,400 M−1 cm−1; (c) rtsv-2.pep (SEQ ID NO:84) where ε579.5=75,000 M−1 cm−1; (B) (d) Misv-F.pep (SEQ ID NO:54) where ε579=111,000 M−1 cm−1 (e) Acasv-C.pep (SEQ ID NO:78) where ε579.5=32,300 M−1 cm−1.FIG.", "13 a graphical representation of examples of absorption scans of two “Type 6” (refer to text in Example 2 and Tables 6 and 7 for further detail) colored proteins, showing extinction coefficients (ελmax) based on the method of Whitaker and Granum (Anal.", "Biochem.", "109: 156-159, 1980) for calculating protein concentration.", "x-axis=relative absorption; y-axis=wavelength (nm); (a) LGAms-5.pep (SEQ ID NO:116) where ε583.5=71,000 M−1 cm−1; (b) Rtms-1.pep (SEQ ID NO:162) where ε554=44,000 M−1 cm−1.FIG.", "14 a graphical representation of (A) Absorption spectra and (B) Chromatogram of gel filtrated protein elution, both showing 95% confidence intervals for N=5, for raw phosphate buffer extract of two colour morphs of Acropora aspera (dark blue pigmented morph; cream morph).", "In (A), the estimation of blue-purple pocilloporin concentration per surface area of coral tissue is based on an extinction coefficient range of 50,000-100,000 M−1 cm−1.In (B), the chromatogram of gel filtrated protein elution is determined from 235 nm chromatograms and 280 nm chromatograms, applying the equation: 235 nm -280 nm)/2.51 (Whitaker and Granum, 1980, supra).", "The total area under the graph represents the total soluble protein.", "Blue-purple pocilloporin concentration is based on the difference between areas under the blue and cream graph in the range of pocilloporin clution (24-26.5 min).", "FIG.", "15 is a representation of multiple alignment of encoded amino acid sequences from T1 (SEQ ID NO:202), D1 (SEQ ID NO:198), S1 (SEQ ID NO:200), T3 (SEQ ID NO:196), D10 (SEQ ID NO:192), S3 (SEQ ID NO:194) and A8 (SEQ ID NO:190).", "FIG.", "16 is a representation of multiple alignment of encoded amino acid sequences from SVIAK (SEQ ID NO:5)-containing peptides T1 (SEQ ID NO:202), D1 (SEQ ID NO:198), S1 (SEQ ID NO:200), T3 (SEQ ID NO:196), D10 (SEQ ID NO:192), S3 (SEQ ID NO:194) and A8 (SEQ ID NO:190), together with the SVIAK (SEQ ID NO:5)-containing peptides shown in FIG.", "1, having SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 70, 72, 74, 76, 78, 80, 82, 84 and 86.FIG.", "17 is a diagrammatic representation of the yeast expression plasmid pCGP3269.The T1 cDNA (SEQ ID NO:201) cloned in a sense orientation behind the yeast glyceraldehyde 3-phosphate dehydrogenase promoter (PGAP) in the expression vector pYE22m.", "Abbreviations are as follows: TRP1=Trp1 gene, TGAP=terminator sequence from the yeast glyceraldehyde 3-phosphate dehydrogenase gene, IR1=inverted repeat of 2 μm plasmid, pBR322=origin of replication from E. coli.", "A selection of restriction enonuclase recognition sites are also marked.", "Refer to Example 7 for further details.", "FIG.", "18 is a diagrammatic representation of the yeast expression plasmid pCGP3270.The A8 cDNA (SEQ ID NO:189) cloned in a sense orientation behind the yeast glyceraldehyde 3-phosphate dehydrogenase promoter (PGAP) in the expression vector pYE22m.", "Abbreviations are as follows: TRP1=Trp1 gene, TGAP=terminator sequence from the yeast glyceraldehyde 3-phosphate dehydrogenase gene, IR1=inverted repeat of 2 μm plasmid, pBR322=origin of replication from E. coli.", "A selection of restriction enonuclase recognition sites are also marked.", "Refer to Example 7 for further details.", "FIG.", "19 is a diagrammatic representation of a plasmid, designated pCGP2756, which comprises a multiple cloning site from pNEB193 (New England Biolabs) between the CaMV (Cauliflower Mosaic Virus) 35S promoter and CaMV 35S terminator sequences, Abbreviations are as follows: Amp=ampicillin resistance gene; p35S=a promoter region from the CaMV 35S gene; t35S=a terminator fragment from the CaMV 35S gene.", "A selection of restriction endonuclease recognition sites are also marked.", "Refer to Example 9 for further details.", "FIG.", "20 is a diagramnmatic representation of the binary plasmid pCGP2757, which comprises the CaMV35S expression cassette of pCGP2756 (FIG.", "19) and a SuRB selectable marker gene.", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border, SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CAMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonay aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 9 for further details.", "FIG.", "21 is a diagrammatic representation of the binary plasmid pCGP2765, which comprises the A8 cDNA from Acropora sp.", "(SEQ ID NO:189) cloned into the binary vector pCGP2757 (FIG.", "20).", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border, SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; A8=cDNA from Acropora sp.", "(SEQ ID NO:189).", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 9 for further details.", "FIG.", "22 is a diagrammatic representation of the binary plasmid pCGP2769, which comprises the D1 cDNA from Discosoma sp.", "(SEQ ID NO:197) cloned into the binary vector pCGP2757 (FIG.", "20).", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; D1=cDNA from Discosoma sp.", "(SEQ ID NO:197).", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 9 for further details.", "FIG.", "23 is a diagrammatic representation of the binary plasmid pCGP2770, which comprises the S1 cDNA from Sinularia sp.", "(SEQ ID NO:199) cloned into the binary vector pCGP2757 (FIG.", "20).", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the: CaMV 35S gene; pVS1 a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; S1=cDNA from Sinulania sp.", "(SEQ ID NO:199).", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 9 for further details.", "FIG.", "24 is a diagrammatic representation of the binary plasmid pCGP2772, which comprises the T1 cDNA from Tubastrea sp.", "(SEQ ID NO:201) cloned into the binary vector pCGP2757 (FIG.", "20).", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; T1=cDNA from Tubastrea sp.", "(SEQ ID NO:201).", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 9 for further details.", "FIG.", "25 is a diagrammatic representation of the plasmid pCGP1116, which comprises a promoter fragment from a chalcone synthase (CHS) gene from Rosa hybrida cv.", "Kardinal.", "Abbreviations are as follows: Rose CHS=Rose chalcone synthase promoter fragment; ori=origin of replication; Amp=ampicillin resistance gene; Several restriction endonuclease recognition sites are also marked.", "Refer to Example 10 for further details.", "FIG.", "26 is a diagrammatic representation of the binary plasmid pCGP3255.The CaMV35S promoter of the 35S expression cassette of pCGP2757 (FIG.", "20) has been replaced with the rose chalcone synthase promoter fragment from pCGP1116 (FIG.", "25) Abbreviations are as follows: rCHS=rose chalcone synthase promoter fragment; TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC on=modified replicon from pACYC184 from E. coli.", "Refer to Example 10 for further details.", "FIG.", "27 is a diagrammatic representation of the bianry plasmid pCGP2782.The T1 cDNA from Tubastrea sp.", "(SEQ ID NO:201) was cloned into binary vector pCGP3255 (FIG.", "26) behind the rose chalcone synthase promoter fragment.", "Abbreviations are as follows: rCHS=rose chalcone synthase promoter fragment; TetR=the tetracycline resistance gene; LB=left border, RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid form Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; T1=cDNA from Tubastrea sp.", "(SEQ ID NO:201).", "A selection of restriction endonuclease recognition sites is also marked.", "Refer to Example 10 for further details.", "FIG.", "28 is a diagrammatic representation of the binary plasmid pCGP2773.The D1 cDNA from Discosoma sp.", "(SEQ ID NO:197) was cloned into binary vector pCGP3255 (FIG.", "26), behind the rose chalcone synthase promoter fragment.", "Abbreviations are as follows: rCHS=rose chalcone synthase promoter fragment; TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; D1=cDNA from Discosoma sp.", "(SEQ ID NO:197).", "A selection of restriction endonuclease recognition sites is also marked.", "Refer to Example 10 for further details.", "FIG.", "29 is a diagrammatic representation of the binary plasmid pCGP2774.The S1 cDNA from Sinularia sp.", "(SEQ ID NO:199) was cloned into binary vector pCGP3255 (FIG.", "26), behind the rose chalcone synthase promoter fragment.", "Abbreviations are as follows: rCHS=rose chalcone synthase promoter fragment; TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; S1=cDNA from Sinularia sp.", "(SEQ ID NO:199).", "A selection of restriction endonuclease recognition sites is also marked.", "Refer to Example 10 for further details.", "FIG.", "30 is a diagrammatic representation of the binary plasmid pCGP2780, which is plasmid pCGP2757 (FIG.", "20) from which has been removed a ˜290 base-pair SalI fragment to allow the creation of a unique BamHI restriction endonuclease site.", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli A selection of restriction endonuclease recognition sites is also marked.", "Refer to Example 11 for further details.", "FIG.", "31 is a diagrammatic representation of the binary plasmid pCGP2784, which is comprised of the ˜0.2 kb chloroplast transit-peptide from the small subunit of ribulose bisphosphate carboxylase gene (RBCase) from Nicotiana sylvestris, cloned into the multiple cloning site of pCGP2780 of FIG.", "30.Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; TSSU=chloroplast transit-peptide from the small subunit of RBCase of Nicotiana sylvestris.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 11 for further details.", "FIG.", "32 is a diagrammatic representation of the binary plasmid pCGP2781, which is plasmid pCGP2772 (FIG.", "24) from which has been removed a ˜290 base-pair SalI fragment to allow the creation of a unique BamHI restriction endonuclease site.", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodornonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli.", "T1=T1 cDNA from Tubastrea sp.", "(SEQ ID NO:201).", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 11 for further details.", "FIG.", "33 is a diagrammatic representation of the binary plasmid pCGP2785, which is comprised of the ˜0.2 kb chloroplast transit peptide from the small subunit of ribulose biphosphate carboxylase (RBCase) from Nicotiana sylvestris inserted into the CaMV 35S expression cassette of binary vector pCGP2781 (FIG.", "32), upstream of the T1 cDNA.", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=led border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli.", "T1=T1 cDNA from Tubastrea sp.", "(SEQ ID NO:201); TSSU=chloroplast transit peptide from the small subunit of RBCase from Nicotiana sylvestris.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 11 for further details.", "FIG.", "34 is a diagrammatic representation of the binary plasmid pCGP2787 which is comprised of the ˜0.2 kb chloroplast transit peptide from the small subunit of ribulose biphosphate carboxylase (RBCase) from Nicotiana sylvestris inselted into the Rose CHS expression cassette of binary vector pCGP2782 (FIG.", "27), upstream of the T1 cDNA.", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; rCHS=rose chalcone synthase promoter fragment; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli.", "T1=T1 cDNA from Tubastrea sp.", "(SEQ ID NO:201); TSSU=chloroplast transit peptide from the small subunit of RBCase from Nicotiana sylvestris.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 11 for further details.", "FIG.", "35 is a diagrammatic representation of the plasmid pCGP3257, which is comprised of the basic chitinase N-terminal endoplasmic reticulum (ER) transit peptide signal sequence from Arabidopsis thaliana inserted into the CaMV 35S expression cassette of binary vector pCGP2780 (FIG.", "30), downstream of the CaMV 35S promoter.", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; ERT=ER transit peptide signal sequence from Arabidopsis basic chitinase gene.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 11 for further details.", "FIG.", "36 is a diagrammatic representation of the binary plasmid pCGP3259.The T1 cDNA from Tubastrea sp.", "(SEQ ID NO:201)with an in-frame HDEL peptide sequence at the 3′ end was cloned into the CaMV 35S expression cassette of binary vector pCGP3257 (FIG.", "35), downstream of the ER transit-peptide signal sequence from Arabidopsis thaliana.", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeniginosa.", "pACYC ori=modified replicon from pACYC184 from E. coli; ERT:T1:HDEL=T1 cDNA clone from Tubastrea (SEQ ID NO:201) with an in-frame ER transit peptide sequence from Arabidopsis basic chitinase gene at the 5′ end and an HDEL ER retention sequence at the 3′ end.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 11 for further details.", "FIG.", "37 is a diagrammatic representation of the binary plasmid pCGP3262 which is comprised of the basic chitinase N-terminal endoplasmic reticulum (ER) transit peptide signal sequence from Arabidopsis thaliana inserted into the Rose CHS expression cassette of binary vector pCGP3255 (FIG.", "26), downstream of the Rose CHS promoter.", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; rCHS rose chalcone synthase promoter fragment; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; ERT=ER transit peptide signal sequence from Arabidopsis basic chitinase gene.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 11 for fisher details.", "FIG.", "38 is a diagrammatic representation of the binary plasmid pCGP3263.The T1 cDNA from Tubastrea sp.", "(SEQ ID NO:201) with an in-frame HDEL peptide sequence at the 3′ end was cloned into the Rose CHS expression cassette of binary vector pCGP3262 (FIG.", "37), downstream of the ER transit-peptide signal sequence from Arabidopsis thaliana.", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding legion and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; ERT:T1:HDEL=T1 cDNA clone from Tubastrea (SEQ ID NO:201) with an in-frame ER transit peptide sequence from Arabidopsis basic chitinase gene at the 5′ end and an HDEL ER retention sequence at the 3′ end; rCHS=Rose chalcone synthase promoter fragment.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 11 for further details.", "FIG.", "39 is a diagrammatic representation of the binary plasmid pCGP3258.An in-frame fusion of the T1 coding sequence (SEQ ID NO:201) and the mgfp4 sequence was cloned into the CaMV 35S expression cassette of pCGP3257 (FIG.", "35).", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; T1:mgfp4=T1 cDNA clone from Tubastrea (SEQ ID NO:201) with an in-frame fusion of the mgfp4 coding sequence.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 12 for further details.", "FIG.", "40 is a representation of an autoradiograph of an RNA blot probed with 32P-labelled fragments of (A) a 0.7 kb BamHI/HindIII fragment of the T1 clone contained in pCGP2921 (FIG.", "10) and (B) 0.8 kb HindIII fragment of SuRB contained in pCGP1651.Each lane contained a 5 to 10 μg sample of total RNA isolated from the leaves and petals of transgenic P. hybrida plants.", "(C) Ethidium bromide staining of the 18S rRNA is shown as an indication of RNA loading levels.", "Lane numbers are marked 1 to 12.The numbers above the lane numbers refer to construct pCGP numbers used in the transformation experiments.", "Refer to Example 15 for further details.", "FIG.", "41 is a representation of an autoradiograph of an RNA blot probed with 32P-labelled fragments of (A) a 0.7 kb BamHI/HindIII fragment of the T1 clone contained in pCGP2921 (FIG.", "10) and (B) 0.8 kb HindIII fragment of SuRB contained in pCGP1651.Each lane contained a 5 μg sample of total RNA isolated from the leaves of non-transgenic arid transgenic A. thaliana plants.", "(C) Ethidium bromide staining of the 25S rRNA is shown as an indication of RNA loading levels.", "Lane numbers are marked 1 to 17.The numbers above the lane numbers refer to construct pCGP numbers used in the transformation experiments with the exception of NTG and 35Smgfp4.NTG=non transgenic; 35Smgfp4=pBIN35Smgfp4.Refer to Example 14 for further details.", "FIG.", "42 is a graphical representation of absorption, excitation and emission spectra for Rtms-5 (SEQ ID NO:166) and its variants.", "(A) Absorption spectra for Rtms-5 (SEQ ID NO:166); (B) Absorption spectra for variants generated via site directed mutagenesis: Rtms5-H142S and Rtms-5v (SEQ ID NO:216); C Excitation (exc) and emission (em) spectra for Rtms5-H142S and Rtms-5v (SEQ ID NO:216) at wavelengths indicated.", "FIG.", "43 is a graphical representation of examples of excitation and emission spectra for two other colored proteins, showing extinction coefficients (ελmax) based on the method of Whitaker and Granum (1980, supra) for calculating protein concentration.", "x-axis=relative absorption; y-axis=wavelength (nm); (A) Aams-4 (SEQ ID NO:90)-H142S, and (B) Rtms-1 (SEQ ID NO:162)-N142S; λmax for each spectrum is shown on the figure.", "FIG.", "44 is a diagrammatic representation of the binary plasmid pCGP2926.A ˜0.1 kb AscI/BamHI fragment (containing sequences to a prokaryotic ribosome binding site (RBS), translational initiation consensus sequence (TICS) and an RGSHHHHHH epitope) generated by ligating the primers TICS-His-FWD (SEQ ID NO:227) and TICS-His-REV (SEQ ID NO:228) was introduced into the binary plasmid pCGP2781 (FIG.", "32).", "Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border, SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodornonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli.", "T1=T1 cDNA from Tubastrea sp.", "(SEQ ID NO:201), His=RGSHHHHHH epitope.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 9 for further details.", "FIG.", "45 is diagrammatic representation of the binary plasmid pCGP3261.An ER targeted T1:mGFP4 fusion was cloned into CaMV 35S expression cassette of the binary vector pCGP3257.Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; ERT:T1 :mGFP4:HDEL=T1 cDNA clone from Tubastrea (SEQ ID NO:201):mGFP4 in-frame fusion with an in-frame ER transit peptide sequence from Arabidopsis basic chitinase gene at the 5′ end and an HDBL ER retention sequence at the 3′ end.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 12 for further details.", "FIG.", "46 is diagrammatic representation of the binary plasmid pCGP3260.An ER targeted mGFP4 coding region was cloned into CAMV 35S expression cassette of the binary vector pCGP2780.Abbreviations are as follows: TetR=the tetracycline resistance gene; LB=left border; RB=right border; SuRB=the coding region and terminator sequence from the acetolactate synthase gene from tobacco; p35S=a promoter region from the cauliflower mosaic virus (CaMV) 35S gene; t35S=a terminator fragment from the CaMV 35S gene; pVS1=a broad host range origin of replication from a plasmid from Pseuodomonas aeruginosa; pACYC ori=modified replicon from pACYC184 from E. coli; ERT:mGFP4:HDEL=mGFP4 coding sequence with an in-frame ER transit peptide sequence from Arabidopsis basic chitinase gene at the 5′ end and an HDEL ER retention sequence at the 3′ end.", "Selected restriction endonuclease recognition sites are also marked.", "Refer to Example 12 for further details.", "FIG.", "47 is a photographic representation of clear nature gel electrophoresis showing separation of fluorescently labeled mitochondrial ATP synthase.", "1.b-gfp fusion protein; 2.b-Rtms-5v fusion protein; 3.b-dsRed fusion protein; 4.GFP not fused to another protein.", "A summary of sequence identifiers used throughout the subject specification is provided in Table 1.TABLE 1 SUMMARY OF SEQUENCE IDENTIFIERS SEQ ID NO.", "NAME DESCRIPTION 1 POC FOR oligonucleotide 2 POC 220 oligonucleotide 3 MSVIAT FOR oligonucleotide 4 POC 231 oligonucleotide 5 SVIAK N-terminal amino acid sequence of a CFM 6 (M)SVIAT N-terminal amino acid sequence of a CFM 7 SGIAT N-terminal amino acid sequence of a CFM 8 SVIVT N-terminal amino acid sequence of a CFM 9 SVSAT N-terminal amino acid sequence of a CFM 10 SVIATQMTYKVYMSGT N-terminal amino acid sequence of a CFM 11 SVIATQMTYKVYMSPT N-terminal amino acid sequence of a CFM 12 SVIATQVTYKVYMSGT N-terminal amino acid sequence of a CFM 13 SGIATQMTYKVYMSGT N-terminal amino acid sequence of a CFM 14 SVIVTQMTYKVYMSGT N-terminal amino acid sequence of a CFM 15 SVSATQMTYKVYMSGT N-terminal amino acid sequence of a CFM 16 SVIAKQMTYKVNMSGT N-terminal amino acid sequence of a CFM 17 SVIAKQMTYKVYMSDT N-terminal amino acid sequence of a CFM 18 SVIAKQMTYX1X2YX3SGT N-terminal amino acid sequence of a CFM 19 Aasv-1 nucleotide sequence of SVIAK-type clone 20 Aasv-1.pep translated amino acid sequence of SVIAK CFM 21 Aasv-3 nucleotide sequence of SVIAK-type clone 22 Aasv-3.pep translated amino acid sequence of SVIAK CFM 23 Aasv-P nucleotide sequence of SVIAK-type clone 24 Aasv-P.pep translated amino acid sequence of SVIAK CFM 25 Acasv-A nucleotide sequence of SVIAK-type clone 26 Acasv-A.pep translated amino acid sequence of SVIAK CFM 27 Acasv-C nucleotide sequence of SVIAK-type clone 28 Acasv-C.pep translated amino acid sequence of SVIAK CFM 29 Acasv-D nucleotide sequence of SVIAK-type clone 30 Acasv-D.pep translated amino acid sequence of SVIAK CFM 31 Ce61-3sv nucleotide sequence of SVIAK-type clone 32 Ce61-3sv.pep translated amino acid sequence of SVIAK CFM 33 Ce61-4sv nucleotide sequence of SVIAK-type clone 34 Ce61-4sv.pep translated amino acid sequence of SVIAK CFM 35 Ce61-5sv nucleotide sequence of SVIAK-type clone 36 Ce61-5sv.pep translated amino acid sequence of SVIAK CFM 37 Ce61-7sv nucleotide sequence of SVIAK-type clone 38 Ce61-7sv.pep translated amino acid sequence of SVIAK CFM 39 GPd58-2sv nucleotide sequence of SVIAK-type clone 40 GPd58-2sv.pep translated amino acid sequence of SVIAK CFM 41 LGAsv-A nucleotide sequence of SVIAK-type clone 42 LGAsv-A.pep translated amino acid sequence of SVIAK CFM 43 LGAsv-C nucleotide sequence of SVIAK-type clone 44 LGAsv-C.pep translated amino acid sequence of SVIAK CFM 45 LGAsv-D nucleotide sequence of SVIAK-type clone 46 LGAsv-D.pep translated amino acid sequence of SVIAK CFM 47 LGAsv-E nucleotide sequence of SVIAK-type clone 48 LGAsv-E.pep translated amino acid sequence of SVIAK CFM 49 Misv-A nucleotide sequence of SVIAK-type clone 50 Misv-A.pep translated amino acid sequence of SVIAK CFM 51 Misv-B nucleotide sequence of SVIAK-type clone 52 Misv-B.pep translated amino acid sequence of SVIAK CFM 53 Misv-F nucleotide sequence of SVIAK-type clone 54 Misv-F.pep translated amino acid sequence of SVIAK CFM 55 PM1Asv-rep nucleotide sequence of SVIAK-type clone 56 PM1Asv-rep.pep translated amino acid sequence of SVIAK CFM 57 PM1Csv-rep nucleotide sequence of SVIAK-type clone 58 PM1Csv-rep.pep translated amino acid sequence of SVIAK CFM 59 PMsv-4 nucleotide sequence of SVIAK-type clone 60 PMsv-4.pep translated amino acid sequence of SVIAK CFM 61 PMsv-5 nucleotide sequence of SVIAK-type clone 62 PMsv-5.pep translated amino acid sequence of SVIAK CFM 63 PPsv-1 nucleotide sequence of SVIAK-type clone 64 PPsv-1.pep translated amino acid sequence of SVIAK CFM 65 PPsv-2 nucleotide sequence of SVIAK-type clone 66 PPsv-2.pep translated amino acid sequence of SVIAK CFM 67 PPsv-3 nucleotide sequence of SVIAK-type clone 68 PPsv-3.pep translated amino acid sequence of SVIAK CFM 69 PPsv-4 nucleotide sequence of SVIAK-type clone 70 PPsv-4.pep translated amino acid sequence of SVIAK CFM 71 PPsv-5 nucleotide sequence of SVIAK-type clone 72 PPsv-5.pep translated amino acid sequence of SVIAK CFM 73 PPsv-6 nucleotide sequence of SVIAK-type clone 74 PPsv-6.pep translated amino acid sequence of SVIAK CFM 75 Pavsv-A nucleotide sequence of SVIAK-type clone 76 Pavsv-A.pep translated amino acid sequence of SVIAK CFM 77 Pavsv-B nucleotide sequence of SVIAK-type clone 78 Pavsv-B.pep translated amino acid sequence of SVIAK CFM 79 Pavsv-C nucleotide sequence of SVIAK-type clone 80 Pavsv-C.pep translated amino acid sequence of SVIAK CFM 81 RTsv-1 nucleotide sequence of SVIAK-type clone 82 RTsv-1.pep translated amino acid sequence of SVIAK CFM 83 RTsv-2 nucleotide sequence of SVIAK-type clone 84 RTsv-2.pep translated amino acid sequence of SVIAK CFM 85 RTsv-3 nucleotide sequence of SVIAK-type clone 86 RTsv-3.pep translated amino acid sequence of SVIAK CFM 87 Aams-2 nucleotide sequence of (M)SVIAT-type clone 88 Aams-2.pep translated amino acid sequence of (M)SVIAT CFM 89 Aams-4 nucleotide sequence of (M)SVIAT-type clone 90 Aams-4.pep translated amino acid sequence of (M)SVIAT CFM 91 Aams-5 nucleotide sequence of SGIAT-type clone 92 Aams-5.pep translated amino acid sequence of SGIAT CFM 93 Aams-6 nucleotide sequence of (M)SVIAT-type clone 94 Aams-6.pep translated amino acid sequence of (M)SVIAT CFM 95 Aams-A nucleotide sequence of (M)SVIAT-type clone 96 Aams-A.pep translated amino acid sequence of (M)SVIAT CFM 97 Aams-B nucleotide sequence of (M)SVIAT-type clone 98 Aams-B.pep translated amino acid sequence of (M)SVIA5 CFM 99 Acams-2 nucleotide sequence of (M)SVIAT-type clone 100 Acams-2.pep translated amino acid sequence of (M)SVIAT CFM 101 Acams-3 nucleotide sequence of (M)SVIAT-type clone 102 Acams-3.pep translated amino acid sequence of (M)SVIAT CFM 103 Acams-4 nucleotide sequence of (M)SVIAT-type clone 104 Acams-4.pep translated amino acid sequence of (M)SVIAT CFM 105 Acams-5 nucleotide sequence of (M)SVIAT-type clone 106 Acams-5.pep translated amino acid sequence of (M)SVIAT CFM 107 Cems-F nucleotide sequence of (M)SVIAT-type clone 108 Cems-F.pep translated amino acid sequence of (M)SVIAT CFM 109 Cems-G nucleotide sequence of (M)SVIAT-type clone 110 Cems-G.pep translated amino acid sequence of (M)SVIAT CFM 111 Cems-H nucleotide sequence of (M)SVIAT-type clone 112 Cems-H.pep translated amino acid sequence of (M)SVIAT CFM 113 Cems-I nucleotide sequence of (M)SVIAT-type clone 114 Cems-I.pep translated amino acid sequence of (M)SVIAT CFM 115 LGAms-5 nucleotide sequence of (M)SVIAT-type clone 116 LGAms-5.pep translated amino acid sequence of (M)SVIAT CFM 117 LGAms-6 nucleotide sequence of (M)SVIAT-type clone 118 LGAms-6.pep translated amino acid sequence of (M)SVIAT CFM 119 Mi68Dms nucleotide sequence of (M)SVIAT-type clone 120 Mi68Dms.pep translated amino acid sequence of (M)SVIAT CFM 121 Mims-A nucleotide sequence of (M)SVIAT-type clone 122 Mims-A.pep translated amino acid sequence of (M)SVIAT CFM 123 Mims-B nucleotide sequence of (M)SVIAT-type clone 124 Mims-B.pep translated amino acid sequence of (M)SVIAT CFM 125 Mims-C nucleotide sequence of (M)SVIAT-type clone 126 Mims-C.pep translated amino acid sequence of (M)SVIAT CFM 127 PMms-A nucleotide sequence of (M)SVIAT-type clone 128 PMms-A.pep translated amino acid sequence of (M)SVIAT CFM 129 PMms-B nucleotide sequence of (M)SVIAT-type clone 130 PMms-B.pep translated amino acid sequence of (M)SVIAT CFM 131 PMms-C nucleotide sequence of (M)SVIAT-type clone 132 PMms-C.pep translated amino acid sequence of (M)SVIAT CFM 133 PMms-D nucleotide sequence of (M)SVIAT-type clone 134 PMms-D.pep translated amino acid sequence of (M)SVIAT CFM 135 PMms-E nucleotide sequence of (M)SVIAT-type clone 136 PMms-E.pep translated amino acid sequence of (M)SVIAT CFM 137 PPd57-1ms nucleotide sequence of (M)SVIAT-type clone 138 PPd57-1ms.pep translated amino acid sequence of (M)SVIAT CFM 139 PPd57-2ms nucleotide sequence of (M)SVIAT-type clone 140 PPd57-2ms.pep translated amino acid sequence of (M)SVIAT CFM 141 PPd57-3 nucleotide sequence of (M)SVIAT-type clone 142 PPd57-3.pep translated amino acid sequence of (M)SVIAT CFM 143 PPd57-4ms nucleotide sequence of (M)SVIAT-type clone 144 PPd57-4ms.pep translated amino acid sequence of (M)SVIAT CFM 145 PPms-1 nucleotide sequence of (M)SVIAT-type clone 146 PPms-1.pep translated amino acid sequence of (M)SVIAT CFM 147 PPms-2 nucleotide sequence of (M)SVIAT-type clone 148 PPms-2.pep translated amino acid sequence of (M)SVIAT CFM 149 PPms-E nucleotide sequence of (M)SVIAT-type clone 150 PPms-E.pep translated amino acid sequence of (M)SVIAT CFM 151 PPms-G nucleotide sequence of (M)SVIAT-type clone 152 PPms-G.pep translated amino acid sequence of (M)SVIAT CFM 153 Pav5ms nucleotide sequence of (M)SVIAT-type clone 154 Pav5ms.pep translated amino acid sequence of (M)SVIAT CFM 155 Pavms-2 nucleotide sequence of (M)SVIAT-type clone 156 Pavms-2.pep translated amino acid sequence of (M)SVIAT CFM 157 Pavms-3 nucleotide sequence of (M)SVIAT-type clone 158 Pavms-3.pep translated amino acid sequence of (M)SVIAT CFM 159 Pavms-4 nucleotide sequence of (M)SVIAT-type clone 160 Pavms-4.pep translated amino acid sequence of (M)SVIAT CFM 161 RTms-1 nucleotide sequence of (M)SVIAT-type clone 162 RTms-1.pep translated amino acid sequence of (M)SVIAT CFM 163 RTms-2 nucleotide sequence of SVSAT-type clone 164 RTms-2.pep translated amino acid sequence of SVSAT CFM 165 RTms-5 nucleotide sequence of (M)SVIAT-type clone 166 RTms-5.pep translated amino acid sequence of (M)SVIAT CFM 167 RTms-6 nucleotide sequence of SVIVT-type clone 168 RTms-6.pep translated amino acid sequence of SVIVT CFM 169 Acasv-B nucleotide sequence of SVIAK-type clone with a stop codon at amino acid position 14 170 Acasv-B.pep translated amino acid sequence of SVIAK CFM 171 GPd58-1sv nucleotide sequence of SVIAK-type clone with a stop codon at amino acid position 14 172 GPd58-1sv.pep translated amino acid sequence of SVIAK CFM 173 GPd58-3sv nucleotide sequence of SVIAK-type clone with a stop codon at amino acid position 14 174 GPd58-3sv.pep translated amino acid sequence of SVIAK CFM 175 GPd58-4sv nucleotide sequence of SVIAK-type clone with a stop codon at amino acid position 14 176 GPd58-4sv.pep translated amino acid sequence of SVIAK CFM 177 Misv-D nucleotide sequence of SVIAK-type clone with a stop codon at amino acid position 14 178 Misv-D.pep translated amino acid sequence of SVIAK CFM 179 Pavsv-D nucleotide sequence of SVIAK-type clone with a stop codon at amino acid position 14 180 Pavsv-D.pep translated amino acid sequence of SVIAK CFM 181 Aapat-1 amino acid sequence of coral protein disclosed in WO00/46233 182 Aapat-2 amino acid sequence of coral protein disclosed in WO00/46233 183 dT(17)Ad2Ad1 oligonucleotide 184 vispro-F1 oligonucleotide 185 vispro-R1 oligonucleotide 186 pQEprom oligonucleotide 187 pQErev oligonucleotide 188 Coral-R1 oligonucleotide 189 A8 (pCGP2918) nucleotide sequence of full-length cDNA clone 190 A8.aa translated amino acid sequence of full-length cDNA clone 191 D10 (pCGP2920) nucleotide sequence of full-length cDNA clone 192 D10.aa translated amino acid sequence of full-length cDNA clone 193 S3 (pCGP2924) nucleotide sequence of full-length cDNA clone 194 S3.aa translated amino acid sequence of full-length cDNA clone 195 T3 (pCGP2922) nucleotide sequence of full-length cDNA clone 196 T3.aa translated amino acid sequence of full-length cDNA clone 197 D1 (pCGP2919) nucleotide sequence of full-length cDNA clone 198 D1.aa translated amino acid sequence of full-length cDNA clone 199 S1 (pCGP2923) nucleotide sequence of full-length cDNA clone 200 S1.aa translated amino acid sequence of full-length cDNA clone 201 T1 (pCGP2921) nucleotide sequence of full-length cDNA clone 202 T1.aa translated amino acid sequence of full-length cDNA clone 203 Kpn.6His.F oligonucleotide 204 T1/A8.Sal.R oligonucleotide 205 TSSU-Fnew oligonucleotide 206 TSSU-R oligonucleotide 207 AscI-ER.F oligonucleotide 208 ER-BamHI.R oligonucleotide 209 CP-HDEL-PacI.R oligonucleotide 210 Pst-mGFP4F oligonucleotide 211 mGFP4-PacIR oligonucleotide 212 visproF1-new oligonucleotide 213 MSV-RBS oligonucleotide 214 SVIAK-RBS oligonucleotide 215 POC 220 H6 oligonucleotide 216 Rtms-5v mutated variant amino acid sequence from Rtms-5 (SEQ ID NO:166) 217 gtCP translated amino acid sequence of SVIAK CFM 218 poc4 translated amino acid sequence of SVIAK CFM 219 baspoc3 translated amino acid sequence of SVIAK CFM 220 dsFP593 translated amino acid sequence of a CFM 221 drFP583, also known translated amino acid sequence of a CFM as dsRed583 222 gfp translated amino acid sequence of a CFM 223 MGFP-4 nucleotide sequence from GFP-4 from Aequorea victoria (jelly fish), mutated for plants 224 MGFP-4.pep translated amino acid sequence of GFP-4 CFM 225 BASPOC4 translated amino acid sequence of a CFM 226 AsFP595 translated amino acid sequence of a CFM 227 TICS-His-FWD oligonucleotide 228 TICS-His-REV oligonucleotide 229 mGFP4-HDEL-PacR oligonucleotide 230 T1.N-QN(AAT)SQ(CAG) oligonucleotide 231 Ti.S-IS(TCC) > I(ATC) oligonucleotide 232 YGFP3UP oligonucleotide 233 YGFP3DO oligonucleotide 234 RFPUP1 oligonucleotide 235 RFPDO1 oligonucleotide 236 MSVIATUP oligonucleotide 237 COFPDO oligonucleotide 238 ATP4PROMUP2 oligonucleotide 239 ATP4DO2 oligonucleotide 240 ATP7TUP oligonucleotide 241 ATP7TDO oligonucleotide 242 SPPDYTLEFP N-terminal amino acid sequence of a CFM 243 SPPDYTLERP N-terminal amino acid sequence of a CFM 244 (D)SS(P)E N-terminal amino acid sequence of a CFM 245 SYLPN N-terminal amino acid sequence of a CFM 246 SYLQN N-terminal amino acid sequence of a CFM 247 MEGIVNG-A oligonucleotide 248 MEGIVNG-T oligonucleotide 249 MEGIVNG-C oligonucleotide 250 REV-MEG-T oligonucleotide 251 REV-MEG-C oligonucleotide DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated on the identification of peptides, polypeptides and proteins having one or more amino acid sequences or one or more amino acid sequences which exhibit color-facilitating properties, either on their own or following interaction with one or more other amino acids and nucleic acid moleclues encoding same.", "Such peptides, polypeptides and proteins are referred to herein as “color-facilitating molecules” or “CFMs”.", "The present invention contemplates a range of uses of CFMs, including their use as color expression markers and as color intensifiers, as well as in gel-like formulations for use as photon traps and in light-filtering formulations such as topically-applied sun screens.", "The present invention further contemplates the use of genetic material encoding CFMs to generate eukaryotic or prokaryotic cells or eukaryotic or prokaryotic cell tissue which, in the presence of the CFMs, exhibit altered visual characteristics to the human eye in the absence of excitation of the CFMs by extraneous non-white light or particle emission.", "Such altered visual characteristics are also referred to as being altered to the naked, unaided eye.", "Reference to “naked” or “unaided” is not to imply that the eye may not require magnification aids such as in the form of spectacles or glasses or a magnifying glass.", "Reference to extraneous light or particle emission includes ultraviolet (UV) light, blue laser light, plasma irradiation, γ-irradiation, particle irradiation, single wavelength light such as 340 nm, 382 nm, 396 nm, 405 nm, 475 nm, 490 mn, 575 nm or other forms of emission or particle bombardment.", "It does not include white light.", "Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a color-facilitating molecule (CFM) which, in a cell, alone or together with one or more other molecules imparts an altered visual characteristic to said cell when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "Preferably, the nucleic acid molecule is derived from Anemonia majano, Anemonia sulcata, Clavularia sp, Zoanthus sp, Discosoma sp (e.g.", "Discosoma striata), Aequorea sp (e.g.", "Aequorea Victoria), Anthozoa sp, Cassiopea sp, (e.g.", "Cassiopea xamachana), Millepora sp, Acropora sp (e.g.", "Acropora aspera and Acropora nobilis), Montipora sp, Porites murrayensis, Pocillopora damicormis, Pavona descussaca, Acanthastrea sp, Platygyra sp or Caulastrea sp.", "In a preferred embodiment, the nucleic acid molecule encodes a CFM with an amino acid at its N-terminal region selected from SVIAK (SEQ ID NO:5), (M)SVIAT (SEQ ID NO:6), SGIAT (SEQ ID NO:7), SVIVT (SEQ ID NO:8) or SVSAT (SEQ ID NO:9).", "Even more particularly, the CFM comprises an amino acid sequence selected from SVIAT QMTY KVYM SGT (SEQ ID NO:10), SVIAT QMTY KVYM PGT (SEQ ID NO:11), SVIAT QVTY KVYM SGT (SEQ ID NO:12), SGIAT QMTY KVYM SGT (SEQ ID NO:13), SVIVT QMTY KVYM SGT (SEQ ID NO:14), SVSAT QMTY KVYM SGT (SEQ ID NO:15), SVIAK QMTY KVNM SGT (SEQ ID NO:16), SVIAK QMTY KVYM SDT (SEQ ID NO:17) and SVIAK QMTY X1X2YX3 SGT (SEQ ID NO:18) wherein X1, X2 and X3 may be any amino acid provided that X1 is not K; X2 is not V; X3 is not M. In a particular embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a CFM or a fragment, variant or derivative thereof, wherein said isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,:139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 189, 191, 193, 195, 197, 199 and 201, or a biologically active fragment or derivative of these.", "Particular preferred nucleic acid molecules comprise the nucleotide sequences set forth in SEQ ID NOs:189, 191, 193, 195, 197, 199 and 201.The nucleic acid molecule is regarded as genetic material and generally comprises a coding region encoding a CFM optionally operably linked to a single or multiple promoters.", "In one embodiment, the nucleic acid molecule is a genetic construct under the control of (i.e.", "operably linked to) a single promoter.", "In another embodiment, the genetic construct is a bicistronic, ticistronic or multicistronic construct carrying the gene encoding the CFM and optionally other genes such as encoding a reporter molecule.", "As used herein, the terms “nucleic acid molecule” including “genetic material” refers to any single-stranded or double-stranded nucleic acid molecule which at least comprises deoxyribonucleotides and/or ribonucleotides, including DNA (cDNA or genornic DNA), RNA, mRNA, or tRNA, amongst others.", "The combination of such molecules with non-nucleotide substituents derived from synthetic means or naturally-occurring sources is also contemplated by the present invention.", "Genetic material may also include sequences optimized for expression of codons in a particular host cell.", "The present invention extends to derivatives of the nucleic acid molecules and such derivatives includes any isolated nucleic acid molecule which comprises at least 10 and preferably at least 20 contiguous nucleotides derived from the genetic sequence as described herein according to any embodiment.", "A derivative includes a part, fragment, portion or analog.", "A derivative also includes a fusion molecule between two or more genetic sequences encoding CFMs.", "The present invention also comprises analogs of the nucleic acid molecules.", "An “analog” means any isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence as described herein according to any embodiment, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to, alkaline phosphatase or horseradish peroxidase, amongst others.", "A “homolog” is a functionally similar molecule from a different species or strain.", "Generally, analogs or derivatives of the nucleic acid molecule of the invention are produced by synthetic means or alternatively, derived from naturally-occurring sources.", "For example, the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions.", "A derivative encompasses a nucleotide sequence modified for optimized or enhanced codon usage in a particular cell.", "The genetic sequence of the present invention may comprise a sequence of nucleotides or be complementary to a sequence of nucleotides which comprise one or more of the following: a promoter sequence, a 5′ non-coding region, a cis-regulatory region such as a functional binding site for transcriptional regulatory protein or translational regulatory protein, an upstream activator sequence, an enhancer element, a silencer element, a TATA box motif, a CCAAT box motif, or an upstream open reading frame, transcriptional start site, translational start site, and/or nucleotide sequence which encodes a leader sequence.", "The genetic sequence also encodes the CFM.", "The term “5′ non-coding region” is used herein in its broadest context to include all nucleotide sequences which are derived from the upstream region of an expressible gene, other than those sequences which encode amino acid residues which comprise the polypeptide product of said gene, wherein 5′ non-coding region confers or activates or otherwise facilitates, at least in part, expression of the gene.", "The nucleic acid molecule may also be regarded as a gene.", "The term “gene” is used in its broadest context to include both a genomic DNA region corresponding to the gene as well as a cDNA sequence corresponding to exons or a recombinant molecule engineered to encode a functional form of a product.", "The term “gene” is used in its broadest sense and includes cDNA corresponding to the exons of a gene.", "Accordingly, reference herein to a gene” is to be taken to include: (i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e.", "introns, 5′- and 3′-untranslated sequences); or (ii) mRNA or cDNA corresponding to the coding regions (i.e.", "exons) and 5′- and 3′-untranslated sequences of the gene.", "The term “gene” is also used to describe synthetic or fusion molecules encoding all or part of a functional product.", "As used herein, the term “cis-acting sequence” or “cis-regulatory region” or similar term shall be taken to mean any sequence of nucleotides which is derived from an expressible genetic sequence wherein the expression of the first genetic sequence is regulated, at least in part, by said sequence of nucleotides.", "Those skilled in the art will be aware that a cis-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of any structural gene sequence.", "Reference herein to a “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e.", "upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner.", "A promoter is usually, but not necessarily, positioned upstream or 5′, of a structural gene, the expression of which it regulates.", "Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.", "In the present context, the term “promoter” is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a structural gene or other nucleic acid molecule, in a plant cell.", "Preferred promoters according to the subject invention may contain additional copies of one or more specific regulatory elements to further enhance expression in a cell, and/or to alter the timing of expression of a structural gene to which it is operably connected.", "In a preferred embodiment, the nucleic acid molecules are expressed in a cell.", "The cell may be a eukaryotic or prokaryotic cell.", "Reference to a eukaryotic cell includes a mammalian animal cell, a non-mammalian animal cell or a plant cell.", "Insofar as the eukaryotic cell is a plant cell, the plant cell may be part of a plant callus or a whole plant.", "Reference to a “plant” includes ornamental or flowering plants or parts thereof such as flowers, roots, leaves, stems, seeds, fruit or fibers.", "Particularly preferred plant cells are those selected from rose, carnation, lisianthus, petunia, lily, tulip, pansy, gerbera or chrysanthemum.", "The CFM is preferably a GFP or a derivative or homolog thereof such as a non-fluorescent GFP homolog.", "Another aspect of the present invention provides an isolated color-facilitating molecule (CFM) comprising a polypeptide which, in a cell, alone or together with one or more other molecules imparts an altered visual characteristic to said cell when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "The CFM of the present invention is preferably a protein comprising a sequence of amino acids such that upon folding, the sequence alone or following interaction with one or more other amino acids which may be within the same molecule or in another molecule such as in a dimer, trimer or oligomer exhibits chromophore or fluorophore properties.", "Particularly useful proteins comprise the contiguous amino acid sequence Gln-Tyr-Gly (QYG).", "Even more preferably, the protein is a GFP or a homolog or derivative thereof.", "An example of a homolog of a GFP is a non-fluorescent GFP homolog.", "An example of a derivative of a GEP or non-fluorescent GFP homolog is a GPP modified to cause a shift in the ratio of excitation or emission peaks.", "Such modifications may result in a more intense fluorescence or may exhibit altered or weaker fluorescence.", "Any number of GFP or non-fluorescent GFP homologs or other derivatives may be employed as CEMs in accordance with the present invention.", "Examples of such molecules are from Anemonia majano, Anemonia sulcata, Clavularia sp, Zoanthus sp, Discosoma sp (e.g.", "Discosoma striata), Aequorea sp (e.g.", "Aequorea Victoria), Anthozoa sp, Cassiopea sp, (e.g.", "Cassiopea xamachana), Millepora sp, Acropora sp (e.g.", "Acropora aspera and Acropora nobilis), Montipora sp, Porites murrayensis, Pocillopora damicormis, Pavona descussaca, Acanthastrea sp, Platygyra sp and Caulastrea sp.", "Particularly preferred protein sequences which constitute CFMs of the present invention comprise one of the following sequences of amino acids towards the amino-terminal end of the polypeptide: “SVIAK” (SEQ ID NO:5), “(M)SVIAT” (SEQ ID NO:6), “SGIAT” (SEQ ID NO:7), “SVIVT” (SEQ ID NO:8), or “SVSAT” (SEQ ID NO:9).", "Examples of such preferred protein sequences may be selected from the group consisting of: SVIAT QMTY KVYM SGT; (SEQ ID NO:10) SVIAT QMTY KVYM PGT; (SEQ ID NO:11) SVIAT QVTY KVYM SGT; (SEQ ID NO:12) SGIAT QMTY KVYM SGT; (SEQ ID NO:13) SVIVT QMTY KVYM SGT; (SEQ ID NO:14) SVSAT QMTY KVYM SGT; (SEQ ID NO:15) SVIAK QMTY KVNM SGT; (SEQ ID NO:16) SVIAK QMTY KVYM SDT; (SEQ ID NO:17) and SVIAK QMTY X1X2YX3 SGT, (SEQ ID NO:18) wherein X1, X2 and X3 may be any amino acid provided that X1 is not K; X2 is not V; X3 is not M. Accordingly, in another aspect of the present invention there is provided an isolated polypeptide, or a biologically active fragment thereof, or a variant or derivative of these, said polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:10, 11, 12, 13, 14, 15, 16, 17 and 18, with the proviso that, in said isolated polypeptide or biologically active fragment or variant or derivative of SEQ ID NO:18, X1 is not lysine, X2 is not valine, and X3 is not methionine.", "Particularly suitable molecules comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 190, 192, 194, 196, 198, 200 and 202.Accordingly, a preferred embodiment of the present invention provides an isolated polypeptide, or a biologically active fragment thereof, or a variant or derivative of these, said polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 190, 192, 194, 196, 198, 200 and 202 provided that, where the said biologically active fragment or variant or derivative comprises the sequence SVLAK QMTY X1X2YX3 SGT, X1 is not lysine, X2 is not valine, and X3 is not methionine.", "Such isolated polypeptides, when present in a prokaryotic or eukaryotic cell or group of prokaryotic or eukaryotic cells such as in plant cells in the form of plant tissue or plant callus, may alone or in combination with one or more other molecules impart an altered visual characteristic to said cell or group of cells when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "Accordingly, another aspect of the present invention provides a prokaryotic or eukaryotic cell or group of prokaryotic or eukaryotic cells in the form of tissue wherein said cell or group of cells or their parent cells are genetically modified to enable the production of a color-facilitating molecule (CFM) which alone or together with one or more other molecules imparts an altered visual characteristic to said cell or group of cells when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "The CFM is as herein defined and in a preferred embodiment includes polypeptides having amino acid sequence selected from the list comprising SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 190, 192, 194, 196, 198, 200 and 202 provided that, where the said amino acid sequence comprises the sequence SVLIK QMTY X1X2YX3SGT, X1 is not lysine, X2 is not valine, and X3 is not methiomine.", "A “eukaryotic” cell is regarded as any cell which is not characterized as being a “prokaryotic” cell.", "Particularly useful eukaryotic cells are plant cells as well as fungi and yeast.", "Other eukaryotic cells, however, are also contemplated such as mammalian cells, non-mammalian animal cells including insect cells as well as plant cells.", "A “plant” may be regarded as a monocotyledonous or dicotyledonous plant and includes ornamental and crop plants.", "Reference to “tissue” includes plant callus.", "A “prokaryotic cell” is generally a cell comprising a nucleus not surrounded by a nuclear membrane and includes bacteria and microbial cells.", "Such prokaryotic cells include Pseudomonas sp., E. coli, Enterobacter sp., Salmonella sp., Kiebsiella sp., Acetobacter sp., Staphylocous sp., Streptococcus sp.", "or Bacillus sp., amongst many others.", "In a preferred embodiment, the present invention provides a plant cell or group of plant cells such as in the form of plant tissue or plant callus wherein said plant cells or group of plant cells or their parent cells are genetically modified to enable production of a CFM which alone or in combination with one or more other molecules imparts an altered visual characteristic to said cell or group of cells when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "Particularly preferred plants are ornamental and flowering plants.", "Particularly useful plants contemplated by the present invention include but are not limited to rose, carnation, lisianthus, petunia, lily, tulip, pansy, gerbera and chrysanthemum.", "Reference herein to a “plant” includes parts of plants.", "Similarly, reference herein to “plant tissue” includes parts of plants.", "Examples of such plant parts, include but are not limited to, flowers, roots, leaves, stems, seeds, fruit and fibres.", "The term “flowers” includes parts of flowers such as petals, petioles, flower heads and flower buds.", "Plant tissue may also include callus material as well as embryogenic or non-embryogenic material.", "The term “fibre” includes cotton and hemp fibres.", "Accordingly, another aspect of the present invention is directed to a plant or part of a plant including a flower, root, leaf, stem, seed, fruit or fibre or reproductive portion of said plant or cells of said plant wherein said plant or plant part comprises cells genetically modified to enable production of a CFM which alone or in combination with one or other molecules imparts an altered visual characteristic to said cells when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "The term “genetically modified” is used in its broadest sense and includes introducing gene(s) into cells, mutating gene(s) in cells and altering or modulating the regulation of gene(s) in cells.", "A “part” of a plant includes flowers (e.g.", "cut or severed flowers), petals, stems, leaves and fibrous material such as cotton and vegetative, propagative and reproductive material such as cuttings, pollen, seeds and callus.", "The altered visual characteristic may be exhibited by all cells in the plant or in selected tissue or plant parts such as flowers, roots, leaves, sterns, seeds, fruit or fibres.", "The production of the CFM may, therefore, be tissue specific or tissue preferential.", "Furthermore, CFM production may be developmentally dependent, determined, influenced or otherwise regulated.", "The CFM may be produced in the whole plant with the use of a constitutive promoter such as cauliflower mosaic virus (CaMV) 35S promoter, operably connected or operably linked to a gene or other nucleic acid molecule encoding the CFM.", "Alternatively, the molecule may be confined to, for example, petal tissue, epidermal cell layers of petals or to different organelles within cells.", "For example, a floral specific promoter such as a chalcone synthase promoter substantially limits a colored protein expression to flower petals.", "The use of some gene promoters (e.g.", "35S) may produce CFM accumulation in the cytoplasm of transformed cells and confer a visible color to the plant tissue.", "The CFM may be targeted to different organelles within the plant cell to confer a color change in the tissue visible to the naked unaided eye under white light illumination.", "The CFM can be targeted to plastids using a chloroplast transit peptide fused in-frame with the colored protein cDNA sequence.", "An example of a plastid transit peptide that can be used is the transit peptide of the small subunit of ribulose-1,5-bisphosphate-carboxylase (e.g.", "InCheol et al., Molecular Breeding 5: 453-461, 1999).", "The targeting of a CFM to plastids can dramatically increase the total amount of protein accumulated (InCheol et al., 1999, supra) and thereby increase color intensity.", "Chromoplasts are numerous in the petals of some flowers, leaves and fruit.", "A chromoplast specific transit peptide fused in-frame with the protein cDNA sequence may be used to modify flower or other tissue color with a much reduced potential for interfering with total plant photosynthetic activity, as may occur if an constitutive promoter and a chloroplast transit peptide were used to target the CFM.", "The use of a chromoplast transit peptide and a floral specific promoter may be optimal for the modification of flower color.", "It may be beneficial to target all CFMs to the vacuole or endoplasmic reticulum to avoid any detrimental effects to the transformed cells or plants.", "An example of an endoplasmic reticulum targeting peptide sequence that can be used is the amino acid sequence HDEL (Haseloff et al., 1997, supra).", "The CFM may also be targeted to the cell wall.", "The term “operably connected” or “operably linked” in the present context means placing a structural gene (e.g.", "a nucleic acid molecule encoding a CFM) under the regulatory control of a promoter which then controls expression of the gene.", "Promoters and the like are generally positioned 5′ (upstream) to the genes which they control.", "In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the genetic sequence or promoter at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting, i.e., the gene from which the genetic sequence or promoter is derived.", "As is known in the art, some variation in this distance can be accommodated without loss of function.", "Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived.", "The cells genetically modified to enable production of a CFM may be the cells into which genetic material has been introduced or they may represent progeny of genetically modified parent cells.", "Accordingly, the present invention contemplates a method for generating a transgenic plant or part of a plant, wherein said plant or plant part comprises cells genetically modified to enable production,of a CFM which alone or in combination with one or other molecules imparts an altered visual characteristic to said cells when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission, said method comprising introducing into said cells an isolated nucleic acid molecule encoding said CFM.", "Preferably, the CFM is derived from Anemonia majano, Anemonia sulcata, Clavularia sp, Zoanthus sp, Discosoma sp (e.g.", "Discosoma striata), Aequorea sp (e.g.", "Aequorea victoria), Anthozoa sp, Cassiopea sp, (e.g.", "Cassiopea xamachana), Millepora sp, Acropora sp (e.g.", "Acropora aspera and Acropora nobilis), Montipora sp, Porites murrayensis, Pocillopora damicormis, Pavona descussaca, Acanthastrea sp, Platygyra sp or Caulastrea sp.", "More preferably, the CFM comprises an amino acid sequence in its N-terminal end selected from SVIAK (SEQ ID NO:5), (M)SVIAT (SEQ ID NO:6), SGIAT (SEQ ID NO:7), SVIVT (SEQ ID NO:8) or SVSAT (SEQ ID NO:9).", "Even more preferably, the CFM comprises an amino acid sequence selected from the list comprising SVIAT QMTY KVYM SGT (SEQ ID NO:10), SVIAT QMTY KVYM PGT (SEQ ID NO:11), SVIAT QVTY KVYM SGT (SEQ ID NO:12), SGIAT QMTY KVYM SGT (SEQ ID NO:13), SVIVT QMTY KVYM SGT (SEQ ID NO:14), SVSAT QMTY KVYM SGT (SEQ ID NO:15), SVIAK QMTY KVNM SGT (SEQ ID NO:16), SVIAK QMTY KVYM SDT (SEQ ID NO:17) and SVLAK QMTY X1X2YX3 SGT (SEQ ID NO:18) wherein X1, X2 and X3 may be any amino acid provided that X1 is not K; X2 is not V; X3 is not M. Most preferably, the CFM is encoded by a nucleotide sequence selected from the list comprising SEQ ID NOs:19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 189, 191, 193, 195, 197, 199 and 201.Another aspect of the present invention provides a transgenic plant wherein said plant or a part thereof such as a flower, leaf, root, stem, seed, fruit or fibre exhibits an altered visual characteristic to a human eye in the absence of extraneous non-white light or particle emission wherein cells of said transgenic plant or of a parent plant have been genetically modified to enable production of a CFM.", "As stated above, the present invention extends to genetically modified mammalian cells, non-mammalian animal cells as well as plant cells.", "Farmers use conventional breeding techniques to develop new colors in animals and animal products for the market, for example, colored wools and leathers or hides.", "Presently the main way of coloring these products to obtain novel colors is by using dyes or tints or paints or pigments on natural colored products.", "However, the use of the CFMs of the present invention can be employed to produce a transgenic animal which exhibits a novel color: for example, sheep with blue or red colored fleece, cows with red colored hide.", "Specifically the CFM can be used in a range of agriculturally important animals such as but not limited to sheep, pigs, cattle, horses, goats, llamas, fish, ostriches, emus, ducks and chickens.", "Accordingly, another aspect of the present invention provides a transgenic mammalian or non-mammalian animal cell or transgenic non-human mammal or non-mammalian animal comprising said cells, said cells exhibiting an altered visual characteristic to a human eye in the absence of extraneous non-white light or particle emission wherein cells of said transgenic plant, mammal or animal or plant cells thereof have been genetically modified to enable production of a CFM.", "The CFM is as herein defined.", "Production of the CFM may be constitutive or developmental or may be inducible in response to internal or external stimulus including stress.", "Reference herein to a “color-facilitating molecule”, “CFM”, “protein”, “GFP” or “non-fluorescent GFP-homolog” includes fragments, derivatives, variants and homologs thereto.", "Examples of derivatives include mutants, parts, fragments and portions of these molecules including single or multiple amino acid substitutions, deletions and/or additions to the molecules.", "Derivatives also include fusion molecules between two or more CPMs or between a CFM and another molecule such as a leader sequence, targeting sequence, expression-facilitating sequence and/or a reporter molecule capable of providing an identifiable signal.", "As stated above a derivative also includes a modified form providing altered ratios of excitation or emission spectra.", "In addition, or as a consequence of the altered ratios of excitation or emission, the modified GFP or their homologs may have a more intense color-producing capacity relative to an unmodified molecule.", "Furthermore, other proteins may be used in conjunction with the CFMs to alter the visual characteristics of the cells.", "Examples of other proteins include copper containing proteins containing one or more type 1 (CuII) motifs as found in the Fet3 protein from Saccharomyces cerevisiae (Hassett et al., Journal of Biological Chemistry 273: 23274-23282, 1998) and other multinuclear copper ferroxidase enzymes such as laccase, ceruloplasmin and ascorbate oxidase (Messerschmidt and Huber, Eur.", "J. Biochem.", "187: 341-352, 1990).", "Similarly, the mononuclear blue or type 1 copper proteins (cupredoxins), such as plastocyanin, azurin, pseudoazurin, plantacyanin, rusticyanin, amicyanin, auracyanin and halocyanin (Nersissian et al., Protein Science 5: 2184-2192, 1996).", "These proteins have not been associated with pigmentation in nature.", "However, when these proteins are concentrated an intense blue color is evident (Hassett et al., 1998, supra; Messerschmidt and Huber, 1990, supra).", "The over-expression of a type 1 (CuII) containing protein in flowers and other plant tissues under conditions that allow correct folding and acquisition of Cu ions can modify or impart a color visible to the naked unaided eye under white light.", "Reference to “in conjunction” includes reference to a fusion protein between a CFM and another protein such as a cuproprotein and well as the expression of nucleotide sequences in multicistronic form encoding a CFM and at least one other protein.", "Another aspect of the present invention provides a eukaryotic or prokaryotic cell or a group of eakaryotic or prokaryotic cells in the form of a tissue wherein said cell or group of cells or their parent cells are genetically modified to produce a GFP or derivative or homolog thereof such as a non-fluorescent GFP homolog which imparts an altered visual characteristic on said cell or group of cells when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "Preferably, the eukaryotic cells are plant cells or plant tissue.", "The eukaryotic cells may, however, be mammalian cells or non-mammalian animal cells.", "Reference to “plant tissue” includes “callus”.", "Accordingly, another aspect of the present invention is directed to a plant or part of a plant including a flower, root, leaf, stem, seed, fruit or fibre or reproductive portion of said plant or cells of said plant wherein said plant or plant part comprises cells genetically modified to enable production of a GFP or a derivative or homolog thereof such as a non-fluorescent GFP homolog which imparts an altered visual characteristic to said cells when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "A particularly preferred embodiment the present invention is directed to a plant or part of a plant including a flower, root, leaf, stem, seed, fruit or fibre or reproductive portion of said plant or cells of said plant wherein said plant or plant part comprises cells genetically modified to comprise a polynucleotide comprising the nucleotide sequence set forth in any one of SEQ ID NOs:19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 189, 191, 193, 195, 197, 199 or 201, or a derivative or homolog of any of these, thereby enabling production of a CFM which alone or in combination with one or more other molecules imparts an altered visual characteristic to said cell or group of cells when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "The present invention particularly provides, in a preferred embodiment, a genetically modified plant carrying flowers having an altered flower color relative to a non-genetically modified plant as well as cut flowers form such a plant.", "Reference herein to a “genetically modified plant” includes progeny of a genetically modified plant as well as hybrids and derivatives of a genetically modified plant.", "The altered coloration of eukaryotic cells such as plant cells is useful not only for the ornamental plant market but also as propriety tags, for example, of seeds, root stock, flowers, crops and whole plants and plant parts.", "This may be particularly important for distinguishing between transgenic and non-transgenic crops, plants and other horticultural products.", "Furthermore, the modification of visible color in cotton fibre or hemp is a useful means of reducing the toxicity of dye processes in color fabric manufacture.", "The modification of visible color in edible and/or ornamental fungal species may also be used to differentiate and enhance marketability.", "The modification of visible color in fruit and vegetables may be used to differentiate and enhance their marketability.", "A suitable gene promoter may be used to control the expression of the CFM to signal optimal time to, for example, harvest crop plants including harvesting plant parts such as flowers or seeds.", "In addition, a stress-inducible promoter may be utilized to promote an early warning of water or pathogen stress, allowing for early intervention by the grower and subsequent reduction in economic loss.", "Other uses for the CFM of the present invention include, for example, the production of novel colored plant extracts wherein the extract includes, for example, a flavoring or food additive or health product or beverage or juice or coloring.", "Beverages may include but are not limited to wines, spirits, beers, teas, coffee, milk and dairy products.", "The CFM may be used to alter the color of many products such as but not limited to foods (e.g.", "breads and yeast products, confectionery), beverages (see above) or novelty items (e.g.", "toys).", "A further aspect of the present invention provides a transfected or transformed cell, tissue, organ or non-cellular material which contains or is capable of producing a CPM or a functional derivative or homolog thereof.", "Preferably, the CFM is a protein such as GFP or a non-fluorescent GFP-homolog.", "The genetic construct(s) of the present invention may be introduced into a cell by various techniques known to those skilled in the art.", "The technique used may vary depending on the known successful techniques for that particular organism.", "Techniques for introducing recombinant DNA into cells include, but are not limited to, transformation using CaCl2 and variations thereof, direct DNA uptake into protoplasts, PEG-mediated uptake to protoplasts, microparticle bombardment, electroporation, microinjection of DNA, microparticle bombardment of tissue explants or cells, vacuum-infiltration of tissue with nucleic acid, and T-DNA-mediated transfer from Agrobacterium to the plant tissue.", "For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell.", "Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention.", "Exemplary apparatus and procedures are disclosed by Stomp et al.", "(U.S. Pat.", "No.", "5,122,466) and Sanford and Wolf (U.S. Pat.", "No.", "4,945,050).", "When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.", "Examples of microparticles suitable for use in such systems include 0.1 to 10 μm and more particularly 10.5 to 5 μm tungsten or gold spheres.", "The DNA construct may be deposited on the microp article by any suitable technique, such as by precipitation.", "Once introduced into cells such as plant tissue, the expression of a CFM may be assayed in a transient expression system or it may be determined after selection for stable integration within for example, the plant genome.", "Hence, a CFM of the present invention may be useful as an expression marker.", "For example, genetic material encoding a CFM of the present invention, optionally operably linked to a single or multiple promoters, may be introduced into cells as a fluorescent “tag” , optionally fused with one or more other nucleic acid sequences that may encode a polypeptide or a regulatory nucleotide sequence.", "In this manner, a CFM fused with another polypeptide may be usefull in assessing subcellular localisation of the fusion or, alternatively, as an expression marker for assessing possible activity of the regulatory nucleotide sequence in a given host cell.", "Host cells may be prokaryolic cells, for example bacterial, or eukaryotic cells, for example yeast, plant, and animal cells, including human.", "Preferred host cells are bacterial or plant.", "Plant tissue capable of subsequent clonal propagation, whether by organogenesis or cmbryogenesis, may be transformed with a genetic construct of the present invention and a whole plant generated therefrom.", "The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.", "Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g.", "apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g.", "cotyledon meristem and hypocotyl meristem).", "The regenerated transformed plants may be propagated by a variety of means, such as by clonal propagation of classical breeding techniques.", "For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformant, and the T2 plants further propagated through classical breeding techniques.", "Any number of GEP or non-fluorescent GFP-homologs may be employed provided that the GFP or its homolog or other CFM imparts on a cell or group of cells an altered visual characteristic to the human eye in the absence of extraneous non-white light or particle emission.", "Examples of CFMs contemplated herein include but are not limited to non-fluorescent GFP-homologs such as that encoded by asFP595 (Lukyanov et al., 2000, supra) and t7SP6BASPOC3 and T7SP6BASPOC4 (Hoegh-Guldberg and Dove, 2000, supra) and fluorescent GFP variants and homologs such as described in Davis and Vierstra, 1996, supra; Haseloff et al., 1997, supra; Lukyanoy et al., 1999, supra; Matz et al., 1999, supra; Fradkov et al., FEBS Letters 479: 127-130, 2000).", "Accordingly, another aspect of the present invention provides a eukaryotic or prokaryotic cell or group of eukaryotic or prokaryotic cells genetically modified to comprise: (i) a nucleotide sequence set forth in SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39 or SEQ ID NO:41 or SEQ ID NO:43 or SEQ ID NO:45 or SEQ.", "ID NO:47 or SEQ ID NO:49 or SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:55 or SEQ ID NO:57 or SEQ ID NO:59 or SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73 or SEQ ID NO:75 or SEQ ID NO:77 or SEQ ID NO:79 or SEQ ID NO:81 or SEQ ID NO:83 or SEQ ID NO:85 or SEQ ID NO:87 or SEQ ID NO:89 or SEQ ID NO:91 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO:101 or SEQ ID NO:103 or SEQ ID NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or SEQ ID NO:111 or SEQ ID NO:113 or SEQ ID NO:115 or SEQ ID NO:117 or SEQ ID NO:119 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:125 or SEQ ID NO:127 or SEQ ID NO:129 or SEQ ID NO:131 or SEQ ID NO:133 or SEQ ID NO:135 or SEQ ID NO:137 or SEQ ID NO:139 or SEQ ID NO:141 or SEQ ID NO:143 or SEQ ID NO:145 or SEQ ID NO:147 or SEQ ID NO;149 or SEQ ID NO:151 or SEQ ID NO:153 or SEQ ID NO:155 or SEQ ID NO:157 or SEQ ID NO:159 or SEQ ID NO:161 or SEQ ID NO:163 or SEQ ID NO:165 or SEQ ID NO:167 or SEQ ID NO:169 or SEQ ID NO:171 or SEQ ID NO:173 or SEQ ID NO:175 or SEQ ID NO:177 or SEQ ID NO:179 or SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or 201; (ii) a nucleotide sequence having at least about 60% similarity after optimal alignment to SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39 or SEQ ID NO:41 or SEQ ID NO:43 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 or SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:55 or SEQ ID NO:57 or SEQ ID NO:59 or SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73 or SEQ ID NO:75 or SEQ ID NO:77 or SEQ ID NO:79 or SEQ ID NO:81 or SEQ ID NO:83 or SEQ ID NO:85 or SEQ ID NO:87 or SEQ ID NO:89 or SEQ ID NO:91 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO:101 or SEQ ID NO:103 or SEQ ID NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or SEQ ID NO:111 or SEQ ID NO:113 or SEQ ID NO:115 or SEQ ID NO:117 or SEQ ID NO:119 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:125 or SEQ ID NO:127 or SEQ ID NO:129 or SEQ ID NO:131 or SEQ ID NO:133 or SEQ ID NO:135 or SEQ ID NO:137 or SEQ ID NO:139 or SEQ ID NO:141 or SEQ ID NO:143 or SEQ ID NO:145 or SEQ ID NO:147 or SEQ ID NO:149 or SEQ ID NO:151 or SEQ ID NO:153 or SEQ ID NO:155 or SEQ ID NO:157 or SEQ ID NO:159 or SEQ ID NO:161 or SEQ ID NO:163 or SEQ ID NO:165 or SEQ ID NO:167 or SEQ ID NO:169 or SEQ ID NO:171 or SEQ ID NO:173 or SEQ ID NO:175 or SEQ ID NO:177 or SEQ ID NO:179 or SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or 201; (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39 or SEQ ID NO:41 or SEQ ID NO:43 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 or SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:55 or SEQ ID NO:57 or SEQ ID NO:59 or SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73 or SEQ ID NO:75 or SEQ ID NO:77 or SEQ ID NO:79 or SEQ ID NO:81 or SEQ ID NO:83 or SEQ ID NO:85 or SEQ ID NO:87 or SEQ ID NO:89 or SEQ ID NO:91 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO:101 or SEQ ID NO:103 or SEQ D NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or SEQ ID NO:111 or SEQ ID NO:113 or SEQ ID NO:115 or SEQ ID NO:117 or SEQ ID NO:119 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:125 or SEQ ID NO:127 or SEQ ID NO:129 or SEQ ID NO:131 or SEQ ID NO:133 or SEQ ID NO:135 or SEQ ID NO:137 or SEQ ID NO:139 or SEQ ID NO:141 or SEQ ID NO:143 or SEQ ID NO:145 or SEQ ID NO:147 or SEQ ID NO:149 or SEQ ID NO:151 or SEQ ID NO:153 or SEQ ID NO:155 or SEQ ID NO:157 or SEQ ID NO:159 or SEQ ID NO:161 or SEQ ID NO:163 or SEQ ID NO:165 or SEQ ID NO:167 or SEQ ID NO:169 or SEQ ID NO:171 or SEQ ID NO:173 or SEQ ID NO:175 or SEQ ID NO:177 or SEQ ID NO:179 or SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or 201; (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39 or SEQ ID NO:41 or SEQ ID NO:43 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 or SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:55 or SEQ ID NO:57 or SEQ ID NO:59 or SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73 or SEQ ID NO:75 or SEQ ID NO:77 or SEQ ID NO:79 or SEQ ID NO:81 or SEQ ID NO:83 or SEQ ID NO:85 or SEQ ID NO:87 or SEQ ID NO:89 or SEQ ID NO:91 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO:101 or SEQ ID NO:103 or SEQ ID NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or SEQ ID NO:111 or SEQ ID NO:113 or SEQ ID NO:115 or SEQ ID NO:117 or SEQ ID NO:119 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:125 or SEQ ID NO:127 or SEQ ID NO:129 or SEQ ID NO:131 or SEQ ID NO:133 or SEQ ID NO:135 or SEQ ID NO:137 or SEQ ID NO:139 or SEQ ID NO:141 or SEQ ID NO:143 or SEQ ID NO:145 or SEQ ID NO:147 or SEQ ID NO:149 or SEQ ID NO:151 or SEQ ID NO:153 or SEQ ID NO:155 or SEQ ID NO:157 or SEQ ID NO:159 or SEQ ID NO:161 or SEQ ID NO:163 or SEQ ID NO:165 or SEQ ID NO:167 or SEQ ID NO:169 or SEQ ID NO:171 or SEQ ID NO:173 or SEQ ID NO:175 or SEQ ID NO:177 or SEQ ID NO:179 or SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or 201; (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 60% similarity after optimal alignment to SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO39 or SEQ ID NO:41 or SEQ ID NO:43 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 or SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:55 or SEQ ID NO:57 or SEQ ID NO:59 or SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73 or SEQ ID NO:75 or SEQ ID NO:77 or SEQ ID NO:79 or SEQ ID NO:81 or SEQ ID NO:83 or SEQ ID NO:85 or SEQ ID NO:87 or SEQ ID NO:89 or SEQ ID NO:91 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO:101 or SEQ ID NO:103 or SEQ ID NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or SEQ ID NO:111 or SEQ ID NO:113 or SEQ ID NO:115 or SEQ ID NO:117 or SEQ ID NO:119 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:125 or SEQ ID NO:127 or SEQ ID NO:129 or SEQ ID NO:131 or SEQ ID NO:133 or SEQ ID NO:135 or SEQ ID NO:137 or SEQ ID NO:139 or SEQ ID NO:141 or SEQ ID NO:143 or SEQ ID NO:145 or SEQ ID NO:147 or SEQ ID NO:149 or SEQ ID NO:151 or SEQ ID NO:153 or SEQ ID NO:155 or SEQ ID NO:157 or SEQ ID NO:159 or SEQ ID NO:161 or SEQ ID NO:163 or SEQ ID NO:165 or SEQ ID NO:167 or SEQ ID NO:169 or SEQ ID NO:171 or SEQ ID NO:173 or SEQ ID.", "NO:175 or SEQ ID NO:177 or SEQ ID NO:179 or SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or 201; (vi) a nucleotide sequence capable of hybriding under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; wherein said nucleotide sequences encode a CFM which imparts an altered visual characterization to said cell or group of cells to a human eye in the absence of extraneous non-white light or particle emission.", "More particularly, the present invention provides a eukaryotic or prokaryotic cell or group of eukaryotic or prokaryotic cells genetically modified to comprise: (i) a nucleotide sequence set forth in SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or SEQ ID NO:201; (ii) a nucleotide sequence having at least about 60% similarity after optimal alignment to SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or SEQ ID NO:201; (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or SEQ ID NO:201 or its complementary form; (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:190 or SEQ ID NO:192 or SEQ ID NO:194 or SEQ ID NO:196 or SEQ ID NO:198 or SEQ ID NO:200 or SEQ ID NO:202; (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 60% similarity after optimal alignment to SEQ ID NO:190 or SEQ ID NO:192 or SEQ ID NO:194 or SEQ ID NO:196 or SEQ ID NO:198 or SEQ ID NO:200 or SEQ ID NO:202; (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; wherein said nucleotide sequences encode a CFM which imparts an altered visual characterization to said cell or group of cells to a human eye in the absence of extraneous non-white light or particle emission.", "Preferably, the eukaryotic cells are plant cells.", "Accordingly, in another aspect of the present invention, there is provided a plant or cells of a plant or parts of a plant or progeny of a plant wherein said plant comprises cells comprising: (i) a nucleotide sequence set forth in SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39 or SEQ ID NO:41 or SEQ ID NO:43 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 or SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:55 or SEQ ID NO:57 or SEQ ID NO:59 or SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73 or SEQ ID NO:75 or SEQ ID NO:77 or SEQ ID NO:79 or SEQ ID NO:81 or SEQ ID NO:83 or SEQ ID NO:85 or SEQ ID NO:87 or SEQ ID NO:89 or SEQ ID NO:91 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO:101 or SEQ ID NO:103 or SEQ ID NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or SEQ ID NO:111 or SEQ ID NO:113 or SEQ ID NO:115 or SEQ ID NO:117 or SEQ ID NO:119 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:125 or SEQ ID NO:127 or SEQ ID NO:129 or SEQ ID NO:131 or SEQ ID NO:133 or SEQ ID NO:135 or SEQ ID NO:137 or SEQ ID NO:139 or SEQ ID NO:141 or SEQ ID NO:143 or SEQ ID NO:145 or SEQ ID NO:147 or SEQ ID NO:149 or SEQ ID NO:151 or SEQ ID NO:153 or SEQ ID NO:155 or SEQ ID NO:157 or SEQ ID NO:159 or SEQ ID NO:161 or SEQ ID NO:163 or SEQ ID NO:165 or SEQ ID NO:167 or SEQ ID NO:169 or SEQ ID NO:171 or SEQ ID NO:173 or SEQ ID NO:175 or SEQ ID NO:177 or SEQ ID NO:179 or SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or 201; (ii) a nucleotide sequence having at least about 60% similarity after optimal alignment to SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39 or SEQ ID NO:41 or SEQ ID NO:43 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 or SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:55 or SEQ ID NO:57 or SEQ ID NO:59 or SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73 or SEQ ID NO:75 or SEQ ID NO:77 or SEQ ID NO:79 or SEQ ID NO:81 or SEQ ID NO:83 or SEQ ID NO:85 or SEQ ID NO:87 or SEQ ID NO:89 or SEQ ID NO:91 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO:101 or SEQ ID NO:103 or SEQ ID NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or SEQ ID NO:111 or SEQ ID NO:113 or SEQ ID NO:115 or SEQ ID NO :117 or SEQ ID NO:119 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:125 or SEQ ID NO:127 or SEQ ID NO:129 or SEQ ID NO:131 or SEQ ID NO:133 or SEQ ID NO:135 or SEQ ID NO:137 or SEQ ID NO:139 or SEQ ID NO:141 or SEQ ID NO:143 or SEQ ID NO:145 or SEQ ID NO:147 or SEQ ID NO:149 or SEQ ID NO:151 or SEQ ID NO:153 or SEQ ID NO:155 or SEQ ID NO:157 or SEQ ID NO:159 or SEQ ID NO:161 or SEQ ID NO:163 or SEQ ID NO:165 or SEQ ID NO:167 or SEQ ID NO:169 or SEQ ID NO:171 or SEQ ID NO:173 or SEQ ID NO:175 or SEQ ID NO:177 or SEQ ID NO:179 or SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or 201; (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39 or SEQ ID NO:41 or SEQ ID NO:43 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 or SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:55 or SEQ ID NO:57 or SEQ ID NO:59 or SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73 or SEQ ID NO:75 or SEQ ID NO:77 or SEQ ID NO:79 or SEQ ID NO:81 or SEQ ID NO:83 or SEQ ID NO:85 or SEQ ID NO:87 or SEQ ID NO:89 or SEQ ID NO:91 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO:101 or SEQ ID NO:103 or SEQ ID NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or SEQ ID NO:111 or SEQ ID NO:113 or SEQ ID NO:115 or SEQ ID NO:117 or SEQ ID NO:119 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:125 or SEQ ID NO:127 or SEQ ID NO:129 or SEQ ID NO:131 or SEQ ID NO:133 or SEQ ID NO:135 or SEQ ID NO:137 or SEQ ID NO:139 or SEQ ID NO:141 or SEQ ID NO:143 or SEQ ID NO:145 or SEQ ID NO:147 or SEQ ID NO:149 or SEQ ID NO:151 or SEQ ID NO:153 or SEQ ID NO:155 or SEQ ID NO:157 or SEQ ID NO:159 or SEQ ID NO:161 or SEQ ID NO:163 or SEQ ID NO:165 or SEQ ID NO:167 or SEQ ID NO:169 or SEQ ID NO:171 or SEQ ID NO:173 or SEQ ID NO:175 or SEQ ID NO:177 or SEQ ID NO:179 or SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ I) NO:199 or 201; (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39 or SEQ ID NO:41 or SEQ ID NO:43 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 or SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:55 or SEQ ID NO:57 or SEQ ID NO:59 or SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73 or SEQ ID NO:75 or SEQ ID NO:77 or SEQ ID NO:79 or SEQ ID NO:81 or SEQ ID NO:83 or SEQ ID NO:85 or SEQ ID NO:87 or SEQ ID NO:89 or SEQ ID NO:91 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO:101 or SEQ ID NO:103 or SEQ ID NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or SEQ ID NO:111 or SEQ ID NO:113 or SEQ ID NO:115 or SEQ ID NO:117 or SEQ ID NO:119 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:125 or SEQ ID NO:127 or SEQ ID NO:129 or SEQ ID NO:131 or SEQ ID NO:133 or SEQ ID NO:135 or SEQ ID NO:137 or SEQ ID NO:139 or SEQ ID NO:141 or SEQ ID NO:143 or SEQ ID NO:145 or SEQ ID NO:147 or SEQ ID NO:149 or SEQ ID NO:151 or SEQ ID NO:153 or SEQ ID NO:155 or SEQ ID NO:157 or SEQ ID NO:159 or SEQ ID NO:161 or SEQ ID NO:163 or SEQ ID NO:165 or SEQ ID NO:167 or SEQ ID NO:169 or SEQ ID NO:171 or SEQ ID NO:173 or SEQ ID NO:175 or SEQ ID NO:177 or SEQ ID NO:179 or SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or 201; (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 60% similarity after optimal alignment to SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39 or SEQ ID NO:41 or SEQ ID NO:43 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 or SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:55 or SEQ ID NO:57 or SEQ ID NO:59 or SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73 or SEQ ID NO:75 or SEQ ID NO:77 or SEQ ID NO:79 or SEQ ID NO:81 or SEQ ID NO:83 or SEQ ID NO:85 or SEQ ID NO:87 or SEQ ID NO:89 or SEQ ID NO:91 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO:101 or SEQ ID NO:103 or SEQ ID NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or SEQ ID NO:111 or SEQ ID NO:113 or SEQ ID NO:115 or SEQ ID NO:117 or SEQ ID NO:119 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:125 or SEQ ID NO:127 or SEQ ID NO:129 or SEQ ID NO:131 or SEQ ID NO:133 or SEQ ID NO:135 or SEQ ID NO:137 or SEQ ID NO:139 or SEQ ID NO:141 or SEQ ID NO:143 or SEQ ID NO:145 or SEQ ID NO:147 or SEQ ID NO:149 or SEQ ID NO:151 or SEQ ID NO:153 or SEQ ID NO:155 or SEQ ID NO:157 or SEQ ID NO:159 or SEQ ID NO:161 or SEQ ID NO:163 or SEQ ID NO:165 or SEQ ID NO:167 or SEQ ID NO:169 or SEQ ID NO:171 or SEQ ID NO:173 or SEQ ID NO:175 or SEQ ID NO:177 or SEQ ID NO:179 or SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or 201; (vi) a nucleotide sequence capable of hybriding under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; wherein said nucleotide sequences encode a CFM which imparts an altered visual characterization to said cell or group of cells to a human eye in the absence of extraneous non-white light or particle emission.", "More particularly, there is provided a plant or cells of a plant or parts of a plant or progeny of a plant wherein said plant comprises cells comprising: (i) a nucleotide sequence set forth in SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or SEQ ID NO:201; (ii) a nucleotide sequence having at least about 60% similarity after optimal alignment to SEQ ID NO;189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or SEQ ID NO:201; (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO:189 or SEQ ID NO:191 or SEQ ID NO:193 or SEQ ID NO:195 or SEQ ID NO:197 or SEQ ID NO:199 or SEQ ID NO:201 or its complementary form; (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:190 or SEQ ID NO:192 or SEQ ID NO:194 or SEQ ID NO:196 or SEQ ID NO:198 or SEQ ID NO:200 or SEQ ID NO:202; (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 60% similarity after optimal ali gmnent SEQ ID NO:190 or SEQ ID NO:192 or SEQ ID NO:194 or SEQ ID NO:196 or SEQ ID NO:198 or SEQ ID NO:200 or SEQ ID NO:202; (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; wherein said nucleotide sequences encode a CFM which imparts an altered visual characterization to said plant or cells of a plant to a human eye in the absence of extraneous non-white light or particle emission.", "In a particularly preferred embodiment, there is provided a use of a CFM such as but not limited to GFP or a non-fluorescent GFP-homolog in the manufacture of a plant exhibiting altered visual characteristics to all or a part of said plant or to cells of said plant to a human eye in the absence of extraneous non-white light or particle emission.", "Reference herein to extraneous light is not to be read as encompassing white light or background irradiation.", "The altered visual characteristics are visualized in the presence of white light, for example the light as generated by an 60 W electric bulb or daylight.", "White light includes light that contains all the wavelengths of the visible spectrum, such as sunlight.", "The term “similarity” as used herein includes exact identity between compared sequences at the nucleotide or amino acid level.", "Where there is non-identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels.", "Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels.", "In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.", "Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.", "A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length.", "Because two polynucleotides may each comprise (1) a sequence (i.e.", "only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.", "A “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.", "The comparison window may comprise additions or deletions (i.e.", "gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.", "Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e.", "resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.", "Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al.", "(Nucl.", "Acids Res.", "25: 3389, 1997).", "A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al.", "(Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15).", "The terms “sequence similarity” and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.", "Thus, a “percentage of sequence identity”, for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g.", "A, T, C, G, I) or the identical amino acid residue (e.g.", "Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.", "For the purposes of the present invention, “sequence identity” will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA) using standard defaults as used in the reference manual accompanying the software.", "Similar comments apply in relation to sequence similarity.", "Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.", "Generally, low stringency is at from about 25-30° C. to about 42° C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.", "Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v fonnamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions.", "In general, washing is carried out Tm=69.3+0.41 (G+C) % (Marnur and Doty, J. Mol.", "Biol.", "5: 109, 1962).", "However, the Tm of a duplex DNA decreases by 1° C. with every increase of 1% in the nunber of mismatch base pairs (Bonner and Laskey, Eur.", "J. Biochem.", "46: 83, 1974).", "Formamide is optional in these hybridization conditions.", "Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6×SSC buffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.; high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C. The tobacco ribosomal DNA spacer element may be used to increase the expression of CFMs or colored proteins in transgenic Arabidopsis, carnation, rose or other plant species.", "The tobacco ribosomal DNA spacer element can be used to increase copy number and expression levels of transgenes in plants (Borisjuk et al., Nat.", "Biotechnol.", "18: 1303-1306, 2000).", "The tobacco ribosomal DNA spacer element may be inserted into pCGP2772, pCGP2785, pCGP3259 or other construct used to express CFMs or colored proteins in plants.", "There is a clear correlation between codon usage and gene expression levels in Arabidopsis, Caenorhabditis and Drosophila (Duret and Mouchiroud, Proc.", "Natl.", "Acad.", "Sci.", "USA 96: 4482-4497, 1999).", "Codon usage within the open reading frames of CFM or colored proteins may be modified to increase levels of CFMs or colored protein in transgenic Arabidopsis, carnation, rose or other plant species.", "A recent study by Stevens et al.", "(Plant Physiology 173-182, 2000) has highlighted the possibility of increasing the stability of recombinant proteins in transgenic plants by modifying protein glycosylation patterns.", "Plant virus gene vectors may be used for high level gene expression of foreign genes in plants (Scholthof and Scholthof, Annu.", "Rev.", "of Phytopathol.", "34: 299-323, 1996; Chapman et al., Plant Journal 2: 549-557, 1992).", "The use of a plant virus expression system may increase levels of CFMs or colored protein in transgenic Arabidopsis, carnation, rose or other plant species.", "Selection of an appropriate virus type or strain may allow the expression of CFMs or colored protein in specific tissues or patterns to produce novel phenotypes.", "For example a CFM or colored protein gene may be incorporated into the genome of tulip breaking virus or tulip chlorotic blotch potyvirus to induce colored sector production in tulip or other flowers.", "The availability of the isolated CFMs of the present invention further provides the possibility for generating antibodies, whether monoclonal or polyclonal, against any or all of these isolated sequences or derivatives or homologs thereof.", "Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al.", "(Current Protocols in Immunology, John Wiley & Sons, N.Y., 1991-94) and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988,which are both herein incorporated by reference.", "Generally, antibodies of the invention bind to or conjugate with a polypeptide, fragment, variant or derivative thereof.", "For example, the antibodies may comprise polyclonal antibodies.", "Such antibodies may be prepared, for example, by injecting a polypeptide, fragment, variant or derivative thereof into a production species, which may include mice or rabbits, to obtain polyclonal antisera.", "Methods for the production of polyclonal antibodies are well known to those skilled in the art.", "Exemplary protocols are descnbed in Coligan et al, 1991-1994, supra and Harlow and Lane, 1988, supra.", "In lieu of polyclonal antisera obtained in a production species, monoclonal antibodies may be produced using the standard method as described by Köhler & Milstein (European Journal of Immunology 6: 511-519, 1976) or by more recent modifications thereof as, for example, described in Coligan et at.", "(1991-1994, supra) by immortalizing spleen or other antibody-producing cells derived from a production species which has been inoculated with one or more of the polypeptides, fragments, variants or derivatives of the present invention.", "The present invention also contemplates antibodies that comprise Fc or Fab fragments of the polyclonal or monoclonal antibodies referred to above.", "Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the peptides of the present invention.", "Such scFvs may be prepared, for example, in accordance with the methods described respectively in U.S. Pat.", "No.", "5,091,513, European Patent No 239,400 or Winter and Milstein (Nature 349: 293, 1991).", "Antibodies produced in accordance with the present invention may be used for affinity chromatography in isolating natural or recombinant pigment polypeptides.", "For appropriate protocols, reference may be made to immunoaffinity chromatographic procedures described in Chapter 9.5 of Coligan et al.", "(1991-1994, supra).", "Accordingly, the present invention provides an antibody specific for a CFM, said CFM comprising an amino acid sequence in its N-terminal end selected from SVIAK (SEQ ID NO:5), (M)SVIAT (SEQ ID NO:6), SGIAT (SEQ ID NO:7), SVIVT (SEQ ID NO:8) or SVSAT (SEQ ID NO:9).", "Preferably, the isolated antibody is specific for a CFM comprising an amino acid sequence selected from the list comprising SVIAT QMTY KVYM SGT (SEQ ID NO:10), SVIAT QMTY KVYM PGT (SEQ ID NO:11), SVIAT QVTY KVYM SGT (SEQ ID NO:12), SGIAT QMTY KVYM SOT (SEQ ID NO:13), SVIVT QMTY KVYM SOT (SEQ ID NO:14), SVSAT QMTY KVYM SGT (SEQ ID NO:15), SVIAK QMTY KVNM SGT (SEQ ID NO:16), SVIAK QMTY KVYM SDT (SEQ ID NO:17) and/or SVIAK QMTY X1X2YX3 SGT (SEQ ID NO:18) wherein X1, X2 and X3 may be any amino acid provided that X1 is not K; X2 is not V; X3 is not M. Most preferably, the antibody is specific for a CFM comprising an amino acid sequence selected from the listing comprising SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 190, 192, 194, 196, 198, 200 and 202.Once antibodies have been produced, one or more polypeptides of the present invention may be conjugated thereto, preferably to a secondary antibody as part of an antibody staining complex, and thereby become useful as a fluorescent marker in microscopy and related procedures.", "Alternatively, or in addition, one or more nucleic acid sequence encoding a polypeptide of the present invention may be expressed as a recombinant polypeptide fused with a secondary antibody.", "These antibodies may be useful for in situ labelling procedures or in other related procedures such as fluorescence in situ hybridization (FISH).", "As already described above, a fusion partner well known in the art is GFP.", "This fusion partner may serve as a fluorescent “tag” which facilitates the identification and/or localization, by fluorescence microscopy or by flow cytometry, of a polypeptide fused thereto.", "Flow cytometric methods such as fluorescence activated cell sorting (FACS) are particularly useful in this regard.", "There is perpetual interest in developing high-sensitivity biochemical assays, which employ luminescence, fluorescence or visible color rather than radioisotopes, for use in research and in medicine.", "Interest in developing assays with visible detection systems is increasing as these often obviate the need for expensive luminescence, fluorescence or isotopic detection equipment.", "Accordingly, the present invention further comprises a diagnostic assay comprising screening for the presence of CFM wherein the nucleic acid molecule encoding said CFM is expressed in a cell.", "The capability of the CFMs to absorb incident light which encompasses the UV range (320-700 nm) makes them useful candidates for inclusion as components in topically-applicable sun screen formulations.", "The purpose of a sun screen is to block the excessive UV radiation from affecting the skin.", "Sun screen formulations act by deflecting and scattering the incident light that produces burning and tanning of the skin or by absorbing this light.", "It is known that careful selection of sun screens can offer this protection to the skin and reduce the darkening and damaging effects of the radiation.", "Such a formulation would include, for example, an effective amount of one or more CFMs of the present invention, optionally admixed with a pharmaceutically acceptable vehicle such as a carrier or excipient that will not harm the skin.", "By “carrier” is meant a solid or liquid filler, diluent or substance that may be safely used in topical administration.", "These carriers may be selected from a group including powder absorbants, creams, oils, synthetic oils, phosphate buffered solutions, emulsifiers, and liquids such as emollients, propellants, solvents, humectants, thickners, isotonic saline, and pyrogen-free water.", "The sun screen formulation may also include other screening agents, well known in the art, such as propyl hydroxybenzoate, dimethylaminobenzoate (PABA), phenyl salicylates and/or octyl methoxycinnamate.", "These formulations may be prepared for topical application to the skin in the form of conventional products such as lotions, creams, ointments and aerosol products.", "A useful sun screen formulation and method of preparing an emulsion therefor are provided in International Patent Publication No.", "WO 00/46233 in Example 4.Accordingly, the present invention provides a biomatrix comprising a CFM, said CFM comprising a polypeptide which, in a cell, alone or together with one or more other molecules imparts an altered visual characteristic to said cell when visualized by a human eye in the absence of excitation by extraneous non-white light or particle emission.", "Reference to a “biomatrix” includes any composition comprising a CFM such as a cell, sun screen, a purified preparation of a CFM or any solid support onto or into which a CFM is immobilized.", "Reference to a biomatrix also includes a bioinstrument.", "Yet another aspect of the present invention contemplates the use of a CFM in a cosmetic or light filtering composition.", "Cosmetics include many products that can be applied to the face or body in order to alter appearance or color.", "New combinations of ingredients may result in cosmetic compositions that protect against environmental stresses such as exposure to the sun.", "The use of a CFM in a cosmetic may provide a visible coloration that is aesthetically desirable and/or it may provide light filtering capability such as may be afforded, for example, by a sun screen.", "Light filtering compositions may also be used to screen out or block UV light or different wavelengths of light within the entire spectrum.", "A cosmetic or light filtering composition according to the invention may also include cosmetically or pharmaceutically compatible carriers, preservatives, emusifiers, thickners, perfume, color, as well as other materials having properties which are beneficial for skin, such as moisturizers, emollients anti-ageing compounds inter alia.", "Other applications of the CFMs of the present invention may also be contemplated.", "Since they are active in affecting the manner in which, and degree to which, various kinds of impinging light/radiation are processed and detected, the CFMs may find application in, for example, transducing or intensifying an image.", "For example, converting less visible wavelengths of light such as w radiation to wavelengths that are more visible might be beneficial.", "A gel or similar material comprising a CFM may be located behind a membrane or selective barrier and combined with an optic fiber probe, such as an optode or micro-electrode.", "Changes in physical and chemical environments into which the probe is inserted may be calibrated to changes in fluorescent intensity and/or fluorescence half-life, to provide micro-scale measurements of parameters such as oxygen concentration and pH.", "Similar applications involving fluorescence intensity and/or half-life fluorescent imaging techniques may also incorporate a CFM of the present invention.", "As stated above, each of the CFMs of the present invention and homologs thereof, has distinct excitation and emission characteristics.", "These may be fluorescently coupled such that captured photons can be passed successively between a plurality of CFMs, for example as many as six.", "This lengthens the pathway and the amount of time that a photon spends within any material comprising the CFMs and may thereby increase light intensity within these environments considerably.", "Such a light enhancement effect may be useful for providing additional light for growing phototrophic organisms, for example plants, algae and/or corals, by increasing the likelihood of a photon's interaction with constituent photosystems.", "This embodiment of the present invention may also be useful for creating light enhancer fluids that may be used to increase light intensity within a medium above that of incident light.", "Furthermore, a CFM embedded in a gel matrix or other useful material may improve image quality in situations of distorted light spectra such as, for example, under water where light is shifted to the blue end of the spectrum.", "A CFM rendered water-soluble may prove useful in a range of different types of liquids.", "Alternatively, or in addition, a derivative or homolog of polypeptide of the present invention may be synthesized by substituting amino acids or adding N- or C-terminal tags to increase their insolubility and hence make them more useful in less polar environments.", "In this embodiment, a CFM, or a CFM modified such as through amino acid inclusion or substitution to make it more hydrophobic, combined with a water-soluble or non-water soluble emulsion, may be used to coat materials that experience UV damage such as, for example, plastics and car upholstery.", "The present invention is further described by the following non-limiting Examples.", "EXAMPLE 1 General Methods In general, the methods followed were as described in Sambrook et al.", "(Molecular Cloning: A Laboratory Manual.", "(2nd edition), Cold Spring Harbor Laboratory Press, USA, 1989).", "The cloning vectors pBluescript and PCR script were obtained from Stratagene.", "pCR7 2.1 was obtained from Invitrogen.", "The bacterial expression vector pQE-30 was obtained from Qiagen.", "E. coli Transformation The Eschenichia coli strains used were: DH5α supE44, Δ (lacZYA-ArgF)U169, (ø801acZΔM15), hsdR17(rk−, mk+), recA1, endA1, gyrA96, thi-1, relA1, deoR.", "(Hanahan, J. Mol.", "Biol.", "166: 557 1983 XL1-Blue supE44, hsdR17(rk−, mk+), recA1, endA1, gyrA96, thi-1, relA1, lac−,[F'proAB, lacIq, lacZΔM15, Tn10(tetR)] (Bullock et al., Biotechniques 5: 376, 1987).", "BL21-Codonlus-RIL strain ompT hsdS(rB-mB-) dcm+Tetr gal endA Hte [argU ileY leuW Camr] M15 E. coli is derived from E.coli K12 and has the phenotype Nals, Strs, Rifs, Thi−, Ara+, Gal+, Mtl−, F−, RecA+, Uvr+, Lon+.", "Transformation of the E. coli strains was performed according to the method of Inoue et al., (Gene 96: 23-28, 1990).", "Agrobactedunm tuinefaciens Strains and Transformations The disarmed Agrobacterium tumefaciens strain used was AGL0 (Lazo et al.", "Bio/technology 9: 963-967, 1991).", "Plasmid DNA was introduced into the Agrobacterium tumefaciens strain AGL0 by adding 5 μg of plasmid DNA to 100 μL of competent AGL0 cells prepared by inoculating a 50 mL LB culture and growing for 16 hours with shaking at 28° C. The cells were then pelleted and resuspended in 0.5 mL of 85% v/v 100 mM CaCl2/15% v/v) glycerol.", "The DNA-Agrobacterium mixture was frozen by incubation in liquid N2 for 2 minutes and then allowed to thaw by incubation at 37° C. for 5 minutes.", "The DNA/bacterial mix was then placed on ice for a further 10 minutes.", "The cells were then mixed with 1 mL of LB (Sambrook et al., 1989 supra) media and incubated with shaking for 16 hours at 28° C. Cells of A. tumefaciens carrying the plasmid were selected on LB agar plates containing 50 μg/mL tetracycline.", "The confirmation of the plasmid in A. tumefaciens was done by restriction enzyme analysis of DNA isolated from the tetracycline-resistant transformants.", "Saccharomyces cerevisiae Strains and Transformations The yeast expression vector used was pYE22m (Tanaka et al., J. Biochem.", "103: 954-961, 1988).", "The yeast strain G-1315 (Mat α, trp1) (Ashikari et al., Appl.", "Microbiol.", "Biotechnol.", "30: 515-520, 1989) was transformed with plasmid DNA according to Ito et al., (J. Bacteriol.", "153: 163-168, 1983).", "The transformants were selected by their ability to restore G-1315 to tryptophan prototrophy.", "DNA Ligations DNA ligations were carried out using the Amersham Ligation Kit according to procedures recommended by the manufacturer.", "Isolation and Purification of DNA Fragments Fragments were generally isolated on a 1% w/v agarose gel and purified using the QIAEX II Gel Extraction kit (Qiagen).", "Reparation of Overhanging Ends After Restriction Dizestion Overhanging 5′ ends were repaired using DNA polymerase (Klenow fragment) according to standard protocols (Sambrook et al., 1989 supra).", "Overhanging 3′ ends were repaired using T4 DNA polymerase according to standard protocols (Sambrook et al, 1989 supra).", "Removal of Phosphoryl Groups from Nucleic Acids Shrimp alkaline phosphatase (SAP) (USB) was typically used to remove phosphoryl groups from cloning vectors to prevent re-circularization according to the manufacturer's recommendations.", "Polymerase Chain Reaction (PCR) Unless otherwise specified, PCR conditions using plasmid DNA as template included using 2 ng plasmid, 100 ng each of primers, 2 μL 10 mM dNTP mix, 5 μL 10× PfuTurbo (registered trademark) DNA polymerase buffer (Stratageme), 0.5 μL PfuTurbo (registered trademark) DNA polymerase (2.5 units/μL) (Stratagene) in a total volume of 50 μL.", "Cycling conditions were an initial denaturation step of 5 min at 94° C., followed by 35 cycles of 94° C. for 20 sec, 50° C. for 30 sec and 72° C. for 1 min with a last treatment of 72° C. for 10 min and then finally storage at 4° C. PCRs were performed in a Perkin Elmer GeneAmp PCR System 9600.32P-Labelling of DNA Probes DNA fragments (50 to 100 ng) were radioactively labelled with 50 μCi of [α-32P]-dCTP using a Gigaprime kit (Geneworks).", "Unincorporated [α-32P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column.", "Plasmid Isolation Single colonies were analyzed for inserts by growing in LB broth (Sambrook et al., 1989, supra) with appropriate antibiotic selection (e.g.", "100 μg/mL ampicillin or 10 to 50 μg/mL tetracycline for binary vector constructs).", "Plasmid DNA was purified using the alkali-lysis procedure (Sambrook et al., 1989, supra) or using The WizardPlus SV minipreps DNA purification system (Promega) or Qiagen Plasmid Mini Kit (Qiagen).", "Once the presence of an insert had been determined, larger amounts of plasmid DNA were prepared from 50 mL overnight cultures using a QIAfilter Plasmid Midi kit (Qiagen).", "DNA Sequence Analysis DNA sequencing was performed using the PRISM (trademark) Ready Reaction Dye Primer Cycle Sequencing Kits from Applied Biosystems.", "The protocols supplied by the manufacturer were followed.", "The cycle sequencing reactions were performed using a Perkin Elmer PCR machine (GeneAmp PCR System 9600).", "Sequencing runs were performed by the Australian Genome Research Facility at The Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia).", "Homology searches against Genbank, SWISS-PROT and EMBL databases were performed using the FASTA and TFASTA programs (Pearson and Lipman, 1988) or BLAST programs (Altschul et al., J. Mol.", "Biol.", "215(3): 403-410, 1990).", "Percentage sequence similarities were obtained using LALIGN program (Huang and Miller, Adv.", "Appl.", "Math.", "12: 373-381, 1991) using default settings.", "Multiple sequence alignments were produced using ClustalW (Thompson et al., Nucleic Acids Research 22: 4673-4680, 1994).", "Petunia Transformations (a) Plant Material Leaf tissue from mature plants of P. hybrida cv Mitchell (or Ba20 or Br140w) was treated in 1.88% w/v sodium hypochlorite for 2 minutes and then rinsed three times in sterile water.", "The leaf tissue was then cut into 25-50 mm2 squares and precultured on MS media (Murashige and Skoog, Physiol.", "Plant 15: 73-97, 1962) supplemented with 1.0 mg/L α-benzylaminopurine (BAP) and 0.1 mg/L α-naphthalene acetic acid (NAA) for 24 hours under white fluorescent lights.", "(b) Co-Cultivation of Agrobacterium and Petunia Tissue A. tumefaciens strain AGL0 containing a binary vector were maintained at 4° C. on LB agar plates with 50 μg/mL tetracycline.", "A single colony was grown overnight in liquid LB medium containing 40 μg/mL tetracycline.", "The following morning 1-2 mL of the overnight culture was added to a fresh batch of 25 mL liquid LB medium and the culture was grown at 37° C. with shaking until an absorbance reading at 650 mn (A650) of 0.4 to 0.8 was reached.", "A final concentration of 5×108 cells/mL was prepared by dilution in liquid MS medium containing 50 μM acetosyringone and 3% w/v sucrose B5 vitamins (Gamborg et al., Exp.", "Cell Res.", "50: 151-158, 1968).", "The leaf discs were dipped for 2 minutes into the inoculum and then blotted dry and placed on co-cultivation media for 5 days.", "The co-cultivation medium consisted of SH medium (Schenk and Hildebrandt, Can.", "J. Bot.", "50: 199-204, 1972) supplemented with 0.05 mg/L kinetin and 1.0 mg/L 2,4-D. (c) Recovery of Transgenic Petunia Plants After co-cultivation, the leaf discs were transferred to selection medium (MS medium supplemented with 3% w/v sucrose, 3 mg/L BAP, 0.2 mg/L IAA, 1 μg/L chlorsulfuron, 300 mg/L timentin and 0.3% w/v Gelrite Gellan Gum (Schweizerhall).", "Regenerating explants were transferred to fresh selection medium after 2 weeks.", "Adventitious shoots which survive the chlorsulfuron selection are isolated and transferred to BPM containing 1 μg/L chlorsulfuron and 300 mg/L timentin for root induction.", "All cultures are maintained under a 16 hour photoperiod (60 μmol.", "m−2, s−1 cool white fluorescent light) at 23±2° C. When roots reach 2-3 cm in length the transgenic petunia plantlets are transferred to autoclaved Debco 51410/2 potting mix in 8 cm tubes.", "After 4 weeks, plants are be replanted into 15 cm pots, using the same potting mix, and maintained at 23° C. under a 14 hour photoperiod (300 μmol.", "m−2, s−1 mercury halide light).", "Arabidopsis Transformations Arabisopsis thaliana ecotype WS-2 seeds were obtained from The University of Melbourne, Parkville, Melbourne, Australia.", "Plant growth conditions and transformation of A. thaliana were as essentially as described by Clough and Bent, (Plant J., 16: 735-743, 1998) except that seeds from the transformed plants were selected on 100 μg/mL chlorsulfuron when binary vectors containing the SuRB selectable marker gene were used for the transformation process.", "EXAMPLE 2 Isolation of New Colored-Protein Sequences from Heron Island Coral Coral samples were collected from Heron Island Reef flat, Queensland, Australia.", "These samples were viewed as whole tissue under a fluorescent microscope, as described herein.", "Assessment of Fluorescence Properties Table 2 shows taxonomic relationships of GFP isolated from the phylum Cnidaria and comparison with one amino acid sequence of the invention (Aams2-pep; SEQ ID NO:88).", "Fluorescent properties were analysed using an Olympus fluorescent microscope (BH2-RFL) with filter combinations, as shown in Table 3.Tables 4 and 5 show fluorescent properties of colors for different species of organisms from Anthozoa and Hydrozoa.", "Total RNA Isolation Plating corals were ground with a mortar and pestle and branching corals were scrubbed with a toothbrush directly into cold solution D, as described in Chomczynski and Sacchi, 1987, supra.", "Solution D-comprising tissue was homogenized using a glass homogenizer and transferred to 1.5 ml eppendorf microcentrifige tubes.", "A 10% w/v 2 M sodium acetate (pH 4) solution was added prior to phenol chloroform extraction and extracted material was precipitated overnight in isopropanol at −20° C. Pellets were resuspended in solution D, and precipitated again in isopropanol.", "Resulting pellets were dissolved in 3 mM EDTA and 50 mM sodium acetate (pH 5) to be finally precipitated and stored at −20° C. in ethanol.", "cDNA Construction RNA isolated from collected coral tissue was used to prepare cDNA.", "cDNA were constructed using a directional cDNA synthesis kit from Clontech Laboratories (Palo Alto, Calif., USA) herein incorporated by reference.", "5′ Forward Primers for PCR Amplification POC FOR TCC GTT ATC GCT AAA CAG ATG ACC TAC AAA SEQ ID NO:1 POC 220 GGC GAC CAC AGG TTT GCG TGT SEQ ID NO:2 MSVIAT(FOR) ATG AGT GTG ATC GCT ACA CAA SEQ ID NO:3 SEQ ID NO:1 was previously designed as a 5′ (or forward primer) for PCR amplification of nucleic acids encoding coral pigment proteins.", "SEQ ID NO:1 was shown to anneal to nucleic acids encoding a polypeptide comprising amino acids, SVIAK (SEQ ID NO:5): Refer to Dove et al.", "(2001; supra) and International Patent Publication No.", "WO 00/46233.SEQ ID NO:2 was originally designed as a 3′ (or reverse primer) for PCR amplification of nucleic acids encoding coral pigment polypeptides as disclosed in WO 00/46233.in addition to annealing to a 3′ region of the nucleic acid as intended, SEQ ID NO:2 also anneals to a 5′ UTR region of pocilloporin from Acropora aspera as disclosed herein.", "SEQ ID NO:3 is newly designed and synthesized based on sequence information from PCR amplification products using SEQ ID NO:1 and SEQ ID NO:2.The amplified products comprise 5′ UTR nucleotide sequence that includes sequence encoding a novel amino terminal end for a polypeptide similar to, but distinct from, the polypeptide disclosed in International Patent Publication No.", "WO 00/46233.This novel polypeptide has an amino terminal end comprising amino acids (M)SVIAT (SEQ ID NO:6; FIG.", "3).", "Accordingly, SEQ ID NO:3 anneals to nucleic acids encoding a peptide comprising (M)SVIAT (SEQ ID NO:6).", "Although peptide sequences SVIAK (SEQ ID NO:5) and (M)SVIAT (SEQ ID NO;6) differ by only one amino acid, the corresponding nucleic acids only share 67% identity (12 nucleic acids of 18).", "Notably, SEQ ID NO:1 cannot be used to amplify sequences starting with the N-terminal peptide (M)SVIAT (SEQ ID NO:6), and SEQ ID NO:3 cannot be used to amplify sequences begining with the SVIAK (SEQ ID NO:5) peptide.", "3′ Reverse Primers for PCR Amplification POC 231 TTT GTG CCT TGA TTT GAC TCT SEQ ID NO:4 SEQ ID NO:2 was also used as a 3′ reverse primer and is described above.", "SEQ ID NO:4 was designed to anneal to a 3′ end of previously isolated pocilloporin from Acropora aspera (Dove et al.", "[2001; supra] and International Patent Publication No.", "WO 00/46233).", "PCR Amplification PCR amplification was performed using a combination of the abovementioned SEQ ID NOs as described in more detail hereinafter.", "Hybaid PCR express (Hybaid PCR Express, Integrated Sciences, Australia) was used according to instructions provided therein.", "Amplification products were separated by gel electrophoresis on a 1.5% w/v agarose gel and nucleic acid bands comprising desired nucleic acids were visualized using standard methods.", "Agarose gel comprising the desired nucleic acids were gel purified and the gel purified nucleic acids were inserted by ligation into pGemT-vector (Promega, Madison, Wis., USA) producing a recombinant vector.", "The inserted nucleic acids were sequenced using T7 and SP6 primers, which flank the inserted nucleic acid (sequencing service provided by AGRF; University of Queensland, Australia).", "Sequencing of the insert Was performed at least twice in both forward and reverse directions until ambiguities were resolved.", "The following sequences were sequenced in a single direction: Ce61-7sv-rep (SEQ ID NO:37); Ce61-5sv-rep (SEQ ID NO:35); PM1Asv-rep (SEQ ID NO:57); PM1Asv-rep (SEQ ID NO:55); Mi68Dms (SEQ ID NO:119); Acams-3 (SEQ ID NO:101).", "Table 6 shows amino acid sequences within 5 Angstroms of the fluorphore which encode possible spectral variants of the polypeptides of the invention comprising an amino acid sequence SGIAT (SEQ ID NO:7), SVIVT (SEQ ID NO:8), SVSAT (SEQ ID NO:9) or (M)SVIAT (SEQ ID NO:6) at the amino terminal end.", "These amino acid sequences were slated from nucleic acid sequences derived by PCR using 5′ primers: SEQ ID NO:2 (5′ UTR) and SEQ ID NO:3 [(M)SVIAT]; and 3′ primers: SEQ ID NO:2 and SEQ ID NO:4.Table 7 shows amino acid sequences within 5 Angstroms of the fluorphore which encode possible spectral variants of the polypeptides of the invention comprising an amino acid sequence SVIAK (SEQ ID NO:5) at the amino terminal end.", "These amino acid sequences were translated from nucleic acid sequences derived by PCR using 5′ primer SEQ ID NO:1 and 3′ primer SEQ ID NO:2, and 3′ SEQ ID NO:3.Polypeptide Modelling A 3-dimensional model of the polypeptides was used to predict those amino acids within 5 Angstroms of the fluorophore “QYG”.", "These amino acids have potential to influence spectral properties (Tsien, 1998, supra and Dove et al., 2001, Yupra) and are shown in Tables 6 and 7.The amino acids which are predicted to be located within 5 Angstroms of the fluorophore correspond to amino acid residues 37, 39, 56-65 (which comprises the fluorophore QYG), 86, 88, 90, 104, 106, 115, 139, 141, 143, 156, 158, 171, 192, 194, 208, 209 and 210.Amino acid residue numbers refer to numbering beginning with amino terminal amino acids S-V-I as residues 1, 2 and 3, respectively.", "Information in relation to amino acid residues within 5 Angstroms of the fluorophore and details of atomic contacts for the polypeptide disclosed in Table 4 of International Patent Publication No.", "WO 00/46233, may be useful with the polypeptides of the present invention.", "In Tables 6 and 7, “Type” refers to a grouping or class of common amino acids within 5 Angstroms of the fluorophore, and “” indicates an internal stop codon.", "“Name” refers to consensus sequence name from multiple repeat sequences.", "FIG.", "9 lists many of the pigment polypeptides of the invention and indicates the amino acid residues that are located within 5 Angstroms of a fluorophore region of the polypeptide.", "In addition, those amino acids residue positions where variation is found throughout the different polypeptides are shown.", "Variable amino acids indicated throughout the polypeptide may be significant, as they may interfere with polypeptide folding.", "Amino Acid and Nucleotide Sequence Comparisons FIGS.", "1 and 3 show amino acid sequences for polypeptides comprising amino terminal SVIAK (SEQ ID NO:5; FIG.", "1) and comprising (M)SVIAT (SEQ ID NO:6), SGIAT (SEQ ID NO:7), SVIVT (SEQ ID NO:8) and SVSAT (SEQ ID NO:9) at or near the terminal amino end (FIG.", "3).", "Aams-2.pep (SEQ ID NO:88) and Aams-4.pep (SEQ ID NO:90) are shown comprising additional amino acids at the amino terminal end.", "Alignments of the corresponding nucleotide sequences of the amino acid sequences shown in FIGS.", "1 and 3 are set forth in FIGS.", "2 and 4, respectively.", "Polypeptides comprising five shared amino acid sequences SVIAK (SEQ ID NO:5), (M)SVIAT (SEQ ID NO:6), SGIAT (SEQ ID NO:7), SVIVT (SEQ ID NO:8) and SVSAT (SEQ ID NO:9) may be grouped accordingly.", "Additional common amino acids immediately adjacent to the abovementioned amino acids are shown below: SVIAT QMTY KVYM SGT; (SEQ ID NO:10) SVIAT QMTY KVYM PGT; (SEQ ID NO:11) SVIAT QVTY KVYM SGT; (SEQ ID NO:12) SGIAT QMTY KVYM SGT; (SEQ ID NO:13) SVIVT QMTY KVYM SGT; (SEQ ID NO:14) SVSAT QMTY KVYM SGT; (SEQ ID NO:15) SVIAK QMTY KVNM SGT; (SEQ ID NO:16) SVIAK QMTY KVYM SDT; (SEQ ID NO:17) and SVIAK QMTY X1X2YX3 SGT, (SEQ ID NO:18) wherein X1, X2 and X3 may be any amino acid provided that X1 is not K; X2 is not V; X3 is not M. FIG.", "5 shows an alignment of amino acid sequences comprising SVIAK (SEQ ID NO:5) at the amino terminus and a stop or termination codon at corresponding amino acid residue 14.The termination codon results from the addition of two nucleic acid residues.", "The resulting polypeptide is much different when compared with polypeptides lacking this termination codon.", "An alignment of the corresponding nucleic acid sequences is shown in FIG.", "6.These nucleic acids are approximately 40 nucleotide bases longer than those lacking the termination codon (FIG.", "6).", "The differences can be more redily seen by referring to FIG.", "7, which shows an alignment of one nucleic acid sequence comprising the termination codon (SEQ ID NO:169) and a nucleic acid sequence lacking the termination codon (SEQ ID NO:19).", "Previously-disclosed SVIAK (SEQ ID NO:5)-containing proteins Aapat-1 (SEQ ID NO:181) and Aapat-2 (SEQ ID NO:182) are also included on an amino acid sequence alignment with many of the SVIAK (SEQ ID NO:5)-containing polypeptides of the present invention, in FIG.", "8.Shaded amino acid residues indicate amino acids unique to SEQ ID NO:181 and/or SEQ ID NO:182.EXAMPLE 3 Isolation of New Colored-Proten Sequences from Melbourne Coral Extraction and Visualization of Colored Proteins from Coral Samples of various coral and algae were purchased from Water World Aquarium (Melbourne, Australia) and Coburg Aquarium (Melbourne, Australia).", "These included Goniopora sp.", "(“flower pot coral”) [brownish tentacles with an iridescent green centre underwater], green Acropora sp.", "coral (“staghorn coral”), brown/light blue Porites sp.", "coral (“finger”), Sinularia sp.", "and Tubastrea sp.", "corals as well as deep blue and bright orange Corallimorphs (Discosoma sp.).", "Small samples of each coral were incubated in 1 M sodium phosphate buffer pH 7.5 at 4° C. A sample of “purple algae” that was growing on dead coral (normally sold as “living rock”) was also collected in buffer.", "After 48 h the Acropora sp.", "extract appeared yellow-brown in color, the Porites sp.", "extract appeared orange in color and the purple algae extract was a clear pink color.", "When the extracts were exposed to UV light the Acropora sp.", "extract contained orange and blue fractions, the Porites sp.", "extract contained pink fractions and the “purple algae” extract was a bright orange color.", "Goniopora sp.", "coral tips were extracted in 1 M Na phosphate buffer pH 7.5.After an overnight incubation at 4° C. the extract was orange-pink under natural light and appeared orange under UV light.", "Fluorescent green fractions were also observed in the solid phase under UV light.", "A 10 μL sample of the crude extracts described above was electrophoresed through precast SDS PAGE gels (12% w/v resolving, 4% w/v stacking gel) (Ready Gels, Biorad) in a running buffer made of 25 mM Tris-HCl, pH 8.3, 192 mM glycine, 0.1% w/v SDS at 100V for 75 min.", "The crude protein extracts were either denatured by boiling in 10% v/v glycerol, 3% w/v SDS, 3% β-mercaptoethanol, 0.025% W/V bromophenol blue or loaded in their native state in 5% v/v glycerol, 0.04% w/v bromophenol blue.", "Standards included pre-stained Low Range markers (Biorad) which contained standard protein samples of 116 kDa, 80 kcDa, 51.8 kDa and 34.7 kDa.", "Prior to staining with Coomassie blue (0.25% (w/v) Coomassie Brilliant Blue, 45% (v/v) methanol, 10% (v/v) acetic acid), PAGE gels were examined under a hand-held UV transilluninator (BLAK-RAY, longwave UV lamp, model B100 AP, UVP Inc).", "The non-denatured crude protein extract from Goniopora sp.", "contained orange bands (running higher than 116 kD marker protein) and blue-green bands (runing between 51.8 kD and 80 kD protein markers).", "The non-denatured crude protein extract from Porites sp.", "contained two orange bands under UV light at approximately the same position as that from Goniopora sp (i.e.", "running higher than 116 kD marker protein).", "The non-denatured crude protein extract from Acropora sp.", "contained a single orange band under UV light at approximately the same position as that from Goniopora sp.", "(i.e.", "running higher than 116 kD marker protein) as well as a green band (running between 80 kD and 116 kD marker proteins).", "These fluorescent bands were not observed in any of the denatured crude protein extracts.", "No protein bands were visible under natural light before Coomasie blue staining.", "Isolation of RNA and Synthesis of cDNA from Coral Total RNA was isolated from the anthozoans Acropora sp., Discosoma sp., Sinularia sp.", "and Tubastrea sp.", "using RNeasy Plant mini kit (Qiagen) or the method of Turpen and Griffith (Biotechniques 4: 11-15, 1986).", "Complementary DNA was synthesized using 1 μg total RNA, 1 μL DNase RQ1 RNase free (Promega), 1 μL 10× buffer (final concentration: 40 mM Tris-HCl pH 8, 10 mM NaCl, 6 mM MgCl2, 10 mM CaCl2).", "The reactions were incubated at 37° C. for 10 min then 65° C. for 10 min.", "One microlitre (1 μg) of primer dT(17)Ad2Ad1 (SEQ ID NO:183) was then added and the reaction was boiled for 5 min and then incubated on ice for 5 min.", "4 μL 5×RT buffer, 2 μL 0.1 M DTT, 1 μL 10 mM dNTPS and 1 μL RNasin (Promega) were then added and the reaction was incubated at 50° C. for 2 min.", "1 μL (200 U) Superscript II reverse trancriptase (Gibco BRL) was then added and the reaction was incubated at 50° C. for 1.5 h. The cDNA was purified using QIAquick PCR purification kit (Qiagen).", "PCR of Colored Protein Sequences Oligonucleotide primers “vispro-F1” (SEQ ID NO:184) and “vispro-R1” (SEQ ID NO:185) were designed to hybridize to the 5′ and 3′ ends of T7SP6BASPOC3 and T7SP6BASPOC4 sequences, respectively (International Patent Application No.", "PCT/AU00/00056).", "The primer “vispro-F1” (SEQ ID NO:184) contained a BamHI site for cloning into the bacterial expression vector pQE-30 (Qiagen) and an AscI site with a translation initiating codon for cloning into binary vectors.", "The primer “vispro-R1” (SEQ ID NO:185) contains a PstI site for cloning into the bacterial expression vector pQE-30 and a PacI site with translation termination codon for cloning into binary vectors.", "vispro-F1 (5′ to 3′) AscI BamHI CAG GGCGCGCC ATG GGA TCC GTT ATC GCT SEQ ID NO:184 M G S V I A AAA CAG ATG ACC K Q M T vispro-R1 (5′ to 3′) PacI PstI GGG TTA ATT AAG CTG CAG GGC GAC CAC SEQ ID NO:185 stop N L Q L A V V AGG TTT GCG TG P K R Polymerase chain reactions were set up using 20 pmole vispro-F1 (SEQ ID NO:184) and 20 pmole vispro-R1 (SEQ ID NO:185) primers and 5 μL cDNA synthesized from coral RNA as template, 2.5 units HotStarTaq (trademark) DNA polymerase (Qiagen), 200 μM dNTP mix and 1×PCR buffer (Qiagen) in a 50 μL reaction.", "PCR conditions included a denaturation step at 95° C. for 15 min, followed by 35 cycles of 94° C. for 30 sec, 50° C. for 30 sec and 72° C. for 1 min with a final treatment at 72° C. for 10 min followed by storage at 4° C. PCR products were electrophoresed through a 1% w/v agarose gel.", "Products of ˜700 bp were excised from the gel and purified using QLKEX II Gel Extraction Kit (Qiagen).", "Purified DNA was digested with BamHI and PstI restriction enzymes and re-purified using a QIAquick PCR purification Kit (Qiagen).", "The purified DNA was ligated with BamHI/PstI ends of the bacterial expression vector pQE-30 (Qiagen).", "Ligated DNA was transformed into Eschericia coli BL21-RIL, M15 (containing pREP4 (Qiagen)) or XLI-blue competent cells and plated onto Luria Broth (LB) agar plates containing 100 μg/mL ampicillin.", "After overnight incubation at 37° C. a colony lift on nylon membrane (DuPont/NEN) was taken and placed colony side up onto LB agar containing 100 μg/mL ampicillin and 1 mM IPTG.", "The plates were incubated overnight at 37° C. or alternatively at room temperature for 2 nights.", "Blue and purple colored colonies that were visible under natural light were obtained from products originating from Acropora sp., Discosoma sp., Sinularia sp.", "and Tubastrea sp.", "Cultures of the purple and blue colonies were initiated and incubated overnight at 37° C. Plasmid DNA was isolated and analyzed by restriction endonuclease digestion.", "Plasmid DNA isolated from purple colonies included pCGP2915 (A10 clone from Acropora sp.", "), pCGP2916 (A11 clone from Acropora sp.", "), pCGP2917 (A12 clone from Acropora sp.", "), pCGP2918 (A8 clone from Acropora sp.", "), pCGP2920 (D10 clone from Discosoma sp.", "), pCGP2922 (T3 clone from Tubastrea sp.", "), pCGP2924 (S3 clone from Sinularia sp.).", "Plasmid DNA isolated from blue colonies included pCGP2919 (D1 clone from Discosoma sp.", "), pCGP2921 (T1 clone from Tubastrea sp.", "), pCGP2923 (S1 clone from Sinularia sp.).", "See FIG.", "10 for all schematics of above mentioned plasmids.", "Sequence Analysis of cDNA Clones Complete sequence analysis of the cDNA clones contained in the pQE-30 vectors was generated using pQEprom (Qiagen) (SEQ ID NO:186), pQErev (Qiagen) (SEQ ID NO:187), Coral-R1 (SEQ ID NO:188), vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) as sequencing primers.", "pQEprom CCC GAA AAG TGC CAC CTG SEQ ID NO:186 pQErev GTT CTG AGG TCA TTA CTG G SEQ ID NO:187 Coral-R1 TCA GGG TAC TTG GTG AAT GG SEQ ID NO:188 Complete nucleotide sequences were generated from the: A8 cDNA clone from Acropora sp.", "contained in pCGP2918 (SEQ ID NO:189); D10 cDNA clone from Discosoma sp contained in pCGP2920 (SEQ ID NO:191); S3 cDNA clone from Sinularia sp contained in pCGP2924 (SEQ ID NO: 193); T3 cDNA clone from Tubastrea sp.", "contained in pCGP2922 (SEQ ID NO: 195); D1 cDNA clone from Discosoma sp.", "contained in pCGP2919 (SEQ ID NO: 197); T1 cDNA clone from Sinularia sp.", "contained in pCGP2923 (SEQ ID NO:199); and T1 cDNA clone from Tubastrea sp.", "contained in pCGP2921 (SEQ ID NO:201).", "The A8 nucleotide sequence contained a putative open reading frame of 669 bases which encodes a putative polypeptide of 223 amino acids (SEQ ID NO:190).", "The D10 nucleotide sequence contained a putative open reading frame of 669 bases which encodes a putative polypeptide of 223 amino acids (SEQ ID NO:192).", "The S3 nucleotide sequence contained a putative open reading frame of 669 bases which encodes a putative polypeptide of 223 amino acids (SEQ ID NO:194).", "The T3 nucleotide sequence contained a putative open reading frame of 669 bases which encodes a putative polypeptide of 223 amino acids (SEQ ID NO:196).", "The D1 nucleotide sequence contained a putative open reading frame of 669 bases which encodes a putative polypeptide of 223 amino acids (SEQ ID NO: 198).", "The S1 nucleotide sequence contained a putative open reading frame of 669 bases which encodes a putative polypeptide of 223 amino acids (SEQ ID NO:200).", "The T1 nucleotide sequence contained a putative open reading frame of 669 bases which encodes a putative polypeptide of 223 amino acids (SEQ ID NO:202).", "Nucleotide and amino acid sequence similarities were determined using LALIGN (Huang and Miller, 1991, supra).", "The sequences isolated from the four species of coral share high nucleic acid and amino acid sequence similarities (Table 8 and Table 9).", "EXAMPLE 4 Colored Protein Expression from Heron Island Coral cDNAs For expression in bacteria, nucleotide sequences encoding CFMs were retrieved from pGEM-T cloning vector using a forward oligonucleotide primer consisting of the NotI restriction binding site, a ribosomal binding site, a spacer and 15 bases encoding the N-terminus of the protein and a reverse oligonucleotide primer encoding H6-tag (POC220-H6; POC220 is SEQ ID NO:2).", "PCR product was gel purified and diluted (×10) prior to cloning into PCRII-TOPO and transformed into Top 10 cells (Invitogen).", "Cells were induced with 0.5 mM IPTG, and protein was purified on Ni-columns (Pro-Bond, Invitrogen) eluting with 50 mM, 200 mM, 350 mM and 500 mM Imidazole in PBS pH 6.0, prior to overnight dialysis against 50 mM Potassium Phosphate pH 6.65.Expression of Examples of Type 1 Peptides Results of expressing sequences of type 1 (as defined in Tables 6 and 7 and in FIG.", "9) in bacteria are set forth in Table 10.Only non-identical sequences are shown.", "Several additional sequences, which are identical to those shown in the Table, are indicated at the top of the Table (i.e.", ": Acasv-D=PavsvB, etc.).", "Sequence alignment is taken from International Patent Publication No.", "WO 00/46233 and Dove et al.", "(2001; supra).", "Horizontal bars above the amino acid sequence indicate β-strands from GFP structure.", "The chromophore “QYG” is shown in white type on black background.", "Amino acid differences in the sequences are grey-shaded.", "The majority of type 1 sequences are deep blue with λmax ranging from 589 nm to 593 nm.", "Naturally-occurring amino acid substitution L161P, as seen in RTms5 (SEQ ID NO:166) compared with Acasv-D (SEQ ID NO:30) leads to clear bacteria that no longer absorb within 520-600 nm range.", "Reverse substitution of P161L re-establishes the ability to absorb in this range.", "The alignment shows amino acids that appear to affect colour of protein and those that do not.", "Absorption scans for examples of expressed type 1 sequences are shown in FIG.", "11.Extinction coefficients at λmax, as shown in this and in subsequent FIGS.", "12 and 13, are based on the method of Whitaker and Granum (1980, supra) for protein detection.", "Extinction coefficient variability is partly due to the state of protein maturation; similar variability has been demonstrated for DsRed (Baird et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 97: 11984-11989,2000).", "Expression of Examples of Types 2 and 14 Peptides Results of expressing sequences of type 2 in bacteria are shown in Table 11.Again, only non-identical sequences are shown.", "Additional sequences, identical to those shown in the Table, are indicated at the top of the Table (i.e.", ": PMms-B=PMms-E=PPd57-4 ms, etc.).", "The majority of type 2 sequences are pinky-purple with λmax ranging from 579 nm to 580 nm.", "Naturally-occurring amino acid substitution P15S leads to clear bacteria that no longer absorb within 520-600 nm range.", "Alignment shows amino acids that do not affect the colour of protein, although it was noted that some of these proteins had a greater tendency to aggregate and precipitate than did others.", "Analogous results, following expressing of type 14 sequences in bacteria, are shown in Table 12.Only non-identical sequences are shown.", "Table formatting is the same as in Tables 10 and 11.The majority of type 14 sequences are pinky-purple, with λmax ranging from 579 nm to 579.5 nm.", "Alignment shows amino acids that do not affect the colour of protein.", "It was noted, however, that MisvF and MisvA, with AA147=F, was more soluble at higher concentrations than at others.", "The spectral properties of Type 2 and Type 14 sequences are similar.", "This may be driven by AA61, which is Ser in both of these cases as opposed to Cys in type 1 and Thr in type 6 sequences.", "FIGS.", "12A and B show absorption scans for examples of expressed type 2 and type 14 sequences.", "As described above for type 1 sequences, observed extinction coefficient variability is partly due to the state of protein maturation.", "Expression of Examples of Type 6 Peptides Examples of Type 6 sequences were similarly expressed in bacteria.", "Again, only non-identical sequences are shown.", "In this case, the majority of sequences are blue-purple, with λmax ranging from 583.5 nm to 585.5 nm.", "Alignment shows that naturally occurring amino acid substitutions V8M and/or T182P lead to colourless bacteria, as does G238E, and that substitutions at AA101 and AA147 have slight effect on λmax.", "Results are shown in Table 13 (see over).", "The format is the same as for Tables 10, 11 and 12.FIG.", "13 shows absorption scans for examples of expressed type 6 sequences.", "As already stated above, extinction coefficient variability is partly due to the state of protein maturation and similar variability has been demonstrated for DsRed (Baird et al.", "2000).", "Expression of Examples of Peptides of Other Types Results of bacterial expression of sequence types other than the major types 1, 2, 6 and 14, are shown in Table 14 (see over).", "Many of the sequences that failed to express blue-purple or pink proteins were isolated from cDNA in which this was not the predominant GFP homolog present.", "EXAMPLE 5 Estimation of Amount of Total Soluble Protein for Colored Proteins Raw phosphate buffer extracts of two colour morphs of Acropora aspera (a dark blue pigmented morph and a cream morph) were used in the determination of the colored protein proportion of total soluble protein.", "Two separate estimations were made—by absorption spectroscopy and by gel filtration (n=5; 95% confidence intervals, in each case).", "Results are set forth in FIGS.", "14A/B.", "FIG.", "14A shows an absorption scan of the two Acropora aspera morphs.", "Estimation of blue-purple pocilloporin concentration (Dove et al., 1995, supra; Dove et al., 2001, supra) per surface area of coral tissue is based on an extinction coefficient range of 50,000-100,000 M−1 cm−1.FIG.", "14B shows the results for chromatograms of gel filtrated protein elution, determined from 235 nm and 280 nm chromatograms, applying the equation (235 nm −280 nm)/2.51 (Whitaker and Granum, 1980, supra).", "The total area under the graph provides a measure of the total soluble protein.", "Blue-purple pocilloporin concentration is based on the difference between areas under the blue and cream graphs in the range of pocilloporin elution (24-26.5 min).", "Notably the independent methods for blue-purple pocilloporin concentration give similar results.", "EXAMPLE 6 Colored Protein Expression from Melbourne Coral cDNAs Colonies of coral EDNA clones isolated from Discosoma sp.", "(D2 (pCGP2925 (blue in color)), Sinularia sp.", "(S1, pCGP2923) and Tubastrea sp.", "(T1, pCGP2921, T3, pCGP2922) were grown overnight with shaking at 37° C. in 2 mL LB media containing 100 μg/mL ampicillin.", "One mL of the overnight culture was then used to inoculate 25 mL LB media containing 100 μg/mL ampicillin.", "This culture was then incubated at 37° C. with shaking until the OD600 was around 0.5.IPTG was added to a final concentration of 1 mM and the cultures were grown overnight with shaking at 37° C. Cells (10 mL) of the incubated cultures were pelleted by centrifugation at 2000 rpm for 10 min.", "The bacterial pellets and supernatant of the D2 (pCGP2925), S1 pCGP2923) and T1 (pCGP2921) were blue those of T3 (pCGP2922) were purple under natural light.", "Bacterial pellets were stored at −20° C. Proteins contained in the supernatant of the cultures were concentrated using Centricon 30 spin columns (Amicon) according to the manufacturer's instructions.", "The final volume of each of the concentrated protein extract was ˜200 μL.", "Aliquots (8 μL) of the concentrated proteins derived from the supernatants of the cultures were electrophoresced through precast SDS PAGE gels (12% w/v resolving, 4% w/v stacking gel) (Ready Gels, BIORAD) in a running buffer made of 25 mM Tris-HCl, Ph 8.3, 192 mM glycine, 0.1% w/v SDS at 100V for 75 min.", "Standards included Biorad Pre-stained Broad Range markers which contained standard protein samples of 206 kDa, 119 kDa, 91 kDa, 51.4 kDa, 34.7 kDa, 28.1 kDa, 20.4 kDa and 7.2k Da.", "Samples were either denatured by boiling in 10% v/v glycerol, 3% w/v SDS, 3% β-mercaptoethanol (BME), 0.025% w/v bromophenol blue or denatured by boiling in 10% v/v glycerol, 3% w/v SDS, 0.025% w/v bromophenol blue or loaded in their native state in 5% v/v glycerol, 0.04% w/v bromophenol blue.", "Prior to staining with Coomassie blue, protein bands were examined under a hand-held UV transilluminator.", "No fluorescent bands were visible under IW light in any of the samples.", "However, under natural light a blue band running at the same position as the 28 kDa protein standard was visible in the concentrated protein sample from the D2 supernatant.", "Blue smears that extended between the 28 kDa and 51 kDa protein standards were visible under natural light in the non-denatured concentrated protein samples from T1 and S1 supernatants.", "A purple smear which extended between the 28 kDa and 51 kDa protein standards was visible under natural light in the non-denatured concentrated protein samples from the S3 supernatant.", "There were no bands observed under natural light in samples that were denatured by boiling.", "Staining the gel with Coomassie blue showed that the proteins produced co-migrated with a 25 kDa protein marker (Biorad Precision Broadrange Prestained Marker).", "Cultures of (E. coli XLI-blue) coral cDNA clones from Discosoma sp.", "(D1 in pCGP2919), Sinularia sp.", "(S1 in pCGP2923) and Tubastrea sp.", "(T1 in pCGP2921 and T3 in pCGP2922) that had grown at 37° C. overnight with shaking were used to inoculate 100 mL LB media containing 100 μg/mL ampicillin and further incubated with shaking at 37° C. until the OD600 was ˜0.5.IPTG was added to a final concentration of 1 mM and the cultures were grown overnight with shaking at 37° C. Proteins expressed by Tubastrea sp.", "clones (T1 and T3) were purified under native conditions using Ni-NTA Superflow resin (Qiagen; QIAexpressionist March 1997) as recommended by the manufacturer.", "The elution buffer was exchanged with 20 mM Tris-HCl pH 8.0 using Sephadex G-25 columns (NAP10; Pharmacia) as per the manufacturer's instructions.", "Proteins expressed by the Discosoma sp.", "clone D1 and the Sinularia sp.", "clone S1 were purified under native conditions using the Ni-NTA method (Qiagen; QIAexpressionist March 1997) except that protein was precipitated from cleared bacterial lysate using 65% isopropanol and centriflged at 10,000 rpm, 4° C., 10 min.", "The colored pellet was resuspended in 20 mM Tris-HCl pH 8.0.The proteins encoded by the Acropora sp.", "A8 clone in pCGP2918, the Discosoma sp.", "D10 clone in pCGP2920, the Sinularia sp S3 clone in pCGP2924 and the Tubastrea sp.", "T3 clone in pCGP2922 were a purple color (Royal Horticultural Society Color Chart (RHSCC) 88A) when concentrated.", "The proteins from Tubastrea sp.", "T3 clone and the Sinularia sp.", "S3 clone bad absorbance peaks at approximately 580 nm.", "The proteins encoded by the Discosoma sp.", "D1 clone in pCGP2919 and the Tubastrea sp.", "T1 clone in pCGP2921 were a blue color (RHSCC 102A) when concentrated and absorbance peaks at approximately 595 nm.", "The protein encoded by Sinularia sp.", "S1 clone in pCGP2923 was a blue-purple color (RHSCC 90A) when concentrated and had an absorbance peak at approximately 590 nm.", "Amino Acid Sequence Alignment A multiple alignment of the encoded amino acid sequence of T1 (SEQ ID NO:202), D1 (SEQ ID NO:198), S1 (SEQ ID NO:200), A8 (SEQ ID NO:190), T3 (SEQ ID NO:196), D10 (SEQ ID NO:192) and S3 (SEQ ID NO:194) was produced using the Clustal W (1.4) program in MacVector (6.5.3; Oxford Molecular Group Plc, 1999) (FIG.", "15).", "The multiple alignment of encoded amino acids showed that there are only 16 amino acid positions that differed between proteins exhibiting blue, blue-purple and purple color.", "From this alignment there appear to be eight amino acid positions that may influence the color of the protein (Table 15).", "The protein encoded by S1 (SEQ ID NO:200) has a color that is intermediate of the blue and purple proteins.", "The amino acid sequence alignment (FIG.", "15) showed that the S1 amino acid sequence contained four amino acid identities characteristic of blue proteins towards the amino-terminal end and four amino acid identities characteristic to purple proteins towards the carboxy-terminal end (Table 15).", "The substitution of one or more amino acids listed in Table 15 may influence the visible color characteristics of the protein.", "Alignment of Melbourne and Heron Island Coral Protein Sequences The amino acid sequences of the above seven polypeptides (SEQ ID NOs 190, 192, 194, 196, 198, 200 and 202) were compared with other SVIAK (SEQ ID NO:5)containing polypeptides, as set forth in FIG.", "1.The resulting alignment is shown in FIG.", "16.EXAMPLE 7 Expression of Colored Proteins in an Eukaryotic Organism Saccharomyces cerevisiae In order to observe whether the colored protein sequences were able to produce color in a eukaryotic cell, the colored protein cDNA clones T1 (SEQ ID NO:201) and A8 (SEQ ID NO:189) were introduced into a yeast expression vector (pYE22m) (Tanaka et al., 1988, supra) and transformed into Saccharomyces cerevisiae strain G1315.Construction of pCGP3269 and PCGP3270 (T1 or A8 in pYE22m) The plasmids pCGP3269 (FIG.", "17) and pCGP3270 (FIG.", "18) were constructed by cloning the T1 or A8 cDNA clones, respectively, in a sense orientation behind the yeast glyceraldehyde 3-phosphate dehydrogenase promoter of pYE22m (Tanaka et al., 1988, supra).", "A forward primer (Kpn.6His.F; SEQ ID NO:203) was designed to amplify the colored protein sequences that would result in 6 x Histidine tag fused in-frame with the colored protein at the N-terminus and a KpnI restriction endonuclease recognition site at the 5′ end.", "A reverse primer (T1/A8.Sal.R; SEQ ID NO:204) included a SalI restriction endonuclease recognition site at the 3′ end Kpn.6His.F KpnI GCAT GGT ACC ATG AGA GGA TCG CAT CAC SEQ ID NO:203 M R G S H H CAT CAC CAT CAC H H H H T1/A8.Sal.R SalI CTGA GTC GAC TCA CTG CAG GGC GAC CAC SEQ ID NO:204 Q L A V V AGG TTT P K The coding regions of T1 (SEQ ID NO:201) and A8 (SEQ ID NO:189) were amplified by PCR using the primers Kpn.6His (SEQ ID NO:203) and T1/A8.Sal.R (SEQ ID NO:204) and the plasmid DNA pCGP2921 (T1) (FIG.", "10) and pCGP2918 (A8) (FIG.", "10) as template.", "The ˜700 bp PCR products were purified using a QIAquick PCR purification kit (Qiagen) and then digested with the restriction endonucleases KpnI and SalI.", "The KpnI/SalI digested products were finally purified using a QIAquick PCR purification kit (Qiagen) and subsequently ligated with the KpnI/SalI ends of the pYE22m yeast expression vector (Tanaka et al., 1988 supra) using a DNA Ligation Kit (Amersham) according to the manufacturer's recommendations.", "Correct insertion of the T1 or A8 cDNA clones into the yeast expression vector was confirmed by visualisation of colour of transformants that were selected by their ability to restore G-1315 to tryptophan prototrophy.", "The T1 clone in the yeast expression vector pYE22m (designated as pCGP3269) produced blue coloured colonies (RHSCC 101C) when introduced into the yeast strain G1315.The A8 clone in the yeast expression vector pYE22m (designated as pCGP3270) produced purple coloured colonies (RHSCC 82B) when introduced into the yeast strain G1315.EXAMPLE 8 Estimation of Colored Protein Amounts Produced by Bacterial and Yeast Cultures Quantitation of colored Protein Expression in Saccaryomyces cerevisiae Pure cultures of yeast cells harbouring pCGP3269 FIG.", "17) or pCGP3270 (FIG.", "18) were grown at 29° C. for 48 hours in 100 mL of YEPD liquid broth (1% yeast extract, 2% bacto-peptone, 2% w/v glucose, pH5.0).", "The cultures were centrifuged at 2000 rpm for 15 min.", "The resulting pellets were blue (pCGP3270) and purple (pCGP3269).", "The His-tagged colored proteins were extracted under native conditions by first resuspending the pellets in 4 mL lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole, 5 mg/mL Yeast Lytic enzyme (IBN)) and incubated at 30° C. for 1 hour.", "The solutions were sonicated on ice 10 times for 10 sec with 15 sec cooling between treatments.", "The lysates were then centrifiged at 10 000 rpm for 10 min and the supernatants (crude extract) collected.", "The His-tagged colored proteins were purified by nickel-nitrilotriacetic acid metal-affinity chromatography (Qiagen) as recommended by the manufacturer.", "The protein content of the crude extracts and purified His-tagged colored proteins were measured using a Bio-Rad Protein Assay using 1, 3 and 5 μL aliquots of extracts as per the manufacturer's instructions (Bio-Rad Microassay Procedure).", "The absorbances at 595 nm were compared with bovine serum albumin (BSA) standard curves (0-10 μg/mL) to obtain estimations of protein concentrations.", "Samples of crude extracts and a dilution series of known amounts of purified His-tagged colored protein were electrophoresed through precast SDS PAGE gels (12% w/v resolving, 4% w/v stacking gel) (Ready Gels, Biorad) as described in Example 3.The gels were then stained with Coomasie blue (0.25% (w/v) Coomassie Brilliant Blue, 45% (v/v) methanol, 10% (v/v) acetic acid) and the amounts of colored protein in the crude extracts were estimated by comparing the intensities of the stained bands with those of the purified His-tagged colored protein dilution series.", "This allowed the estimation of expression of colored protein in yeast as a percentage of total soluble protein (Table 16).", "Quantitation of Colored Protein Expression in Escherichia coli One mL of an overnight Escherichia coli XL1blue culuture harbouring the plasmid pCGP2921 (T1) FIG.", "10) (Example 3) was used to inoculate 100 mL LB broth (containing 50 μg/mL ampicillin) and incubated 37° C. with shaking at 200 rpm until the OD600 was between 0.5-0.7.Protein production was induced with the addition of IPTG to 1 mM and incubation overnight at 29° C. with shaking at 200 rpm.", "The cells were pelleted by centrifugation at 2000 rpm for 15 min.", "The resulting pellet was blue.", "The pellet was resuspended in 4 mL lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole) and sonicated on ice 6 times for 10 sec with 15 see cooling between treatments.", "The solution was centrifuged at 10 000 rpm for 10 min and the (crude extract) supernatant collected.", "The His-tagged colored protein (T1) was extracted under native conditions by nickel-nitrilotriacetic acid metal-affinity chromatography (Qiagen) as recommended by the manufacturer.", "The protein content of the crude extract and purified His-tagged colored protein was measured using a Bio-Rad Protein Assay using 1, 3 and 5 μL of extracts as per the manufacturers instructions (Bio-Rad Microassay Procedure).", "The absorbances at 595 nm were compared with BSA standard curves (0-10 μg/mL) to obtain estimations of protein concentrations.", "Samples of crude extract and a dilution series of known amounts of purified His-tagged colored protein were electrophoresed through SDS PAGE gels as per the crude extract from yeast cultures (as described above).", "The amounts of colored protein in the crude extracts were estimated by comparing the intensites of the stained bands with those of the purified His-tagged colored protein dilution series.", "This allowed the estimation of expression of colored protein in E. coli as a percentage of total soluble protein (Table 16).", "EXAMPLE 9 Expression of Colored Proteins in Plants Under the Control of a Constitutive Promoter Construction of pCGP2756 (35S: MCS: 35S Expression Cassette) Plasmid pCGP2756 (FIG.", "19) was constructed by cloning the multicloning site (MCS) (containing the rare restriction endonuclease sites PacI and AscI) from pNEB193 (New England Biolabs) into the CaMV35S expression cassette of pRTppoptcAFP (Wnendt et al., Curr Genet 25: 510-523, 1994).", "The plasmid pRTppoptcAFP was digested with EcoRI and XhaI to release 300 bp AFP (antifungal protein) insert and the 3.3 kb vector containing the CaMV 35S expression cassette.", "The plasmid pNEB193 was digested with EcoRI and XbaI to release the 40 bp fragment containing the multicloning site.", "The 40 bp EcoRI/XbaI fragment from pNEB193 and the 3.3 kb vector containing the CaMV35 expression cassette from pRTppoptcAFP were isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and ligated together.", "The ligation was carried out using the Amersham ligation kit.", "Correct insertion of the fragment in pCGP2756 was established by restriction enzyme analysis (SalI, KpnI, BamHI, XbaI, AscI, PacI, HindIII/BamHI) of DNA isolated from ampicillin-resistant transformants.", "Construction of pCGP2757 (35S: MCS: 35S Binary Vector) Plasmid pCGP2757 (FIG.", "20) was constructed by cloning the CaMV35S expression cassette of pCGP2756 (described above) into the binary vector pWTT2132 (DNAP).", "The plasmid pCGP2756 was digested with PstI to release the 0.7 kb CaMV35S expression cassette containing the multicloning site from pNEB193.The 0.7 kb fragment was isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and ligated with PstI ends of pWTT2132 binary vector.", "Correct insertion of the fragment in a tandem orientation to the CaMV35S: surB cassette in pWTT2132 was established by restriction enzyme analysis (KpnI, PacI/AscI, EcoRI, XbaI, PstI) of DNA isolated from tetracycline-resistant transformants.", "PCR products of CFMs or colored proteins derived using the primers vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) or using any primers containing AscI and PacI restriction endonuclease recognition sites, can be digested with AscI and PacI and ligated with AscI/PacI ends of pCGP2757.Construction of pCGP2765 (35S: A8: 35S Binary) Plasmid pCGP2765 (FIG.", "21) was constructed by cloning the A8 PCR clone amplified from Acropora sp.", "into the CaMV35S expression cassette contained in the binary vector of pCGP2757 (described above).", "The A8 PCR product generated using the vispro-F1.", "(SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) primers and cDNA synthesized from Acropora sp.", "total RNA as template (see Example 1), was digested with AscI and PacI.", "The ˜0.7 kb fragment was isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and ligated with AscI/PacI ends of pCGP2757 binary vector.", "Correct insertion of the fragment in a sense orientation behind the CaMV35S promoter was established by restriction enzyme analysis (EcoRI, PstI, BstXI) of DNA isolated from tetracycline-resistant transformants.", "Construction of pCGP2769 (35S: D1: 35S Binary) (FIG.", "22) Plasmid pCGP2769 (FIG.", "22) was constructed by cloning the D1 PCR clone amplified from Discosoma sp.", "into the CaMV35S expression cassette contained in the binary vector of pCGP2757 (described above).", "The PCR product generated using the primers vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) and the template pCGP2919 (containing the D1 cDNA clone) was digested with AscI and PacI.", "PCR was carried out in 50 μL reactions with 200 μM dNTPs, 20 pmol vispro-F1 (SEQ ID NO:184), 20 pmol visproR1 (SEQ ID NO:185), 1×Pfu buffer (Stratagene), 2.5 units Pfu trubo DNA Polymerase (Stratagene) and ˜2 ng pCGP2919 plasmid DNA as template.", "The ˜0.7 kb fragment was isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and ligated with AscI/PacI ends of pCGP2757 binary vector.", "Correct insertion of the fragment in a sense orientation behind the CaMV35S promoter was established by restriction enzyme analysis (EcoRI, PstI, BstXI, BamHI) of DNA isolated from tetracycline-resistant transformants.", "Construction of pCGP2770 (35S: S1: 35S Binary) (FIG.", "23) Plasmid pCGP2770 (FIG.", "23) was constructed by cloning the S1 PCR clone amplified from Sinularia sp.", "into the CaMV35S expression cassette contained in the binary vector of pCGP2757 (described above).", "The PCR product generated using the primers vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) and the template pCGP2923 (containing the S1 cDNA clone) was digested with AscI and PacI.", "PCR was carried out in 50 μL reactions with 200 μM dNTPs, 20 pmol vispro-F1 (SEQ ]D NO:184), 20 pmol vispro-R1 (SEQ ID NO:185), 1×Pfu buffer (Stratagene), 2.5 units Pfu trubo DNA Polymerase (Stratagene) and ˜2 ng pCGP2923 plasmid DNA as template.", "The ˜0.7 kb fragment was isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and ligated with AscI/PacI ends of pCGP2757 binary vector.", "Correct insertion of the fragment in a sense orientation behind the CaMV35S promoter was established by restriction enzyme analysis (EcoRI, PstI, BstXI, BamHI) of DNA isolated from tetracycline-resistant transformants.", "Construction of pCGP2772 (35S: T1: 35S Binary) (FIG.", "24) Plasmid pCGP2772 (FIG.", "24) was constructed by cloning the T1 PCR clone amplified from Tubastrea sp.", "into the CaMV35S expression cassette contained in the binary vector of pCGP2757 (described above).", "The PCR product generated using the primers vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) and the template pCGP2921 (containing the T1 cDNA clone) was digested with AscI and PacI.", "PCR was carried out in 50 μL reactions with 200 μM dNTPs, 20 pmol vispro-F1 (SEQ ID NO:184), 20 pmol vispro-R1 (SEQ ID NO:185), 1×Pfu buffer (Stratagent), 2.5 units Pfu trubo DNA Polymerase (Stratagene) and 2 ng pCGP2921 plasmid DNA as template.", "The ˜0.7 kb fragment was isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and ligated with AscI/PacI ends of pCGP2757 binary vector.", "Correct insertion of the fragment in a sense orientation behind the CaMV35S promoter was established by restriction enzyme analysis (EcoRI, PstI, BstXI, BamHI) of DNA isolated from tetracycline-resistant transformants.", "Construction of PCGP2926 (35S:His T1: 35S Binary) A histidine-tagged version of T1 was also produced for expression in the CaMV 35S gene expression cassette.", "The expression of this modified version of T1 will allow for a way of easily concentrating the expressed T1 protein to calculate the amount being produced in plants.", "The RGS-His epitope was created by, ligation of the 2 complementary primers TICS-His-FWD (SEQ ID NO:227) and TICS-His-REV (SEQ ID NO:228).", "This ligation resulted in a fragment containing the sequences to a prokaryotic ribosome binding site (RBS), a translational initiation consensus sequence (TICS) (for optimal translation in plants), the RGS-His epitope (consisting of sequences that encode the amino acids RGSHHHHHH) and overhanging AscI (at 5′ end) and BamHI (at 3′ end).", "This AscI/BamHI fragment was ligated with AscI/BamHI ends of plasmid pCGP2781 (FIG.", "32).", "Correct ligation of the insert into pCGP2781 was established by restriction enzyme analysis of DNA isolated from tetracycline-resistant transformants.", "The plasmid was designated as pCGP2926 (FIG.", "44).", "TICS-His-FWD (5′ to 3′) SEQ ID NO:227 CGCGCC AAGGAGATAT AACA ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC G RBS TICS M R G S H H H H H H RGS-His epitope TICS-His-REV (5′ to 3′) SEQ ID NO:228 GATCC GTG ATG GTG ATG GTG ATG CGA TCC TCT CAT TGTT ATATCTCCTT GG RGS-His epitope TICS RBS A. tumefaciens Transformations The plasmids pCGP2772 and pCGP2765 were introduced into the Agrobacterium tumefaciens strain AGL0 by adding 5 μg of plasmid DNA to 100 μL of competent AGL0 cells prepared by inoculating a 50 mL LB culture and growing for 16 hours with shaking at 28° C. The cells were then pelleted and resuspended in 0.5 mL of 85% v/v 100 mM CaCl2/15% v/v) glycerol.", "The DNA-Agrobacterium mixture was frozen by incubation in liquid N2 for 2 minutes and then allowed to thaw by incubation at 37° C. for 5 minutes.", "The DNA/bacterial mix was then placed on ice for a further 10 minutes.", "The cells were then mixed with 1 mL of LB (Sambrook et al., 1989, supra) media and incubated with shaking for 16 hours at 28° C. Cells of A. tumefacien carrying pCGP2772 and pCGP2765 were selected on LB agar plates containing 50 μg/mL tetracycline.", "The presence of pCGP2772 and pCGP2765 were confirmed by restriction enzyme analysis of DNA isolated from the tetracycline-resistant transformants.", "EXAMPLE 10 Spatial and Temporal Expression of Colored Proteins in Plants The use of constitutive promoters such as CaMV35S can be used to direct expression of CFM or colored proteins throughout the whole plant and may be useful in cases where a novel phenotype is sought with respect to the whole plant.", "However in some cases novel color is sought in specific tissues such as floral, seeds, leaves, fibre (e.g.", "cotton fibre), stems, roots, pollen, etc.", "In these cases tissue-specific promoters can be used to target expression of CFM or colored proteins to specific tissues.", "There are many cases in the literature, which describe the use of promoters to direct spatial and temporal expression.", "These promoters include, but are not limited to, the examples of a seed specific promoters (Song et al., Journal of Cotton Science 4: 217-223, 2000), leaf and chlorophyll containing tissue specific promoters (Song et al., 2000, supra), and tuber specific promoters (Rocha-Sosa et al., EMBO J 8: 23-29, 1989).", "Isolation of Rose CHS Promoter A rose genomic DNA library was prepared from Rosa hybrida cv.", "Kardinal.", "The rose library was screened with rose CHS cDNA clone.", "A 6.6kb fragment upstream from the translational initiation site was cloned into pBluescript KS (−) (Stratagene) and the plasmid designated pCGP114.The plasmid pCGP1114 was digested with HindIII and EcoRV to release a ˜2.7-3.0kb fragment which was purified using a Bresaclean kit (Geneworks) and ligated with HindIII/SmaI ends of pUC19 (New England Biolabs).", "Correct insertion of the Rose CHS promoter fragment was established by restriction enzyme analysis of DNA isolated from ampicillin-resistant transformants.", "The resulting plasmid was designated as pCGP1116 (FIG.", "25).", "Construction of pCGP3255 (Rose CHS 5′: 35S 3′ Pre-Binary) The plasmid pCGP3255 (FIG.", "26) was constructed by replacing the CaMV 35S promoter in the binary vector pCGP2757 with the Rose CHS promoter fragment from pCGP1116.Plasmid pCGP1116 was initially digested with HindIII.", "The overhanging 5′ ends were filled-in using DNA polymerase (Klenow fragment) (Promega) according to the manufacturer's recommendation.", "The linearized vector was then digested with Asp718 to release a ˜2.7 kb rose CHS promoter fragment.", "The plasmid pCGP2757 was initially digested with SalI.", "The overhanging 5′ ends were filled-in using DNA polymerase (Klenow fragment) (Promega) according to the manufacturer's recommendation.", "The SalI digested pCGP2757 was then digested with Asp718 to release the ˜19 kb binary vector fragment and the CaMV 35S promoter fragment.", "The SalI (filled-in)/Asp718 ˜19 kb vector fragment was purified using QIAEX II Gel Extraction kit (Qiagen) and ligated with the HindIII (filled-in)/Asp718 ends of the rose CHS promoter fragment.", "Correct insertion of the rose CHS promoter was established by restriction enzyme analysis (BglII, PstI, EcoRI, HindIII, XbaI, EcoRV) of DNA isolated from tetracycline-resistant transformants.", "PCR products of CFMs or colored proteins derived using the primers vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) or using any primers containing AscI and PacI restriction endonuclease recognition sites, can be digested with AscI and PacI and ligated with AscI/PacI ends of pCGP3255.Construction of pCGP2782 (Rose CHSS: T1: 35S 3′ Binary) The plasmid pCGP2782 (FIG.", "27) was constructed by inserting the cDNA of the T1 coral protein contained in pCGP2921 (Example 1) behind the Rose CHS promoter contained in pCGP3255.The PCR product generated using the primers vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) and the template pCGP2921 (containing the T1 cDNA clone) was digested with AscI and PacI.", "PCR was carried out in 50 μL reactions with 200 μM dNTPs, 20 pmol vispro-F1 (SEQ ID NO:184), 20 pmol vispro-R1 (SEQ ID NO:185), 1×Pfu buffer (Stratagene), 2.5 units Pfu trubo DNA Polymerase (Stratagene) and ˜2 ng pCGP2921 plasmid DNA as template.", "The resulting product was purified using QIAquick Gel Extraction (Qiagen) and ligated with AscI/PacI ends of pCGP3255.Correct insertion of the T1 coding region behind the Rose CHS promoter was established by restriction endonuclease digestion (HindIII, EcoRI, PstI, XbaI, BstX1) of tetracycline-resistant transformants.", "Construction of pCGP2773 (Rose CHS: D1: 35S 3′ Binary) The plasmid pCGP2773 (FIG.", "28) was constructed by inserting the cDNA of the D1 coral protein (Example 1) contained in pCGP2919 behind the Rose CHS promoter contained in pCGP3255.The PCR product generated using the primers vispro-F1 (SEQ ID NO: 184) and vispro-R1 (SEQ ID NO:185) and the template pCGP2919 (containing the D1 cDNA clone) was digested with AscI and PacI.", "The PCR product generated using the primers vispro-F1 (SEQ BD NO:184) and vispro-R1 (SEQ ID NO:185) and the template pCGP2919 (containing the D1 cDNA clone) was digested with AscI and PacI.", "PCR was carried out in 50 μL reactions with 200 μM dNTPs, 20 pmol vispro-F1 (SEQ ID NO:184), 20 pmol vispro-R1 (SEQ ID NO:185), 1×Pfu buffer (Stratagene), 2.5 units Pfu trubo DNA Polymerase (Stratagene) and ˜2 ng pCGP2919 plasmid DNA as template.", "The resulting fragment was purified using QIAquick Gel Extraction (Qiagen) and ligated with AscI/PacI ends of pCGP3255.Correct insertion of the D1 coding region behind the Rose CHS promoter was established by restriction endonuclease digestion (HindIII, EcoRI, PstI, XbaI) of tetracycline-resistant transformants.", "Construction of pCGP2774 (Rose CHS: S1: 35S 3′ Binary) The plasmid pCGP2774 (FIG.", "29) was constructed by inserting the cDNA of the S1 coral protein (Example 1) contained in pCGP2923 behind the Rose CHS promoter contained in pCGP3255.The PCR product generated using the primers vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) and the template pCGP2923 (containing the S1 cDNA clone) was digested with AscI and PacI.", "The PCR product generated using the primers vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) and the template pCGP2923 (containing the S1 cDNA clone) was digested with AscI and PacI.", "PCR was carried out in 50 μL reactions with 200 μM dNTPs, 20 pmol vispro-F1 (SEQ ID NO:184), 20 pmol vispro-R1 (SEQ ID NO:185), 1×Pfu buffer (Stratagene), 2.5 units Pfu trubo DNA Polymerase (Stratagene) and ˜2 ng pCGP2923 plasmid DNA as template.", "The resulting fragment was purified using QIAquick Gel Extraction (Qiagen) and ligated with AscI/PacI ends of pCGP3255.Correct insertion of the S1 coding region behind the Rose CHS promoter was established by restriction endonuclease digestion (HindIII, EcoRI, PstI, XbaI) of tetracycline-resistant transformants.", "EXAMPLE 11 Targeting of Colored Proteins to Increase Expression in Plants The levels of some CFMs or colored proteins produced in the cytosol of cells may have to be elevated in order to impart a visible color or a phenotype with commercial value.", "It is expected that targeting the CFM or colored proteins to different organelles within transgenic cells will significantly increase CFM or colored protein levels.", "The increased accumulation of transgene products by targeting to organelles has been demonstrated previously.", "For example, see Table 17.It is also expected that plastid transformation of Arabidopsis, carnation, rose or other plant species will significantly increase CFM or colored protein levels.", "Increased accumulation of transgene products by plastid transformation has been demonstrated previously.", "For example, see Table 18.Cloning of the Chloroplast/Plastid Transit Peptide Sequence from Tobacco CFMs or colored proteins may be targeted to plastids with the inclusion of N-terminal plastid or chloroplast targeting peptides.", "The 57 amino acid transit peptide of small subunit (SSU) of ribulose biphosphate carboxylase from Nicotiana sylvestris (Pinck et al., Biochimie 66: 539-545, 1984) was selected to target coral colored proteins to plastids of transgenic Arabidopsis, carnation, rose or other plant species.", "The primers TSSU-Fnew (SEQ ID NO:205) and TSSU-R (SEQ ID NO:206) were used to amplify the tobacco chloroplast transit-peptide coding region using the plasmid pCGN5075 (Calgene) as template.", "TSSU-Fnew CAG GGCGCGCC AAGGAGATAT AACA ATG GCT SEQ ID NO:205 AscI RBS TICS M A TCC TCA GTT CTT TCC S S V L S TSSU-R CACT GGATCC GCA TTG CAC TCT TCC GCC SEQ ID NO:206 BamHI C Q V R G G GTT GC N TSSU-Fnew (SEQ ID NO:205) contains an AscI site for cloning into 35S and Rose CHS expression vectors, a prokaryotic ribosomal binding site (RBS) for bacterial expression and a plant translational initiation context sequence (TICS) for improved translation in plants, TSSU-R (SEQ ID NO:206) contains a BamHI site to allow the cloning of the transit peptide in frame with coral colored protein sequences produced using vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) primers.", "PCR conditions included 1 μL TSSU-Fnew (20 pmol/μL) (SEQ ID NO:205), 1 μL TSSU-R (20 pmol/μL) (SEQ ID NO:206), 5 μL 10×pfu buffer (Stratagene), ˜20 ng pCGN5075 plasmid DNA as template, 1 μL 10 mM dNTP mix, 0.5 μL Pfu turbo DNA polymerase (2.5 U/μL) (Stratagene) in a 50 μL reaction.", "The cycling conditions were 94° C. for 5 minutes, followed by 35 cycles of 94° C. for 30 min, 50° C for 30 min and 72° C. for 60 min, and a final incubation at 72° C. for 10 min.", "After completion of the PCR the products were stored at 4° C. PCR products were purified using a QIAquick PCR purification Kit (Qiagen) and cloned into pUC18 SmaI vector (Pharmacia/Amersham).", "The resulting plasmid was designated pCGP2783.The sequence of the transit peptide (TSSU) was confirmed by sequencing across both strands.", "Construction of pCGP2780 (355 Expression Binary with Unique BamHI Site) Plasmid pCGP2780 (FIG.", "30) was constructed by removing a ˜290 bp SalI fragment from pCGP2757.The plasmid pCGP2757 was digested with SalI to release a ˜290 bp fragment and ˜19 kb binary vector.", "The ˜19 kb binary vector was isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and self-ligated using the Amersham Ligation Kit.", "Correct religation of the SalI ends was established by restriction enzyme analysis (PvuII, BamHI, SalI) of DNA isolated from tetracycline-resistant transformants.", "Construction of pCGP2784 (35S Expression Pre-Binary Containing Plastid Transit Peptide) The plasmid pCGP2784 (FIG.", "31) was constructed by inserting the chloroplast transit peptide from tobacco contained in pCGP27.83 into the binary vector pCGP2781.Plasmid pCGP2783 was digested with AscI and BamHI to release the ˜0.2 kb TSSU fragment.", "The 0.2 kb TSSU fragment was isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and ligated with AscI/BamHI ends of pCGP2781 binary vector.", "Correct insertion of the transit peptide in frame and upstream of the T1 coding sequence was established by restriction enzyme analysis (EcoRI, PstI, XbaI, AscI/PacI) of DNA isolated from tetracycline-resistant transformants.", "PCR products of CFMs or colored proteins derived using the primers vispro-F1 (SEQ ID NO:184) and vispro-R1 (SEQ ID NO:185) or using any primers containing BamHI and PacI restriction endonuclease recognition sites, can be digested with BamHI and PacI and ligated with BamHI/PacI ends of pCGP2784.The coding region of the CFMs or colored proteins will then be in-frame with the plastid targeting peptide to allow expression of the proteins in the plastids or chloroplasts.", "Construction of PCGP2781 (35S: T1: 35S Binary with Unique BamHI Site) Plasmid pCGP2781 (FIG.", "32) was constructed by removing a 290 bp SalI fragment from pCGP2772.The plasmid pCGP2772 was digested with SalI to release a ˜290 bp fragment and ˜19 kb binary vector.", "The ˜19 kb binary vector was isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and self-ligated using the Amersham Ligation Kit.", "Correct religation of the SalI ends was established by restriction enzyme analysis (PvuII, BamHI, SalI, XbaI) of DNA isolated from tetracycline-resistant transformants.", "Construction of pCGP2785 (35S: TSSU.", ": T1: 3S Binary) The plasmid pCGP2785 (FIG.", "33) was constructed by inserting the chloroplast transit peptide from tobacco contained in pCGP2783 into the binary vector pCGP2781.Plasmid pCGP2783 was digested with AscI and BamHI to release the ˜0.2 kb TSSU fragment.", "The 0.2 kb TSSU fragment was isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and ligated with AscI/BamHI ends of pCGP2781 binary vector.", "Correct insertion of the transit peptide in frame and upstream of the T1 coding sequence was established by restriction enzyme analysis (EcoRI, PstI, XbaI, AscI/PacI) of DNA isolated from tetracycline-resistant transformants.", "Construction of pCGP2787 (Rose CHS: TSSU: T1: 35S Binary) The plasmid pCGP2787 (FIG.", "34) was constructed by inserting the chloroplast transit peptide from tobacco contained in pCGP2783 (Example 11) into the binary vector pCGP2782 (FIG.", "27).", "Plasmid pCGP2783 was digested with AscI and BamHI to release the ˜0.2 kb TSSU fragment.", "The 0.2 kb TSSU fragment was isolated and purified using the QIAEX II Gel Extraction kit (Qiagen) and ligated with AscI/BamHI ends of pCGP2782 binary vector.", "Correct insertion of the transit peptide in frame and upstream of the T1 coding sequence was established by restriction enzyme analysis of DNA isolated from tetracycline-resistant transformants (FIG.", "34) Targeting of CFMs or Colored Proteins to Endoplasmic Reticulum CFMs or colored proteins are targeted to endoplasmic reticulum with the inclusion of N-terminal endoplasmic reticulum (ER) targeting peptides and C-terminal ER retaining signals.", "The Arabidopsis thaliana basic chitinase N-terminal signal sequence was isolated to target CFMs and colored proteins to the ER (Haseloff et al., 1997, supra).", "To retain the proteins in the ER an HDEL peptide sequence was generated to be cloned in at the 3′ end of the coding region (Haseloff et al., 1997, supra).", "These ER-targeting and ER-retention signals are used to increase levels of CFMs and colored protein in trasgenic Arabidopsis, carnation, rose or other plant species.", "The plasmid pBIN35Sm-GFP4-ER (Haseloff et al., 1997, supra) (http://www.plantsci.cam.ac.uk/Haseloff/GFP/mgfp4.html) was used as the source of Arabidopsis thaliana basic chitinase N-terminal signal sequence and HDEL ER-retention signal.", "A PCR based approach was used to generate AscI and BamHI sites flanking the N-terminal ER transit peptide sequence.", "The primers AscI-ER.F (SEQ ID NO:207) and ER-BamHI.R (SEQ ID NO:208) were used to amplify the N-terminal ER sequence contained in pBIN35Sm-GFP4-ER.", "Primer AscI-ER.F (SEQ ID NO:207) contains an AscI site for cloning into 35S and Rose CHS expression binaries (see Examples 9 and 10), a prokaryotic ribosome binding site (RBS) to allow for bacterial expression and a plant translational initiation context sequence (TICS).", "AscI-ER.F (5′ to 3′) GCAT GGCGCGCC AAGGAGATAT AACA ATG AAG SEQ ID NO:207 AscI RBS TICS M K ACT AAT CTT TTT C T N L F ER-BamHI.R (5′ to 3′) BamHI EcoRI GCAT GGA TCC GAA TTC GGC CGA GGA TAA SEQ ID NO:208 S G F E A S S L TGA TAG S L PCR conditions included using 1 ng plasmid pBIN35Sm-GFP4-ER template, 100 ng each of primers AscI-ER.F (SEQ ID NO:207) and ER-BamHI.R (SEQ ID NO:208), 2.5 μL 10×pfu turbo buffer (Stratagene), 1 μL pfu turbo (Stratagene) in a total volume of 25 μL.", "Cycling conditions were an initial denaturation step of 5 min at 94° C., followed by 35 cycles of 94° C. for 30 sec, 50° C. for 30 sec and 72° C. for 1 min with a last treatment of 72° C. for 5 min and then finally storage at 4° C. An expected product of ˜100 bp was amplified and purified using the QIAEX II Gel Extraction kit (Qiagen) according to procedures recommended by the manufacturer.", "The 100 bp fragment was then cloned into pCR2.1 (Invitrogen) and the plasmid was designated pCGP3256.The sequence of the N-terminal ER transit peptide fragment was confirmed by sequence analysis using the M13 reverse and M13-20 primers.", "Construction of pCGP3257 (35S:ER:MCS:35S Pre-Binary) The N-terminal ER transit peptide fragment was cloned downstream of the 35S promoter contained in the pre-binary pCGP2780 (FIG.", "30) to produce pCGP3257 (FIG.", "35).", "Plasmid pCGP3256 was digested with AscI and BamHI to release the ˜100 bp N-terminal ER transit peptide fragment.", "The fragment was isolated and purified using QIAEX II Gel Extraction kit (Qiagen) and ligated with AscI/BamHI ends of pCGP2780.Correct insertion of the N-terminal ER transit peptide fragment was established by restriction endonuclease analysis of DNA isolated from tetracycline-resistant transformants.", "PCR products of CFMs or colored proteins derived using the primers vispro F1 (SEQ ID NO:185) and CP-HDEL-PacI.R (described in this Example below) can be digested with BamHI and PacI and ligated with BamHI/PacI ends of pCGP3257.The coding region of the CFMs or colored proteins will be under the control of the CaMV 35S promoter and in-frame with the ER transit targeting peptide to allow targeting of the proteins to the ER.", "The coding region of the CFMs or colored proteins will also contain the HDEL sequence at the C-terminal end to allow retention of the proteins in the ER.", "Construction of pCGP3259 (35S: ER: T1.HDEL: 35S Binary) The coding sequence of the colored protein T1 was amplified by PCR using the primers vispro-F1 (SEQ ID NO:184) and CP-HDEL-PacI.R (SEQ ID NO:209) and the plasmid pCGP2779 as template.", "The primer CP-HDEL-PacI.R was designed to include a PacI site with a translational termination codon for cloning into the binary vectors described in this specification, a HDEL peptide sequence in-frame with the colored protein sequence and a PstI site for cloning into the bacterial expression vector pQE-30 (Qiagen).", "CP-HDEL-PacI.R (5′ to 3′) PacI GATCTTAAT TAA AGC TCA TCA TGC TGC AGG SEQ ID NO:209 * L E D H Q L PstI GCG ACC ACA GGT TTG C A V V P K PCR conditions included using 2 ng plasmid pCGP2779 as template, 100 ng each of primers vispro-F1 (SEQ ID NO:184) and CP-HDEL-PacI.R (SEQ ID NO,209), 2 μL 10 mM dNTP mix, 5 μL 10× PfuTurbo (registered trademark) DNA polymerase buffer (Stratagene), 0.5 μL PfuTurbo (registered trademark) DNA polymerase (2.5 units/μL) (Stratagene) in a total volume of 50 μL.", "Cycling conditions were an initial denaturation step of 5 min at 94° C., followed by 35 cycles of 94° C. for 20 sec, 50° C. for 30 see and 72° C. for 1 min with a last treatment of 72° C. for 10 min and then finally storage at 4° C. The resulting ˜700 bp product was digested with BamHI and PacI, isolated and purified using QIAEXII Gel Extraction kit (Qiagen) and ligated with BamHI/PacI ends of pCGP3257.Correct insertion of the T1 coding region and HDEL sequence in-frame with the ER transit peptide sequence under the control of the 35S promoter was established by restriction endonuclease analysis (BamHI, EcoRI, AscI, PacI) of DNA isolated from tetracycline-resistant transformants.", "The resulting plasmid was designated pCGP3259 FIG.", "36).", "Construction of pCGP3262 (RoseCHS.ER.MCS:35S Pre-Binary) The N-terminal ER transit peptide fragment was cloned downstream of the Rose CHS promoter contained in the pre-binary pCGP3255 to produce pCGP3262 (FIG.", "37).", "Plasmid pCGP3256 was digested with AscI and BamHI to release the ˜100 bp N-terminal ER transit peptide fragment.", "The fragment was isolated and purified using QIAEX II Gel Extraction kit (Qiagen) and ligated with AscI/BamHI ends of pCGP3255.Correct insertion of the N-terminal ER transit peptide fragment was established by restriction endonuclease analysis of DNA isolated from tetracycline-resistant transformants.", "PCR products of CFMs or colored proteins derived using the primers vispro-F1 (SEQ ED NO:184) and CP-HDEL-PacI.R (SEQ ID NO:209) can be digested with BamHI and PacI and ligated with BamHI/PacI ends of pCGP3262.The coding region of the CFMs or colored proteins will be under the control of the Rose CHS promoter and in-frame with the ER transit targeting peptide to allow targeting of the proteins to the ER.", "The coding region of the CFMs or colored proteins will also contain the HDEL sequence at the C-terminal to allow retention of the proteins in the ER of floral tissues.", "Constructions of pCGP3263 (Rose CHS:ER: T1-HDEL:35S Binary) The coding sequence of the colored protein T1 was amplified by PCR using the primers vispro-F1 (SEQ ID NO:184) and CP-HDEL-PacI.R (SEQ ID NO:209) and the plasmid pCGP2779 as template.", "PCR conditions were as described above for construction of pCGP3259.The resulting ˜700 bp product was digested with BamHI and PacI, isolated and purified using QIAEXII Gel Extraction kit (Qiagen) and ligated with BamHI/PacI ends of pCGP3262.Correct insertion of the T1 coding region and HDEL sequence in-frame with the ER transit peptide sequence under the control of the Rose CHS promoter was established by restriction endonuclease analysis (BamHI, EcoRI, AscI, PacI) of DNA isolated from tetracycline-resistant transformants.", "The resulting plasmid was designated pCGP3263 (FIG.", "38).", "A site predicting N-glycosylation was identified within the coloured protein T1 (‘NDS’—surrounding amino acid 107) (SEQ ID NO:202).", "This site is conserved among the colored protein clones D1, D10, T1, T3, S3 and A8 and these include both purple and blue varieties.", "Comparison of this region in sequences of other coloured and fluorescent varieties in the GenBank database (e.g., asCP562, asFP499, Clavularia FP484, Discosoma FP483 etc) indicate the presence of two alternative sequences in this position—QDS or NDI.", "The first converts an asparagine residue (N) to a glutamine (Q) (a conservative change given both residues are polar) and the second changes the serine (S) to an isoluecine (I) (a non conservative change from a polar to a non polar residue).", "Both naturally occurring sequence alternatives for this region of the protein were be performed separately.", "That is, mutation of the T1 sequence from NDS to QDS and a separate mutation from NDS to NDI.", "The plasmid pCGP2921 (FIG.", "10) was used as a source of the coding sequence for T1 blue protein.", "A BamHI/HindIII fragment was isolated from pCGP2921 and cloned with BamHI/HindIII ends of pBluescript to produce pCGP3268.The GeneEditor in vitro Site Directed Mutagenesis Kit (Promega) was used following the manufacturer's instructions along with the following oligonucleotides (T1.N-Q N(AAT)>Q(CAG) SEQ ID NO:230) and T1.S-IS(TCC)>I(ATC) SEQ ID NO:231) to introduce the mutations in pCGP3268.T1.N-Q N(AAT) > Q(CAG) GTG TGT ACT GTC AGC CAG GAT TCC AGC SEQ ID NO:230 V C T V S Q D S S ATC CAA G I Q T1.S-I S(TCC) > I(ATC) CT GTC AGC AAT GAT ATC AGC ATC CAA SEQ ID NO:231 GGC AAC The resultant plasmids pCGP3271 and pCGP3272 containing the N107Q and S109I mutated forms of T1 blue protein in pBluescript were sequenced thoroughly to confirm the presence of the mutated sequence.", "Construction of pCGP3273 (pQE30:T1(N107Q) and pCGP3274 (pQE30:T1(S109I) E. coli expression of the mutated forms of T1 in pCGP3271 and pCGP3272 was necessary to determine if the mutations had any effect on the colour of the expressed protein.", "Thus, BamHI/HindIII fragments pCGP3271 and pCGP3272 were subcloned with BamHI/HindIII ends of pQE30.The resultant plasmids were designated pCGP3273 (T1-N107Q) and pCGP3274 (T1-S109I) and were expressed in E. coli as previously described (Example 3 and 6) to determine the colour of the expressed protein.", "The protein expressed by the sequence encoded in pCGP3273 was found to retain the original colour of T1 as expressed by pCGP2921, while the protein expressed by pCGP3274 was not coloured.", "This suggested that the S109I mutation may have had a deleterious effect on the color of the protein.", "Investigation of this protein will provide information on the amino acids that are critical to color formation of colored proteins.", "Construction of pCGP3275 (35S: ER:T1(N107Q).HDEL:35S Binary) and pCGP3276 (355: ER:T1(S109I).HDEL:35S Binary) The coding sequence of the coloured protein T1(N107Q) was amplified by PCR using the primers vispro-F1 (SEQ ID NO:184) and CP-HDEL-PacI.R (SEQ ID NO:207) and the plasmids pCGP3271 (described above) and pCGP3272 (described above) as template essentially as described in the construction of pCGP3259 (Example 11).", "The resulting ˜700 bp products were digested with BamHI and PacI, isolated and purified using QIAEXII Gel Extraction kit (Qiagen) and ligated with BamHI/PacI ends of pCGP3257 (FIG.", "35).", "Correct insertion of the coding regions of T1(N107Q) and T1(S109I) and HDEL sequence in-fame with the ER transit peptide sequence under the control of the CaMV 35S promoter was established by restriction endonuclease analysis (BamHI, EcoRI, AscI, PacI, EcoRV) of DNA isolated from tetracycline resistant transformants.", "The resulting plasmids were designated pCGP3275 and pCGP3276.Construction of pCGP3277 (RoseCHS: ER:T1(N107Q).HDEL:35S Binary) and pCGP3276 Rose CHS: ER:T1(S109I).HDEL:35S Binary) The coding sequence of the coloured protein T1(N107Q) was amplified by PCR using the primers vispro F1 (SEQ ID NO:184) and CP-HDEL-PacI.R (SEQ ID NO:207) and the plasmids pCGP3271 and pCGP3272 as template essentially as described in the construction of pCGP3259 (Example 11).", "The resulting ˜700 bp products were digested with BamHI and PacI, isolated and purified using QIAEXII Gel Extraction kit (Qiagen) and ligated with BamHI/PacI ends of pCGP3262 (FIG.", "37).", "Correct insertion of the coding regions of T1(N107Q) and T1(S109I) and HDEL sequence in-frame with the ER transit peptide sequence under the control of the Rose CHS promoter was established by restriction endonuclease analysis (BamHI, EcoRI, AscI, PacI, EcoRV) of DNA isolated from tetracycline resistant transformants.", "The resulting plasmids were designated pCGP3277 and pCGP3278.EXAMPLE 12 Fusion Proteins Wills GFP Construction of pCGP3258 (35S:T1/mGFP4:35S Binary) As a way of tracking the expression and localisation of the T1 coloured protein the T1 coding region was fused with the N-terminus of mGFP4 (Haseloff et al., PNAS 94: 2122-2127, 1997).", "The mGFP4 coding sequence was amplified using the primers PstI-mGFP4F (SEQ ID NO:210) and mGFP4-PacIR (SEQ ID NO:211) and pBIN35SmGFP4ER (Haseloff et al., 1997) as template.", "A ˜700 bp product was gel purified and then digested with the restriction endonucleases PstI and PacI.", "The T1 coding sequence was amplified using the primers visproF1-new (SEQ ID NO:212) and visproR1 (SEQ ID NO:185) and pCGP2779 as template.", "Pst-mGFP4F (5′ to 3′) PstI linker sequences GCAT CTG CAG GTC GCC ACC AGT AAA GGA SEQ ID NO:210 L Q V A T S K G GAA GAA CTT TTC AC E E L F mGFP4-PacIR PacI CTGA TTAATTAA TTA TTT GTA TAG TTC ATC SEQ ID NO:211 * K Y L E D CAT GCC ATG M G H visproF1-new AscI RBS TICS CAG GGCGCGCC AAGGAGATAT AACA ATG SEQ ID NO:212 M BamHI GGA TCC GTT ATC GCT AAA CAG ATG ACC G S V I A K Q M T A ˜700 bp product was gel purified and then digested with the restriction endonucleases AscI and PstI.", "The PstI/PacI mGFP4 fragment was ligated with the AscI/PstI T1 fragment.", "The resulting ligated fragment was then ligated with the AscI/PacI ends of the binary vector pCGP3257 (FIG.", "35) to produce pCGP3258 (FIG.", "39).", "Correct insertion of the T1:mGFP4 fusions was established by restriction endonuclease analysis (BstXI, EcoRI, NcoI, PstI) of DNA isolated from tetracycline-resistant transformants.", "The resulting plasmid was designated pCGP3258 (FIG.", "39).", "Construction of pCGP3261 (35S.ER:T1:GFP: 35S Binary) An ER targeted version of the T1:mGFP4 fusion in pCGP3258 under the control of the CaMV 35S promoter was also prepared.", "This plasmid was designated pCGP3261 FIG.", "45).", "The T1:mGFP4 fusion was amplified using the primers vispro-F1 (SEQ ID NO:184) and mGFP4-HDEL-PacR (SEQ ID NO:229) and pCGP3258 (FIG.", "39) as template.", "A ˜1.4 kb product was gel purified and then digested wit the restriction endonucleases BamHI and PacI.", "The resulting fragment was then ligated with BamHI/PacI ends of the binary vector pCGP3257 (FIG.", "35) to produce pCGP3261 (FIG.", "45).", "Correct insertion of the T1:mGFP4 fusion was established by restriction endonuclease analysis (BstX1, EcoRI, NcoI, PstI, AscI/PacI, XbaI) of DNA isolated from tetracycline-resistant transformants.", "The resulting plasmid was designated pCGP3261 (FIG.", "45).", "mGFP4-HDEL-PacR (5′ TO 3′) CTG ATT AAT TAA AGC TCA TCA TGT TTG SEQ ID NO:229 TAT AGT TCA TCC ATG CCA TG Construction of pCGP3260 (35S.ER:GFP: 35S Binary) An ER targeted version of the mGFP4 in pBIN35SmGFP4ER (Haseloff et al., 1997 supra) under the control of the CaMV 35S promoter and CaMV 35S terminator was prepared to use as a control for the binaries pCGP3258 (FIG.", "39) and pCGP3261 (FIG.", "45).", "The plasmid pBIN35SmGFP4ER (Haseloff et al., 1997 supra) was initially digested with the restriction endonuclease SacI.", "The resulting overhang was repaired and the linearized vector was then digested with BamHI to release a ˜0.7 kb fragment containing the mGFP4 coding sequence.", "The resulting SacI(blunt)/BamHI mGFP4 fragment was gel purified and then ligated with BamHI/PacI (blunt) ends of the binary vector pCGP2780 (FIG.", "30).", "Correct insertion of the mGFP4 coding sequence was established by restriction endonuclease analysis (EcoRI, NcoI, PstI, BamHI, XbaI) of DNA isolated from tetracycline-resistant transformants.", "The resulting plasmid was designated pCGP3260 (FIG.", "46).", "EXAMPLE 13 Reconstruction of Color In order to determine whether rose petals or plant material in general conatin proteases that may degrade colored proteins reconstructions of rose petal extracts with the T1 colored protein were set up.", "Petals of Rosa hybrida cultivar Medeo are generally white to pale apricot.", "Expression of colored proteins in a white flower should allow visualisation of color when colored proteins are expressed in flowers.", "One gram amounts of Medeo rose petals were ground in 500 μL water using a mortar and pestle.", "The resultant slurries were centrifuged at 14 000 rpm for 5 min in 1.5 mL centrifuge tubes.", "The supernatants were collected and 100 μL of the extracts were aliquoted into the wells of a microtiter tray.", "Ten microliters aliquots containing ˜30 μg of His-tag purified T1 protein (purified as described in Example 8) were added to the Medeo extracts.", "In order to determine whether the color of the colored protein is affected by pH, the pH of some of the reconstructions was modified by addition of NaOH so that the final pH was 7.0, 8.5 or 10.0.The pH of Medeo petal extract alone was pH 4.5 and 4,6.The pH of Medeo petal extract mixed with TI protein was pH 5.2, 5.8 and 6.1.The color of reconstructions of Medeo petal extract mixed with T1 protein at pH 5.2, 5.8 and 6.1 was light blue (RHSCC 101C/RHSCC 115B).", "However the color at pH 7.0 and 8.5 was a pale blue-green (RHSCC 122C) and that at pH 10.0 was yellow.", "The colors were still evident after 5 hours incubation at room temperature as well as 48 hours at room temperature indicating that the colored protein was stable in petal extract.", "An interesting and unexpected observation was that the color of the T1 protein changed to yellow when in a high pH solution.", "Analysis of the conformation of the protein at this high pH provides information that allows for the design of targeted mutations to T1 or other colored protein sequence and thus allows for the production of a yellow color in a low to neutral pH environment such is found in plant cells.", "Alternatively random shuffling (U.S. Pat.", "No.", "6,132,970) using selections of the vast number of colored protein sequences isolated and then expressing these mutated versions in E. coli or yeast as described in Examples 3, 4, 6 and 7 will provide a means of selecting for altered or improved colors and/or brightness of the proteins expressed.", "Incubation of Petunia Petals with T1 Protein The flowers of Petunia hybrida cultivar Mitchell are white.", "Mitchell petal sections were incubated with the T1 protein to determine the color that would be produced in white petals upon production of the colored proteins.", "Petal sections (including part of the tube and limb) were incubated in 200 μL His-tag purified T1 protein (from E. coli cultures as described in Example 8) (6 mg/mL in 20 mM Tris HCl pH 8.0) and His-tag purified A8 protein (from yeast cultures as described in Example 8) (1 mg/mL in 20 mM Tris HCl pH 8.0).", "In both cases the colored proteins were taking up by the petal fragments within a few minutes as visualised by coloration of the cut surface of the petal.", "Incubation of white petals in the T1 protein solution resulted in petals of a pale blue (RHSCC 112D) color whereas incubation of white petals in the A8 protein solution resulted in a pale purple color in the petal tissue.", "This experiment showed that the protein is stable in petal tissue and that the color produced will not be masked or quenched by other plant compounds.", "EXAMPLE 14 Expression of Colored Proteins in Arabidopsis Transformation of Arabidopsis Construction of pCGP960 (35S:gus:ocs Binary) The binary vector pCGP960 was prepared to use as a control in plant transformation experiments.", "A CaMV35S:GUS:ocs3′ expression cassette was isolated from pKMI101 (Klee et al., Bio/Technology 3: 637-642, 1985) and inserted into the pWTT2132 (DNAP) binary vector backbone which contains a CaMV 35S:SuRB selectable marker gene.", "The binary vectors pCGP2772 (FIG.", "24), pCGP2765 FIG.", "21), pCGP3259 (FIG.", "36), pCGP2785 FIG.", "33), pCGP3258 (FIG.", "39), pCGP2926 (FIG.", "44), pCGP3263 (FIG.", "38), pCGP2787 (FIG.", "34), pCGP2782 (FIG.", "27), pCGP960 (see above), pCGP3261 (FIG.", "45), pCGP3260 (FIG.", "46), pBINmGFP4ER (Haseloff et al., 1997, supra) were introduced into Agrobacterium tumefaciens strain AGL0 as described in Example 1.Arabidopsis thaliana ecotype WS-2 was transformed with the above constructs using the floral dip method as mentioned in Example 1.Seeds from dipped plants were plated on selection and transgenic plants were allowed to grow until flowering.", "Plants can be allowed to self-fertilize to produce seed.", "The T2 seed can then be germinated on selection (e.g.", "100 μg/mL chlorsulfuron selection for those transformed with a CaMV 35S: SuRB selectable marker gene) and allowed to grow to flowering.", "A number of the T2 generation would be expected to be homozygous for the introduced transgenes with the expectation that these plants would have increased coloured protein gene expression and protein production than the heterozygous parental lines.", "Northern Analysis Leaves from a random selection of 2 events per construct (pCGP2772, pCGP2765, pCGP3259, pCGP2785, pCGP3258, pCGP3261, pCGP960, pBIN35Smgfp4ER, pCGP3260) were analysed for the presence of transcripts of the introduced T1 or A8 colored protein genes.", "Total RNA was isolated from these events using a Plant RNAeasy kit (QIAGEN) following procedures recommended by the manufacturer.", "RNA samples (5 μg) were electrophoresed through 2.2 M formaldehyde/1.2% w/v agarose gels using running buffer containing 40 mM morpholinopropanesulphonic acid (pH 7.0), 5 mM sodium acetate, 0.1 mM EDTA (pH 8.0).", "The RNA was tansferred to Hybond-N filters (Amersham) as described by the manufacturer.", "The RNA blot was initially probed with 32P-labelled fragments of a BamHI/HindIII fragment isolated from pCGP2921 (T1) FIG.", "10) (108 cpm/μg, 2×106 cpm/mL).", "Prehybridization (1 hour at 42° C.) and hybridization (16 hours at 42° C.) of the membrane were carried out in 50% v/v formamide, 1 M NaCl, 1% w/v SDS, 10% w/v dextran sulphate.", "The filter was washed in 2×SSC, 1% w/v SDS at 65° C. for between 1 to 2 hours and then 0.2×SSC, 1% w/v SDS at 65° C. for between 0.5 to 1 hour.", "The filter was exposed to Kodak XAR film with an intensifying screen at −70° C. for 22 hours.", "The T1 probe hybridized with transcripts of expected sizes (see Table 20) in RNA of transgenic plants that had been transformed with constructs carrying the T1 or A8 clones (lanes 1, 2, 5, 6, 7, 8, 13, 16 and 17) (eg.", "pCGP2772, pCGP2765, pCGP3259, pCGP2785, pCGP3258, pCGP3261) (FIG.", "41A) (Table 20).", "Under the conditions used, no hybridizing transcript was detected by Northern analysis of total RNA isolated from non transgenic control plants (lanes 9 and 10) or transgenic plants transformed with non-T1 carrying constructs (lanes 3, 4, 11, 12, 14 and 15) (e.g.", "pCGP960 (GUS), pBIN35Smgfp4, pCGP3260 (ER:mGFP4).", "The 32P-labelled T1 DNA probe was then stripped from the RNA blot by soaking the membrane in 0.1% SDS at 100° C. and incubating it in a 65° C. oven for 30 minutes with a final incubation step at room temperature for around 30 minutes.", "The RNA blot was then probed with 32P-labelled fragments of a ˜0.8 kb HindIII fragment from pCGP1651 (SuRB) (108 cpm/μg, 2×106 cpm/mL).", "Prehybridization and hybridization were carried out as described above.", "The plasmid pCGP1651 contains a 0.8 kb HindIII fragment from the SuRB coding region contained in the binary plasmid vector pWTT232 (DNAP).", "The SuRB probe hybridized with a 2.2 kb transcript in transgenic plants that had been transformed with the constructs carrying the CaMV 35S: SuRB tansgene (FIG.", "41B) (lanes 1 to 8, 13 to 17) (eg.", "pCGP2772, pCGP2765, pCGP3259, pCGP2785, pCGP3258, pCGP3261) (Table 20).", "Under the conditions used, no hybridizing transcript was detected by Northern analysis of total RNA isolated from non transgenic control plants (lanes 9 and 10) or transgenic plants transformed with non-SuRB constructs (lanes 11 and 12) (e.g.", "pBIN35Smgfp4ER).", "Detection of Colored Proteins in, Transgenic Arabidopsis Polyclonal Rabbit Antibodies to T1 Protein T1 protein was extracted from cultures of E. coli harbouring pCGP2921 (FIG.", "10) as described previously in Example 6.Polyclonal rabbit antibodies against the T1 protein were produced by Institute of Medical and Veterinary Sciences, Veterinary Services Division, 101 Blacks Rd, Gilles Plains, South Australia 5086, Australia.", "An amount of 300 μg of T1 protein (with Freunds complete adjuvent) was initially administered.", "Serial doses of 300 μg T1 protein (with Freunds incomplete adjuvent) were subsequently administered 22 days and 36 days after the initial dose.", "Antibodies collected in the first bleed (which was taken at 45 days after the initial dose) were used to probe Western blots in the first instance.", "Protein Extraction from Plants Leaf material (20-120 mg) was collected from Arabidopsis plants, snap frozen in liquid nitrogen and then ground to a fine powder using a mortar and pestle.", "An equal volume (w/v) of extraction buffer (100 mM Na2PO4 pH 6.8, 150 mM NaCl, 10 mM EDTA, 10 mM DTT, 0.3% Tween 20, 0.05% Triton X) was then added to the fine powder and the mixture was flirter ground using the mortar and pestle.", "The resultant slurry was centrifuged at 10 000 rpm for 10 min and the supernatant was collected.", "Western Blot Analysis of Proteins Extracted from Transgenic Arabidopsis Aliquots (8 μL) of the protein extracts were mixed with 2 μL of 5×SDS loading buffer 10% v/v glycerol, 3% w/v SDS, 3% β-mercaptoethanol, 0.025% w/v bromophenol blue) electrophoresed through precast SDS PAGE gels (12% w/v resolving, 4% w/v stacldng gel) (Ready Gels, Biorad) at 100 V for lh 15 min in a Min-Protean System (Bio-Rad) using conditions as described previously in Example 6.The proteins were then transferred to Lmnun-Blot PVDF membrane (Bio-Rad) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) in Towbin buffer (25 mM Tris, 20% methanol, 192 mM glycine) at 100 V for 1 h. PVDF membranes were incubated in blocking buffer (5% non-fat dry milk, 0.2% Tween-20, 75 mM NaPi pH 7.4, 68 mM NaC]) at room temperature for 1 h. Membranes were then flirter incubated with Rabbit anti-T1 antibody (diluted 1/200 in blocking buffer) for 2 h at room temperature then washed twice for 5 min in wash buffer (0.2% Tween, NaPi pH 7.4, 68 mM NaCl).", "The membranes were finally incubated with goat anti-rabbit-IgG-alkaine phosphatase congugate (Bio-Rad) (diluted 1/300 in blocking buffer) for 1 h at room temperature followed by 4 washes for 10 min each in wash buffer.", "Colorimetric detection was carried out with Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega).", "The polyclonal T1 antibody detected a protein band running at the same position as T1 protein extracted from E.coli cultures harbouring pCGP2921 in extracts from Arabidopsis/2772 event 1.2, Arabidopsis/3259 event 1.5.The same T1 protein band was not detected in extracts from the non-transgenic controls.", "The protein content in a 2 μL sample of the protein extracts was estimated using a Bio-Rad Protein Assay as per the manufacturers instructions (Microassay Procedure).", "The absorbance of each extract at 595 min was compared with BSA standard curves (0-10 μg/mL) to estimate protein concentrations.", "Samples of protein extract and a dilution series of known amounts of purified His-tagged colored protein (T1) were electrophoresed through SDS PAGE gels as described previously.", "The proteins were transferred to PVDF membranes (as described above) and probed with rabbit anti-T1 antibodies.", "The amounts of T1 colored protein in the protein extracts was estimated by comparison with the purified His-tagged colored protein dilution series.", "This allowed an estimation of expression of colored protein in Arabidopsis leaf as a percentage of total soluble protein (Table 21).", "EXAMPLE 15 Expression of Colored Proteins in Petunia Transformation of petunia Petunia hybyida cultivar Mitchell produces white flowers.", "Mitchell was transformed with the binary constructs pCGP2772 (FIG.", "24), pCGP2765 FIG.", "21), pCGP3259 (FIG.", "36) pCGP2785 (FIG.", "33) and pCGP2926 (FIG.", "44) via Agrobacterium-mediated transformation as described in Example 1.Northern Analysis Flowers from a random selection of events transformed with the T-DNAs of pCGP2772 and pCGP2765 were analysed for the presence of transcripts of the introduced T1 or A8 colored protein.", "Total RNA was isolated using a Plant RNAeasy kit (Qiagen) following procedures recommended by the manufacturer.", "Northern analysis was performed as described above for analysis of the Arabidopsis transgenic plants.", "The T1 probe hybridized with transcripts of around 0.9 kb in petal RNA of transgenic Mitchell plants that had been transformed with constructs carrying the T1 or A8 clones (FIG.", "40A) (pCGP2772 (lanes 7 to 12) and pCGP2765 (lanes 1 to 6), respectively).", "Under the conditions used no hybridising transcript was detected in RNA isolated from petals of a non transgenic control (data not shown).", "The SuRB probe hybridized with a 2.2 kb transcript in transgenic plants that had been transformed with the constructs carrying the CaMV 35S: SuRB transgene (FIG.", "40B).", "Under the conditions used no hybridizing transcript was detected in RNA isolated from petals of a non transgenic control (data not shown).", "Detection of Colored Proteins in Transgenic P. hybrida Western Blot Analysis of Proteins Extracted from Transgenic Petunia Proteins were extracted from leaf and flower material petal tube, petal limb, anthers, pistil, stigma and style) (100-300 mg) of transgenic and non-transgenic P. hybrida cv, Mitchell plants as described for Arabidopsis.", "Western blot analysis of these protein extracts was performed as described for Arabidopsis.", "The polyclonal T1 antibody detected a protein band running at the same position as T1 protein extracted from E.coli cultures harbouring pCGP2921 in extracts from Petunia accession 24534 (pCGP2765) and Petunia accession 24444 (pCGP2772).", "The same T1 protein band was not detected in the non-transgenic controls.", "An estimation of expression of colored protein in Petunia leaf and petal as a percentage of total soluble protein was made as described above for Arabidopsis extracts (Table 22).", "The T1 protein was produced in Arabidopsis leaf (example 14) and Petunia leaf and flower tissue (Example 15).", "It is expected that an increase in protein accumulation will produce stronger colours in flower and leaf tissue.", "The first generation of transformed plants are selfed to give homozygous second generation transformants with higher T1 protein or other CFM accumulation and stronger colour.", "Alternatively, different transgenic events are crossed to produce second generation transformants with higher T1 protein or other CFM accumulation and stronger colour.", "Methods envisaged to increase total T1 protein or other CFM accumulating in transformed plants include targeting T1 or other CFM to the chloroplast using a chloroplast transit peptide such as that from the small subunit of ribulose-bisphosphate from tobacco (see Example 11 or Table 17).", "These chloroplast transit peptides will facilitate the movement and accumulation of CFMs into chloroplasts which are abundant in leaves and chromoplasts which are abundant in flowers petals.", "Another method envisaged to produce higher levels of CFMs in plant tissues is the use of chloroplast/plastid transformation techniques which have been used in the past to generate plants expressing recombinant proteins at levels of up to 46% of total soluble protein (De Cosa et al., Nat.", "Biotechnol.", "19, 71-74, 2001; Daniell et al., Trends in Plant Sci.", "7: 84-91, 2002, see Example 11, Table 18).", "It is also envisaged that the co-expression of a suitable chaperonin in conjunction with one or more CFMs allows the efficient folding and packaging of CFMs into stable structures which are accumulated in higher amounts than would normally be expected.", "It is also envisaged that producing a fusion of CFM with ubiquitin in plants will increase levels of accumulated CFMs in transgenic plants as has been demonstrated in yeast (Baker, Curr.", "Opinions in Biotech, 7.541-546, 1996 and references within).", "It is also envisaged that targeting Ti or other CFM to the endoplasmic reticulum (see Example 1 1) will increase the levels of accumulated recombinant protein in plant tissues (Haseloff et al., 1997, supra).", "Detection of Correctly Folded CFMs in Plant Extracts.", "CFMs that are folded correctly in heterologous systems (such as when expressed in flowers or other plant tissues) are expected to retain characteristic absorbance and corresponding colour (see Example 13).", "The level of CFM production or accumulation may initially be too low for significant color change in plant tissue.", "A method for detecting low levels of correctly folded CFMs in plant extracts is described for leaf material from Petunia transformed with pCGP2772 and pCGP2765, however, this method can be used with other plant tissues such as but not limited to Petunia or rose or gerbera.", "Total soluble proteins were extracted from transgenic leaves of Mitchell/pCGP2772 and Mitchell/pCGP2765) (see Example 15).", "These samples were frozen in liquid nitrogen and ground using a mortar and pestle.", "An equal volume (w/v) of extraction buffer (100 mM NaPO4 pH 6.8, 150 mM NaCl, 10 mM EDTA, 10 mM DTT, 0.3% Tween 20, 0.05% Triton X) was added to the sample and flirther ground.", "The resultant slurry was centrifmged at 10000 rpm for 10 min and the supernatant collected.", "The extracts were used undiluted or diluted 1:2 in water and their absorbance characteristics determined between 400 nm and 700 nm using a Varian Cary 50 Bio UV-Visible Spectrophotometer.", "The absorbance spectra were compared to those of extracts of non-transgenic control tissue and non-transgenic control tissue spiked with either T1 or T3 His-tagged purified protein (see Example 8).", "Detectable color was observed through the detection of peaks at approximately 580-590 nm in the extracts from transgenic plant tissue that were not evident in non-transgenic control tissue.", "Methods envisaged to increase protein levels are as described above or by Bailey-Serres and Gallie (American Society of Plant Physiologists, Look beyond transcription, UCLA, USA, 1998) or by modification of mRNA sequence to optimize 5′ and 3′ untranslated sequences thereby improving message stability and/or translation efficiency, optimisation of codon usage in the introduced gene to more closely match that found in highly expressed genes (that is genes which give rise to high levels or encoded protein synthesis) in particular those of target crops, augmentation of protein stability via the attachment for example of stabilising sequences such as ubiquitin, changes to specific N-terminal amino acid residues to promote altered aggregation of monomeric forms of the protein, more effective targeting of the synthesized polypeptide to intracellular organelles or compartments, duplication and there for amplification of introduced genes leading to increased levels of protein biosynthesis for example using ‘Gene Amplification Technology’ (Borisjuk et al., Nature Biotechnology 18: 1303-1306, 2000).", "EXAMPLE 16 Expression of Colored Proteins in Other Plants The horticultural industry relies on the production of novel traits such as new colors, fragrances, productivity and disease resistance.", "Introduction of colored protein sequences (via genetic engineering) into commercially important plant lines such as, for example, but not limited to roses, carnations and gerberas provides a means to produce novel colors in flowers or plants that lack such colors.", "Introduction of colored protein genes into roses is achieved using methods such as those described, for example, in International Patent Application Number PCT/US91/04412, or by Robinson and Firoozabady (Scientia Horticulturae, 55: 83-99, 1993), Rout et al.", "(Scientia Horticulturae, 81: 201-238, 1999) or Marchant et al.", "(Molecular Breeding 4: 187-194, 1998) or by any other method well known in the art.", "Introduction of colored protein genes into carnations is achieved using methods such as those described, for example, in International Patent Application Number PCT/US92/02612 or by Lu et al.", "(Bio/Technology 9: 864-868, 1991), Robinson and Firoozabady (1993, supra) or by any other method known in the art.", "Introduction of colored protein genes into carnations is achieved using methods such as those described, for example, by Robinson and Firoozabady (1993, supra).", "The cotton industry relies on the production of dyed cotton, using dyes that can have concomitant detrimental effects on the environment.", "Introduction of colored protein sequences (via genetic engineering) into commercially important cotton lines, or other plant lines that allow for production of fabrics (such as, but not limited to, hemp), and also relies on use of colored dyes to dye said fabrics, is achieved using methods such as those described, for example, in an International Patent Application having Publication Number WO 00/77230.EXAMPLE 17 Generation of Transformed Animals The use of the CFMs of the present invention are employed to produce transgenic animals which exhibit novel color, for example, sheep with blue or red colored fleece, cows with red colored hide inter alia.", "The transgenic animals of the present invention can be produced by any number of method know in the art.", "Such as, but not limited to transgenic animals are produced by any number of methods, for example, microinjection of constructs comprising a CFM nucleotide sequence into the pronucleus of a fertilized ovum, or injection of embryonic stem (ES) cells into embryos.", "Microinjection Following fertilization a single celled embryo is removed from the animal (e.g.", "sheep, cow, pig, goat).", "Micromanipulators on a specially equipped microscope are used to grasp each embryo.", "A glass pipette drawn to a fine point immobilizes the embryo on one side.", "On the opposite side, a construct containing a CFM nucleotide sequence is injected into the embryo's pronucleus with a second finely drawn injection needle.", "Following the injection, the embryos are transferred back into the hormonally prepared or pseudopregnant recipient females or foster mothers.", "The recipients follow normal pregnancy and deliver fu ill-term young.", "Injection of Embryonic Stem Cells ES cells are isolated from the inner cell mass of blastocyst-stage embryos (about 7 days postfertilization), ES cells are grown in the lab for many generations to produce an unlimited number of identical cells capable of developing into fully formed adults.", "These ES cells are altered genetically by injection of a construct containing a CFM nucleotide sequence.", "Transgenic individuals are produced by microinjection of embryonic stem (ES) cells containing the CFM construct into embryos to produce “hybrid” embryos of two or more distinct cell types.", "Following the injection, the embryos are transferred back into the hormonally prepared or pseudopregnant recipient females or foster mothers.", "The recipients follow normal pregnancy and deliver full-term young.", "EXAMPLE 18 Generation of a Far Red Fluorescent Monomeric Protein Cloning and Expression cDNA encoding the colored protein Rtms-5 (SEQ ID NO:166) was isolated from Montipora efflorescens (Scleractina Acropodiae).", "Under daylight illumination, Montipora efflorescens was a purply-red colour, but fluoresced yellow under blue illumination and red under green illumination.", "To further characterise the protein, the cDNA was tagged with hexahistidine at its C-terminus and expressed at high levels in Escherichia coli.", "For expression in bacteria, the nucleotide sequence encoding Rtms-5.pep (SEQ ID NO:166) was retrieved from pGEM-T cloning vector (Promega) using forward oligonucleotide primers consisting of the NotI restriction binding site, a ribosomal binding site, a spacer and 15 bases encoding the N-terminus of the protein (MSV-RBS, SEQ ID NO:213; SVIAK-RBS, SEQ ID NO:214) and a reverse oligonucleotide primer encoding H6-tag (POC220-H6, SEQ ID NO:215).", "MSV-RBS GGC TCT AGA AAG GAG ATA TAC AAG TGT SEQ ID NO:213 GAT CGC TAC ACA AAT GA SVIAK-RBS GGC TCT AGA AAG GAG ATA TAC AAT GTC SEQ ID NO:214 CGT TAT CGC TAA ACA GAT POC220-H6 GGC AAG CTT TCA GTG GTG GTG GTG GTG SEQ ID NO:215 GTG GGC GAC CAC AGG TTT GCG TG PCR product was gel purified and diluted (×10) prior to cloning into pCRII-TOPO (Invitrogen) and transforming into Top 10 cells (Invitogen).", "Cells were induced with 0.5 mM IPTG, and protein was purified on Ni-columns (Pro-Bond, Invitrogen) eluting with 50 mM, 200 mM, 350 mM and 500 mM Imidazole in PBS pH 6.0, prior to overnight dialysis against 50 mM Potassium Phosphate pH 6.65.Fluorescence Charcteristics of Rtms-5 E. coli colonies were blue in colour in daylight, and weakly red fluorescent when excited with light of wavelength 595 nm.", "An alignment of the amino acid sequence of Rtms-5 (SEQ ID NO:166) with other fluorescent proteins was constructed (Table 19).", "Rtms-5 (SEQ ID NO:166) contains the key amino acids (Tyr-66 and Gly-67) that correspond to those that form the fluorophore in other well-characterised proteins, dsRed583 (also known herein as drFP583, SEQ ID NO:221) and GFP (SEQ ID NO:222).", "Overall, 67% and 20% of the Rtms-5 (SEQ ID NO:166) sequence is identical to dsRed583 (SEQ ID NO:221) and GFP (SEQ ID NO:222), respectively.", "The protein shares a high degree of identity with a number of chromoproteins recently isolated from the Anthozoa species (Gurskaya et al., FEBS Lett.", "507: 16-20, 2001).", "The absorption and excitation emission spectra were measured for the purified “wild-type” Rtms-5 (SEQ ID NO:166).", "The protein displays a major absorption peak at 592 nm, with an extinction that is highly variable (ε592=53,000 M−1 cm−1 111,000 M−1 cm−1) and a shoulder peak at 454 nm (FIG.", "42.The variability in the extinction coefficient is similar to that observed for drFP583 (SEQ ID NO:221) and, similarly, it is dependent on the state of maturity, as well as the conditions under which the protein is expressed (Baird et al., 2000, supra).", "Site Directed Mutagenesis Rtms-5 (SEQ ID NO:166) was only weakly fluorescent.", "To enhance this, site-directed mutagenesis was carried out.", "The alignment of the Rtms-5 sequence (SEQ ID NO:166) with other sequences (Table 19) indicated that position 142 was occupied by the residue histine.", "A variant Rtms-5-H142S, containing the substitution H142S, was engineered by mutagenesis of pCRII-TOPO::RTms5 to produce pCRII-TOPO::RTms5-H142S.", "This single substitution increased the quantum yield of far-red fluorescence by 170-fold to a quantum yield of less than 0.02.Minor effects on the excitation and emission spectra and the absorption spectra were observed (4 nm shift towards the blue end of the spectrum, refer to FIGS.", "42A,B,C).", "Analysis of Oligomeric Structure dsRed583 (SEQ ID NO:221) is known to be an obligate tetramer.", "The formation of oligomers by fluorescent proteins can present a serious problem when expressed fused to other proteins of interest.", "Consequently, it was important to establish the degree of oligomerisation of Rtms-5 (SEQ ID NO:166).", "The protein has a predicted size of 25,820 Da (with H6).", "When subjected to SDS-PAGE under reducing conditions, purified Rtms-5 (SEQ ID NO:166) migrated with an Mr of 26,900, However, under non-reducing conditions the majority of the protein migrated with an Mr of 114,000.These results indicated that native Rtms-5 (SEQ ID NO:166) was predominantly a tetramer.", "Further Site Directed Mutagenesis and Analysis of Structure A second round of site-directed mutagenesis was carried out, to mutagenise CRII-TOPO::RTms5-H142S to produce the variant pCRII-TOP-RTms5-H142S-F158H (pCRII::Rtms-5v).", "This colored peptide contained the additional substitutions F158H and R145H, and is designated Rtms-5v (SEQ D NO:216).", "Rtms-5v (SEQ ID NO:216) was expressed in E. coli and the purified six His-tagged protein was subjected to analytical ultracentrifugation.", "The results indicated that the mutagenised variant sedimented predominantly as a monomer (82%, 30,700 Da) with the remaining proportion sedimenting as a dimer (18%, 50,800 Da).", "This colored protein fluoresced in the far-red range (see FIG.", "42C), and can be used effectively in yeast cells and mammalian cells.", "Effect of Site Directed Mutagenesis of Other Colored Proteins Site directed mutagenesis of residue H or N 142 to S, in other colored protein sequences, also leads to the generation of far-red fluorescence.", "Examples of the excitation and emission spectra for two other colored proteins, Aams-4 (SEQ ID NO:90)-H142S, and Rtms-1 (SEQ ID NO.1162)-N142S are shown in FIG.", "43.EXAMPLE 19 Expression in Yeast, Mammals and as a Fusion Protein The subject inventors sought to demonstrate that the instant CFMs can be expressed in yeast and mammalian cells and can be used as fusion proteins for genetic marking of cells.", "(a) Expression in Yeast For expression in yeast cells a BamHI/Not1 DNA cassette encoding dsRed or YGFP3 (an enhanced variant for expression in yeast) or a BglII/Not1 cassette encoding the novel fluorescent protein, Rtms-5v (SEQ ID NO:216), were retrieved using the pair of oligonucleotide primers RFPUP1 (SEQ ID NO:234), /RFPDO1 (SEQ ID NO:235), YGFP3UP (SEQ ID NO:232), /YGFP3DO (SEQ ID NO:233), or MSVIATUP (SEQ ID NO:236)/COFPDO (SEQ ID NO:237), respectively, using as templates the vectors pYGFP3 (Cormack et al., Microbiology 143: 303-11, 1977), pDsRed-1 [Clontech Industries] or cDNA for pCRII-TOPO::RTms-5v.", "In the case of YGFP3UP, the Not1 site was retrieved after digesting the PCR product from pGEM-T (Promega).", "The PCR product was cloned into the BamHI/NotI site of the multi-copy yeast expression vector pAS1NB to produce pAS1NB::dsRedL, pAS1NB::YEGFP3L or pAS1NB::Rtms-5v from which the DNA cassette encoding wild-type GFP had been removed but retaining the multiple cloning sites of that vector and linker sequence of that vector [Prescott et al., FEBS Letts.", "411: 97-101, 1997].", "pASN1B is a derivative of pAS1N (Prescott et al., 1997, supra) in which a BamH1 restriction site has been removed from the PGK promoter region.", "This series of vectors allows the expression of fluorescent proteins not fused to a partner protein and provides.", "YGFP3UP 5′-GGATCCATCGCCACCATGTCTAAAGGTGAAGAATTATTCACTGG SEQ ID NO:232 YGFP3DO 5′-CAGCTGTTATTTGTACAATTCATCCATACCATGG SEQ ID NO:233 RFPUP1 5′-CGGGATCCATCGCCACCATGAGGTCTTCCAAGAATGTTATC SEQ ID NO:234 RFPDO1 5′-GAGGATCCGCGGCCGCTAAAGGAACAGATGG SEQ ID NO:235 MSVIATUP 5′-GAAGATCTAAAACAATGAGTGTGATCGCTACACAAATG SEQ ID NO:236 COFPDO 5′-TATCAAATCGCCGGCGTCAGGCGACCACAGGTTTG SEQ ID NO:237 (b) Expression as a Fusion Protein Two DNA cassettes encompassing segments of the yeast genes ATP4 and ATP7 for subunit b and d of ATP synthase, respectively, were recovered by PCR from YRD15 genomic DNA using the oligonucleotide primer pairs ATP4PROMUP2 (SEQ ID NO:238)/ATP4DO2 (SEQ ID NO:239), or ATP7TUP (SEQ ID NO:240)/ATP3TDO (SEQ ID NO:241), respectively.", "The first, ATP4PO, encompasses the open reading frame for ATP4 and 500 bp of sequence upstream of the initiation codon flanked by BglII and BamHI restriction sites at the 5′ and 3′, respectively.", "The BamHI restriction site allows for an in frame-fusion between the C-terminus of subunit b and each of the three fluorescent protein cassettes.", "The second, ATP7T, encompasses the transcription terminator cassette representing the terminator region of the ATP7 gene flanked at the 5′ and 3′ ends by restriction sites for NotI and SacII, respectively.", "These restriction sites were obtained on cloning the PCR product into GEM-T.", "The ATP4PO & ATP7T DNA cassettes were cloned sequentially into the BamHI and NotI/SacII sites, respectively of the yeast expression vector pRS413 to produce the expression vector construction denoted pRS413::ATP4PO:ATP7T.", "A BglIIHI/NotI DNA fragment encoding YGFP3L was excised from pAS1NB::YEGFP3L and then cloned into the BglII/NotI site of pRS413::ATP4PO:ATP7T to produce a vector (pRS306::ATP4PO:YEGFP3L:ATP7T) encoding subunit b fused to YEGFP3 with a polypeptide linker of 25 amino acids.", "A vector (pRS413::AT?4PO:RTms-5 :ATP7T or pRS413 :A:ATP4PO:dsRed:ATP7T) encoding subunit b fused to RTms-5B or dsRed with a polypeptide linker of 27 amino acids was derived from pRS306::ATP4PO:YGFP3L:ATP7T by replacing the BamHI/NotI fragment encoding YEGFP3 with an equivalent fragment encoding Rtms-5v or dsRed.", "ATP4PROMUP2 5′-AGATCTGTGTTGTGACGCAACTGCAACTCC SEQ ID NO:238 ATP4DO2 5′-GTGATCAGCGGATCCCTTCAATTTAGAAAGCAATTGTTC SEQ ID NO:239 ATP7TUP 5′-CCTCTATATATTACGCACCATATTC SEQ ID NO:240 ATP7TDO 5′-ATACGTGACGACATTGGTAGTC SEQ ID NO:241 (c) Results were Visualised using Clear Native Gels.", "These were run essentially as described hereinafter.", "Briefly, 200 μg of mitochondrial protein was pelleted for 5 min at 100,000 g. Yeast mitochondrial were isolated from spheropblasts (Law et al., Methods in Enzymol.", "260: 122-163, 1995).", "The pellet was solubilized in buffer (40 μl) containing in dodceyl β-maltoside to isolate the monomer form or digitonin (20 g/g protein) to isolate the diner form and incubated on ice for 20 min and centrifuged 100,000 g for 30 min.", "Supernatants (30 μl) were loaded into wells of 4-16% gradient gels (13 cm×10 cm×0.075 cm).", "After running and while still between the glass plates, gels were imaged for fluorescence using a Perkin-Elmer multi-wavelength imager in ‘edge-illumination mode’ using appropriate filters for excitation (GFP, 480±20 nm; dsRed and Rtms-5v, 540±25 nm) and emission (GFP, 535±20 nm; dsRed, 590±35 nm; Rtms-5v, 620±30 nm).", "DNA cassettes encoding subunit b fused to the N-terminus of each of the three fluorescent proteins were expressed in a yeast strain lacking expression of endogenous subunit b.", "The ATP synthase in each of these strains was established to be assembled and functional as cells of each strain were able to grow using the non-fermentable substrate ethanol as carbon source.", "Yeast cells lacking endogenous subunit b do not assemble functional mtATPase and cannot grow using ethanol as the sole carbon source.", "Yeast cells of each strain expressing the individual fusion proteins were visualized using fluorescence microscopy.", "For cells of each strain the distribution of fluorescence in the cell was similar and consistent with localisation to the mitochondrion.", "Mitochondria were isolated from cells of each of the strains and, after extraction, ATP synthase complexes were subjected to analysis by clear native gel electrophoresis (CNGE).", "ATP synthase isolated from yeast is a large membrane bound complex (˜800 kDa for the monomeric form) made up of 20 different types of subunits some of which are present in the complex as more than one copy.", "The complex can be isolated as a monomer or a dimer depending on the detergent, dodceyl β-maltoside or digitonin, respectively, used to extract the complex from mitochondrial membranes.", "Subunit b is present in a single copy in the monomer.", "ATP synthase in this experiment was extracted from each preparation of mitochondria under conditions that favour the isolation of the monomer.", "Subunit b is present in a single copy in the monomer.", "Samples were subjected to analysis by CNGE and the gel imaged for fluorescence using conditions of illumination and light detection specific for each fluorescent protein (FIG.", "47).", "A single fluorescent band corresponding to the position of assembled monomeric ATP synthase was observed for complexes containing the b-GFP fusion protein (FIG.", "47, lane 1).", "The position of GFP not fused to another protein is shown (FIG.", "47, lane 4).", "A single fluorescent band was seen for complexes containing the fusion protein b-Rtms-5v (FIG.", "47, lane 2).", "However, multiple bands were observed for samples containing b-dsRed FIG.", "47, lane 3).", "It is possible that, in order of decreasing mobility, each fluorescent band corresponds to a monomer, dimer, trimer and tetramer.", "(d) For Expression in Mammals For expression in mammalian cells, a SmaI/NotI fragment encoding Rtms-5v (SEQ ID NO:216) was excised from pAS1NB::RTms-5v and cloned into the expression site of the mammalian expression vector pCI-Neo (Promega Corporation, Madison USA).", "This vector allows the expression of Rtms-5v not fused to a partner protein.", "A major benefit of fluorescent protein technology is the ability to simultaneously monitor using spectrally distinct variants more than one event in the living cell.", "The spectral properties of Rtms-5v suggest that should be feasible to image both dsRed and Rtms-5v expressed in the same cell.", "This would allow Rtms-5 to be used in combination with dsRed rather than substitute for dsRed.", "The emission maxima for dsRed and Rtms-5v are separated by 50 nm.", "We tested the possibility of imaging dsRed, RTms-5v and EGFP expressed in the same cell.", "Three individual DNA cassettes were constructed encoding dsRed fused at its N-terminus to the 16 amino acid mitochondrial targeting sequence of human 3-oxoacyl-CoA thiolase, EGFP fused to the C-terminus of Rab6 and Rtms-5v not fused to any other protein.", "Cells were imaged using a Zeiss 510 Meta confocal laser scanning microscopy (Zeiss).", "The distribution of fluorescence arising from each of the Rtms-5v, dsRed and EGFP fusions was consistent with the locations expected (cytosol/nucleus, mitochondrion and golgi, respectively).", "These results show that Rtms-5v is able to fluorescently label other compartments of the cell such as the mitochondrion in addition to the cytoplasm.", "The position of a non-transfected and, therefore, non-fluorescent cell is shown in the transmitted light image by the white arrow Rtms-5v showed no evidence of aggregation.", "Similar results were observed for the expression of Rtms-5v not targeted in yeast cells.", "Multiple fluorescent proteins are commonly (eg.", "GFP, dsRed, CFP) imaged in the same cell.", "EXAMPLE 20 Additional Color Proteins from Coral The inventors sought additional color proteins from two corals, Montipora efforescens and Pavona decussaca (a) Montipora efforescens Standard purification techniques (Dove et al., 2001, supra) were adopted for the purification of a red fluorescent protein from phosphate buffer extract of M. efforescens.", "A protein was purified using gel filtration and subject to N-terminal amino acid sequencing.", "A polymorphism was identified, comprising F and R residues.", "The N-terminal amino acid sequences are represented as follows: SPPDY TLEP KKXVA SEQ ID NO:242 SPPDY TLEP KKGVA SEQ ID NO:243 The polymorphism is indicated in bold larger type.", "(b) Pavona decussaca Similar techniques as those described in (a), above, were used to identify and purify a green fluorescent protein from P. decussaca.", "Gel electrophoresis showed that the proteins ran as two bands and N-terminal amino acid sequencing identified polymorphic variants, shown in bold larger type, below: Top band: (D)SS(P)E SYLN GIAEE MKTDV MEGI SEQ ID NO:244 Lower band: SN GIAEE MKTDL MEGIV NG SEQ ID NO:245 SN GIAEE MKTDL MEGIV NG SEQ ID NO:246 The protein fraction was generating these N-terminal sequences had absorbed maximally at 440 nm with maximal excitation at 440 nm and emission at 488 nm.", "Oligonucleotide probes were designed in both forward and reverse directions for PCR amplification from a ZAP express cDNA library of Acropora millepora (Scleractinian coral).", "The oligonucleotide primers used were as follows: Forward MEGIVNG-A ATG GAA GGG ATA GTC GAT GG SEQ ID NO:247 MEGIVNG-T ATG GAA GGG ATT GTC GAT GG SEQ ID NO:248 MEGIVNG-C ATG GAA GGG ATC GTC GAT GG SEQ ID NO:249 Reverse REV-MEG-T CCT CGA CAA TCC CTT CCA T SEQ ID NO:250 REV-MEG-C CCT CGA CGA TCC CTT CCA T SEQ ID NO:251 DNA was amplified and separated using gel electrophoresis.", "Bands were purified and cloned into pCRII-TOPO and transfected into TOP 10 cells (Invitrogen).", "Plasmids were then purified and subjected to nucleotide sequencing.", "The complete sequence is shown in Table 23.In this experiment, therefore, a protein identified from P. decussaca was used to identify a clone from Acropora millepora.", "Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described.", "It is to be understood that the invention includes all such variations and modifications.", "The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.", "TABLE 2 Class SubClass Order GFP Initial studies Sequence information % identity(length)* Hydrozoa Hydroida yes Shimomura et al., J.", "Cell.", "Prasher et al.", "(1992) Gene 111: 23(238) Comp.", "Physiol.", "59: 223-239, 229-33; Chalfie et al.", "(1994) Science 1962; Morise et al., 263: 802-5; Inouye & Tsuji (1994) Biochemistry 13: 2656-2662, FEBS Lett.", "341: 277-80.1974; Morin & Hastings, J.", "Cell Physiol.", "77: 313-318, 1971 Milliporina yes Invention 95.0(220)-97.3(221) Stylasterine Trachylina Siphonophora Chondrophora Actinulida Anthozoa Octocorallia Gorgonacea (=Alcyonaria) Telestacea Pennatulacea yes Morin & Hastings, J.", "Cell WO 99/49019 43.5(225) & 43.5(225) (see pens & Physiol.", "77: 313-318, 1977 sea pansies) Alcyonacea Helioporacea Stolonifera yes Matz et al., Nature Biotechnology 52.7(226) 17: 969-973, 1999 Hexacorallia Actiniaria (sea yes Matz et al., Nature Biotechnology 46.2(227) & 47.2(223) (=Zoantharia) anemones) 17: 969-973; 1999; Lukyanov et 46.2(227) & 47.2(223) al., JBC 275: 25879-25882, 2000 Scleractinia yes Kawaguti, Paloa Trop.", "Boil.", "WO 00/46233; Dove et al., Coral 93.2(220)-100(255) (true or Stn.", "Stud.", "2: 617-673, 1994; Reefs 19: 197-204, 2001; Invention.", "stony corals) Dove et al.", "Biol.", "Bull.", "189: 288-297, 1995 Zoanthidea yes Matz et al., Nature Biotechnology 44.1(231) & 45.5(236) 17: 969-973, 1999 Corallimorpharia yes Matz et al., Nature Biotechnology 57.8(225) & 62.5(224) (coral-like 17: 969-973, 1999 anemones) Ceriantipatharia Antipatharia Ceriantharia Cubozoa Scyphozoa Stauromedusae no (jellyfish) Coronatae Semaeostomae Rhizostaomae Best fit in relation to Aams2-pep (SEQ ID NO: 88) over 220-238 amino acids as indicated in length TABLE 3 Fluorescent properties Additional Excitation region Exciter filter Dichroic mirror barrier filter Ultra-violet UG-1 DM400 + L420 L435 Violet BP 405 DM455 + Y455 Y475 Blue BP 490 DM500 + O515 O515 Green BP 545 DM580 + O590 R610 TABLE 4 Class: Anthozoa; Order: Scleractinia Color Family Genus, Species morph Fluorescent properties Pocilloporidae Pocillopora Pink Faintgreen fluorescence under blue light damicormis Pocillopora Green Fluoresce blue-green, green, green and red damicormis under UV, violet, blue and green light, respectively Acroporidae Acropora aspera Blue tipped Tentacles and calices fluoresce violet, blue, green, and faint red under UV, violet, blue and green light, respectively Acropora aspera Blue light Fluoresces green under UV and violet light fluorescent Acropora nobilis Green Calices and tentacles fluorescent violet, blue, green and red under UV, violet, blue and green light, respectively Montipora sp.", "Red/red Yellow fluorescence under blue light, red (plating) fluorescent fluorescence under green light Poritidae Porites Purple Calices fluoresce faint green under violet murrayensis and blue light Agariciidae Pavona Green Blue under UV light; green under violet decussaca light; and blue and moderate red under green light Mussidae Acanthasthastrea Green Calices and polyps fluorescent violet, blue, green and faint under UV, violet, blue and green light, respectively Faviidae Platygyra sp.", "Green Blue under UV and violet light; green under blue light Caulastrea sp.", "Green Blue under UV light; green under violet and blue light TABLE 5 Class: Hydrozoa; Order: Milleporina Genus Color Fluorescent properties Millepora Green Blue under UV and violet light; green under blue light TABLE 6 Amino acids within N-terminus Genus species Name Type 5 A of fluorophore sgiat Acropora aspera Aams-5.pep 1 QVLSPQCQYGSIFWRNSYEHENMERLQCE sviat Acropora aspera Aams-2.pep 1 QVLSPOCQYGSIFWRNSYEHENMERLQCE sviat Acropora aspera Aams-4.pep 1 QVLSPQCQYGSIPWRNSYEHENMERLQCE sviat Acropora aspera Aams-6.pep 1 QVLSPQCQYGSIFWRNSYEHENMERLQCE sviat Acanthastrea sp.", "Acams-2.pep 6 QVLSPQYQYGSIYWRNSYENENMERLQCE sviat Acanthastrea sp.", "Acams-3.pep 6 QVLSPQYQYGSIYWRNSYBNENMERLQCE sviat Acanthastrea sp.", "Acams-4.pep 3 QVLSPQYQYGSIYWRNSHENENMERLQCE sviat Acanthastrea sp.", "Acams-5.pep * sviat Caulastrea sp.", "Cerns-F.pep 5 QVLSPQCQYGNIFWRNSYEHENMGRLQCE sviat Caulastrea sp.", "Cerns-G.pep 5 QVLSPQCQYGNIFWRNSYEHENMGRLQCE sviat Caulastrea sp.", "Cerns-H.pep 5 QVLSPQCQYGNIFWRNSYEHENMGRLQCE sviat Caulastrea sp.", "Cerns-L.pep 16* QVLSPQCQYGNIFWRNSYEHBNMERLQCE sviat Acropora nobilis LGAms-5.pep 6 QVLSPQYQYGSIYWRNSYENENMERLQCE sviat Acropora nobilis LGAms-6.pep 18* QVLSPQYQYGSIFWRNSYENENMERLQCE sviat Millepora sp.", "Mims.pep 1 QVLSPQCQYGSIFWRNSYEEENMERLQCE sviat Millepora sp.", "Mims-A.pep 1 QVLSPCGQYGSIFWRNSYEHENMERLQCE sviat Millepora sp.", "Mims-B.pep 1 QVLSPQCQYGSIFWRNSYEHENMERLQCE sviat Millepora sp.", "Mims-C.pep 1 QVLSPQCQYGSIFWRNSYEHENMERLQCE sviat Pavona decussata Pav5ms.pep 6 QVLSPQYQYGSIYWRNSYENENMERLQCE sviat Pavona decussata Pavms-2.pep 6* QVLSPQYQYGSIYWRNSYENENMERLQCE sviat Pavona decussata Pavms-3.pep 6 QVLSPQYQYGSIYWRNSYENENMERLQCE sviat Pavona decussata Pavms-4.pep 11 QVLSPQYQYGSIYWGNSYENENMERLQCE sviat Porites murrayensis PMms-A.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviat Porites murrayensis PMms-B.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviat Porites murrayensis PMms-C.pep 9 QVLSPQTQYGSIYWRNSYENGNMERLQGE sviat Porites murrayensis PMms-D.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviat Porites murrayensis PMms-E.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviat Pink Pocillopora PPd57-1ms.pep 12 QVLSPQTQYGSIYWRNSYENENMERLQCE sviat Pink Pocillopora PPd57-2ms.pep 1 QVLSPQCQYGSIFWRNSYEHENMBRLQCE sviat Pink Pocillopora PPd57-3.pep 1 QVLSPQCQYGSIFWRNSYEHENMBRLQCE sviat Pink Pocillopora PPd57-4ms.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviat Platygyra sp.", "PPms-1.pep 8 QVLSPQCQYGNIFWRNSYBHENMGRLQCE sviat Platygyra sp.", "PPms-2.pep 19* QVLSPQYQYGSIFWRNSYENENMERLQCE sviat Platygyra sp.", "PPms-E.pep 5 QVLSPQCQYGNIFWRNSYEHENMGRLQGE sviat Platygyra sp.", "PPms-G.pep 13 QVLSPQCQYGNIFWGNSYEHENMGRLQCE sviat Montipora sp.", "RTms-1.pep 6 QVLSPQYQYGSIYWRNSYENENMERLQCE sviat Montipora sp.", "RTms-5.pep 1 QVLSPQCQYGSIFWRNSYEHENMERLQCE svivt Montipora sp.", "RTms-6.pep 6 QVLSPQYQYGSIYWRNSYENENMERLQCE svsat Montipora sp.", "RTms-2.pep 6 QVLSPQYQYGSIYWRNSYENENMERLQCE TABLE 7 Amino acids within N-terminus Species Name Type 5 A of fluorophore sviak Acropora aspera Aasv-1.pep 15 QVLSPQSQYGSIYWRNSYENGNMERLQCE sviak Acropora aspera Aasv-3.pep 14 QVLSPQSQYGSIYWRNSYENENMERLQRE sviak Acropora aspera Aasv-P.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviak Acanthastria sp.", "Acasv-A.pep 4 QVLSPRCQYGNIFWRNSYEHENMGRLQCE sviak Acanthasiria sp.", "Acasv-C.pep 14 QVLSPQSQYGSIYWRNSYENENMERLQRE sviak Acanthastria sp.", "Acasv-D.pep 1 QVLSPQCQYGSIFWRNSYEHENMERLQCE sviak Caulastrea ap.", "Ce61-3sv.pep 14 QYLSPQSQYGSIYWRNSYENENMERLQRE sviak Caulcstrea ap.", "Ce61-4sv.pep 20 QVXSPQSQYGSXYWRNSYEHENMERLQCE sviak Caulaslrea ap.", "Ce61-5sv-rep.pep 18 QVLSPQCQYGSIFWRNSYEHENMESIQCE sviak Caulastrea ap.", "Ce61-7sv-rep.pep 1 QVLSPQCQYGSIFWRNSYEHENMERLQCE sviak Green Pocillopora GPd58-2sv.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviak Acropora nobilis LGAsv-A.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviak Acropora nobilis LGAsv-C.pep 1 QVLSPQCQYGSIFWRNSYEHENMERLQCE sviak Acropora nobilis LGAsv-D.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviak Acropora nobilis LGAsv-E.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviak Millepora sp.", "(Hydrozoan) Misv-A.pep 14 QVLSPQSQYGSIYWRNSYENENMERLQRE sviak Millepora sp.", "(Hydrozoan) Misv-B.pep 14 QVLSPQSQYGSIYWRNSYENENMERLQRE sviak Millepora sp.", "(Hydrozoan) Misv-F.pep 14 QVLSPQSQYGSIYWRNSYENENMERLQRE sviak Pavona decussaca Pavsv-A.pep 7 QVLSPQSQYGSVYWRNSYVNENMERLQCE sviak Pavona decussaca Pavsv-B.pep 1 QVLSPQCQYGSIFWRNSYEHENMERLQCE sviak Pavona decussaca Pavsv-C.pep 17 QVLSPQSQYGSVYWRNSYENENMERLQRE sviak Porites Murrayensis PM1Asv-rep.pep 15 QVLSPQSQYGSIYWRNSYENGNMERLQCE sviak Porites Murrayensis PM1Csv-rep.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviak Porites Murrayensis PMsv-4.pep 2* QVLSPQSQYGSIYWRNSYENENM* sviak Porites Murrayensis PMsv-5.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviak Platygyra sp.", "PPsv-1.pep 5 QVLSPQCQYGNIFWRNSYEHENMGRLQCE sviak Platygyra sp.", "PPsv-2.pep 5 QVLSPQCQYGNIFWRNSYEHENMGRLQCE sviak Platygyra sp.", "PPsv-3.pep 5 QVLSPQCQYGNIFWRNSYEHENMGRLQCE sviak Platygyra sp.", "PPsv-4.pcp 4 QVLSPRCQYGNIFWRNSYEHENMGRLQCE sviak Platygyra sp.", "PPsv-5.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviak Platygyra sp.", "PPsv-6.pep 10 QVLSPQSQYGSIYWRNSYENENMERLQCG sviak Montipora sp.", "RTsv-1 .pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviak Montipora sp.", "RTsv-2.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE sviak Montipora sp.", "RTsv-3.pep 2 QVLSPQSQYGSIYWRNSYENENMERLQCE TABLE 8 Percentage DNA sequence similarities generated using LALIGN A8 D1 D10 S1 S3 T1 T3 A8 97.3 98.7 97.7 99.6 97.5 99.9 D1 97.5 98.1 96.9 99.6 97.2 D10 98.2 98.2 97.6 98.5 S1 97.9 98.2 97.5 S3 97.0 99.4 T1 97.3 T3 TABLE 9 Percentage amino acid sequence similarities generated using LALIGN A8 D1 D10 S1 S3 T1 T3 A8 95.5 98.2 97.3 99.1 96.0 100.0 D1 94.6 96.4 94.6 98.7 95.5 D10 96.4 97.3 95.1 98.2 S1 97.3 96.9 97.3 S3 95.1 99.1 T1 96.0 T3 TABLE 15 Conserved amino acid differences between blue and purple colored proteins Amino acid Amino acid in blue Amino acid in purple in blue-purple Position proteins (n = 2) proteins (n = 4) protein (n = 1) 41 Arg (charged, polar) Lys (charged, polar)* Arg 43 Ala (nonpolar) Thr (uncharged, polar) Ala 61 Cys (uncharged, polar) Ser (uncharged, polar) Cys 87 Phe (nonpolar) Tyr (uncharged, polar) Phe 142 His (charged, polar) Asn (uncharged, polar) Asn 143 Ser (uncharged, polar) Thr (uncharged, polar) Thr 175 Thr (uncharged, polar) Ser (uncharged, polar) Ser 198 Ile (nonpolar) Thr (uncharged, polar) Thr *Amino acid position 41 of the purple protein encoded by D10 (SEQ ID NO: 192) is Arg.", "TABLE 16 Amount of colored protein (expressed as a percentage of total soluble protein) produced in cultures of E. coli and S. cerevisiae.", "Species Plasmid CP clone Colour RHSCC % CP E. coli pCGP2921 T1 blue 102A 50% S. cerevisiae pCGP3269 A8 purple 82B 8% S. cerevisiae pCGP3270 T1 blue 101C 6% RHSCC = Royal Horticultural Society Colour Chart (Kew, UK) TABLE 17 Summary of recombinant protein accumulation levels in plants after nuclear DNA transformation.", "Protein Plant Targeting Accumulation Reference Endoglucanase Tobacco Chloroplast (Tomato- Up to 1.35% TSP in Transgenic Res 9: Rubisco small leaves.", "43-54, 2000 subunit protein) PEPC Rice cytosol Up to 12% TSP in Nat Biotechnol.", "17: leaves.", "22-23, 1999 Cystatin Rice cytosol Up to 2% TSP.", "Plant Mol Biol 30: 149-157, 1996 Antibody Arabid.", "ER-retained Up to 6% TSP Eur J Biochem (DIKDEL), ER- secreted Spider Silk Tobacco ER-retained 2% + TSP in leaves and Nat Biotechnol 19: Potato potato tubers 573-577.2000 Cry9Aa Tobacco cytosol 0.3% TSP in Tobacco Plant Sci 160: 341-353, Potato leaves.", "Expression in 2001 Cauliflower other plants 0.1-0.03%.", "Turnip Xylanase Tobacco ER-excreted in 3% TSP (alk.", "phos.)", "Plant Physiol 124: GFP guttation fluid 927-934, 2000 Alkaline phosphatase GUS- ?", "cytosol Up to 3% TSP FEBS Lett 488: 13-17, Peptide vaccine 2001 Bt, NPTII Tobacco cytosol Bt: 0.02% TSP Nature 328: 33-37, NPTII: 0-07-0.27% TSP 1987 AIMV-CP Tobacco cytosol 0.1-0.4% TSP Tobacco EMBO 6: 1181-1 Tomato 0.1-0.8% TSP Tomato 1188, 1987 sGFP Rice Chloroplast (rbsS-Tp) 10% of TSP.", "Much Plant & Animal higher than cytoplasmic Genome VII control (0.5% TSP) Conference 1999 abstract TSP = total soluble protein TABLE 18 Summary of recombinant protein accumulation levels in plants after Plastid DNA transformation.", "Protein Plant Targeting Accumulation Reference GFP tobacco Chloroplast expression 5% TSP in leaves Plant Journal 27: 257-265 Bt (cry2Aa2) tobacco Chloroplast expression 2-3% TSP in leaves Proc Natl Acad Sci 1840-1845, 1999 Bt (cry2Aa2) ?", "Chloroplast expression 45.3% TSP Nat Biotechnol 19: 71-74, 2001 Somatotropin Tobacco Chloroplast expression 7% + “more than 300- Nat Biotechnol 18: fold higher than a similar 333-338, 2000 gene expressed using a nuclear transgenic approach” Bt (cry1Ac) Tobacco Chloroplast expression 3-5% TSP in leaves Biotechnology 13: 362-365, 1995 EPSPS Tobacco Chloroplast expression 10% + TSP in leaves Plant J 25: 261-270, 2001 TSP = total soluble protein TABLE 20 Summary of Northern analysis of Arabidopsis transgenic plants Selectable Construct number CF Cassette marker T1 SuRB pCGP2772 35S:T1:35S 35S:SuRB ˜0.9 kb ˜2.2 kb pCGP2765 35S:A8:35S 35S:SuRB ˜0.9 kb ˜2.2 kb pCGP3259 35S:ER:T1:35S 35S:SuRB ˜1.0 kb ˜2.2 kb pCGP2785 35S:SSU:T1:35S 35S:SuRB ˜1.1 kb ˜2.2 kb pCGP3258 35S:T1:GFP:35S 35S:SuRB ˜1.6 kb ˜2.2 kb pCGP3261 35S:ER:T1:GFP:35S 35S:SuRB ˜1.7 kb ˜2.2 kb pCGP960 355:GUS 35S:SuRB none ˜2.2 kb pBINmgfp4 35S:mGFP4:nos 35S:nptII none none non transgenic NA NA none none CP cassette = Colored protein cassette contained in construct; SM cassette = the selectable marker gene contained in construct; NA = not applicable; none = no transcripts detected TABLE 21 Estimations of T1 protein in leaf samples from 2 transgenic Arabidopsis events (expressed as a percentage of total protein) Construct Cassette Acc # Sample % T1 pCGP3259 35S:ERT:T1:35S 1.5 leaf 0.005% pCGP2772 35S:T1:35S 1.2b leaf 0.005% Construct = Binary vector used in transformation; Cassette refers to the chimaerie T1 transgene contained in the T-DNA; Acc# refers to the accession number of the transgenic plant.", "TABLE 22 Estimations of T1 protein in petal and/or leaf samples from 2 transgenic P. hybrida events (expressed as a percentage of total protein) Construct Cassette Acc # Sample % T1 pCGP3259 35S:ERT:T1:35S 24444 leaf 0.009% pCGP2765 35S:A8:35S 24534 petal 0.02% pCGP2765 35S:A8:35S 24534 leaf 0.006%" ] ]	Patent_10469661
[ [ "Self-foaming or foamy preparations comprising particulate hydrophobic and/or hydrophobized and/or oil-absorbent solid substances", "Self-foaming and/or foam-like cosmetic or dermatological preparations which comprise I. an emulsifier system which consists of A. at least one emulsifier A chosen from the group of wholly neutralized, partially neutralized or unneutralized branched and/or unbranched, saturated and/or unsaturated fatty acids having a chain length of from 10 to 40 carbon atoms, B. at least one emulsifier B chosen from the group of polyethoxylated fatty acid esters having a chain length of from 10 to 40 carbon atoms and a degree of ethoxylation of from 5 to 100 and C. at least one coemulsifier C chosen from the group of saturated and/or unsaturated, branched and/or unbranched fatty alcohols having a chain length of from 10 to 40 carbon atoms, II.", "up to 30% by weight—based on the total weight of the preparation—of a lipid phase, III.", "1 to 90% by volume, based on the total volume of the preparation, of at least one gas chosen from the group consisting of air, oxygen, nitrogen, helium, argon, nitrous oxide (N2O) and carbon dioxide (CO2) IV.", "0.01-10% by weight of one or more particulate hydrophobic and/or hydro-phobicized and/or oil-absorbing solid-body substances." ], [ "1-12.", "(canceled) 13.A cosmetic or dermatological composition which is at least one of self-foaming and foam-like, wherein the composition comprises: I. an emulsifier system comprising A. an emulsifier A comprising one or more C10-40 fatty acids and salts thereof, B. an emulsifier B comprising one or more polyethoxylated C10-40 fatty acid esters having a degree of ethoxylation of from 5 to 100, and C. a coemulsifier C comprising one or more C10-40 fatty alcohols, II.", "up to 30% by weight of a lipid phase, III.", "from 1% to 90% by volume of a gas comprising at least one of air, oxygen, nitrogen, helium, argon, nitrous oxide (N2O) and carbon dioxide, IV.", "from 0.01% to 10% by weight of one or more particulate solid substances which are at least one of hydrophobic, hydrophobicized and oil-absorbing.", "14.The composition of claim 13, wherein emulsifier A, emulsifier B and coemulsifier C are present in weight ratios of a:b:c, where a, b and c independently are rational numbers of from 1 to 5.15.The composition of claim 14, wherein a, b and c independently are rational numbers of from 1 to 3.16.The composition of claim 14, wherein a:b:c is 1:1:1.17.The composition of claim 13, wherein emulsifier A, emulsifier B and coemulsifier C are present in a total concentration of from 2% to 20% by weight.", "18.The composition of claim 15, wherein emulsifier A, emulsifier B and coemulsifier C are present in a total concentration of from 5% to 15% by weight.", "19.The composition of claim 14, wherein emulsifier A comprises at least one of stearic acid, isostearic acid, palmitic acid, myristic acid and salts thereof.", "20.The composition of claim 14, wherein emulsifier B comprises at least one of PEG-9 stearate, PEG-8 distearate, PEG-20 stearate, PEG-8 stearate, PEG-8 oleate, PEG-25 glyceryl trioleate, PEG-40 sorbitan lanolate, PEG-15 glyceryl ricinoleate, PEG-20 glyceryl stearate, PEG-20 glyceryl isostearate, PEG-20 glyceryl oleate, PEG-20 stearate, PEG-20 methylglucose sesquistearate, PEG-30 glyceryl isostearate, PEG-20 glyceryl laurate, PEG-30 stearate, PEG-30 glyceryl stearate, PEG-40 stearate, PEG-30 glyceryl laurate, PEG-50 stearate, PEG-100 stearate, and PEG-150 laurate.", "21.The composition of claim 19, wherein emulsifier B comprises at least one polyethoxylated stearic ester.", "22.The composition of claim 14, wherein coemulsifier C comprises at least one of butyloctanol, butyldecanol, hexyloctanol, hexyldecanol, octyldodecanol, behenyl alcohol, cetearyl alcohol, and lanolin alcohols.", "23.The composition of claim 19, wherein coemulsifier C comprises at least one of cetyl alcohol and cetearyl alcohol.", "24.The composition of claim 13, wherein the composition further comprises one or more hydrophilic emulsifiers.", "25.The composition of claim 24, wherein the one or more hydrophilic emulsifiers comprise at least one of a mono-, di-, and tri-fatty acid ester of sorbitol.", "26.The composition of claim 24, wherein the one or more hydrophilic emulsifiers are present in a total concentration of less than 5% by weight.", "27.The composition of claim 13, wherein the composition is free of mono- and diglyceryl fatty acid esters.", "28.The composition of claim 13, wherein the lipid phase II comprises nonpolar lipids having a polarity of at least 30 mN/m.", "29.The composition of claim 14, wherein the composition comprises at least 2.5% by weight of the lipid phase II.", "30.The composition of claim 17, wherein the composition comprises from 5% to 15% by weight of the lipid phase II.", "31.The composition of claim 13, wherein the composition comprises from 10% to 80% by volume of the gas III.", "32.The composition of claim 31, wherein the gas III comprises carbon dioxide.", "33.The composition of claim 13, wherein the one or more particulate solid substances IV are selected from modified and unmodified phyllosilicates, modified carbohydrate derivatives, inorganic fillers, inorganic pigments based on metal oxides, metal compounds which are insoluble or sparingly soluble in water, inorganic pigments based on silicon oxides, silicate derivatives, and microspherical particles based on crosslinked polymethyl methacrylates.", "34.The composition of claim 33, wherein the one or more particulate solid substances IV comprise at least one of cellulose and derivatives thereof, microcrystalline cellulose, starch and derivatives thereof, talc, kaolin, a zeolite, boron nitride, an oxide of titanium, zinc, iron, manganese, aluminium or cerium, a sodium silicoaluminate, a magnesium silicate, a sodium magnesium silicate, a magnesium aluminum silicate, a fluoromagnesium silicate, a calcium aluminum borosilicate, and a silica dimethyl silylate.", "35.The composition of claim 34, wherein the one or more particulate solid substances IV comprise at least one of wheat starch, corn starch, rice starch, manioc starch, hydroxypropyl starch phosphate, distarch phosphate, sodium corn starch octenyl succinate, and aluminum starch octenyl succinate.", "36.The composition of claim 13, wherein the one or more particulate solid substances IV comprise at least one of talc, silica, titanium dioxide, kaolin, alumina, aluminum starch ocentyl succinate, a zeolite, distarch phosphate, boron nitride and a silica dimethyl silylate.", "37.The composition of claim 13, wherein the composition further comprises one or more moisturizers.", "38.The composition of claim 13, wherein the composition further comprises at least one antioxidant.", "39.The composition of claim 38, wherein the at least one antioxidant comprises at least one of vitamin E, vitamin A and derivatives thereof.", "40.A skin care product which comprises the composition of claim 13.41.A cosmetic or dermatological composition which is at least one of self-foaming and foam-like, wherein the composition comprises: I. from 8% to 13% by weight of an emulsifier system comprising A. an emulsifier A comprising one or more C10-40 fatty acids and salts thereof, wherein the C10-40 fatty acids comprises at least one of stearic acid, isostearic acid, palmitic acid and myristic acid, B. an emulsifier B comprising one or more polyethoxylated C10-40 fatty acid esters having a degree of ethoxylation of from 5 to 100, wherein the polyethoxylated C10-40 fatty acid esters comprise at least one of PEG-9 stearate, PEG-8 distearate, PEG-20 stearate, PEG-8 stearate, PEG-8 oleate, PEG-25 glyceryl trioleate, PEG-40 sorbitan lanolate, PEG-15 glyceryl ricinoleate, PEG-20 glyceryl stearate, PEG-20 glyceryl isostearate, PEG-20 glyceryl oleate, PEG-20 stearate, PEG-20 methylglucose sesquistearate, PEG-30 glyceryl isostearate, PEG-20 glyceryl laurate, PEG-30 stearate, PEG-30 glyceryl stearate, PEG-40 stearate, PEG-30 glyceryl laurate, PEG-50 stearate, PEG-100 stearate, and PEG-150 laurate, C. a coemulsifier C comprising one or more C10-40 fatty alcohols which comprise at least one of butyloctanol, butyldecanol, hexyloctanol, hexyldecanol, octyldodecanol, behenyl alcohol, cetyl alcohol, cetearyl alcohol, and lanolin alcohols, wherein emulsifier A, emulsifier B and coemulsifier C are present in weight ratios of a:b:c, where a, b and c independently are rational numbers of from 1 to 3, II.", "from 5% to 15% by weight of a lipid phase which comprises nonpolar lipids having a polarity of at least 30 mN/m, III.", "from 30% to 80% by volume of a gas which comprises carbon dioxide, IV.", "from 0.01% to 10% by weight of one or more particulate solid substances which are at least one of hydrophobic, hydrophobicized and oil-absorbing and comprise at least one of modified and unmodified phyllosilicates, modified carbohydrate derivatives, inorganic fillers, inorganic pigments based on metal oxides, metal compounds which are insoluble or sparingly soluble in water, inorganic pigments based on silicon oxides, silicate derivatives, and microspherical particles based on crosslinked polymethyl methacrylates.", "42.A cosmetic or dermatological base composition for gaseous active ingredients which comprises: I. an emulsifier system comprising A. an emulsifier A comprising one or more C10-40 fatty acids and salts thereof, B. an emulsifier B comprising one or more polyethoxylated C10-40 fatty acid esters having a degree of ethoxylation of from 5 to 100, and C. a coemulsifier C comprising one or more C10-40 fatty alcohols, II.", "up to 30% by weight of a lipid phase, III.", "from 0.01% to 10% by weight of one or more particulate solid substances which are at least one of hydrophobic, hydrophobicized and oil-absorbing.", "43.The composition of claim 42, wherein emulsifier A, emulsifier B and coemulsifier C are present in weight ratios of a:b:c, where a, b and c independently are rational numbers of from 1 to 5.44.The composition of claim 43, wherein emulsifier A, emulsifier B and coemulsifier C are present in a total concentration of from 2 to 20% by weight.", "45.The composition of claim 44, wherein emulsifier A comprises at least one of stearic acid, isostearic acid, palmitic acid, myristic acid and salts thereof.", "46.The composition of claim 42, wherein emulsifier B comprises at least one polyethoxylated stearic ester.", "47.The composition of claim 43, wherein coemulsifier C comprises at least one of butyloctanol, butyldecanol, hexyloctanol, hexyldecanol, octyldodecanol, behenyl alcohol, cetyl alcohol, cetearyl alcohol, and lanolin alcohols.", "48.The composition of claim 44, wherein the lipid phase II comprises nonpolar lipids having a polarity of at least 30 mN/m.", "49.The composition of claim 43, wherein the composition comprises at least 2.5% by weight of the lipid phase II.", "50.The composition of claim 42, wherein the composition comprises from 5% to 15% by weight of the lipid phase II.", "51.The composition of claim 50, wherein the of one or more particulate solid substances comprise at least one of talc, silica, titanium dioxide, kaolin, alumina, aluminum starch ocentyl succinate, a zeolite, distarch phosphate, boron nitride and a silica dimethyl silylate.", "52.The composition of claim 42, wherein the composition comprises: I. from 2% to 20% by weight of an emulsifier system comprising A. an emulsifier A comprising one or more C10-40 fatty acids and salts thereof, wherein the C10-40 fatty acids comprises at least one of stearic acid, isostearic acid, palmitic acid and myristic acid, B. an emulsifier B comprising one or more polyethoxylated C10-40 fatty acid esters having a degree of ethoxylation of from 5 to 100, wherein the polyethoxylated C10-40 fatty acid esters comprise at least one of PEG-9 stearate, PEG-8 distearate, PEG-20 stearate, PEG-8 stearate, PEG-8 oleate, PEG-25 glyceryl trioleate, PEG-40 sorbitan lanolate, PEG-15 glyceryl ricinoleate, PEG-20 glyceryl stearate, PEG-20 glyceryl isostearate, PEG-20 glyceryl oleate, PEG-20 stearate, PEG-20 methylglucose sesquistearate, PEG-30 glyceryl isostearate, PEG-20 glyceryl laurate, PEG-30 stearate, PEG-30 glyceryl stearate, PEG-40 stearate, PEG-30 glyceryl laurate, PEG-50 stearate, PEG-100 stearate, and PEG-150 laurate, C. a coemulsifier C comprising one or more C10-40 fatty alcohols which comprise at least one of butyloctanol, butyldecanol, hexyloctanol, hexyldecanol, octyldodecanol, behenyl alcohol, cetyl alcohol, cetearyl alcohol, and lanolin alcohols, II.", "from 5% to 15% by weight of a lipid phase, III.", "from 0.01% to 10% by weight of one or more particulate solid substances which are at least one of hydrophobic, hydrophobicized and oil-absorbing and comprise at least one of cellulose and derivatives thereof, microcrystalline cellulose, starch and derivatives thereof, talc, kaolin, a zeolite, boron nitride, an oxide of titanium, zinc, iron, manganese, aluminium or cerium, a sodium silicoaluminate, a magnesium silicate, a sodium magnesium silicate, a magnesium aluminum silicate, a fluoromagnesium silicate, a calcium aluminum borosilicate, and a silica dimethyl silylate." ], [ "The present invention relates to self-foaming and/or foam-like cosmetic and derma-tological preparations, in particular to skincare cosmetic and dermatological preparations.", "Foams or foam-like preparations are a type of disperse system.", "By far the most important and best known disperse systems are emulsions.", "Emulsions are two- or multi-phase systems of two or more liquids which are insoluble or only slightly soluble in one another.", "The liquids (pure or as solutions) are present in an emulsion in a more or less fine distribution, which generally has only limited stability.", "Foams are structures of gas-filled, spherical or polyhedral cells which are delimited by liquid, semiliquid, high-viscosity or solid cell ribs.", "The cell ribs, connected via points of intersection form a continuous framework.", "The foam lamellae stretch between the cell ribs (closed-cell foam).", "If the foam lamellae are disturbed or if they flow back into the cell rib at the end of foam formation, an open-cell foam is obtained.", "Foams are also thermo-dynamically unstable since a reduction in the surface area leads to the production of surface energy.", "The stability and thus the existence of a foam is thus dependent on to what extent it is possible to prevent its self-destruction.", "Cosmetic foams are usually dispersed systems of liquids and gases, where the liquid represents the dispersant and the gas represents the dispersed substance.", "Foams of low-viscosity liquids are temporarily stabilized by surface-active substances (surfactants, foam stabilizers).", "Because of their large internal surface area, such surfactant foams have a high adsorption capacity, which is utilized, for example, in cleaning and washing operations.", "Accordingly, cosmetic foams are used, in particular, in the fields of cleansing, for example as shaving foam, and of haircare.", "To generate foam, gas is bubbled into suitable liquids, or foam formation is achieved by vigorously beating, shaking, spraying or stirring the liquid in the gas atmosphere in question, provided that the liquids comprise suitable surfactants or other interface-active substances (“foam formers”), which, apart from interfacial activity, also have a certain film-forming ability.", "Cosmetic foams have the advantage over other cosmetic preparations of permitting a fine distribution of active ingredients on the skin.", "However, cosmetic foams can generally only be achieved using particular surfactants, which, moreover, are often not well tolerated by the skin.", "A further disadvantage of the prior art is that such foams have only low stability, for which reason they usually collapse within approximately 24 hours.", "A requirement of cosmetic preparations, however, is that they have stability for years, as far as possible.", "This problem is generally taken into account by the fact that the consumer produces the actual foam himself just before use using a suitable spray system for which purpose, for example, it is possible to use spray cans in which a liquefied pressurized gas serves as propellant gas.", "Upon opening the pressure valve, the propellant liquid mixture escapes through a fine nozzle, and the propellant evaporates, leaving behind a foam.", "After-foaming cosmetic preparations are also known per se.", "They are firstly applied to the skin from an aerosol container in flowable form and, after a short delay, develop the actual foam only once they are on the skin under the effect of the after-foaming agent present, for example a shaving foam.", "After-foaming preparations are often in specific formulation forms, such as, for example, after-foaming shaving gels or the like.", "However, the prior art does not include any sort of cosmetic or dermatological preparations which could be foamed as early as during the preparation and nevertheless have a sufficiently high stability in order to be packaged in the usual manner, stored and put onto the market.", "An object of the present invention was therefore to enrich the prior art and to provide cosmetic or dermatological self-foaming and/or foam-like preparations which do not have the disadvantages of the prior art.", "German laid-open specification DE 197 54 659 discloses that carbon dioxide is a suitable active ingredient for stabilizing or increasing the epidermal ceramide synthesis rate, which may serve to enhance the permeability barrier, reduce the transepidermal water loss and increase the relative skin moisture.", "To treat the skin, the CO2 is, for example, dissolved in water, which is then used to rinse the skin.", "However, the prior art hitherto does not include any sort of cosmetic or dermatological bases in which a gaseous active ingredient could be incorporated in an adequate, i.e.", "effective, concentration.", "It was thus a further object of the present invention to find cosmetic or dermatological bases into which effective amounts of gaseous active ingredients can be incorporated.", "It was surprising and could not have been foreseen by the person skilled in the art that self-foaming and/or foam-like cosmetic or dermatological preparations which comprise I. an emulsifier system which consists of A. at least one emulsifier A chosen from the group of wholly neutralized, partially neutralized or unneutralized branched and/or unbranched, saturated and/or unsaturated fatty acids having a chain length of from 10 to 40 carbon atoms, B. at least one emulsifier B chosen from the group of polyethoxylated fatty acid esters having a chain length of from 10 to 40 carbon atoms and a degree of ethoxylation of from 5 to 100 and C. at least one coemulsifier C chosen from the group of saturated and/or unsaturated, branched and/or unbranched fatty alcohols having a chain length of from 10 to 40 carbon atoms, II.", "up to 30% by weight—based on the total weight of the preparation—of a lipid phase, III.", "1 to 90% by volume, based on the total volume of the preparation, of at least one gas chosen from the group consisting of air, oxygen, nitrogen, helium, argon, nitrous oxide (N2O) and carbon dioxide (CO2) IV.", "0.01-10% by weight of one or more particulate hydrophobic and/or hydrophobicized and/or oil-absorbing solid-body substances, overcome the disadvantages of the prior art.", "According to the prior art to date, foam-like cosmetic emulsions which are characterized by a high introduction of air cannot be formulated or prepared industrially without propellent gas.", "This is true in particular for systems which are based on classic emulsifiers and develop a foam with an extraordinarily high stability as a result of shearing (stirring, homogenization).", "As a result of the powder raw materials and pigments (fillers) used according to the invention, the introduction of the gases is extraordinarily increased.", "This achieves foam boosting with up to 100% increased gas volume without a content of foaming agents customary according to the prior art, such as surfactants.", "As a result of this, it is possible for the first time to prepare formulations with an excellent, novel type of cosmetic performance and with an extraordinarily high gas volume (air and/or other gases, such as oxygen, carbon dioxide, nitrogen, helium, argon, nitrous oxide etc.", "), which are characterized by above-average good skin care and very good sensory properties.", "For the purposes of the present invention, “self-foaming” or “foam-like” are understood as meaning that the gas bubbles are present in (any) distributed form in one (or more) liquid phase(s) where the preparations do not necessarily have to have the appearance of a foam in macroscopic terms.", "Self-foaming and/or foam-like cosmetic or dermatological preparations according to the invention can, for example, be macroscopically visibly dispersed systems of gases dispersed in liquids.", "The foam character can, however, for example, be visible also only under a (light) microscope.", "Moreover, self-foaming and/or foam-like preparations according to the invention are, particularly when the gas bubbles are too small to be recognized under a light microscope, also recognizable from the sharp increase in volume of the system.", "The preparations according to the invention are entirely satisfactory preparations in every respect.", "It was particularly surprising that the foam-like preparations according to the invention are extraordinarily stable, even in cases of an unusually high gas volume.", "Accordingly, they are particularly suitable for use as bases for preparation forms having diverse use purposes.", "The preparations according to the invention have very good sensory properties, such as, for example, distributability on the skin or the ability to be absorbed into the skin, and are, moreover, characterized by above-average skincare.", "The invention further provides for the use of self-foaming and/or foam-like cosmetic or dermatological preparations which comprise I. an emulsifier system which consists of A. at least one emulsifier A chosen from the group of wholly neutralized, partially neutralized or unneutralized branched and/or unbranched, saturated and/or unsaturated fatty acids having a chain length of from 10 to 40 carbon atoms, B. at least one emulsifier B chosen from the group of polyethoxylated fatty acid esters having a chain length of from 10 to 40 carbon atoms and a degree of ethoxylation of from 5 to 100 and C. at least one coemulsifier C chosen from the group of saturated and/or unsaturated, branched and/or unbranched fatty alcohols having a chain length of from 10 to 40 carbon atoms, II.", "up to 30% by weight—based on the total weight of the IV.", "0.01-10% by weight of one or more particulate hydrophobic and/or hydro-phobicized and/or oil-absorbing solid-body substances, as cosmetic or dermatological bases for gaseous active ingredients.", "The emulsifier(s) A is/are preferably chosen from the group of fatty acids which have been wholly or partially neutralized with customary alkalis (such as, for example, sodium hydroxide and/or potassium hydroxide, sodium carbonate and/or potassium carbonate, and mono- and/or triethanolamine).", "Stearic acid and stearates, isostearic acid and isostearates, palmitic acid and palmitates, and myristic acid and myristates, for example, are particularly advantageous.", "The emulsifier(s) B is/are preferably chosen from the following group: PEG-9 stearate, PEG-8 distearate, PEG-20 stearate, PEG-8 stearate, PEG-8 oleate, PEG-25 glyceryl trioleate, PEG-40 sorbitan lanolate, PEG-15 glyceryl ricinoleate, PEG-20 glyceryl stearate, PEG-20 glyceryl isostearate, PEG-20 glyceryl oleate, PEG-20 stearate, PEG-20 methylglucose sesquistearate, PEG-30 glyceryl isostearate, PEG-20 glyceryl laurate, PEG-30 stearate, PEG-30 glyceryl stearate, PEG-40 stearate, PEG-30 glyceryl laurate, PEG-50 stearate, PEG-100 stearate, PEG-150 laurate.", "Particularly advantageous are, for example, polylethoxylated stearic esters.", "The coemulsifier(s) C is/are preferably chosen according to the invention from the following group: butyloctanol, butyldecanol, hexyloctanol, hexyldecanol, octyldodecanol, behenyl alcohol (C22H450H), cetearyl alcohol [a mixture of cetyl alcohol (C16H33OH) and stearyl alcohol (C18H37OH)], lanolin alcohols (wool wax alcohols, which are the unsaponifiable alcohol fraction of wool wax which is obtained following the saponification of wool wax).", "Particular preference is given to cetyl alcohol and cetylstearyl alcohol.", "It is advantageous according to the invention to choose the weight ratios of emulsifier A to emulsifier B to coemulsifier C (A:B:C) as a:b:c, where a, b and c, independently of one another, may be rational numbers from 1 to 5, preferably from 1 to 3.Particular preference is given to a weight ratio of approximately 1:1:1.It is advantageous for the purposes of the present invention to choose the total amount of emulsifiers A and B and of coemulsifier C from the range from 2 to 20% by weight, advantageously from 5 to 15% by weight, in particular from 8 to 13% by weight, in each case based on the total weight of the formulation.", "For the purposes of the present invention, it is particularly preferred if the gas phase of the preparations comprises carbon dioxide or consists entirely of carbon dioxide.", "It is particularly advantageous if carbon dioxide is a or the active ingredient in the preparations according to the invention.", "Compositions according to the invention develop, even during their preparation—for example during stirring or upon homogenization—into fine-bubble foams.", "According to the invention, fine-bubble, rich foams of excellent cosmetic elegance are obtainable.", "Furthermore, preparations which are particularly well tolerated by the skin are obtainable according to the invention, where valuable ingredients can be distributed on the skin in a particularly good manner.", "It may be advantageous, although it is not necessary, for the formulations according to the present invention to comprise further emulsifiers.", "Preference is given to using those emulsifiers which are suitable for the preparation of W/O emulsions, it being possible for these to be present either individually or else in any combinations with one another.", "The further emulsifier(s) is/are advantageously chosen from the group which comprises the following compounds: polyglyceryl-2 dipolyhydroxystearate, PEG-30 dipolyhydroxystearate, cetyldimethicone copolyol, glycol distearate, glycol dilaurate, diethylene glycoldilaurate, sorbitan trioleate, glycol oleate, glyceryl dilaurate, sorbitan tristearate, propylene glycol stearate, propylene glycol laurate, propylene glycol distearate, sucrose distearate, PEG-3 castor oil, pentaerythrityl monostearate, pentaerythrityl sesquioleate, glyceryl oleate, glyceryl stearate, glyceryl diisostearate, pentaerythrityl monooleate, sorbitan sesquioleate, isostearyl diglyceryl succinate, glyceryl caprate, palm glycerides, cholesterol, lanolin, glyceryl oleate (with 40% monoester), polyglyceryl-2 sesquiisostearate, polyglyceryl-2 sesquioleate, PEG-20 sorbitan beeswax, sorbitan oleate, sorbitan isostearate, trioleyl phosphate, glyceryl stearate and ceteareth-20 (Teginacid from.", "Th.", "Goldschmidt), sorbitan stearate, PEG-7 hydrogenated castor oil, PEG-5-soyasterol, PEG-6 sorbitan beeswax, glyceryl stearate SE, methylglucose sesquistearates, PEG-10 hydrogenated castor oil, sorbitan palmitate, PEG-22/dodecyl glycol copolymer, polyglyceryl-2 PEG-4 stearate, sorbitan laurate, PEG-4 laurate, polysorbate 61, polysorbate 81, polysorbate 65, polysorbate 80, triceteareth-4 phosphate, triceteareth-4 phosphate and sodium C14-17 alkyl sec sulfonate (Hostacerin CG from Hoechst), glyceryl stearate and PEG-100 stearates (Arlacel 165 from ICI), polysorbate 85, trilaureth-4 phosphate, PEG-35 castor oil, sucrose stearate, trioleth-8 phosphate, C12-15 pareth-12, PEG-40 hydrogenated castor oil, PEG-16 soyasterol, polysorbate 80, polysorbate 20, polyglyceryl-3 methylglucose distearate, PEG-40 castor oil, sodium cetearyl sulfate, lecithin, laureth-4 phosphate, propylene glycol stearate SE, PEG-25 hydrogenated castor oil, PEG-54 hydrogenated castor oil, glyceryl stearate SE, PEG-6 caprylic/capric glycerides, glyceryl oleate and propylene glycol, glyceryl lanolate, polysorbate 60, glyceryl myristate, glyceryl isostearate and polyglyceryl-3 oleate, glyceryl laurate, PEG-40 sorbitan peroleate, laureth-4, glycerol monostearate, isostearyl glyceryl ether, cetearyl alcohol and sodium cetearyl sulfate, PEG-22 dodecyl glycol copolymer, polyglyceryl-2 PEG-4 stearate, pentaerythrityl isostearate, polyglyceryl-3-diisostearate, sorbitan oleate and hydrogenated castor oil and Cera alba and stearic acid, sodium dihydroxycetyl phosphate and isopropyl hydroxycetyl ether, methylglucose sesquistearate, methylglucose dioleate, sorbitan oleate and PEG-2 hydrogenated castor oil and ozokerite and hydrogenated castor oil, PEG-2 hydrogenated castor oil, PEG-45/dodecyl glycol copolymer, methoxy PEG-22/dodecyl glycol copolymer, hydrogenated cocoglycerides, polyglyceryl-4 isostearate, PEG-40 sorbitan peroleate, PEG-40 sorbitan perisostearate, PEG-8 beeswax, laurylmethicone copolyol, polyglyceryl-2 laurate, stearamidopropyl PG dimonium chloride phosphate, PEG-7 hydrogenated castor oil, triethyl citrate, glyceryl stearate citrate, cetyl phosphate, polyglycerol methyl-glucose distearate, poloxamer 101, potassium cetyl phosphate, glyceryl isostearate, polyglyceryl-3 diisostearates.", "Preferably, for the purposes of the present invention, the further emulsifier(s) is/are chosen from the group of hydrophilic emulsifiers.", "According to the invention, particular preference is given to mono-, di- and tri-fatty acid esters of sorbitol.", "The total amount of further emulsifiers is, according to the invention, advantageously chosen to be less than 5% by weight, based on the total weight of the formulation.", "The list of given further emulsifiers which can be used for the purposes of the present invention is not of course intended to be limiting.", "Particularly advantageous self-foaming and/or foam-like preparations for the purposes of the present invention are free from mono- or diglyceryl fatty acid esters.", "Particular preference is given to preparations according to the invention which comprise no glyceryl stearate, glyceryl isostearate, glyceryl diisostearate, glyceryl oleate, glyceryl palmitate, glyceryl myristate, glyceryl lanolate and/or glyceryl laurate.", "The oil phase of the preparations according to the invention is advantageously chosen from the group of nonpolar lipids having a polarity ≧30 mN/m.", "Particularly advantageous nonpolar lipids for the purposes of the present invention are those listed below.", "Polarity Manufacturer Trade name INCI name mN/m Total SA Ecolane 130 Cycloparaffin 49.1 Neste PAO N.V. Nexbase 2006 FG Polydecene 46.7 (Supplier Hansen & Rosenthal) Chemische Fabrik Polysynlane Hydrogenated 44.7 Lehrte Polyisobutene Wacker Wacker Silicone Polydimethylsiloxane 46.5 oil AK 50 EC Erdölchemie Solvent ICH Isohexadecane 43.8 (Supplier Bayer AG) DEA Mineral oil (Supplier Pionier 2076 Mineral Oil 43.7 Hansen & Rosenthal) Tudapetrol DEA Mineral oil (Supplier Pionier 6301 Mineral Oil 43.7 Hansen & Rosenthal) Tudapetrol Wacker Wacker Silicone Polydimethylsiloxane 42.4 oil AK 35 EC Erdölchemie GmbH Isoeicosane Isoeicosane 41.9 Wacker Wacker Silicone Polydimethylsiloxane 40.9 oil AK 20 Condea Chemie Isofol 1212 Carbonate 40.3 Gattefossé Softcutol O Ethoxydiglycol Oleate 40.5 Creaderm Lipodermanol OL Decyl Olivate 40.3 Henkel Cetiol S Dioctylcyclohexane 39.0 DEA Mineral oil (Supplier Pionier 2071 Mineral Oil 38.3 Hansen & Rosenthal) Tudapetrol WITCO BV Hydrobrite 1000 PO Paraffinum Liquidum 37.6 Goldschmidt Tegosoft HP Isocetyl Palmitate 36.2 Condea Chemie Isofol Ester 1693 33.5 Condea Chemie Isofol Ester 1260 33.0 Dow Corning Dow Corning Fluid Cyclopentasiloxane 32.3 245 Unichema Prisorine 2036 Octyl Isostearate 31.6 Henkel Cognis Cetiol CC Dicaprylyl Carbonate 31.7 ALZO (ROVI) Dermol 99 Trimethylhexyl 31.1 Isononanoate ALZO (ROVI) Dermol 89 2-Ethylhexyl 31.0 Isononanoate Unichema Estol 1540 EHC Octyl Cocoate 30.0 Of the hydrocarbons, paraffin oil, and further hydrogenated polyolefins, such as hydrogenated polyisobutenes, squalane and squalene, in particular, are to be used advantageously for the purposes of the invention.", "The content of the lipid phase is advantageously chosen to be less than 30% by weight, preferably between 2.5 and 30% by weight, particularly preferably between 5 and 15% by weight, in each case based on the total weight of the preparation.", "It may also be advantageous, although it is not obligatory, for the lipid phase to comprise up to 40% by weight, based on the total weight of the lipid phase, of polar lipids (having a polarity of ≦20 mN/m) and/or medium-polarity lipids (having a polarity of from 20 to 30 mN/m).", "For the purposes of the present invention, particularly advantageous polar lipids are all native lipids, such as, for example, olive oil, sunflower oil, soybean oil, groundnut oil, rapeseed oil, almond oil, palm oil, coconut oil, castor oil, wheatgerm oil, grapeseed oil, thistle oil, evening primrose oil, macadamia nut oil, corn oil, avocado oil and the like and those listed below.", "Polarity Manufacturer Trade name INCI name mN/m Condea Chemie Isofol 14 T Butyl Decanol (+) Hexyl 19.8 Octanol (+) Hexyl Decanol (+) Butyl Octanol Lipochemicals Lipovol MOS-130 Tridecyl Stearate(+) 19.4 INC./USA Tridecyl (Induchem) Trimellitate(+) Dipentaerythrityl Hexacaprylate/Hexacaprate Castor oil 19.2 CONDEA Isofol Ester 19.1 Chemie 0604 Huels CONDEA Miglyol 840 Propylene Glycol 18.7 Chemie Dicaprylate/Dicaprate CONDEA Chemie Isofol 12 Butyl Octanol 17.4 Goldschmidt Tegosoft SH Stearyl Heptanoate 17.8 Avocado oil 14.5 Henkel Cognis Cetiol B Dibutyl Adipate 14.3 ALZO (ROVI) Dermol 488 PEG 2 Diethylene Hexanoate 10.1 Condea Augusta Cosmacol ELI C12-13 Alkyl Lactate 8.8 S.P.A. ALZO (ROVI) Dermol 489 Diethylene Glycol 8.6 Dioctanoate(/Diisononanoate Condea Augusta Cosmacol ETI Di-C12/13 Alkyl Tartrate 7.1 S.P.A. Henkel Cognis Emerest 2384 Propylene Glycol 6.2 Monoisostearate Henkel Cognis Myritol 331 Cocoglycerides 5.1 Unichema Prisorine 2041 Triisostearin 2.4 GTIS Particularly advantageous medium-polar lipids for the purposes of the present invention are those listed below Polarity (Water) Manufacturer Trade name INCI name mN/m Henkel Cognis Cetiol OE Dicaprylyl Ether 30.9 Dihexyl carbonate Dihexyl Carbonate 30.9 Albemarle S.A. Silkflo 366 NF Polydecene 30.1 Stearinerie DUB VCI 10 Isodecyl Neopentanoate 29.9 Dubois Fils ALZO (ROVI) Dermol IHD Isohexyl Decanoate 29.7 ALZO (ROVI) Dermol 108 Isodecyl Octanoate 29.6 Dihexyl Ether Dihexyl Ether 29.2 ALZO (ROVI) Dermol 109 Isodecyl 3,5,5 Trimethyl 29.1 Hexanoate Henkel Cognis Cetiol SN Cetearyl Isononanoate 28.6 Unichema Isopropyl Isopropyl Palmitate 28.8 palmitate Dow Corning DC Fluid 345 Cyclomethicone 28.5 Dow Corning Dow Corning Cyclopolydimethylsiloxane 28.5 Fluid 244 Nikko Chemicals Jojoba oil Gold 26.2 Superior Jojoba Oil Gold Wacker Wacker AK 100 Dimethicone 26.9 ALZO (ROVI) Dermol 98 2-Ethylhexanoic Acid 26.2 3,5,5 Trimethyl Ester Dow Corning Dow Corning Open 25.3 Fluid 246 Henkel Cognis Eutanol G Octyldodecanol 24.8 Condea Chemie Isofol 16 Hexyl Decanol 24.3 ALZO (ROVI) Dermol 139 Isotridecyl 3,5,5 24.5 Trimethylhexanonanoate Henkel Cognis Cetiol PGL Hexyldecanol (+) 24.3 Hexyl Decyl Laurate Cegesoft C24 Octyl Palmitate 23.1 Gattefossé M.O.D.", "Octyldodeceyl Myristate 22.1 Macadamia 22.1 Nut Oil Bayer AG, Dow Silicone oil Phenyl Trimethicone 22.7 Corning VP 1120 CONDEA Chemie Isocarb 12 Butyl Octanoic Acid 22.1 Henkel Cognis Isopropyl Isopropyl Stearate 21.9 stearate WITCO, Goldschmidt Finsolv TN C12-15 Alkyl Benzoate 21.8 Dr. Straetmans Dermofeel BGC Butylene Glycol 21.5 Caprylate/Caprate Unichema Hüls Miglyol 812 Caprylic/Capric 21.3 Triglyceride Trivent (via Trivent OCG Tricaprylin 20.2 S. Black) ALZO (ROVI) Dermol 866 PEG ,, 20.1 Diethylhexanoate/ Diisononanoate/Ethylhexyl Isononanoate The inorganic particulate hydrophobic and/or hydrophobicized and/or oil-absorbing solid-body substances can, for example, advantageously be chosen from the group of modified or unmodified phyllosilicates.", "modified carbohydrate derivatives, such as cellulose and cellulose derivatives, microcrystalline cellulose, starch and starch derivatives (distarch phosphate, sodium and aluminum starch octenyl succinate, wheat starch, corn starch (Amidon De Mais MST (Wackherr), Argo Brand corn starch (Corn Products), Pure-Dent (Grain Processing), Purity 21C (National Starch), rice starch (D.S.A.", "7 (Agrana Starch), Oryzapearl (Ichimaru Pharcos), hydroxypropylstarch phosphate distarch phosphate (Corn PO4 (Agrana Starch) Corn PO4 (Tri-K)) sodium corn starch octenyl succinate (C* EmCap—Instant 12639 (Cerestar USA)) Aluminum starch octenyl succinate (Covafluid AMD (Wackherr) Dry Flo-PC (National Starch) Dry Flo Pure-(National Starch) Fluidamid DF 12 (Roquette)) inorganic fillers (such as talc, kaolin, zeolites, boron nitride) inorganic pigments based on metal oxides and/or other metal compounds which are insoluble or sparingly soluble in water (in particular oxides of titanium, zinc, iron, manganese, aluminum, cerium) inorganic pigments based on silicon oxides (such as, in particular, the Aerosil-200, Aerosil 200 V grades).", "silicate derivatives (such as sodium silicoaluminates, magnesium silicates, sodium magnesium silicates (Laponite grades), magnesium aluminum silicates (Sebumasse) or Fluoro Magnesium Silicate (Submica grades), Calcium Aluminum Borosilicate).", "Preference is given here in particular to Silica Dimethyl Silylate (Aerosil R972).", "Microcrystalline cellulose is an advantageous solid-body substances for the purposes of the present invention.", "It is available, for example, from “FMC Corporation Food and Pharmaceutical Products” under the trade name Avicel®.", "A particularly advantageus product for the purposes of the present invention is the grade Avicel® RC-591, which is modified microcrystalline cellulose which is composed of 89% of microcrystalline cellulose and of 11% of sodium carboxymethylcellulose.", "Further commercial products of this class of raw material are Avicel® RC/CL, Avicel® CE-15, Avicel® 500.Further oil-absorbing solid-body substances which are advantageous according to the invention are microspherical particles based on crosslinked polymethyl methacrylates (INCI: Crosslinked Methylmethacrylate).", "These are sold by SEPPIC under the trade names Micropearl® M305, Micropearl® 201, Micropearl® M 310 and Micropearl® MHB and are characterized by an oil-uptake capacity of 40-100 g/100 g. Aerosils (fumed silica)=silicon dioxide obtained by thermal decomposition of ethyl silicate) are highly disperse silicas with an often irregular shape whose specific surface area is usually very large (200-400 m2/g) and can be controlled depending on the preparation process.", "Aerosils for use advantageously according to the invention are obtainable, for example, under the trade names: Aerosil® 130 (Degussa Huls) Aerosil®) 200 (Degussa Huls) Aerosil 255 (Degussa Huls) Aerosil® 300 (Degussa Huls) Aerosil® 380 (Degussa Huls) B-6C (Suzuki Yushi) CAB-O-SIL Fumed Silica (Cabot) CAB-O-SIL EH-5 (Cabot) CAB-O-SIL HS-5 (Cabot) CAB-O-SIL LM-130 (Cabot) CAB-O-SIL MS-55 (Cabot) CAB-O-SIL M-5 (Cabot) E-6C (Suzuki Yushi) Fossil Flour MBK (MBK) MSS-500 (Kobo) Neosil CT 11 (Crosfield Co.) Ronasphere (Rona/EM Industries) Silica, Anhydrous 31 (Whittaker, Clark & Daniels) Silica, Crystalline 216 (Whittaker, Clark & Daniels) Silotrat-1 (Vevy) Sorbosil AC33 (Crosfield Co.) Sorbosil AC 35 (Crosfield Co.) Sorbosil AC 37 (Crosfield Co.) Sorbosil AC 39 (Crosfield Co.) Sorbosil AC77 (Crosfield Co.) Sorbosil TC 15 (Crosfield Co.) Spherica (Ikeda) Spheriglass (Potters-Ballotini) Spheron L-1500 (Presperse) Spheron N-2000 (Presperse) Spheron P-1500 (Presperse) Wacker HDK H 30 (Wacker-Chemie) Wacker HDK N 20 (Wacker-Chemie) Wacker HDK P 100H (Wacker Silicones) Wacker HDK N 20P (Wacker-Chemie) Wacker HDK N 25P (Wacker-Chemie) Wacker HDK S 13 (Wacker-Chemie) Wacker HDK T 30 (Wacker-Chemie) Wacker HDK V 15 (Wacker-Chemie) Wacker HDK V 15 P (Wacker-Chemie) Zelec Sil (DuPont).", "In addition, it is advantageous to use SiO2 pigmente in which the free OH groups have been (completely or partially) organically modified on the particle surface.", "For example, the addition of dimethylsilyl groups gives silica dimethylsilylates (e.g.", "Aerosil® R972 (Degussa Hüls) Aerosil® R974 (Degussa Hüls) CAB-O-SIL TS-610 (Cabot) CAB-O-SIL TS-720 (Cabot) Wacker HDK H15 (Wacker-Chemie) Wacker HDK H18 (Wacker-Chemie) Wacker HDK H2O (Wacker-Chemie)).", "The addition of trimethylsilyl groups gives silica silylates (e.g.", "Aerosil R 812 (Degussa Huls) CAB-O-SIL TS-530 (Cabot) Sipernat D 17 (Degussa Hüls) Wacker HDK H2000 (Wacker-Chemie)).", "Polymethylsilsesquioxanes are offered, for example, under the trade names Tospearl® 2000 B from GE Bayer Silikones, Tospearl 145A from Toshiba, AEC Silicone Resin Spheres from A & E Connock or Wacker—Belsil PMS MK from Wacker-Chemie.", "The cosmetic and/or dermatological preparations according to the invention can have the customary composition.", "For the purposes of the present invention, skincare preparations are particularly advantageous: they can be used for cosmetic and/or dermatological light protection, and also for the treatment of the skin and/or of the hair and as make-up products in decorative cosmetics.", "A further advantageous embodiment of the present invention consists in aftersun products.", "Corresponding to their structure, cosmetic or topical dermatological compositions can be used, for the purposes of the present invention, for example as skin protection cream, day cream or night cream etc.", "It may be possible and advantageous to use the compositions according to the invention as a base for pharmaceutical formulations.", "Just as emulsions of liquid and solid consistency are used as cosmetic cleansing lotions or cleansing creams, the preparations according to the invention can also be “cleansing foams” which can be used, for example, for the removal of make-up or as a mild washing foam, possibly also for bad skin.", "Such cleansing foams can advantageously also be used as “rinse-off” preparations, which are rinsed from the skin following application.", "The cosmetic and/or dermatological preparations according to the invention can also advantageously be in the form of a foam for care of the hair or of the scalp, in particular a foam for arranging the hair, a foam which is used when blow-drying the hair, a styling foam and treatment foam.", "For use, the cosmetic and dermatological preparations according to the invention are applied to the skin and/or the hair in an adequate amount in the manner customary for cosmetics.", "The cosmetic and dermatological preparations according to the invention can comprise cosmetic auxiliaries, as are customarily used in such preparations, e.g.", "preservatives, preservative assistants, bactericides, perfumes, dyes, pigments which have a coloring action, moisturizers and/or humectants, fillers which improve the feel on the skin, fats, oils, waxes or other customary constituents of a cosmetic or dermatological formulation, such as alcohols, polyols, polymers, foam stabilizers, electrolytes, organic solvents or silicone derivatives.", "Advantageous preservatives for the purposes of the present invention are, for example, formaldehyde donors (such as, for example, DMDM hydantoin), iodopropylbutyl carbamates (e.g.", "those available under the trade names Koncyl-L, Koncyl-S and Konkaben LMB from Lonza), parabens, phenoxyethanol, ethanol, benzoic acid and the like.", "According to the invention, the preservative system usually also advantageously comprises preservative assistants, such as, for example, octoxyglycerol, glycine soybean etc.", "Particularly advantageous preparations are also obtained if antioxidants are used as additives or active ingredients.", "According to the invention, the preparations advantageously comprise one or more antioxidants.", "Favorable, but nevertheless optional antioxidants which may be used are all antioxidants customary or suitable for cosmetic and/or dermatological applications.", "The antioxidants are advantageously chosen from the group consisting of amino acids (e.g.", "glycine, histidine, tyrosine, tryptophan) and derivatives thereof, imidazoles (e.g.", "urocanic acid) and derivatives thereof, peptides such as D,L-carnosine, D-carnosine, L-carnosine and derivatives thereof (e.g.", "anserine), carotenoids, carotenes (e.g.", "α-carotene, β-carotene, lycopene) and derivatives thereof, lipoic acid and derivatives thereof (e.g.", "dihydrolipoic acid), aurothioglucose, propylthiouracil and other thiols (e.g.", "thioredoxin, glutathione, cysteine, cystine, cystamine and the glycosyl, N-acetyl, methyl, ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl, γ-linoleyl, cholesteryl and glyceryl esters thereof) and salts thereof, dilauryl thiodipropionate, distearyl thiodipropionate, thiodipropionic acid and derivatives thereof (esters, ethers, peptides, lipids, nucleotides, nucleosides and salts) and sulfoximine compounds (e.g.", "buthionine sulfoximines, homocysteine sulfoximine, buthionine sulfones, penta-, hexa-, heptathionine sulfoximine) in very low tolerated doses (e.g.", "pmol to μmol/kg), and also (metal) chelating agents (e.g.", "α-hydroxy fatty acids, palmitic acid, phytic acid, lactoferrin), α-hydroxy acids (e.g.", "citric acid, lactic acid, malic acid), humic acid, bile acid, bile extracts, bilirubin, biliverdin, EDTA, EGTA and derivatives thereof, unsaturated fatty acids and derivatives thereof (e.g.", "γ-linolenic acid, linoleic acid, oleic acid), folic acid and derivatives thereof, ubiquinone and ubiquinol and derivatives thereof, vitamin C and derivatives (e.g.", "ascorbyl palmitate, Mg ascorbyl phosphate, ascorbyl acetate), tocopherols and derivatives (e.g.", "vitamin E acetate), vitamin A and derivatives (vitamin A palmitate) and coniferyl benzoate of benzoin resin, rutinic acid and derivatives thereof, ferulic acid and derivatives thereof, butylhydroxytoluene, butylhydroxyanisole, nordihydroguaiacic acid, nordihydroguaiaretic acid, trihydroxybutyrophenone, uric acid and derivatives thereof, mannose and derivatives thereof, zinc and derivatives thereof (e.g.", "ZnO, ZnSO4), selenium and derivatives thereof (e.g.", "selenomethionine), stilbenes and derivatives thereof (e.g.", "stilbene oxide, trans-stilbene oxide) and the derivatives (salts, esters, ethers, sugars, nucleotides, nucleosides, peptides and lipids) of these listed active ingredients which are suitable according to the invention.", "For the purposes of the present invention, water-soluble antioxidants, such as, for example, vitamins, e.g.", "ascorbic acid and derivatives thereof, can be used particularly advantageously.", "A surprising property of the preparations according to the invention is that they are very good vehicles for cosmetic or dermatological active ingredients into the skin, preferred active ingredients being antioxidants which can protect the skin against oxidative stress.", "Preferred antioxidants here are vitamin E and derivatives thereof, and vitamin A and derivatives thereof.", "The amount of antioxidants (one or more compounds) in the preparations is preferably 0.001 to 30% by weight, particularly preferably 0.05 to 20% by weight, in particular 0.1 to 10% by weight, based on the total weight of the preparation.", "If vitamin E and/or derivatives thereof are the antioxidant(s), it is advantageous to choose their respective concentrations from the range from 0.001 to 10% by weight, based on the total weight of the formulation.", "If vitamin A or vitamin A derivatives, or carotenes or derivatives thereof are the anti-oxidant(s), it is advantageous to choose their respective concentrations from the range from 0.001 to 10% by weight, based on the total weight of the formulation.", "The active ingredients (one or more compounds) can also very advantageously be chosen according to the invention from the group of lipophilic active ingredients, in particular from the following group: acetylsalicylic acid, atropine, azulene, hydrocortisone and derivatives thereof, e.g.", "hydrocortisone-17 valerate, vitamins of the B and D series, very favorably vitamin B1, vitamin B12 and vitamin D1, but also bisabolol, unsaturated fatty acids, namely the essential fatty acids (often also called vitamin F), in particular gamma-linolenic acid, oleic acid, eicosapentaenoic acid, docosahexaenoic acid and derivatives thereof, chloroamphenicol, caffeine, prostaglandins, thymol, camphor, extracts or other products of a vegetable and animal origin, e.g.", "evening primrose oil, borage oil or currant seed oil, fish oils, cod-liver oil and also ceramides and ceramide-like compounds, etc.", "It is also advantageous to choose the active ingredients from the group of refatting substances, for example purcellin oil, Eucerit® and Neocerit®.", "The active ingredient(s) is/are also particularly advantageously chosen from the group of NO synthase inhibitors, particularly if the preparations according to the invention are to be used for the treatment and prophylaxis of the symptoms of intrinsic and/or extrinsic skin aging and for the treatment and prophylaxis of the harmful effects of ultraviolet radiation on the skin.", "A preferred NO synthase inhibitor is nitroarginine.", "The active ingredient(s) is/are also advantageously chosen from the group which includes catechins and bile esters of catechins and aqueous or organic extracts from plants or parts of plants which have a content of catechins or bile esters of catechins, such as, for example, the leaves of the Theaceae plant family, in particular of the species Camellia sinensis (green tea).", "Particularly advantageous are typical ingredients thereof (such as e.g.", "polyphenols or catechins, caffeine, vitamins, sugars, minerals, amino acids, lipids).", "Catechins are a group of compounds which are to be regarded as hydrogenated flavones or anthocyanidines and are derivatives of “catechin” (catechol, 3,3′,4′,5,7-flavanpentaol, 2-(3,4-dihydroxyphenyl)chroman-3,5,7-triol).", "Epicatechin ((2R,3R)-3,3′,4′,5,7-flavanpentaol) is also an advantageous active ingredient for the purposes of the present invention.", "Also advantageous are plant extracts with a content of catechins, in particular extracts of green tea, such as e.g.", "extracts from leaves of plants of the species Camellia spec., very particularly the types of tea Camellia sinenis, C. assamica, C. taliensis and C. irrawadiensis and hybrids of these with, for example, Camellia japonica.", "Preferred active ingredients are also polyphenols or catechins from the group (−)-catechin, (+)-catechin, (−)-catechin gallate, (−)-gallocatechin gallate, (+)-epicatechin, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin and (−)-epigallocatechin gallate.", "Flavone and its derivatives (also often collectively called “flavones”) are also advantageous active ingredients for the purposes of the present invention.", "They are characterized by the following basic structure (substitution positions are shown): Some of the more important flavones which can also preferably be used in preparations according to the invention are given in the table below: OH substitution positions 3 5 7 8 2′ 3′ 4′ 5′ Flavone − − − − − − − − Flavonol + − − − − − − − Chrysin − + + − − − − − Galangin + + + − − − − − Apigenin − + + − − − + − Fisetin + − + − − + + − Luteolin − + + − − + + − Kaempferol + + + − − − + − Quercetin + + + − − + + − Morin + + + − + − + − Robinetin + − + − − + + + Gossypetin + + + + − + + − Myricetin + + + − − + + + In nature, flavones are usually in glycosylated form.", "According to the invention, the flavonoids are preferably chosen chosen from the group of substances of the generic structural formula where Z1 to Z7, independently of one another, are chosen from the group consisting of H, OH, alkoxy and hydroxyalkoxy, where the alkoxy and hydroxyalkoxy groups can be branched or unbranched and have 1 to 18 carbon atoms, and where Gly is chosen from the group of mono- and oligoglycoside radicals.", "According to the invention, the flavonoids can however, also advantageously be chosen from the group of substances of the generic structural formula where Z1 to Z6, independently of one another, are chosen from the group consisting of H, OH, alkoxy and hydroxyalkoxy, where the alkoxy and hydroxyalkoxy groups can be branched or unbranched and have 1 to 18 carbon atoms, and where Gly is chosen from the group of mono and oligoglycoside radicals.", "Preferably, such structures can be chosen from the group of substances of the generic structural formula where Gly1, Gly2 and Gly3, independently of one another, are m onoglycoside radicals.", "Gly2 and Gly3 can also, individually or together, represent saturations by hydrogen atoms.", "Preferably, Gly1, Gly2 and Gly3, independently of one another, are chosen from the group of hexosyl radicals, in particular of rhamnosyl radicals and glucosyl radicals.", "However, other hexosyl radicals, for example allosyl, altrosyl, galactosyl, gulosyl, idosyl, mannosyl and talosyl, can also be used advantageously in some circumstances.", "It may also be advantageous according to the invention to use pentosyl radicals.", "Z1 to Z5 are, independently of one another, advantageously chosen from the group consisting of H, OH, methoxy, ethoxy and 2-hydroxyethoxy, and the flavone glycosides have the structure The flavone glycosides according to the invention are particularly advantageously chosen from the group given by the following structure: where Gly1, Gly2 and Gly3, independently of one another, are monoglycoside radicals.", "Gly2 and Gly3 can also, individually or together, represent saturations by hydrogen atoms.", "Preferably, Gly1, Gly2 and Gly3, independently of one another, are chosen from the group of hexosyl radicals, in particular of rhamnosyl radicals and glucosyl radicals.", "However, other hexosyl radicals, for example allosyl, altrosyl, galactosyl, gulosyl, idosyl, mannosyl and talosyl, can also advantageously be used in some circumstances.", "It may also be advantageous according to the invention to use pentosyl radicals.", "For the purposes of the present invention, it is particularly advantageous to choose the flavone glucoside(s) from the group consisting of α-glucosylrutin, α-glucosylmyricetin, α-glucosylisoquercitrin, α-glucosylisoquercetin and α-glucosylquercitrin.", "Particular preference is given, according to the invention, to α-glucosylrutin.", "Also advantageous according to the invention are naringin (aurantin, naringenin-7-rhamnoglucoside), hesperidin (3′,5,7-trihydroxy-4′-methoxyflavanone-7-rutinoside, hesperidoside, hesperetin-7-O-rutinoside), rutin (3,3′,4′,5,7-pentahydroxyflyvone-3-rutinoside, quercetin-3-rutinoside, sophorin, birutan, rutabion, taurutin, phytomelin, melin), troxerutin (3,5-dihydroxy-3′,4′,7-tris(2-hydroxyethoxy)flavone-3-(6-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranoside)), monoxerutin (3,3′,4′,5-tetrahydroxy-7-(2-hydroxyethoxy)flavone-3-(6-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranoside)), dihydrorobinetin (3,3′,4′,5′,7-pentahydroxyflavanone), taxifolin (3,3′,4′,5,7-pentahydroxy-flavanone), eriodictyol-7-glucoside (3′,4′,5,7-tetrahydroxyflavanone-7 glucoside), flavanomarein (3′,4′,7,8-tetrahydroxyflavanone-7 glucoside) and isoquercetin (3,3′,4′,5,7-pentahydroxyflavanone-3-(β-D-glucopyranoside).", "It is also advantageous to choose the active ingredient(s) from the group of ubiquinones and plastoquinones.", "Ubiquinones are distinguished by the structural formula and are the most widespread and thus the most investigated bioquinones.", "Ubiquinones are referred to depending on the number of isoprene units linked in the side chain as Q-1; Q-2, Q-3 etc., or depending on the number of carbon atoms, as U-5, U-10, U-15 etc.", "They preferably appear with certain chain lengths, e.g.", "in some microorganisms and yeasts where n=6.In most mammals including man, Q10 predominates.", "Coenzyme Q10 is particularly advantageous and is characterized by the following structural formula: Plastoquinones have the general structural formula Plastoquinones differ in the number n of isoprene radicals and are referred to accordingly, e.g.", "PQ-9 (n=9).", "In addition, other plastoquinones with varying substituents on the quinone ring exist.", "Creatine and/or creatine derivatives are preferred active ingredients for the purposes of the present invention.", "Creatine is characterized by the following structure: Preferred derivatives are creatine phosphate and creatine sulfate, creatine acetate, creatine ascorbate and the derivatives esterified at the carboxyl group with mono- or polyfunctional alcohols.", "A further advantageous active ingredient is L-carnitine [3-hydroxy-4-(trimethylammonio)butyrobetaine].", "Acylcarnitines which chosen from the group of substances of the following general structural formula where R is chosen from the group of branched and unbranched alkyl radicals having up to 10 carbon atoms, are advantageous active ingredients for the purposes of the present invention.", "Preference is given to propionylcarnitine and, in particular, acetylcarnitine.", "Both enantiomers (D and L form) are to be used advantageously for the purposes of the present invention.", "It may also be advantageous to use any enantiomer mixtures, for example a racemate of D and L form.", "Further advantageous active ingredients are sericoside, pyridoxol, vitamin K, biotin and aroma substances.", "The list of said active ingredients and active ingredient combinations which can be used in the preparations according to the invention is, of course, not intended to be limiting.", "The active ingredients can be used individually or in any combinations with one another.", "Skin aging is caused e.g.", "by endogenous, genetically determined factors.", "As a result of aging, the epidermis and dermis experience e.g.", "the following structural damage and functional disorders, which can also be covered by the term “senile xerosis”: a) dryness, roughness and formation of (dryness) wrinkles, b) itching and c) reduced refatting by sebaceous glands (e.g.", "after washing).", "Exogenous factors, such as UV light and chemical noxae, can have a cumulative effect and, for example, accelerate or add to the endogenous aging processes.", "The epidermis and dermis experience, in particular as a result of exogenous factors, e.g.", "the following structural damage and functional disorders in the skin, which go beyond the degree and quality of the damage in the case of chronological aging: d) visible vascular dilations (telangiectases, cuperosis); e) flaccidity and formation of wrinkles; f) local hyperpigmentation, hypopigmentation and abnormal pigmentation (e.g.", "age spots) and g) increased susceptibility to mechanical stress (e.g.", "cracking).", "Surprisingly, selected formulations according to the invention can also have an anti-wrinkle action or considerably increase the action of known antiwrinkle active ingredients.", "Accordingly, for the purposes of the invention, formulations are particularly advantageously suitable for the prophylaxis and treatment of cosmetic or dermatological skin changes, as arise, for example, during skin aging.", "They are also advantageously suitable for combating the development of dry or rough skin.", "In a particular embodiment, the present invention thus relates to products for the care of skin aged in a natural manner, and for the treatment of the secondary damage of light aging, in particular the phenomena listed under a) to g).", "The water phase of the preparations according to the invention can advantageously comprise customary cosmetic auxiliaries, such as, for example, alcohols, in particular those of low carbon number, preferably ethanol and/or isopropanol, diols or polyols of low carbon number, and ethers thereof, preferably propylene glycol, glycerol, ethylene glycol, ethylene glycol monoethyl or monobutyl ether, propylene glycol monomethyl, monoethyl or monobutyl ether, diethyleneglycol monomethyl or monoethyl ether and analogous products, polymers, foam stabilizers, electrolytes and moisturizers.", "Moisturizers is the term used to describe substances or mixtures of substances which, following application or distribution on the surface of the skin, confer on cosmetic or dermatological preparations the property of reducing the moisture loss by the horny layer (also called transepidermal water loss (TEWL)) a nd/or have a beneficial effect on the hydration of the horny layer.", "Advantageous moisturizers for the purposes of the present invention are, for example, glycerol, lactic acid, pyrrolidonecarboxylic acid and urea.", "In addition, it is particularly advantageous to use polymeric moisturizers from the group of polysaccharides which are soluble in water and/or swellable in water and/or gellable using water.", "Particularly advantageous are, for example, hyaluronic acid, chitosan and/or a fucose-rich polysaccharide which is listed in Chemical Abstracts under the registry number 178463-23-5 and is available, for example, under the name Fucogel®1000 from SOLABIA S.A.", "The cosmetic and dermatological preparations according to the invention can comprise dyes and/or color pigments, particularly when they are in the form of decorative cosmetics.", "The dyes and color pigments can be chosen from the corresponding positive list of the Cosmetics Directive or the EC list of cosmetic colorants.", "In most cases they are identical to the dyes approved for foods.", "Advantageous color pigments are, for example, titanium dioxide, mica, iron oxides (e.g.", "Fe2O3, Fe3O4, FeO(OH)) and/or tin oxide.", "Advantageous dyes are, for example, carmine, Berlin blue, chrome oxide green, ultramarine blue and/or manganese violet.", "It is particularly advantageous to choose the dyes and/or color pigments from the following list.", "The Colour Index Numbers (CIN) are taken from the Rowe Colour Index, 3rd Edition, Society of Dyers and Colourists, Bradford, England, 1971.Chemical or other name CIN Color Pigment Green 10006 green Acid Green 1 10020 green 2,4-Dinitrohydroxynaphthalene-7-sulfonic acid 10316 yellow Pigment Yellow 1 11680 yellow Pigment Yellow 3 11710 yellow Pigment Orange 1 11725 orange 2,4-Dihydroxyazobenzene 11920 orange Solvent Red 3 12010 red 1-(2′-Chloro-4′-nitro-1′-phenylazo)- 12085 red 2-hydroxynaphthalene Pigment Red 3 12120 red Ceres red; Sudan red; Fat Red G 12150 red Pigment Red 112 12370 red Pigment Red 7 12420 red Pigment Brown 1 12480 brown 4-(2′-Methoxy-5′-sulfodiethylamido-1′- 12490 red phenylazo)-3-hydroxy-5″-chloro-2″,4″- dimethoxy-2-naphthanilide Disperse Yellow 16 12700 yellow 1-(4-Sulfo-1-phenylazo)-4-aminobenzene-5-sulfonic 13015 yellow acid 2,4-Dihydroxyazobenzene-4′-sulfonic acid 14270 orange 2-(2,4-Dimethylphenylazo-5-sulfo)-1- 14700 red hydroxynaphthalene-4-sulfonic acid 2-(4-Sulfo-1-naphthylazo)-1-naphthol-4-sulfonic 14720 red acid 2-(6-Sulfo-2,4-xylylazo)-1-naphthol-5-sulfonic 14815 red acid 1-(4′-Sulfophenylazo)-2-hydroxynaphthalene 15510 orange 1-(2-Sulfo-4-chloro-5-carboxy-1-phenylazo)-2- 15525 red hydroxynaphthalene 1-(3-Methylphenylazo-4-sulfo)-2-hydroxynaphthalene 15580 red 1-(4′,(8′)-Sulfonaphthylazo)-2- 15620 red hydroxynaphthalene 2-Hydroxy-1,2′-azonaphthalene-1′-sulfonic 15630 red acid 3-Hydroxy-4-phenylazo-2-naphthylcarboxylic acid 15800 red 1-(2-Sulfo-4-methyl-1-phenylazo)-2- 15850 red naphthylcarboxylic acid 1-(2-Sulfo-4-methyl-5-chloro-1-phenylazo)-2- 15865 red hydroxynaphthalene-3-carboxylic acid 1-(2-Sulfo-1-naphthylazo)-2-hydroxynaphthalene-3- 15880 red carboxylic acid 1-(3-Sulfo-1-phenylazo)-2-naphthol-6-sulfonic acid 15980 orange 1-(4-Sulfo-1-phenylazo)-2-naphthol-6-sulfonic acid 15985 yellow Allura Red 16035 red 1-(4-Sulfo-1-naphthylazo)-2-naphthol-3,6-disulfonic 16185 red acid Acid Orange 10 16230 orange 1-(4-Sulfo-1-naphthylazo)-2-naphthol-6,8-disulfonic 16255 red acid 1-(4-Sulfo-1-naphthylazo)-2-naphthol-3,6,8- 16290 red trisulfonic acid 8-Amino-2-phenylazo-1-naphthol-3,6-disulfonic acid 17200 red Acid Red 1 18050 red Acid Red 155 18130 red Acid Yellow 121 18690 yellow Acid Red 180 18736 red Acid Yellow 11 18820 yellow Acid Yellow 17 18965 yellow 4-(4-Sulfo-1-phenylazo)-1-(4-sulfophenyl)-5- 19140 yellow hydroxy-pyrazolone-3-carboxylic acid Pigment Yellow 16 20040 yellow 2,6-(4′-Sulfo-2″,4″- 20170 orange dimethyl)bisphenylazo)-1,3-dihydroxybenzene Acid Black 1 20470 black Pigment Yellow 13 21100 yellow Pigment Yellow 83 21108 yellow Solvent Yellow 21230 yellow Acid Red 163 24790 red Acid Red 73 27290 red 2-[4′-(4″-Sulfo-1″-phenylazo)-7′- 27755 black sulfo-1′-naphthylazo]-1-hydroxy-7- aminonaphthalene-3,6-disulfonic acid 4′-[(4″-Sulfo-1″-phenylazo)-7′- 28440 black sulfo-1′-naphthylazo]-1-hydroxy-8- acetylaminonaphthalene-3,5-disulfonic acid Direct Orange 34, 39, 44, 46, 60 40215 orange Food Yellow 40800 orange trans-β-Apo-8′-carotenaldehyde (C30) 40820 orange trans-Apo-8′-carotenic acid (C30)-ethyl ester 40825 orange Canthaxanthin 40850 orange Acid Blue 1 42045 blue 2,4-Disulfo-5-hydroxy-4′-4″- 42051 blue bis(diethylamino)triphenylcarbinol 4-[(4-N-Ethyl-p-sulfobenzylamino)phenyl(4- 42053 green hydroxy-2-sulfophenyl)(methylene)-1-(N-ethyl- N-p-sulfobenzyl)-2,5-cyclohexadienimine] Acid Blue 7 42080 blue (N-Ethyl-p-sulfobenzylamino)phenyl(2- 42090 blue sulfophenyl)methylene-(N-ethyl-N-p- sulfobenzyl)Δ2,5-cyclohexadienimine Acid Green 9 42100 green Diethyldisulfobenzyl-di-4-amino-2-chloro-di-2- 42170 green methyl-fuchsonimmonium Basic Violet 14 42510 violet Basic Violet 2 42520 violet 2′-Methyl-4′-(N-ethyl-N-m-sulfobenzyl)amino- 42735 blue 4″-(N-diethyl)amino-2-methyl-N-ethyl-N-m- sulfobenzylfuchsonimmonium 4′-(N-Dimethyl)amino-4″-(N- 44045 blue phenyl)aminonaphtho-N-dimethyl-fuchsonimmonium 2-Hydroxy-3,6-disulfo-4,4′- 44090 green bisdimethylaminonaphtho-fuchsonimmonium Acid Red 52 45100 red 3-(2′-Methylphenylamino)-6-(2′-methyl-4′- 45190 violet sulfophenylamino)-9-(2″- carboxyphenyl)xanthenium salt Acid Red 50 45220 red Phenyl-2-oxyfluorone-2-carboxylic acid 45350 yellow 4,5-Dibromofluorescein 45370 orange 2,4,5,7-Tetrabromofluorescein 45380 red Solvent Dye 45396 orange Acid Red 98 45405 red 3′,4′,5′,6′-Tetrachloro-2,4,5,7- 45410 red tetrabromofluorescein 4,5-Diiodofluorescein 45425 red 2,4,5,7-Tetraiodofluorescein 45430 red Quinophthalone 47000 yellow Quinophthalonedisulfonic acid 47005 yellow Acid Violet 50 50325 violet Acid Black 2 50420 black Pigment Violet 23 51319 violet 1,2-Dioxyanthraquinone, calcium-aluminum complex 58000 red 3-Oxypyrene-5,8,10-sulfonic acid 59040 green 1-Hydroxy-4-N-phenylaminoanthraquinone 60724 violet 1-Hydroxy-4-(4′-methylphenylamino)anthraquinone 60725 violet Acid Violet 23 60730 violet 1,4-Di(4′-methylphenylamino)anthraquinone 61565 green 1,4-Bis(o-sulfo-p-toluidino)anthraquinone 61570 green Acid Blue 80 61585 blue Acid Blue 62 62045 blue N,N′-Dihydro-1,2,1′,2′-anthraquinone azine 69800 blue Vat Blue 6; Pigment Blue 64 69825 blue Vat Orange 7 71105 orange Indigo 73000 blue Indigo-disulfonic acid 73015 blue 4,4′-Dimethyl-6,6′-dichlorothioindigo 73360 red 5,5′-Dichloro-7,7′-dimethylthioindigo 73385 violet Quinacridone Violet 19 73900 violet Pigment Red 122 73915 red Pigment Blue 16 74100 blue Phthalocyanine 74160 blue Direct Blue 86 74180 blue Chlorinated phthalocyanine 74260 green Natural Yellow 6, 19; Natural Red 1 75100 yellow Bixin, Norbixin 75120 orange Lycopene 75125 yellow trans-alpha-, beta- and gamma-carotene 75130 orange Keto- and/or hydroxyl derivatives of carotene 75135 yellow Guanine or pearlescent agent 75170 white 1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene- 75300 yellow 3,5-dione Complex salt (Na, Al, Ca) of carminic acid 75470 red Chlorophyll a and b; copper compounds of 75810 green chlorophylls and chlorophyllins Aluminum 77000 white Hydrated alumina 77002 white Hydrous aluminum silicates 77004 white Ultramarine 77007 blue Pigment Red 101 and 102 77015 red Barium sulfate 77120 white Bismuth oxychloride and its mixtures with mica 77163 white Calcium carbonate 77220 white Calcium sulfate 77231 white Carbon 77266 black Pigment Black 9 77267 black Carbo medicinalis vegetabilis 77268:1 black Chromium oxide 77288 green Chromium oxide, hydrous 77289 green Pigment Blue 28, Pigment Green 14 77346 green Pigment Metal 2 77400 brown Gold 77480 brown Iron oxides and hydroxides 77489 orange Iron oxide 77491 red Hydrated Iron oxide 77492 yellow Iron oxide 77499 black Mixtures of iron (II) and iron(III)hexacyanoferrate 77510 blue Pigment White 18 77713 white Manganese animonium diphosphate 77742 violet Manganese phosphate; Mn3(PO4)2.7H20 77745 red Silver 77820 white Titanium dioxide and its mixtures with mica 77891 white Zinc oxide 77947 white 6,7-Dimethyl-9-(1′-D-ribityl)isoalloxazine, yellow lactoflavine Sugar coloring brown Capsanthin, capsorubin orange Betanin red Benzopyrylium salts, anthocyans red Aluminum, zinc, magnesium and calcium stearate white Bromothymol blue blue Bromocresol green green Acid Red 195 red If the formulations according to the invention are in the form of products, which are intended for use in the facial area, it is favorable to choose one or more substances from the following group as the dye: 2,4-dihydroxyazobenzene, 1-(2′-chloro-4′-nitro-1′-phenyl-azo)-2-hydroxynaphthalene, Ceres Red, 2-(4-sulfo-1-naphthylazo)-1-naphthol-4-sulfonic acid, calcium salt of 2-hydroxy-1,2′-azonaphthalene-1′-sulfonic acid, calcium and barium salts of 1-(2-sulfo-4-methyl-1-phenylazo)-2-naphthylcarboxylic acid, calcium salt of 1-(2-sulfo-1-naphthylazo)-2-hydroxynaphthalene-3-carboxylic acid, aluminum salt of 1-(4-sulfo-1-azo)-2-naphthol-6-sulfonic acid, aluminum salt of 1-(4-sulfo-1-naphthylazo)-2-naphthol-3,6-disulfonic acid, 1-(4-sulfo-1-naphthylazo)-2-naphthol-6,8-disulfonic acid, aluminum salt of 4-(4-sulfo-1-phenylazo)-1-(4-sulfophenyl)-5-hydroxypyrazolone-3-carboxylic acid, aluminum and zirconium salts of 4,5-dibromofluorescein, aluminum and zirconium salts of 2,4,5,7-tetrabromofluorescein, 3′,4′,5′,6′-tetrachloro-2,4,5,7-tetra-bromofluorescein and its aluminum salt, aluminum salt of 2,4,5,7-tetraiodofluorescein, aluminum salt of quinophthalone disulfonic acid, aluminum salt of indigo disulfonic acid, red and black iron oxide (CIN: 77 491 (red) and 77 499 (black)), iron oxide hydrate (CIN: 77 492), manganese ammonium diphosphate and titanium dioxide.", "Also advantageous are oil-soluble natural dyes, such as, for example, paprika extracts, β-carotene or cochenille.", "Also advantageous for the purposes of the present invention are formulations with a content of pearlescent pigments.", "Preference is given in particular to the types of pearlescent pigments listed below: 1.Natural pearlescent pigments, such as, for example “pearl essence” (guanine/hypoxanthin mixed crystals from fish scales) and “mother of pearl” (ground mussel shells) 2.Monocrystalline pearlescent pigments, such as, for example, bismuth oxychloride (BiOCl) 3.Layer-substrate pigments: e.g.", "mica/metal oxide Bases for pearlescent pigments are, for example, pulverulent pigments or castor oil dispersions of bismuth oxychloride and/or titanium dioxide, and bismuth oxychloride and/or titanium dioxide on mica.", "The luster pigment listed under CIN 77163, for example, is particularly advantageous.", "Also advantageous are, for example, the following types of pearlescent pigment based on mica/metal oxide: Coating/layer Group thickness Color Silver-white TiO2: 40-60 nm silver pearlescent pigments Interference TiO2: 60-80 nm yellow pigments TiO2: 80-100 nm red TiO2: 100-140 nm blue TiO2: 120-160 nm green Color luster Fe2O3 bronze pigments Fe2O3 copper Fe2O3 red Fe2O3 red-violet Fe2O3 red-green Fe2O3 black Combination TiO2/Fe2O3 gold shades pigments TiO2/Cr2O3 green TiO2/Berlin blue deep blue TiO2/carmine red Particular preference is given, for example, to the pearlescent pigments obtainable from Merck under the trade names Timiron, Colorona or Dichrona.", "The list of given pearlescent pigments is not of course intended to be limiting.", "Pearlescent pigments which are advantageous for the purposes of the present invention are obtainable by numerous methods known per se.", "For example, other substrates apart from mica can be coated with further metal oxides, such as, for example, silica and the like.", "SiO2 particles coated with, for example, TiO2 and Fe2O3 (“ronaspheres”), which are marketed by Merck and are particularly suitable for the optical reduction of fine lines are advantageous.", "It can moreover be advantageous to dispense completely with a substrate such as mica.", "Particular preference is given to iron pearlescent pigments prepared without the use of mica.", "Such pigments are obtainable, for example, under the trade name Sicopearl Kupfer 1000 from BASF.", "In addition, also particularly advantageous are effect pigments which are obtainable under the trade name Metasome Standard/Glitter in various colors (yellow, red, green, blue) from Flora Tech.", "The glitter particles are present here in mixtures with various auxiliaries and dyes (such as, for example, the dyes with the Colour Index (CI) Numbers 19140, 77007, 77289, 77491).", "The dyes and pigments may be present either individually or in a mixture, and can be mutually coated with one another, different coating thicknesses generally giving rise to different color effects.", "The total amount of dyes and color-imparting pigments is advantageously chosen from the range from e.g.", "0.1% by weight to 30% by weight, preferably from 0.5 to 15% by weight, in particular from 1.0 to 10% by weight, in each case based on the total weight of the preparations.", "For the purposes of the present invention, it is also advantageous to provide cosmetic and dermatological preparations whose main purpose is not protection against sunlight, but which nevertheless have a content of UV protection substances.", "Thus, for example, UV-A and/or UV-B filter substances are usually incorporated into day creams or make-up products.", "UV protection substances, like antioxidants, and, if desired, preservatives, also constitute effective protection of the preparations themselves against spoilage.", "Also favorable are cosmetic and dermatological preparations in the form of a sunscreen.", "Accordingly, for the purposes of the present invention, as well as comprising one or more UV filter substances according to the invention, the preparations additionally comprise at least one further UV-A and/or UV-B filter substance.", "The formulations may, although not necessarily, optionally also comprise one or more organic and/or inorganic pigments as UV filter substances which may be present in the water and/or oil phase.", "Preferred inorganic pigments are metal oxides and/or other metal compounds which are insoluble or virtually insoluble in water, in particular oxides of titanium (TiO2), zinc (ZnO), iron (e.g.", "Fe2O3), zirconium (ZrO2), silicon (SiO2), manganese (e.g.", "MnO), aluminum (Al2O3), cerium (e.g.", "Ce2O3), mixed oxides of the corresponding metals and mixtures of such oxides.", "For the purposes of the present invention, such pigments may advantageously be surface-treated (“coated”), the intention being to form or retain, for example, an amphiphilic or hydrophobic character.", "This surface treatment can consist in providing the pigments with a thin hydrophobic layer by processes known per se.", "Advantageous according to the invention are e.g.", "titanium dioxide pigments which have been coated with octylsilanol.", "Suitable titanium dioxide particles are available under the trade name T805 from Degussa.", "Also particularly advantageous are TiO2 pigments coated with aluminum stearate, e.g.", "those available under the trade name MT 100 T from TAYCA.", "A further advantageous coating of the inorganic pigments consists of dimethyl-polysiloxane (also: dimethicone), a mixture of completely methylated, linear siloxane polymers which have been terminally blocked with trimethylsiloxy units.", "Particularly advantageous for the purposes of the present invention are zinc oxide pigments which have been coated in this way.", "Also advantageous is a coating of the inorganic pigments with a mixture of dimethyl-polysiloxane, in particular dimethylpolysiloxane having an average chain length of from 200 to 350 dimethylsiloxane units, and silica gel, which is also referred to as simethicone.", "In particular, it is advantageous for the inorganic pigments to be additionally coated with aluminum hydroxide or aluminum oxide hydrate (also: alumina, CAS No.", ": 1333-84-2).", "Particularly advantageous are titanium dioxides which have been coated with simethicone a nd a lumina, it also being possible for the coating to comprise water.", "An example thereof is the titanium dioxide available under the trade name Eusolex T2000 from Merck.", "An advantageous organic pigment for the purposes of the present invention is 2,2′-methylenebis(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) [INCI: bisoctyltriazole], which is characterized by the chemical structural formula and is available under the trade name Tinosorb® M from CIBA-Chemikalien GmbH.", "Preparations according to the invention advantageously comprise substances which absorb UV radiation in the UV-A and/or UV-B range, the total amount of filter substances being, for example, from 0.1% by weight to 30% by weight, preferably from 0.5 to 20% by weight, in particular from 1.0 to 15.0% by weight, based on the total weight of the preparations, in order to provide cosmetic preparations which protect the hair and the skin from the entire range of ultraviolet radiation.", "They can also be used as sunscreens for the hair or the skin.", "Advantageous UV-A filter substances for the purposes of the present invention are dibenzoylmethane derivatives, in particular 4-(tert-butyl)-4′-methoxydibenzoylmethane (CAS No.", "70356-09-1), which is sold by Givaudan under the name Parsol® 1789 and by Merck under the trade name Eusolex® 9020.Further advantageous UV-A filter substances are phenylene-1,4-bis(2-benzimidazyl)-3,3′-5,5′-tetrasulfonic acid: and its salts, particularly the corresponding sodium, potassium or triethanolammonium salts, in particular phenylene-1,4-bis(2-benzimidazyl)-3,3′-5,5′-tetrasulfonic bis-sodium salt: with the INCI name Bisimidazylate, which is available, for example, under the trade name Neo Heliopan AP from Haarmann & Reimer.", "Also advantageous are 1,4-di(2-oxo-10-sulfo-3-bornylidenemethyl)benzene and salts thereof (in particular the corresponding 10-sulfato compounds, in particular the corresponding sodium, potassium or triethanolammonium salt), which is also referred to as benzene-1,4-di(2-oxo-3-bornylidenemethyl-10-sulfonic acid) and is characterized by the following structure: Advantageous UV filter substances for the purposes of the present invention are also broadband filters, i.e.", "filter substances which absorb both UV-A and also UV-B radiation.", "Advantageous broadband filters or UV-B filter substances are, for example, bisresorcinyltriazine derivatives having the following structure: where R1, R2 and R3 independently of one another are chosen from the group of branched and unbranched alkyl groups having 1 to 10 carbon atoms, or are a single hydrogen atom.", "Particular preference is given to 2,4-bis{[4-(2-ethylhexyloxy)-2-hydroxy]-phenyl}-6-(4-methoxyphenyl)-1,3,5-triazine (INCI: Aniso Triazine), which is available under the trade name Tinosorb® S from CIBA-Chemikalien GmbH.", "For the purposes of the present invention, particularly advantageous preparations which are characterized by high or very high UV-A protection preferably comprise two or more UV-A and/or broadband filters, in particular dibenzoylmethane derivatives [for example 4-(tert-butyl)-4′-methoxydibenzoylmethane], benzotriazole derivatives [for example 2,2′-methylenebis(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol)], phenylene-1,4-bis(2-benzimidazyl)-3,3′-5,5′-tetrasulfonic acid and/or its salts, 1,4-di(2-oxo-10-sulfo-3-bornylidenemethyl)benzene and/or salts thereof and/or 2,4-bis{[4-(2-ethylhexyloxy)-2-hydroxy]phenyl}-6-(4-methoxyphenyl)-1,3,5-triazine, in each case individually or in any combinations with one another.", "Other UV filter substances, which have the structural formula are also advantageous UV filter substances for the purposes of the present invention, for example the s-triazine derivatives described in European laid-open specification EP 570 838 A1, whose chemical structure is expressed by the generic formula where R is a branched or unbranched C1-C18-alkyl radical, a C5-C12-cycloalkyl radical, optionally substituted with one or more C1-C4-alkyl groups, X is an oxygen atom or an NH group, R1 is a branched or unbranched C1-C18-alkyl radical, a C5-C12-cycloalkyl radical, optionally substituted by one or more C1-C4-alkyl groups, or a hydrogen atom, an alkali metal atom, an ammonium group or a group of the formula in which A is a branched or unbranched C1-C18-alkyl radical, a C5-C12-cycloalkyl or aryl radical, optionally substituted by one or more C1-C4-alkyl groups, R3 is a hydrogen atom or a methyl group, n is a number from 1 to 10, R2 is a branched or unbranched C1-C1-8-alkyl radical, a CS-C1-2-cycloalkyl radical, optionally substituted by one or more C1-C4-alkyl groups, when X is the NH group, and a branched or unbranched C1-C18-alkyl radical, a C5-C12-cycloalkyl radical, optionally substituted by one or more C1-C4-alkyl groups, or a hydrogen atom, an alkali metal atom, an ammonium group or a group of the formula in which A is a branched or unbranched C1-C18-alkyl radical, a C5-C12-cycloalkyl or aryl radical, optionally substituted by one or more C1-C4-alkyl groups, R3 is a hydrogen atom or a methyl group, n is a number from 1 to 10, when X is an oxygen atom.", "A particularly preferred UV filter substance for the purposes of the present invention is also an unsymmetrically substituted s-triazine, the chemical structure of which is expressed by the formula and which is also referred to below as dioctylbutylamidotriazone (INCI: Dioctylbutamidotriazone), and is available under the trade name UVASORB HEB from Sigma 3V.", "Also advantageous for the purposes of the present invention is a symmetrically substituted s-triazine, tris(2-ethylhexyl) 4,4′,4″-(1,3,5-triazine-2,4,6-triyltriimino)tris-benzoate, synonym: 2,4,6-tris[anilino-(p-carbo-2′-ethyl-1′-hexyloxy)]-1,3,5-triazine (INCI: Octyl Triazone), which is marketed by BASF Aktiengesellschaft under the trade name UVINUL® T 150.European laid-open spacification 775 698 also describes preferred bisresorcinyltriazine derivatives, the chemical structure of which is expressed by the generic formula where R1, R2 and A1 represent very different organic radicals.", "Also advantageous for the purposes of the present invention are 2,4-bis{[4-(3-sulfonato)-2-hydroxypropyloxy)-2-hydroxy]phenyl}-6-(4-methoxyphenyl)-1,3,5-triazine sodium salt, 2,4-bis{([4-(3-(2-propyloxy)-2-hydroxypropyloxy)-2-hydroxy]pheny}-6-(4-methoxyphenyl)-1,3,5-triazine, 2,4-bis{[4-(2-ethylhexyloxy)-2-hydroxy]phenyl}-6-[4-(2-methoxyethylcarboxyl)phenylamino]-1,3,5-triazine, 2,4-bis{[4-(3-(2-propyloxy)-2-hydroxypropyloxy)-2-hydroxy]phenyl}-6-[4-(2-ethylcarboxyl)phenylamino]-1,3,5-triazine, 2,4-bis{[4-(2-ethylhexyloxy)-2-hydroxy]phenyl}-6-(1-methylpyrrol-2-yl)-1,3,5-triazine, 2,4-bis{[4-tris(trimethylsiloxysilylpropyloxy)-2-hydroxy]phenyl}-6-(4-methoxyphenyl)-1,3,5-triazine, 2,4-bis{([4-(2″-methylpropenyloxy)-2-hydroxy]phenyl}-6-(4-methoxyphenyl)-1,3,5-triazine and 2,4-bis{[4-(1′,1′,′,3′,5′,5′,5′-heptamethylsiloxy-2″-methylpropyloxy)-2-hydroxy]phenyl}-6-(4-methoxyphenyl)-1,3,5-triazine.", "An advantageous broadband filter for the purposes of the present invention is 2,2′-methylenebis(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol), which is characterized by the chemical structural formula and is available under the trade name Tinosorb® M from CIBA-Chemikalien GmbH.", "Another advantageous broadband filter for the purposes of the present invention is 2-(2H-benzotriazol-2-yl)-4-methyl-6-[2-methyl-3-[1,3,3,3-tetramethyl-1-[(trimethylsilyl)oxy]disiloxanyl]propyl]phenol (CAS No.", ": 155633-54-8) having the INCI name Drometrizole Trisiloxane, which is characterized by the chemical structural formula The UV-B and/or broadband filters can be oil-soluble or water-soluble.", "Examples of advantageous oil-soluble UV-B and/or broadband filter substances are: 3-benzylidenecamphor derivatives, preferably 3-(4-methylbenzylidene)camphor, 3-benzylidenecamphor; 4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4-(dimethylamino)benzoate, amyl 4-(dimethylamino)benzoate; 2,4,6-trianilino(p-carbo-2′-ethyl-1′-hexyloxy)-1,3,5-triazine; esters of benzalmalonic acid, preferably di(2-ethylhexyl) 4-methoxybenzalmalonate, esters of cinnamic acid, preferably 2-ethylhexyl 4-methoxycinnamate, isopentyl 4-methoxycinnamate; derivates of benzophenone, preferably 2-hydroxy4-methoxybenzophenone, 2-hydroxy-4-methoxy-4′-methylbenzophenone, 2,2′-dihydroxy-4-methoxybenzophenone and UV filters bonded to polymers.", "Examples of advantageous water-soluble UV-B and/or broadband filter substances are: salts of 2-phenylbenzimidazole-5-sulfonic acid, such as its sodium, potassium or its triethanolammonium salt, and also the sulfonic acid itself; sulfonic acid derivatives of 3-benzylidenecamphor, such as, for example, 4-(2-oxo-3-bornylidenemethyl) benzenesulfonic acid, 2-methyl-5-(2-oxo-3-bornylidenemethyl)sulfonic acid and salts thereof.", "A further light protection filter substance which can be used advantageously according to the invention is ethylhexyl 2-cyano-3,3-diphenylacrylate (octocrylene), which is available from BASF under the name Uvinul® N 539 and is characterized by the following structure: It can also be of considerable advantage to use polymer-bonded or polymeric UV filter substances in the preparations according to the present invention, in particular those described in WO-A-92/20690.In some instances, it can also be advantageous to incorporate further UV-A and/or UV-B filters in accordance with the invention into cosmetic or dermatological preparations, for example certain salicylic acid derivatives, such as 4-isopropylbenzyl salicylate, 2-ethylhexyl salicylate (=octyl salicylate), homomenthyl salicylate.", "The list of given UV filters which can be used for the purposes of the present invention is, of course, not intended to be limiting.", "The preparations according to the invention advantageously comprise the substances which absorb UV radiation in the UV-A and/or UV-B region in a total amount of, for example, 0.1% by weight to 30% by weight, preferably 0.5 to 20% by weight, in particular 1.0 to 15.0% by weight, in each case based on the total weight of the preparations, in order to provide cosmetic preparations which protect the hair or the skin from the entire range of ultraviolet radiation.", "They can also be used as sunscreens for the hair or the skin.", "The examples below serve to illustrate the present invention without limiting it.", "Unless stated otherwise, all amounts, proportions and percentages are based on the weight and the total amount or on the total weight of the preparations.", "EXAMPLE 1 Foam-Like O/W Cream Emulsion I % by wt.", "% by vol.", "Stearic acid 3.00 Cetyl alcohol 8.50 PEG-20 stearate 8.50 Talc 2.00 SiO2 2.00 C12-15alkyl benzoate 4.00 Paraffin oil 5.00 Isohexadecane 2.00 Glycerol 5.00 Sodium hydroxide q.s.", "Preservative q.s.", "Perfume q.s.", "Water, demineralized ad 100.00 pH adjusted to 6.5-7.5 Emulsion I 70 Nitrogen 30 Combining of the fatty phase heated to 75° C. with the water phase heated to 70° C. Addition of the particulate hydrophobic and/or hydrophobicized and/or oil-absorbing solid-body substances (pigments) with stirring.", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 65° C. Stirring for 45 min with gassing with nitrogen at 0.7 bar and cooling.", "Addition of the additives at 30° C. (perfume, active ingredients).", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 27° C. EXAMPLE 2 Foam-Like O/W Lotion Emulsion II % by wt.", "% by vol.", "Stearic acid 2.00 Myristyl alcohol 1.50 Cetylstearyl alcohol 0.50 PEG-100 stearate 3.00 TiO2 1.00 Kaolin 5.00 Mineral oil 5.00 Hydrogenated 15.00 polyisobutene Glycerol 3.00 Sodium hydroxide q.s.", "Preservative q.s.", "Perfume q.s.", "Water, demineralized ad 100.00 pH adjusted to 5.0-6.5 Emulsion II 50 Gas (carbon dioxide) 50 Combining of the fatty phase heated to 80° C. with the water phase heated to 72° C. Addition of the particulate hydrophobic and/or hydrophobicized and/or oil-absorbing solid-body substances (pigments) with stirring.", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 65° C. Stirring for 45 min with gassing with carbon dioxide at 1.2 bar and cooling.", "Addition of the additives at 30° C. (perfume).", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 30° C. EXAMPLE 3 Foam-Like O/W Lotion Emulsion III % by wt.", "% by vol.", "Stearic acid 5.00 Cetylstearyl alcohol 5.50 PEG-30 stearate 1.00 Aluminum starch 3.00 octenyl succinate Al2O3 0.50 Cyclomethicone 3.00 Isoeicosane 10.00 Polydecene 10.00 Citric acid 0.10 Glycerol 3.00 Perfume, preservative q.s.", "Sodium hydroxide q.s.", "Dyes etc.", "q.s.", "Water ad 100.00 pH adjusted to 6.0-7.5 Emulsion III 65 Gas (air) 35 Combining of the fatty phase heated to 80° C. with the water phase heated to 75° C. Addition of the particulate hydrophobic, hydrophobicized solid-body substances (aluminum oxide) with stirring.", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 65° C. Stirring for 45 min in an open reactor up to 30° C. Addition of the particulate hydrophobicized solid-body substances (aluminum starch octenyl succinate) and further additives (perfume, active ingredients) at 30° C. Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 25° C. EXAMPLE 4 Foam-Like O/W Emulsion Make-Up Emulsion IV % by wt.", "% by vol.", "Palmitic acid 2.00 Cetyl alcohol 2.00 PEG-100 stearate 2.00 Talc 0.50 Zeolites 0.75 Dimethicone 0.50 Paraffin oil 9.50 Dicaprylyl ether 2.00 Glycerol 3.00 Mica 1.00 Iron oxides 1.00 Titanium dioxide 4.50 Vitamin A palmitate 0.10 Sodium hydroxide q.s.", "Preservative q.s.", "Perfume q.s.", "Water demineralized ad 100.00 pH adjusted to 6.0-7.5 Emulsion IV 37 Gas (oxygen) 63 Combining of the fatty and color pigment phase heated to 78° C. with the water phase heated to 75° C. Addition of the particulate hydrophobic, hydrophobicized solid-body substances (talc, zeolites) with stirring.", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 65° C. Stirring for 45 min in the Becomix with gassing with oxygen at 1.3 bar with cooling to 30° C. Addition of the additives at 30° C. (perfume, active ingredients).", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 25° C. EXAMPLE 5 Foam-Like O/W Cream Emulsion V % by wt.", "% by vol.", "Stearic acid 4.00 Cetyl alcohol 2.00 PEG-30 stearate 2.00 Sorbitan monostearate 1.50 Talc 2.50 Paraffin oil 5.00 Cyclomethicone 1.00 Vitamin E acetate 1.00 Retinyl palmitate 0.20 Glycerol 3.00 BHT 0.02 Na2H2EDTA 0.10 Perfume, preservative q.s.", "Dyes q.s.", "Potassium hydroxide q.s.", "Water ad 100.00 pH adjusted to 5.0-7.0 Emulsion V 43 Gas (nitrous oxide) 57 Combining of the fatty phase heated to 80° C. with the water phase heated to 75° C. Addition of the particulate hydrophobic, hydrophobicized solid-body substances (talc) with stirring.", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 65° C. Stirring for 45 minutes in the Becomix with gassing with nitrous oxide at 0.7 bar with cooling to 30° C. Addition of the additives at 30° C. (perfume, active ingredients).", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 26° C. EXAMPLE 6 Foam-Like O/W Lotion Emulsion VI % by wt.", "% by vol.", "Stearic acid 4.00 Cetylstearyl alcohol 1.00 PEG-100 stearate 1.00 Distarch phosphate 0.50 SiO2 Paraffin oil 6.50 Dimethicone 0.50 Vitamin E acetate 2.00 Glycerol 3.00 Perfume, preservative dyes, etc.", "q.s.", "Sodium hydroxide q.s.", "Water ad 100.00 pH adjusted to 6.0-7.5 Emulsion VI 35 Gas (argon) 65 Combining of the fatty phase heated to 80° C. with the water phase heated to 75° C. Addition of the particulate hydrophobic, hydrophobicized solid-body substances with stirring.", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 65° C. Stirring for 45 min in an open reactor down to 30° C. Addition of the particulate hydrophobicized solid-body substances (distarch phosphate) and further additives (perfume, active ingredients) at 30° C. Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 23° C. EXAMPLE 7 Foam-Like Sunscreen Cream Emulsion VII % by wt.", "% by vol.", "Stearic acid 1.00 Cetylstearyl alcohol 4.00 Myristyl alcohol 1.00 Boron nitride 1.00 Silica dimethyl silylate 1.50 Kaolin 0.50 PEG-20 stearate 1.00 Caprylic/capric 2.00 triglycerides Paraffin oil 15.50 Dimethicone 0.50 Octyl isostearate 5.00 Glycerol 3.00 Octyl methoxycinnamate 4.00 Butyl methoxydibenzoylmethane 3.00 Ethylhexyltriazone 3.00 BHT 0.02 Disodium EDTA 0.10 Perfume, preservative q.s.", "Dyes, etc.", "q.s.", "Potassium hydroxide q.s.", "Water ad 100 pH adjusted to 5.0-6.0 Emulsion VII 35 Gas (helium) 65 Combining of the fatty phase and light protection filter phase heated to 78° C. with the water phase and light protection filter phase heated to 75° C. Addition of the particulate hydrophobic, hydrophobicized solid-body substances with stirring.", "Homogenization by means of a toothed-rim dispersing machine (rotor-stator principle) at 65° C. Stirring for 45 min in the Becomix with gassing with helium at 1 bar with cooling to 30° C. Addition of the additives at 30° C. (perfume).", "Homogenization by means of a toothed-dm dispersing machine (rotor-stator principle) at 23° C." ] ]	Patent_10469704
[ [ "Method for the detection of nucleic acid molecules", "There is provided a method of simultaneously detecting at least two mutually different nucleic acid molecules in a sample, wherein in a first step a multiplex-PCR and in a second step a hybridizing reaction is carried out with probes immobilized on a microarray, whereupon the hybridized PCR products are detected and optionally quantitated, wherein the probes employed for the hybridizing reaction which in each case will hybridize specifically with the mutually different nucleic acid molecules have melting temperatures which differ from each other by 2° C. at the most, preferably by 1° C. at the most." ], [ "1-27.", "(canceled) 28.A method of simultaneously detecting mutually different nucleic acid molecules in a sample comprising: obtaining a sample comprising at least two mutually different nucleic acid molecules each with a mutually different nucleic acid sequence; amplifying the at least two mutually different nucleic acid molecules in a multiplex-PCR reaction to obtain at least two mutually different amplified nucleic molecules, each comprising an amplified portion having the nucleic acid sequence of one of the at least two mutually different nucleic acid molecules; hybridizing the amplified portions of the at least two mutually different amplified nucleic acid molecules with at least two mutually different probes immobilized on a microarray to obtain at least two mutually different hybridized amplified nucleic acid molecules with melting temperatures that differ by at most 2° C.; and detecting the at least two mutually different hybridized nucleic acid molecules.", "29.The method of claim 28, further comprising quantifying the mutually different hybridized nucleic acid molecules.", "30.The method of claim 28, wherein each of the at least two mutually different probes comprises a hybridizing sequence adapted to hybridize, during use, to the amplified portion of one of the at least two mutually different amplified nucleic acid molecules to produce a hybridized amplified nucleic acid molecule having a melting temperature which differs from the melting temperature of one or more other hybridized amplified nucleic acid molecules by at most 2° C. 31.The method of claim 28, wherein the at least two mutually different hybridized amplified nucleic acid molecules have melting temperatures which differ by at most 1° C. 32.The method of claim 28, wherein at least six mutually different nucleic acid molecules are simultaneously detected in the sample.", "33.The method of claim 32, wherein at least eight mutually different nucleic acid molecules are simultaneously detected in the sample.", "34.The method of claim 33, wherein at least twelve mutually different nucleic acid molecules are simultaneously detected in the sample.", "35.The method of claim 28, wherein the mutually different nucleic acid molecules are comprised in at least two antibiotic resistance genes.", "36.The method of claim 35, wherein at least one of the antibiotic resistance genes is the gene for beta-lactamase blaZ, chloramphenicol acetyltransferase, fosB protein, adenin methylase ermC, aacA-aphD aminoglycoside resistance, 3′5′-aminoglycoside phosphotransferase aphA-3, mecR, penicillin binding protein PBP2′, aminoglycoside-3′-adenyltransferase aadA, tetracycline-resistance protein tetC, DHFR DfrA, or D-Ala:D-Ala ligase vanB.", "37.The method of claim 28, wherein the hybridizing reaction is carried out at 30-80° C. 38.The method of claim 37, wherein the hybridizing reaction is carried out at 40-70° C. 39.The method of claim 28, wherein the hybridizing reaction is carried out at 55-65° C. 40.The method of claim 28, wherein the hybridizing reaction is carried out under highly stringent conditions.", "41.The method of claim 28, wherein the multiplex PCR is carried out with labeled probes.", "25332285.1 42.The method of claim 28, further comprising the separation of “+” and “−” individual strands of the at least two mutually different amplified nucleic acid molecules prior to the hybridizing step.", "43.The method of claim 42, wherein “+” individual strands of the at least two mutually different amplified nucleic acid molecules that have sequences identical to the probes are separated after the amplifying step.", "44.The method of claim 43, wherein primers employed for the elongation of the “+” individual strands are coupled to a substance that ensures the separation of the “+” individual strands.", "45.The method of claim 44, wherein the primers are coupled to the substance at their 5′ termini.", "46.The method of claim 44, wherein the substance is at least one biotin molecule.", "47.The method of claim 46, wherein the “+” individual strands are separated by means of streptavidin bound to beads.", "48.The method of claim 28, further comprising purifying the at least two mutually different amplified nucleic acid molecules before the hybridizing step.", "49.The method of claim 48, wherein the purification step occurs before separation of “+” and “−” individual strands of the at least two mutually different amplified nucleic acid molecules.", "50.The method of claim 48, wherein the purification step occurs after separation of “+” and “−” individual strands of the at least two mutually different amplified nucleic acid molecules.", "51.A microarray adapted to, during use, hybridize to amplified portions of at least two mutually different amplified nucleic acid molecules, the microarray comprising at least two mutually different probes immobilized on the microarray, wherein each probe comprises a hybridizing sequence adapted to hybridize, during use, to the amplified portion of one of at least two mutually different amplified nucleic acid molecules to produce a mutually different hybridized amplified nucleic acid molecule having a melting temperature which differs from the melting temperature of one or more other mutually different hybridized amplified nucleic acid molecules by at most 2° C. 52.The microarray of claim 51, wherein each probe comprises a hybridizing sequence adapted to hybridize, during use, to the amplified portion of one of the at least two mutually different amplified nucleic acid molecules to produce a hybridized amplified nucleic acid molecule having a melting temperature which differs from the melting temperature of one or more other hybridized amplified nucleic acid molecule by at most 1° C. 53.The microarray of claim 51, further defined as comprising at least six probes.", "54.The microarray of claim 53, further defined as comprising at least twelve probes 55.The microarray of claim 51, wherein the probes are adapted to hybridize to amplified portions of mutually different nucleic acid molecules comprised in at least two antibiotic resistance genes.", "56.The microarray of claim 55, wherein at least one of the antibiotic resistance genes is the gene for beta-lactamase blaZ, chloramphenicol acetyltransferase, fosB protein, adenin methylase ermC, aacA-aphD aminoglycoside resistance, 3′5′-aminoglycoside phosphotransferase aphA-3, mecR, penicillin binding protein PBP2′, aminoglycoside-3′-adenyltransferase aadA, tetracycline-resistance protein tetC, DHFR DfrA, or D-Ala:D-Ala ligase vanB.", "57.The microarray of claim 51, wherein the probes are bound to the surface of the microarray in spots having a diameter of from 100 to 500 μm.", "58.The microarray of claim 57, wherein the probes are bound to the surface of the microarray in spots having a diameter of from 200 to 300 μm.", "59.The microarray of claim 58, wherein the probes are bound to the surface of the microarray in spots having a diameter of about 240 μm.", "60.The microarray of claim 57, wherein the spots are from 100 to 500 μm apart.", "61.The microarray of claim 60, wherein the spots are from 200 to 300 μm apart.", "62.The microarray of claim 61, wherein the spots are 280 μm apart.", "63.The microarray of claim 51, further defined as made of glass, a synthetic material, or a membrane.", "64.The microarray of claim 51, wherein the probes are covalently bound to the surface of the microarray.", "65.The microarray of claim 51, wherein the hybridizing sequences comprise 15 to 25 nucleotides.", "66.The microarray of claim 65, wherein the hybridizing sequences comprise 20 nucleotides.", "67.The microarray of claim 51, wherein the probes are bound to the microarray via dT sequences at their 5′ termini.", "68.The microarray of claim 51, wherein the hybridizing sequence of at least one probe comprises a sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.69.The microarray of claim 68, wherein the hybridizing sequence of each probe comprises a sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.70.A probe set comprising at least two mutually different probes, wherein each probe comprises a hybridizing sequence adapted to hybridize, during use, to an amplified portion of one of at least two mutually different amplified nucleic acid molecules to produce a hybridized amplified nucleic acid molecule having a melting temperature which differs from the melting temperature of one or more other mutually different hybridized amplified nucleic acid molecule by at most 2° C. 71.The probe set of claim 70, further defined as comprising at least six probes.", "72.The probe set of claim 71, further defined as comprising at least twelve probes.", "73.The probe set of claim 70, wherein the hybridizing sequences comprise 15 to 25 nucleotides.", "74.The probe set of claim 70, wherein the hybridizing sequences comprise 20 nucleotides.", "75.The probe set of claim 70, wherein the probes comprise dT sequences at their 5′ termini.", "76.The probe set of claim 70, wherein the hybridizing sequence of at least one probe comprises a sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.77.The probe set of claim 76, wherein the hybridizing sequence of each probe comprises a sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.78.The probe set of claim 70, wherein the probes are adapted to hybridize to amplified portions of mutually different nucleic acid molecules comprised in at least two antibiotic resistance genes.", "79.The probe set of claim 78, wherein at least one of the antibiotic resistance genes is the gene for beta-lactamase blaZ, chloramphenicol acetyltransferase, fosB protein, adenin methylase ermC, aacA-aphD aminoglycoside resistance, 3′5′-aminoglycoside phosphotransferase aphA-3, mecR, penicillin binding protein PBP2′, aminoglycoside-3′-adenyltransferase aadA, tetracycline-resistance protein tetC, DHFR DfrA, or D-Ala:D-Ala ligase vanB.", "80.A kit for simultaneously detecting at least two mutually different nucleic acid molecules in a sample, wherein the kit comprises: a microarray adapted to, during use, hybridize to amplified portions of at least two mutually different amplified nucleic acid molecules, the microarray comprising at least two mutually different probes immobilized on the microarray, wherein each probe comprises a hybridizing sequence adapted to hybridize, during use, to the amplified portion of one of at least two mutually different amplified nucleic acid molecules to produce a mutually different hybridized amplified nucleic acid molecule having a melting temperature which differs from the melting temperature of one or more other mutually different hybridized amplified nucleic acid molecules by at most 2° C.; and at least one container with primers for the specific amplification of nucleic acid molecules to be detected via multiplex-PCR during use.", "81.The kit of claim 80, further comprising a probe set comprising at least two mutually different probes, wherein each probe comprises a hybridizing sequence adapted to hybridize, during use, to an amplified portion of one of at least two mutually different amplified nucleic acid molecules to produce a hybridized amplified nucleic acid molecule having a melting temperature which differs from the melting temperature of one or more other mutually different hybridized amplified nucleic acid molecule by at most 2° C. 82.The kit of claim 80, further comprising at least one container comprising at least one nucleic acid molecule to be detected as a positive sample.", "83.The kit of claim 80, further comprising a container comprising streptavidin bound to beads." ], [ "The present invention relates to a method of simultaneously detecting at least two mutually different nucleic acid molecules in a sample, wherein in a first step a multiplex PCR and in a second step a hybridizing reaction is carried out with probes immobilized on a microarray, whereupon the hybridized PCR products are detected and optionally quantified, as well as a microarray and a set for hybridizing multiplex-PCR products, and a kit for the simultaneous detection of at least two mutually different nucleic acid molecules in a sample.", "The detection of nucleic acid molecules in a sample is carried out in the most various areas, e.g.", "in medicine, in quality check-ups, in research etc.", "Often it is necessary to detect at least two mutually different nucleic acid molecules, often 20, 50, 100 or more, in a sample.", "For reasons of time and costs it is desirable to detect the different nucleic acid molecules simultaneously in one sample.", "A series of publications relate to the detection of nucleic acid molecules and disclose various methods for carrying out the detection: In U.S. Pat.", "No.", "5,994,066, a method for detecting bacterial or antibiotic resistances, respectively, in biological samples is described.", "According to a first method, a multiplex-PCR is carried out for the simultaneous detection of several antibiotic resistances.", "As an example of the detection of the amplified products, agarose gel electrophoresis, fluorescence polarization and the detection by means of fluorescence labeling have been mentioned.", "A hybridization method is described as a further, second method of detecting the sequences searched for in samples, hybridization being carried out at 65° C., and the hybridization of a sample with the specific target DNA indicating a high degree of identity between the two nucleotide sequences.", "U.S. Pat.", "No.", "6,045,996 describes a method for hybridizing a nucleotide sequence on a microarray.", "Temperatures of between 20 and 75° C. are indicated as the hybridization temperature.", "As an example of target nucleotides, amplification products of a multiplex PCR are mentioned.", "According to U.S. Pat.", "No.", "5,614,388, specific nucleotide sequences are amplified by means of PCR, whereupon the amplification products are detected by hybridizing.", "As the preferred embodiment, a multiplex PCR is carried out.", "The detection may be specifically carried out by adjusting stringent conditions.", "As stringent hybridizing conditions, temperatures are stated which allow for a specific hybridization.", "As an example of a hybridizing temperature, 50 to 55° C. are indicated.", "U.S. Pat.", "No.", "5,846,783 relates to a method of detecting nucleotide sequences, wherein following a multiplex PCR, a detection by means of hybridizing is carried out.", "For example, the hybridization is carried out at a temperature of 55° C. WO 98/48041 A2 relates to a method for identifying antibiotic-resistant bacterial strains, wherein the genes are amplified via PCR and detected by means of hybridizing probes.", "In doing so, hybridizing is to be carried out under stringent conditions, such as 20° C. below the melting point of the hybridizing DNA.", "The oligonucleotides preferably are chosen such that they have similar melting temperatures and thus several genes in the same hybridizing mixture can be tested by the same conditions.", "As an example, furthermore, the hybridization on an oligonucleotide microarray is described.", "As the hybridizing temperature, a temperature of from 45 to 60° C. is indicated.", "However, all these above-mentioned methods have the disadvantage that there are limits as regards the specificity and the maximum number of nucleic acid molecules that are simultaneously detectable.", "In some of these methods, a multiplex PCR is carried out in a first step, whereby the simultaneous amplification of several nucleotide sequences is ensured.", "The subsequent detection of the various nucleotide sequences is, however, a problem, since according to this method it is not possible to simultaneously specifically detect a larger number of nucleotide sequences.", "If a hybridizing reaction is carried out after the PCR reaction, specific, stringent hybridizing conditions must be adjusted for each nucleotide sequence, a lower temperature being adjusted for shorter sequences than for longer sequences, cf.", "e.g.", "U.S. Pat.", "No.", "6,045,996, whereby, however, the possible number of simultaneously detectable nucleotide sequences decreases.", "In WO 98/48041 A2, it has, e.g., been suggested to select oligonucleotides which have similar melting temperatures so that several genes can be tested in the same hybridizing mixture, wherein, however, a maximum of eight oligonucleotides is tested on one array.", "Thus, these methods are not suitable to carry out methods for the detection of several or a large number of nucleic acid molecules, e.g.", "for the detection of antibiotic resistances.", "For such detection methods, a method which is restricted to a simultaneous detection of merely a few oligonucleotides is insufficient and too labor intensive and time-consuming in practice, in particular for screens.", "Therefore, the present invention has as its object to provide a method in which a large number of nucleic acid molecules can be detected simultaneously, so that a detection of certain oligonulceotides or genes, respectively, in a sample can be carried out quickly, cost-efficiently and with little work involved.", "The initially indicated method of the present invention is characterized in that the probes employed for the hybridizing reaction which in each case will hybridize specifically with the mutually different nucleic acid molecules have melting temperatures (Tm) which differ from each other by 2° C. at the most, preferably 1° C. at the most.", "By the fact that the melting temperatures of the probes used for the hybridizing reaction differ from each other by 2° C. at the most, or preferably, by 1° C., at the most, it has become possible for the first time to simultaneously detect a large number of nucleic acid molecules in one sample, since the same conditions as regards temperature and also salt concentration, pH, etc., will be adjusted for the hybridizing reaction for all the probes.", "The melting temperature Tm is defined as that temperature at which (under given parameters, such as, e.g., salt concentration), half of all the molecules will be in the helical state.", "It is possible to provide sequences with a certain melting temperature for nearly all nucleic acid molecules: One possible way of calculating the melting temperature of a sequence is by means of the commercial software “Gene Runner 3.0” (© 1994, Hastings Software, Inc.).", "This software allows the Tms to be determined by means of various methods/algorithms.", "The statements in the present patent application are values of the so-called “nearest-neighbor thermodynamic melting temperature”-method according to Breslauer et al.", "(Proc.", "Natl.", "Acad.", "Sciences 83: 3746-3750, Predicting DNA duplex stability from the base sequence).", "The parameters for the calculation may, e.g.", "be 660 mM for the salt concentration and 7.5 pM for the sample concentration.", "For determining the Tms of several probes for a simultaneous hybridizing experiment, it is not the absolute values (which may be higher or lower, depending on salt and DNA concentration) which are decisive, but the method chosen (i.e.", "for probes having a length of between 15 and 30 bases, the “thermodynamic one”) and the values for the Tms of the individual probes in relationship relative to each other.", "In this manner, the sequence to be hybridized, “hybridizing sequence”, for the nucleic acid molecules or genes, respectively, to be tested can be calculated and chosen so that specific probes therefor can be prepared.", "Within the scope of the present invention, by nucleic acid molecules, portions of sequences are to be understood which are, e.g., certain genes, parts of a gene or genome, an mRNA or parts of an mRNA, etc.", "By the term “multiplex-PCR” within the scope of the present invention, a PCR is to be understood in which simultaneously at least two mutually different nucleic acid molecules are amplified, i.e.", "that with the assistance of different primers, different sequences can be amplified simultaneously in one reaction.", "By “microarray” a carrier is to be understood on which a high number of probes are immobilized in high density so that under the same conditions, simultaneously a large number of nucleic acid molecules can be hybridized.", "Microarrays usually are used for the detection of DNA molecules, yet microarrays already are also being used for the detection of peptides.", "With the assistance of microarrays, the in vitro DNA-diagnosis has been substantially simplified so that complex tests can be carried out very rapidly in one single working step, since several thousands of specifically designed oligonucleotides can be immobilized on the relatively small microarrays.", "For instance, the hybridization on a microarray ensures the simultaneous examination of tens of thousands of genes.", "A series of different microarrays have already been used for the detection of nucleiotide sequences, the different parameters being chosen by the person skilled in the art in a wide range (cf.", "e.g., Lockhart et al., Nature Biotechnology, vol.", "14, December 1996, pp.", "1675-1679).", "Within the scope of the present invention, it is e.g., possible to adapt and vary the material, size, structure etc.", "of the microarray to the probes to be immobilized as regards the number, length and sequence thereof.", "On the one hand, it is possible to merely detect the nucleic acid molecules, i.e.", "to test whether or not they are present in a sample, and this test will yield a YES/NO result.", "According to the invention, however, it is also possible to quantify the amount of the nucleic acid molecules in the sample, and this can be carried out highly specifically because of the use of the microarrays.", "For this, any detection method known to the person skilled in the art may be used, e.g., chemical, enzymatic, physico-chemical or antigen-antibody binding processes may be employed.", "The nucleic acid to be detected can be labeled, e.g.", "with a radioactive, fluorescent or chemoluminescent molecule.", "These detection methods are very well known to the person skilled in the art and therefore need not be discussed here in detail, the choice of the respective method depending on the nucleic acid molecules to be detected and on whether the product is merely to be detected or to be quantified.", "The preparation of the probes is effected according to methods known per se.", "The less the melting temperatures of the probes differ from each other, the more specific the nucleic acid molecules can be detected since by this hybridization conditions can be adjusted which will merely ensure a highly specific hybridization, yet not a hybridization of not completely complementary sequences, whereby the risk of the falsely positive, but also of falsely negative results is lowered or completely eliminated.", "The primer and probes can be chosen such that nucleic acid molecules are amplified which have a sequence longer than the hybridizing sequence, i.e.", "that sequence which hybridizes with the probes.", "It is however, also possible that merely the hybridizing sequence is amplified, i.e.", "that the nucleic acid molecule only consists of that sequence with which the respective probes hybridize.", "Preferably, according to the invention at least 6, preferably at least 8, particularly preferred at least 12 nucleic acid molecules which differ from each other are simultaneously detected in a sample.", "The number of mutually different nucleic acid molecules detected in the sample will depend on the specific case, there being practically no upward limits.", "Particularly preferably, nucleic acid molecules are detected which are contained in antibiotic resistance genes.", "A large number of antibiotic resistance genes is known, the detection methods as a rule being carried out by long and error-prone microbiological growth tests on antibiotic-containing nutrient media and subsequent determination of the viable germs.", "Even though methods for the identification of antibiotic resistances with the assistance of gene amplifications and subsequent hybridizing have already been described (cf.", "WO 98/48041 A2), it has not been possible to test one sample for several antibiotic resistance genes simultaneously, without a reduction of the specificity.", "With the method according to the invention it has now become possible to detect an unlimited number of antibiotic resistance genes in a sample, which is of particular importance in the field of hospitals since an accumulation of antibiotic-resistant bacterial strains will occur there.", "All the standard DNA isolation methods are functional.", "In any event, it should be ensured that smaller molecules (such as plasmids, e.g.)", "are copurified so as not to lose episomally encoded resistances.", "As the nucleic acid molecules, parts of sequences from the antibiotic resistance genes are chosen which are specific of the respective gene and do not occur in other genes.", "In this manner, falsely positive test results can be even better prevented.", "Preferably, the antibiotic resistance genes are selected from the group consisting of genes for the beta-lactamase blaZ, chloramphenicol acetyltransferase, the fosB protein, the adenin methylase ermC, aacA-aphD aminoglycoside resistance, 3′5′-aminoglycoside phosphotransferase aphA-3, mecR, the penicillin binding protein PBP2′, the aminoglycoside-3′-adenyltransferase aada, the tetracycline-resistance protein tetc, DHFR DfrA and the D-Ala:D-Ala ligase vanB.", "These are frequently occurring antibiotic resistances which cause severe medical difficulties, and thus it is particularly important for these antibiotic resistances to provide a rapid and highly specific test method.", "It is particularly suitable if all these said antibiotic resistances can be tested simultaneously in one sample, i.e.", "that the nucleic acid molecules which are respectively specific of each of these antibiotic resistance genes are simultaneously amplified in a multiplex PCR and subsequently hybridize with probes on a microarray, wherein at least one probe each is specific for a nucleic acid molecule and thus, for an antibiotic resistance gene.", "It is particularly suitable if the hybridizing reaction is carried out at 30-80° C., preferably at 40-70° C., particularly preferred at 55-65° C. The hybridizing temperature to be adjusted is dependent on the melting temperature of the probes and, according to the invention, may be calculated and adjusted for each hybridizing reaction, it being particularly important that the temperature be held constant during the hybridizing reaction.", "It has been shown that it is particularly suitable for the present method to adjust temperatures of between 55 and 65° C., since in this temperature range probes have melting temperatures which are particularly well suited for the present method, in particular as regards specificity and length.", "For a particularly precise detection, it is advantageous if the hybridizing reaction is carried out under highly stringent conditions.", "This means that hybridizing conditions are adjusted which will ensure a hybridizing of highly complementary sequences, yet not of sequences which differ in a few nucleotides.", "It is particularly advantageous if hybridizing conditions are chosen under which only completely complementary sequences will bind to each other, yet not sequences which differ merely in one single nucleotide.", "In this manner, a method is provided which ensures a highly specific detection of nucleic acid molecules in a sample and which will not give false positive results.", "The highly stringent conditions are adjusted by choosing the temperature and ionic strength in the reaction mixture.", "For instance, the hybridizing temperature is adjusted to 5 to 100 below the melting temperature of the probes; the buffer(s) will be chosen according to the desired ionic strength or pH in dependence on the hybridizing temperature.", "Preferably, the multiplex-PCR is carried out with primers that are labeled.", "In this manner, it is ensured that the amplified PCR products will have a labeling that can be detected after the hybridizing reaction.", "As has already been described above, the labeling may consist in a molecule, a chemically, physico-chemically or enzymatically detectable signal, which can be determined and quantified, e.g., via a color reaction by measuring the fluorescence, luminescence, radioactivity etc.", "For a particularly specific method it is suitable if the hybridizing reaction is carried out after separation of the “+” and “−” strands.", "Thereby it is avoided that the strands which have a sequence identical to the probes will competitively bind with these probes to the individual strand molecules to be detected, which would lead to falsified results particularly in case of a quantitative detection.", "By separating the “+” and the “−” individual strands, merely the individual strands complementary to the probes will be present in the hybridizing mixture.", "In doing so, it is particularly advantageous if the “+” individual strands which have sequences identical to the probes are separated after the multiplex-PCR.", "In this manner, the “−” individual strands which have sequences complementary to the probes will remain in the hybridizing mixture so that the hybridizing reaction can be carried out immediately thereafter.", "A particularly advantageous separating procedure is characterized in that primers are used for the elongation of the “+” individual strands which, preferably at their 5′ terminus, each are coupled to a substance, in particular at least one biotin molecule, which ensures the separation of the “+” individual strands.", "In this manner, the “+” individual strands can be changed already in the amplification step of the PCR so that their complete separation will be specifically ensured without having to incorporate additional intermediate steps into the method.", "In this manner, the risk that also the “−” individual strands will be separated is eliminated.", "Biotin is particularly suitable since it can easily be coupled to a DNA sequence and can be separated specifically.", "For this purpose, it is particularly suitable if biotin molecules are coupled to the primers for the elongation of the “+” individual strands, the “+” individual strands being separated after the multiplex-PCR by means of streptavidin bound to beads.", "By means of the beads it is made possible that a large area of streptavidin is introduced into the sample, whereby the biotin molecules will completely bind to the streptavidin.", "Furthermore, by using the beads it is ensured that the streptavidin-biotin compound will be separated again from the sample.", "The beads used therefor are known per se and may, e.g., be made of glass or with a magnetic core, respectively.", "Preferably, a purification step precedes the hybridizing step.", "In this manner substances which possibly could interfere in the hybridization are removed from the hybridizing mixture, this purification step optionally occurring during or after the separation of the “+” individual strands.", "The purification may, e.g., be carried out by precipitation of the DNA and re-uptake of the DNA in a buffer.", "According to a further aspect, the present invention relates to a microarray for hybridizing multiplex-PCR products according to any one of the above-described inventive methods, wherein at least two, preferably at least six, particularly preferred at least twelve probes which each specifically hybridize with the mutually different nucleic acid molecules to be detected, are bound to its surface and have melting temperatures which differ from one another by 2° C. at the most, preferably by 1° C. at the most.", "As regards the microarray and the probes, the definitions already set out above for the method also apply here.", "Again, the number of probes bound to the microarray will depend on the number of the nucleic acid molecules to be detected, wherein, of course, also additional probes which do not hybridize with the nucleic acid molecules to be detected may be bound to the microarray as a negative test.", "What is important is, as has already been described above, that the melting temperatures of the probes differ from one another by merely 2° C. at the most, preferably by 1° C. at the most, whereby it is ensured that conditions can be adjusted for the hybridizing reaction under which all the nucleic acid molecules which have a sequence that is complementary to the probes will hybridize equally specifically and tightly with the probes.", "Preferably, the probes are bound to the surface of the microarray in spots having a diameter of from 100 to 500 μm, preferably from 200 to 300 μm, particularly preferred 240 μm.", "It has been found that spots having this diameter are particularly well suited for the above-described method according to the invention, a detection following the hybridizing reaction yielding particularly clear and unmistakable results.", "One spot each exhibits one type of probe, i.e.", "probes having the same sequence.", "It is, of course, also possible to provide several spots with the same type of probe on the microarray, as parallel tests.", "Furthermore, it is advantageous if the spots have a distance from each other of from 100 to 500 μm, preferably from 200 to 300 μm, particularly preferred 280 μm.", "In this manner it will be ensured that a maximum number of spots is provided on the microarray, it being possible at the same time to clearly distinguish in the detection procedure between the various spots and, thus, probes and bound nucleic acid molecules to be detected.", "Preferably, the microarray is made of glass, a synthetic material or a membrane, respectively.", "These materials have proven particularly suitable for microarrays.", "It is particularly suitable if the probes are covalently bound to the surface of the microarray.", "In this manner, a tight bond of the probes to the microarray will be ensured without a detachment of the probe-microarray bond and, thus, a falsified result occurring in the course of the hybridizing and washing steps.", "If the microarray is made of coated glass, e.g., the primary amino groups can react with the free aldehyde groups of the glass surface under formation of a Schiff's base.", "It has proven to be suitable if the probes have a hybridizing sequence comprising 15 to 25, preferably 20, nucleotides.", "By hybridizing sequence, as has already been described above, that sequence is to be understood with which the nucleic acid molecules to be detected will hybridize.", "Of course, the probes may be made longer than the hybridizing sequence, yet with the increase in the additional length of the probe, an undesired bond with other nucleic acid molecules could occur, which would falsify the result.", "Therefore, it is advantageous if the probes—besides the parts which are required for the binding to the surface of the microarray—merely consist of the hybridizing sequence.", "The length of from 15 to 25, preferably 20, nucleotides has proven suitable since in this length range it is possible to find hybridizing sequences with the above-described methods, which have the required melting temperature.", "This length is sufficient to allow for a specific binding and to eliminate the risk that also other DNA molecules by coincidence have the same sequence as the nucleic acid molecules to be detected.", "Preferably, the probes at their 5′ terminus each have a dT10 sequence via which they can be bound to the microarray.", "In this manner, the distance between the microarray and the hybridizing sequence will be sufficient so that the latter will be freely accessible to the nucleic acid molecules.", "The number of the Tm may, e.g., be from five to fifteen, preferably ten.", "For the simultaneous detection of antibiotic resistance genes it is suitable if as the hybridizing sequence, the probes comprise a sequence selected from the group consisting of No.", "25, No.", "26, No.", "27, No.", "28, No.", "29, No.", "30, No.", "31, No.", "32, No.", "33, No.", "34, No.", "35 and No.", "36.These sequences occur in antibiotic resistance genes which especially frequently occur in bacterial strains and are medically important.", "These are the antibiotic resistance genes for the beta-lactamase blaZ, chloramphenicol acetyltransferase, the fosB protein, the adenin-methylase ermC, aacA-aphD aminoglycoside resistance, 3′5′-aminoglycoside phosphotransferase aphA-3, mecR, the penicillin binding protein PBP2′, the aminoglycoside-3′-adenyltrasnferase aada, the tetracycline-resistance protein tetC, DHFR DfrA and the DAla:D-Ala ligase vanB and have melting temperatures which differ from one another by about 1° C. at the most.", "According to a further aspect, the present invention relates to a set for hybridizing multiplex-PCR products according to any one of the above-described methods of the invention, which set comprises at least two, preferably six, particularly preferred at least twelve probes, each specifically hybridizing with the mutually different nucleic acid molecules to be detected and having melting temperatures that differ from each other (i.e.", "from the respective other probe molecules/detected nucleic acid pairs in the set) by 2° C. at the most, preferably by 1° C. at the most.", "The probes may be dissolved in a buffer.", "Furthermore, the set may comprise several containers, probes with the same sequence being present per container.", "By this it will be possible to apply probes of the same sequence on the microarray per spot.", "It is, of course, also possible to provide probes with two or more sequences that differ from each other in one container.", "Preferably, the probes have a hybridizing region comprising 15 to 25, preferably 20, nucleotides.", "Furthermore, it is suitable if the probes each have a dT sequence at their 5′ terminus, the number of the Tm preferably being between 5 and 15, e.g.", "10.It is particularly advantageous if the probes in their hybridizing region each have a sequence which is selected from the group consisting of No.", "25, No.", "26, No.", "27, No.", "28, No.", "29, No.", "30, No.", "31, No.", "32, No.", "33, No.", "34, No.", "35 and No.", "36.According to another aspect, the present invention relates to a kit for simultaneously detecting at least two mutually different nucleic acid molecules in a sample, the kit comprising a microarray according to the invention, as described above, at least one container with primers for the specific amplification of the nucleic acid molecules to be detected and optionally, a set according to the invention as described above.", "A container may comprise primers with the same sequence, but also a primer pair for amplification of a nucleic acid molecule, or finally also several primer pairs for the amplification of several mutually different nucleic acid molecules, wherein, however, the primers should be present at a certain concentration.", "The kit may, of course, also further comprise user's instructions with a protocol for carrying out the above-described inventive method, as well as possible further buffers, salts, solutions etc.", "which are necessary for the amplification reaction, hybridizing reaction, and detection, respectively.", "The microarray may comprise probes already immobilized thereon.", "The set comprising the probes may be present separate from the microarry (in case that the microarray is blank, i.e.", "that it does not contain any bound probes), yet it may also be an integrated component of the microarray.", "Preferably, the kit further comprises at least one container with at least one nucleic acid molecule to be detected, as positive sample.", "Also here, a container again may comprise nucleic acid molecules with the same sequence, it being possible that several containers are provided in the kit, yet it is also possible to provide nucleic acid molecules with several, mutually different sequences in one container.", "For instance, if the kit is provided for the detection of antibiotic resistance genes, the kit may provide nucleic acid molecules with the sequences with the hybridizing sequence SEQ ID No.", "25 to SEQ ID No.", "36, as a positive sample.", "It is particularly suitable if the kit further comprises a container with streptavidin bound to beads.", "This allows for a separation of the amplified “+” and “−”individual strands, if the “+” or “−” individual strand is coupled to biotin, e.g.", "by using primers coupled to biotin.", "In the following, the invention will be explained in more detail by way of the example given as well as by way of the figures to which, however, it shall not be restricted.", "FIG.", "1 shows the separation of the PCR products of all twelve ABR targets by means of gel electrophoresis; FIG.", "2 shows the microarray layout of the ABR chip; FIG.", "3 shows the diagram of the test course; FIG.", "4 shows an illustration of the control hybridization on the ABR chip; and FIG.", "5 shows the result of the ABR chip detection after the multiplex amplification.", "EXAMPLE 1.Gene Synthesis of Reference “ABR Targets” A series of antibiotic resistance (ABR) sequences was prepared by gene synthesis in vitro, since either “type strains” were not available or working with the organisms in question was not possible for safety reasons (bio-safety level 2 or higher).", "In Table 1 all the targets are summarized and provided with a number (No.", "), the control resistances being derived from vectors and primarily serving to validate the chip.", "For these targets, probes are provided on the ABR chip prototype.", "TABLE 1 Antibiotic Target No.", "Resistance Species (resistance gene) 8 Ampicillin S. aureus, E. faecalis beta-lactamase blaZ (control) 9 Chloram- Bacillus sp., Chloramphenicol acetyl- phenicol Corynebacterium sp.", "transferase (control) 11 Fosformycin S. epidermidis, fosB protein Staphylococcus sp.", "7 Erythromycin Staphylococcus sp.", "Adenin methylase ermC 12 Gentamycin S. aureus aacA-aphD Aminoglyco- side resistance gene 2 Kanamycin S. aureus, S. faecalis, 3′5′-Aminoglycoside E. faecalis phosphotransferase aphA-3 3 Methicillin S. aureus mecR 1 Penicillin S. aureus Penicillin binding protein PBP2′ 5 Streptomycin, Salmonella Aminoglycoside-3′- Spectinomycin adenyltransferase aadA 10 Tetracycline Salmonella sp.", "Tetracycline resistance (control) protein tetC 4 Trimethoprim S. aureus DHFR DfrA 6 Vancomycin- Enterococcus, D-Ala:D-Ala ligase vanB VanB-Type Streptococcus Table 2 gives the sequences of the PCR primers and the lengths of the PCR products which were developed for the prototype, in FIG.", "1 all 12 PCR products after agarose gel electrophoresis can be seen.", "TABLE 2 No.", "Name PCR Primer (SEQ ID No.)", "PCR Product 1 PBP2 1 + 2 423 bp 2 KanR 3 + 4 532 bp 3 MecR 5 + 6 517 bp 4 DhfrA 7 + 8 279 bp 5 StrR 9 + 10 549 bp 6 VanB 11 + 12 498 bp 7 MlsR 13 + 14 564 bp 8 AmpR 15 + 16 219 bp 9 CmR 17 + 18 247 bp 10 TetR 19 + 20 245 bp 11 FosB 21 + 22 304 bp 12 AacA 23 + 24 497 bp 2.Microarray Design For each available (PCR) nucleic acid molecule of the antibiotic resistance (ABR) genes in question, highly specific DNA probes were located by means of bioinformatic standard methods.", "Generally, the software “Gene Runner 3.0” (© 1994, Hastings Software, Inc.) was used, for the PCR and Hyb Primer selection “Primer 3”, Steve Rozen & Helen J. Skaletsky (1998) Primer 3 was used, for homology search and database cross-checks “Fasta3”, W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448, W. R. Pearson (1990), “Rapid and Sensitive Sequence Comparison with FASTP and FASTA” Methods in Enzymology 183:63-98 was used, for alignments “ClustalX 1.8”, Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G. (1997), The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.", "Nucleic Acids Research, 24:4876-4882, was used.", "Particular attention was paid to the fact that potential cross-hybridizations with other possible ABR targets can be excluded.", "Extensive EMBL and GenBank database searches were employed so as to make sure that the respective probes do not allow hybridizations in error with “foreign” sequences.", "The probes are localized in A/T rich regions of the PCR fragments so as to ensure optimum conditions during hybridization with dsPCR products.", "Optimum conditions in this instance mean that hybridizations are generally more efficient if the probe “recognizes” a region in the dsDNA which denatures more easily (because, e.g., in a region richer in A/T.", "Each probe has a Ts value of 65° C.±1 and has an extra dT10 sequence at the 5′ terminus as a spacer between the chip surface and the hybridizing sequence (cf.", "Table 3).", "All the oligonucleotides were synthesized with a 5′ (CH2)6—NH2 modification and purified by means of a reversed phase chromatography HPLC protocol.", "The probes are adjusted to a concentration of 1 mM and stored at −20° C. in MT plates.", "TABLE 3 Sequence (SEQ ID No.", "Name No.)", "Tm 1 PBP2 25 64.8° C. 2 KanR 26 65.1° C. 3 MecR 27 64.8° C. 4 DhfrA 28 65.5° C. 5 StrR 29 63.7° C. 6 VanB 30 64.4° C. 7 MlsR 31 64.9° C. 8 AmpR 32 65.4° C. 9 CmR 33 64.9° C. 10 TetR 34 65.9° C. 11 FosB 35 64.2° C. 12 AacA 36 65.0° C. Co BSreverse 37 65.9° C. hCo AT-M33 38 65.3° C. 3.Array Layout The probes are covalently bound to the glass surface, face, and in doing so, the 5′ primary amino groups react with free aldehyde groups of the glass surface under formation of a Schiff's base (“Silylated Slides”, CEL Associates).", "The probes were applied to the glass carriers by means of a spotter (Affymetrix 417 Arrayer).", "In doing so, the spotting protocols were optimized for a good reproducibility and spot consistence.", "Spotting was effected in 3×SSC 0.1% SDS with hits/dot.", "The spots have a diameter of approximately 240 μm and are applied on the microarray with a spotto-spot distance of 280 μm.", "There exist two replicas for each spot.", "For validating the chip, control probes (Bluescript polylinker sequence) are applied in a typical pattern (“guide dots”) and negative controls (blank values, so-called “buffer dots”).", "FIG.", "2 shows an array layout of the ABR chip.", "The position of the 12 ABR targets is denoted with the respective numbers (No.).", "“Guide dots” are black, “buffer dots” are white, the position of the heterologous controls is marked in gray.", "4.Chip Validation and Control Hybridization The hybridizing conditions are mainly optimized on the microarray with the help of the control probe set.", "Six spots on the microarray contain a control probe with a BS polylinker-specific sequence.", "Hybridization was carried out in a 7 μl volume with a 3′ terminal Cy5-dCTP labeled oligonucleotide (BSrevco, 5′ AAGCTCACTGGCCGTCGTTTTAAA SEQ ID No.", "39) in SSARC buffer under a 15×15 mm (2.25 mm2) cover slip for 1 hour at 55° C. The chip was washed according to standard protocols (2×SSC 0.1% SDS, then 0.2×SSC 0.1% SDS, then with 0.2×SSC and finally with 0.1×SSC, 2 min each).", "Then the glass carrier was scanned in a confocal fluorescence scanner (Affymetrix 418 Array Scanner) with a suitable laser output and suitable PMT voltage adjustments.", "In FIG.", "3, the control hybridization on the ABR chip is shown.", "The result of a total of 12 individually effected hybridizing experiments with one of the specific targets each (No.", "1 to no.", "12) with the “guide dot” controls (from left to right in each case No.", "1 to No.", "3, No.", "4 to No.", "6, No.", "7 to No.", "9 and No.", "10 to No.", "12) can be seen.", "5.Pilot Studies with the ABR Chip Prototype In a first functional test of the ABR chip, two different sample mixtures (“MixA” and “MixB”) of three different ABR targets each and two control targets (Ampicillin No.", "8 and Tetracycline No.", "10) were prepared: “MixA” contained kanamycin (aphA-3 No.", "2), trimethroprim (dhfrA No.", "4) and gentamycin (aacA No.", "12) in addition to the control targets, “MixB” contained vancomycin (vanB No.", "6), erythromycin (ermC No.", "7) and fosfomycin (fosB No.", "11) in addition to the control targets.", "The synthetic templates were used so as to allow for as exact an adjustment of the template amounts as possible.", "The multiplex amplification was carried out under standard PCR conditions in 35 cycles, wherein the primers for the amplification of the “+” individual strands which are identical to the probes were coupled to biotin molecules.", "The primers for the “−” individual strands which have sequences complementary to the probes were coupled to marker molecules 5′Cy-5: The reaction formulation was purified, individual strands were isolated by means of alkaline denaturing on dynabeads and hybridized in SSARC buffer for 1 hour at 55° C. on the ABR prototype arrays (cf.", "FIG.", "4): A) PCR (Controls in the Individual Formulation) 25 μl Volume: 1.0 μl template (3 fmol/μl) 1.0 μl primer plus (25 μM) 5′-biotin [VBC-GENOMICS] 1.0 μl primer minus (25 μM) 5′-Cy5 [VBC-GENOMICS] 2.5 μl HotStar™ buffer (10×) [Qiagen] 0.5 μl dNTPs (10 mM) [Roche] 0.1 μl HotStar™ Taq DNA polymerase [Qiagen] ad 25 μl with aqua bidest.", "Cycling: 15 min 95° C. 30×[30 sec 95° C. 20 sec 60° C. 40 sec 72° C.] 10 min 72° C. Purification of the PCR formulation by means of QIA-quick™ PCR-Purification Kit B) Multiplex-PCR 50 μl Volume: ×μl template (×fmol/μl) 6.0 μl primer “cocktail” plus (je 25 μM) 5′-biotin [VBC-GENOMICS] 6.0 μl primer “cocktail” minus (je 25 μM) 5′-Cy5 [VBC-GENOMICS] 5.0 μl HotStar™ buffer (10×) [Qiagen] 1.0 μl dNTPs (10 mM) [Roche] 0.2 μl HotStar™ Taq DNA polymerase [Qiagen] ad 50 pl with aqua bidest.", "Cycling: 15 min 95° C. 35×[30 sec 95° C. 20 sec 55° C. 40 sec 72° C.] 10 min 72° C. Purification of the PCR formulation by means of QIAquick™ PCR Purification Kit C) Single-Strand Isolation Wash 20 μl Dynabeads (10 μg/μl) [Roche] 2× with 200 μl 1×TS buffer take up in 8 μl 6×TS buffer incubate with 40 μl of the PCR formulation for 30 min at 37° C. wash Dynabeads 2× with 200 μl 1×TS buffer denature DNA 2× with 20 μl 0.2 N NaOH for 5 min at RT precipitate with 120 μl 90% EtOH/0.3 M NaOAC (20 min −20° C., 30 min at 16,500 rpm 4° C.) wash pellet with 70% EtOH, dry and take up in 14 μl SSARC buffer D) Hybridizing Denature 7 μl of hybridizing sample in SSARC buffer with 0.1 μl BSrevco-Cy5 (1 μM) for 3 min at 98° C. and put on ice immediately hybridize for 1 h at 55° C., under a 15×15 mm cover slip Wash slide (2×SCC 0.1% SDS 5 min RT, 0.2×SSC 5 min RT, 0.2×SSC 5 min RT, 0.1×SSC 2 min RT) scan slide E) Buffer TS buffer: 100 mM Tris-Cl, pH 7.6, 150 mM NaCl; autoclaved SSARC buffer: 4×SSC, 0.1% (w/v) Sarkosyl; 0.2 μm filtered SSC buffer: 20×: 3 M NaCl, 0.3 M trisodium citrate (dihydrate), pH 7.0 The chips were washed and scanned.", "FIG.", "5 shows a multiplex amplification and subsequent ABR chip detection of two different synthetic targets and two control targets, “MixA” on the left, “MixB” on the right.", "“False color” images of the fluorescent scan can be seen, each under the negative with the correct allocation of the ABR target.", "As can be seen in FIG.", "5, a clear allocation of the correct target is possible in the two different sample mixtures.", "This shows that the simultaneous detection of 12 nucleic acid molecules according to the method of the invention yields unambiguous results." ] ]	Patent_10469713
[ [ "Ligand sensing fluorescent acetylcholinesterase for detection of organophosphate activity", "Disclosed are methods for the preparation and use of labeled AChE and labeled AChE inhibitory conjugate compositions for detecting accumulation of toxic materials such as organophosphates, insecticides, and other nerve agents.", "Also disclosed are methods for the use of labeled AChE and labeled AChE inhibitory conjugate compositions in a variety of areas, including the detecting of toxic materials in biological samples, in the area of food and water analysis, in environmental monitoring, and in industrial settings." ], [ "1.An in vitro method for identifying an acetylcholinesterase (AChE) cognate partner comprising; a) contacting a sample suspected of containing a cognate partner with a labeled AChE and b) measuring accumulation of a conjugate between the AChE and the partner, wherein detection of the conjugate is indicative of the presence of the cognate partner.", "2.The method of claim 1, wherein the cognate partner is selected from the group consisting of a ligand, a modulator and an inhibitor of AChE.", "3.The method of claim 2, wherein the inhibitor is a carbamylating inhibitor or a phosphorylating inhibitor.", "4.The method of claim 3, wherein the inhibitor is a nerve toxin.", "5.The method of claim 4, wherein the inhibitor is selected from the group consisting of insecticides and organophosphates.", "6.The method of claim 1, wherein the AChE is labeled with a fluorophore.", "7.The method of claim 6, wherein AChE comprises at least one fluorophore.", "8.The method of claim 7, wherein at least one label is peripheral to the active center of the AChE.", "9.The method of claim 6, wherein the fluorophore shows a Stokes' shift upon binding of ligand, modulator or inhibitor to the labeled enzyme.", "10.The method of claim 1, further comprising measuring the ratio of conjugated to unconjugated AChE.", "11.The method of claim 10, wherein the measuring is by chromatography or spectroscopy.", "12.The method of claim 11, wherein chromatography comprises separation of conjugated and unconjugated AChE by capillary electrophoresis.", "13.The method of claim 11, wherein spectroscopy comprises detecting conjugated and unconjugated AChE by fluorescence.", "14.The method of claim 6, wherein the fluorophore is selected a dimethoxyphosphoryl compound or a diethoxyphosphoryl compound.", "15.A device for measuring accumulation of a conjugate between acetylcholinesterase (AChE) and a cognate partner comprising labeled AChE, wherein at least one site of the AChE is labeled peripheral to the active site of the enzyme.", "16.The device of claim 15, wherein the cognate partner is selected from the group consisting of a ligand, a modulator and an inhibitor of AChE.", "17.The device of claim 15, wherein the label is a fluorophore.", "18.The device of claim 17, wherein the fluorophore shows a Stokes' shift upon binding of a ligand, modulator or inhibitor to AChE.", "19.The device of claim 15, wherein the AChE is compartmentalized in a mobile or stationary phase.", "20.The device of claim 19, wherein the mobile phase is a suspension.", "21.The device of claim 19, wherein the stationary phase is a chip.", "22.The device of claim 21, wherein the AChE is covalently or non-covalently immobilized on the stationary phase.", "23.The device of claim 15, wherein the AChE can detect multiple AChE cognate partners.", "24.The device of claim 15, wherein the cognate partner is a nerve toxin.", "25.The device of claim 24, wherein the nerve toxin is selected from the group consisting of insecticides and organophosphates.", "26.A labeled acetylcholinesterase (AChE) molecule comprising at least one fluorophore present at the periphery of the active site of AChE, wherein the at least one site is selected from the group consisting of residues 76, 81, 84, 124, 262 and 287 of AChE or equivalents thereof.", "27.The labeled AChE of claim 26, wherein the fluorophore shows a Stokes' shift upon binding of a ligand to the labeled enzyme.", "28.The labeled AChE of claim 26, wherein the enzyme is recombinantly produced.", "29.The labeled AChE of claim 28, modified by mutagenesis or evolution methods, wherein the modification comprises substitution of one or more selected amino acid residues with cysteine residues.", "30.The labeled AChE of claim 26, wherein the fluorophore is a dimethoxyphosphoryl compound or a diethoxyphosphoryl compound.", "31.The labeled AChE of claim 30, wherein the enzyme is conjugated to an inhibitor.", "32.The labeled AChE of claim 31, wherein the conjugate is detectable by antibody.", "33.A conjugate comprising a labeled acetylcholinesterase (AChE) molecule, an inhibitor of AChE and an antibody.", "34.A method for evaluating the presence of an inhibitor of acetylcholinesterase (AChE), wherein the inhibitor is selected from the group consisting of carbamates and organophosphates, comprising the steps of: a) providing a biological sample; b) contacting the sample with a labeled AChE molecule comprising at least one fluorophore present at the periphery of the active site of the enzyme under conditions sufficient for binding of an inhibitor to the enzyme; and c) measuring accumulation of a conjugate between the AChE and the inhibitor.", "35.The method of claim 34, wherein the biological sample is selected from the group consisting of an integumentary system sample, sputum, feces, blood, urine, plasma, lacrimal secretions, cerumen, and semen.", "36.The method of claim 35, wherein the inhibitor is a carbamylating inhibitor.", "37.The method of claim 35, wherein the inhibitor is a phosphorylating inhibitor.", "38.The method of claim 34, wherein the inhibitor is a nerve toxin.", "39.The method of claim 34, wherein in the fluorophore shows a Stokes' shift upon binding of a ligand, modulator or inhibitor to the labeled enzyme.", "40.The method of claim 34, further comprising measuring the ratio of conjugated to unconjugated AChE.", "41.The method of claim 40, wherein the measuring is by chromatography or spectroscopy.", "42.The method of claim 41, wherein chromatography comprises separation of conjugated and unconjugated AChE by capillary electrophoresis.", "43.The method of claim 41, wherein spectroscopy comprises detecting conjugated and unconjugated AChE by fluorescence.", "44.The method of claim 34, comprising a labeled AChE, wherein the fluorophore is a dimethoxyphosphoryl compound or a diethoxyphosphoryl compound.", "45.A method for evaluating the presence of an inhibitor of acetylcholinesterase (AChE), wherein the inhibitor is selected from the group consisting of carbamates and organophosphates, comprising the steps of: a) providing a non-biological sample; b) contacting the sample with a labeled AChE molecule comprising at least one fluorophore present at the periphery of the active site of the enzyme under conditions sufficient for binding of an inhibitor to the enzyme; and c) measuring accumulation of a conjugate between the AChE and the inhibitor.", "46.The method of claim 45, wherein the non-biological sample is selected from the group consisting of soil, water, air and non-biological surfaces.", "47.The method of claim 46, wherein the inhibitor is a carbamylating inhibitor.", "48.The method of claim 46, wherein the inhibitor is a phosphorylating inhibitor.", "49.The method of claim 45, wherein the inhibitor is a nerve toxin.", "50.The method of claim 45, wherein in the fluorophore shows a Stokes' shift upon binding of an inhibitor to the labeled enzyme.", "51.The method of claim 45, comprising measuring the ratio of conjugated to unconjugated AChE.", "52.The method of claim 51, wherein the measuring is by chromatography or spectroscopy.", "53.The method of claim 52, wherein chromatography comprises separation of conjugated and unconjugated AChE by capillary electrophoresis.", "54.The method of claim 52, wherein the spectroscopy comprises detecting conjugated and unconjugated AChE by fluorescence.", "55.The method of claim 45, wherein the fluorophore is a dimethoxyphosphoryl compound or a diethoxyphosphoryl compound.", "56.A kit comprising: a) a labeled acetylcholinesterase (AChE) molecule comprising at least one fluorophore present at the periphery of the active site of AChE and b) a container comprising the labeled enzyme.", "57.The kit of claim 56, wherein the container is selected from the group consisting of a glass vial, a glass jar, a plastic pack, a plastic tube, glass tube, a metal tube, a foil pouch and a plastic pouch.", "58.The kit of claim 56, wherein the labeled AChE is immobilized on a microtitre plate or a chip.", "59.The kit of claim 56, further comprising: c) a control sample.", "60.The kit of claim 59, wherein the control sample is a negative control sample.", "61.The kit of claim 59, wherein the control sample is a positive control sample." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention This invention relates generally to the field of detecting hazardous chemicals and chemical analysis, to include portable automatic sensing devices.", "More particularly, the present invention relates to methods, compositions, devices and kits thereof useful in the detection of chemical agents, insecticides and other acetylcholinesterase (AChE) inhibitors, modifiers and ligands.", "2.Background Information Acetylcholinesterase (AChE), a serine hydrolase in the α/β-fold hydrolase protein superfamily, terminates nerve signals by catalyzing hydrolysis of the neurotransmitter acetylcholinesterase at a diffusion-limited rate.", "A number of nerve toxins, including insecticides and organophosphates, act through binding to and inhibiting AChE.", "Organophosphorus and organosulfur compounds, are used extensively in insecticides and are highly toxic to many organisms including humans.", "Insecticide residues are found in soil and groundwater, and the detection of these residues is important for their elimination from the environment and to protect the health of both humans and animals.", "Organophosphorus compounds are also used in nerve agents, such as sarin, phosphine, soman, and tabun, for chemical warfare purposes.", "These agents are some of the most potent toxic agents and are specific inhibitors of acetylcholinesterase (AChE).", "Acetylcholine is an essential neurotransmitter that affects parasympathetic synapses (autonomic and CNS), sympathetic preganglionic synapses, and the neuromuscular junction (see, e.g., Taylor et al., in Basic Neurochemistry, 5th ed., 1993, (Siegal et al., eds.", "), Chapter 11, pp.", "231-260, Raven Press, New York, N.Y.).", "Hydrolysis of acetylcholine by acetylcholinesterase, present in nervous tissue, normally limits the duration of action function.", "Organophosphate (e.g., Malathion, Parathion, Diasinon, Dursban) and carbamate (e.g., Sevin, Furadan) insecticides exert their toxicity by inhibiting the action of acetylcholinesterase and thereby causing a pronounced cholinergic response (Arron et al., Insecticides: Organophosphate and Carbamates in Goldfrank's Toxicologic Emergencies, 1994, (Goldfrank et al., eds.", "), Appleton & Lange, Norwalk, Conn.).", "Enzyme inhibition is the consequence of phosphorylation (organophosphates) or carbamylation (carbamates) of the cholinesterase-active site serine residue.", "The resulting phosphoroyl-serine bond is stable; therefore, enzyme inhibition is physiologically irreversible, whereas the carbamyl-serine bond undergoes spontaneous hydrolysis with regeneration of enzyme activity (24-48 h).", "For this reason and because of poor CNS penetration, carbamate insecticide neurotoxicity is less severe and of shorter duration than that for the organophosphates ( Tietz Textbook of Clinical Chemistry, 1999, (Burtis et al., eds.", "), W. B. Saunders Company, Philadelphia, Pa.).", "Excess synaptic acetylcholine stimulates muscarinic receptors (peripheral and CNS) and stimulates but then depresses and paralyzes nicotinic receptors.", "The CNS neurotoxic effects include restlessness, agitation, lethargy, confusion, slurred speech, seizures, coma, cardiorespiratory depression, or death.", "The need for the reliable determination of these cholinesterase inhibitors has led to the development of a number of sophisticated instrumental methods, mostly involving the use of gas and liquid chromatography and mass spectrometry.", "Also a number of liquid phase chemiluminescence procedures have been developed for the determination of inorganic and organic species mostly utilizing the luminol and peroxyoxalate reactions.", "See Robards K. and Worsfold P. J., Anal Chem Acta (1992) 266:147.These traditional methods are not practical for individual use as the methods are time consuming and complicated and the instruments utilized are expensive, non-portable and require high maintenance.", "Additionally, the measurement of nerve agents in mixtures with these traditional methods requires cumbersome extraction and manipulation procedures.", "Thus, biosensors were developed as an alternative to the traditional gas and liquid chromatography and mass spectrometry technology.", "Generally, biosensors include those which are enzyme-based and bioaffinity-based.", "An enzymatic biosensor uses an enzymatic or metabolic process to detect a reaction product which occurs between an incoming substrate and an immobilized enzyme.", "A bioaffinity sensor relies on a biological binding event of a target substance.", "Many existing methods for the detection of organophosphates and cumulative inhibition of cholinesterases lack sensitivity since they are based on inhibition of basal activities rather than accumulation of the inhibitory conjugate.", "Basal activities vary substantially between subjects resulting in inconsistency in present assays.", "Existing monitoring methods routinely require expensive laboratory procedures involving sample transport or preparations of samples for assay.", "Rapid analysis of toxic materials in the areas of food and water analysis, environmental monitoring, and in industrial settings is a problem that continues to exist and is currently addressed by time-consuming, expensive methods or by techniques that may be described as inadequate.", "Many problems associated with exposure to toxic materials could be avoided or minimized by a detection procedure which gives near “real-time” indication of the presence of toxic gases.", "Equally important are the characteristics of economy, small size, and ease of use for the successful application of such devices.", "Accordingly, there is a need for a method of detecting, quantifying, and evaluating hazards which provides for early detection and which can detect low levels of toxic materials.", "The present invention satisfies this need, as well as others." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention overcomes one or more of the drawbacks in the prior art by providing compositions and methods for their use in the detection of specific inhibitors, modifiers, or ligands of acetylcholinesterase (AChE).", "Disclosed are methods for the preparation and use of labeled AChE and labeled AChE inhibitory conjugate compositions which are useful in detecting accumulation of toxic materials, which include but are not limited to, organophosphates, insecticides, nerve agents, such as sarin, phosphine, soman, and tabun, and other materials used for chemical warfare purposes.", "Also disclosed are methods for the use of labeled AChE and labeled AChE inhibitory conjugate compositions in a variety of areas, including the detecting of toxic materials in biological samples, the areas of food and water analysis, environmental monitoring, and in industrial settings.", "Embodiments are disclosed which describe methods for making and using labeled AChE and labeled AChE inhibitory conjugate compositions comprising fluorescing compounds, including but not limited to, dimethoxyphosphoryl and diethoxyphosphoryl labels.", "In one embodiment of the invention, methods are disclosed for measuring accumulation of inhibitory conjugates comprising labeled AChE and an inhibitor, modulator or ligand (i.e., cognate partner).", "In a related aspect, such methods may comprise contacting a sample suspected of containing such cognate partners with labeled AChE in order for labeled AChE binding to occur between cognate partners in the sample and the enzyme.", "In a further related aspect, conjugated and unconjugated AChE may be separated by chromatographic methods.", "For example, such chromatographic methods may include, but are not limited to, capillary electrophoresis.", "In another embodiment, the conjugated and unconjugated, labeled AChE are detected and differentiated by fluorochromic emission shift, where conjugated AChE shows a detectable shift in emission signal.", "In a related aspect, the shift in emission is a Stokes' shift upon conjugate formation.", "In a related aspect, the cognate partner is an inhibitor, where the inhibitor may be designated as a carbamylating inhibitor or a phosphorylating inhibitor.", "In a further related aspect, the inhibitor may be an insecticide or an organophosphate.", "In another related aspect, the presence of the cognate partner is estimated by determining the ratio of conjugated to unconjugated AChE.", "In another embodiment, the sample may be obtained from the atmosphere, soil, water, industrial sites or environmental sites.", "Further, the sample may be biological or non-biological.", "In a related aspect, a biological sample may include, but is not limited to, the integumentary system, sputum, feces, blood, urine, plasma, lacrimal secretions, cerumen, and semen.", "In one embodiment, the AChE is labeled on at least one site.", "In a related aspect, the AChE is labeled at multiple sites.", "In a further related aspect, the at least one label is peripheral to the active center of the AChE.", "In one embodiment, a device comprising AChE is envisaged, where the AChE is compartmentalized in a mobile or stationary phase.", "In a related aspect, the stationary phase is a chip.", "In another related aspect, the mobile phase is a suspension.", "In one embodiment, the device is a biosensor for analyzing a sample for at least one organophosphorous, nerve agent and/or insecticide, where at least one enzyme is immobilized on or within the device.", "In a related aspect, the immobilized enzyme is either covalently or non-covalently bound to the device.", "In a further related aspect, the biosensor is divided into multiple zones, where each zone differentiates between one or more organophosphorous, nerve and/or insecticide agents.", "In another embodiment, the present invention also provides kits which contain the present labeled AChE for use in the present identification method.", "In another embodiment, a labeled AChE composition comprising at least one fluorophore located peripherally to the active center of the AChE, where the at least one fluorophore possesses an emission signal that shows a Stokes' shift upon conjugate formation.", "In a related aspect, the fluorophore comprises a dimethoxyphosphoryl label or a diethoxyphosphoryl label.", "In another embodiment, a labeled AChE molecule comprises at least one fluorophore on at least one site present at the periphery of the active site of the enzyme, wherein the at least one site is selected from the group consisting of residues 76, 81, 84, 124, 262 and 287 of AChE or equivalents thereof.", "Exemplary methods and compositions according to this invention, are described in greater detail below." ], [ "This invention was made in part with government support under Grant Nos.", "R37-GM18360 and 17-1-8014 awarded by the United States Public Health Service (USPHS) and the Department of the Army Medical Defense Command, respectively.", "The government may have certain rights in this invention.", "BACKGROUND OF THE INVENTION 1.Field of the Invention This invention relates generally to the field of detecting hazardous chemicals and chemical analysis, to include portable automatic sensing devices.", "More particularly, the present invention relates to methods, compositions, devices and kits thereof useful in the detection of chemical agents, insecticides and other acetylcholinesterase (AChE) inhibitors, modifiers and ligands.", "2.Background Information Acetylcholinesterase (AChE), a serine hydrolase in the α/β-fold hydrolase protein superfamily, terminates nerve signals by catalyzing hydrolysis of the neurotransmitter acetylcholinesterase at a diffusion-limited rate.", "A number of nerve toxins, including insecticides and organophosphates, act through binding to and inhibiting AChE.", "Organophosphorus and organosulfur compounds, are used extensively in insecticides and are highly toxic to many organisms including humans.", "Insecticide residues are found in soil and groundwater, and the detection of these residues is important for their elimination from the environment and to protect the health of both humans and animals.", "Organophosphorus compounds are also used in nerve agents, such as sarin, phosphine, soman, and tabun, for chemical warfare purposes.", "These agents are some of the most potent toxic agents and are specific inhibitors of acetylcholinesterase (AChE).", "Acetylcholine is an essential neurotransmitter that affects parasympathetic synapses (autonomic and CNS), sympathetic preganglionic synapses, and the neuromuscular junction (see, e.g., Taylor et al., in Basic Neurochemistry, 5th ed., 1993, (Siegal et al., eds.", "), Chapter 11, pp.", "231-260, Raven Press, New York, N.Y.).", "Hydrolysis of acetylcholine by acetylcholinesterase, present in nervous tissue, normally limits the duration of action function.", "Organophosphate (e.g., Malathion, Parathion, Diasinon, Dursban) and carbamate (e.g., Sevin, Furadan) insecticides exert their toxicity by inhibiting the action of acetylcholinesterase and thereby causing a pronounced cholinergic response (Arron et al., Insecticides: Organophosphate and Carbamates in Goldfrank's Toxicologic Emergencies, 1994, (Goldfrank et al., eds.", "), Appleton & Lange, Norwalk, Conn.).", "Enzyme inhibition is the consequence of phosphorylation (organophosphates) or carbamylation (carbamates) of the cholinesterase-active site serine residue.", "The resulting phosphoroyl-serine bond is stable; therefore, enzyme inhibition is physiologically irreversible, whereas the carbamyl-serine bond undergoes spontaneous hydrolysis with regeneration of enzyme activity (24-48 h).", "For this reason and because of poor CNS penetration, carbamate insecticide neurotoxicity is less severe and of shorter duration than that for the organophosphates (Tietz Textbook of Clinical Chemistry, 1999, (Burtis et al., eds.", "), W. B. Saunders Company, Philadelphia, Pa.).", "Excess synaptic acetylcholine stimulates muscarinic receptors (peripheral and CNS) and stimulates but then depresses and paralyzes nicotinic receptors.", "The CNS neurotoxic effects include restlessness, agitation, lethargy, confusion, slurred speech, seizures, coma, cardiorespiratory depression, or death.", "The need for the reliable determination of these cholinesterase inhibitors has led to the development of a number of sophisticated instrumental methods, mostly involving the use of gas and liquid chromatography and mass spectrometry.", "Also a number of liquid phase chemiluminescence procedures have been developed for the determination of inorganic and organic species mostly utilizing the luminol and peroxyoxalate reactions.", "See Robards K. and Worsfold P. J., Anal Chem Acta (1992) 266:147.These traditional methods are not practical for individual use as the methods are time consuming and complicated and the instruments utilized are expensive, non-portable and require high maintenance.", "Additionally, the measurement of nerve agents in mixtures with these traditional methods requires cumbersome extraction and manipulation procedures.", "Thus, biosensors were developed as an alternative to the traditional gas and liquid chromatography and mass spectrometry technology.", "Generally, biosensors include those which are enzyme-based and bioaffinity-based.", "An enzymatic biosensor uses an enzymatic or metabolic process to detect a reaction product which occurs between an incoming substrate and an immobilized enzyme.", "A bioaffinity sensor relies on a biological binding event of a target substance.", "Many existing methods for the detection of organophosphates and cumulative inhibition of cholinesterases lack sensitivity since they are based on inhibition of basal activities rather than accumulation of the inhibitory conjugate.", "Basal activities vary substantially between subjects resulting in inconsistency in present assays.", "Existing monitoring methods routinely require expensive laboratory procedures involving sample transport or preparations of samples for assay.", "Rapid analysis of toxic materials in the areas of food and water analysis, environmental monitoring, and in industrial settings is a problem that continues to exist and is currently addressed by time-consuming, expensive methods or by techniques that may be described as inadequate.", "Many problems associated with exposure to toxic materials could be avoided or minimized by a detection procedure which gives near “real-time” indication of the presence of toxic gases.", "Equally important are the characteristics of economy, small size, and ease of use for the successful application of such devices.", "Accordingly, there is a need for a method of detecting, quantifying, and evaluating hazards which provides for early detection and which can detect low levels of toxic materials.", "The present invention satisfies this need, as well as others.", "SUMMARY OF THE INVENTION The present invention overcomes one or more of the drawbacks in the prior art by providing compositions and methods for their use in the detection of specific inhibitors, modifiers, or ligands of acetylcholinesterase (AChE).", "Disclosed are methods for the preparation and use of labeled AChE and labeled AChE inhibitory conjugate compositions which are useful in detecting accumulation of toxic materials, which include but are not limited to, organophosphates, insecticides, nerve agents, such as sarin, phosphine, soman, and tabun, and other materials used for chemical warfare purposes.", "Also disclosed are methods for the use of labeled AChE and labeled AChE inhibitory conjugate compositions in a variety of areas, including the detecting of toxic materials in biological samples, the areas of food and water analysis, environmental monitoring, and in industrial settings.", "Embodiments are disclosed which describe methods for making and using labeled AChE and labeled AChE inhibitory conjugate compositions comprising fluorescing compounds, including but not limited to, dimethoxyphosphoryl and diethoxyphosphoryl labels.", "In one embodiment of the invention, methods are disclosed for measuring accumulation of inhibitory conjugates comprising labeled AChE and an inhibitor, modulator or ligand (i.e., cognate partner).", "In a related aspect, such methods may comprise contacting a sample suspected of containing such cognate partners with labeled AChE in order for labeled AChE binding to occur between cognate partners in the sample and the enzyme.", "In a further related aspect, conjugated and unconjugated AChE may be separated by chromatographic methods.", "For example, such chromatographic methods may include, but are not limited to, capillary electrophoresis.", "In another embodiment, the conjugated and unconjugated, labeled AChE are detected and differentiated by fluorochromic emission shift, where conjugated AChE shows a detectable shift in emission signal.", "In a related aspect, the shift in emission is a Stokes' shift upon conjugate formation.", "In a related aspect, the cognate partner is an inhibitor, where the inhibitor may be designated as a carbamylating inhibitor or a phosphorylating inhibitor.", "In a further related aspect, the inhibitor may be an insecticide or an organophosphate.", "In another related aspect, the presence of the cognate partner is estimated by determining the ratio of conjugated to unconjugated AChE.", "In another embodiment, the sample may be obtained from the atmosphere, soil, water, industrial sites or environmental sites.", "Further, the sample may be biological or non-biological.", "In a related aspect, a biological sample may include, but is not limited to, the integumentary system, sputum, feces, blood, urine, plasma, lacrimal secretions, cerumen, and semen.", "In one embodiment, the AChE is labeled on at least one site.", "In a related aspect, the AChE is labeled at multiple sites.", "In a further related aspect, the at least one label is peripheral to the active center of the AChE.", "In one embodiment, a device comprising AChE is envisaged, where the AChE is compartmentalized in a mobile or stationary phase.", "In a related aspect, the stationary phase is a chip.", "In another related aspect, the mobile phase is a suspension.", "In one embodiment, the device is a biosensor for analyzing a sample for at least one organophosphorous, nerve agent and/or insecticide, where at least one enzyme is immobilized on or within the device.", "In a related aspect, the immobilized enzyme is either covalently or non-covalently bound to the device.", "In a further related aspect, the biosensor is divided into multiple zones, where each zone differentiates between one or more organophosphorous, nerve and/or insecticide agents.", "In another embodiment, the present invention also provides kits which contain the present labeled AChE for use in the present identification method.", "In another embodiment, a labeled AChE composition comprising at least one fluorophore located peripherally to the active center of the AChE, where the at least one fluorophore possesses an emission signal that shows a Stokes' shift upon conjugate formation.", "In a related aspect, the fluorophore comprises a dimethoxyphosphoryl label or a diethoxyphosphoryl label.", "In another embodiment, a labeled AChE molecule comprises at least one fluorophore on at least one site present at the periphery of the active site of the enzyme, wherein the at least one site is selected from the group consisting of residues 76, 81, 84, 124, 262 and 287 of AChE or equivalents thereof.", "Exemplary methods and compositions according to this invention, are described in greater detail below.", "BRIEF DESCRIPTION OF THE DRAWINGS The drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention.", "The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.", "FIGS.", "1A-D show locations of introduced cysteines for fluorophore modification.", "Residues 76, 81, and 84 are at the tip (76) and outer portion (81, 84) of the Q loop.", "Residues 124 and 287 are on an opposing face of the gorge and make up part of the peripheral anionic site.", "Residue 262 is on a peripheral disulfide loop and in the crystal has a large thermal factor.", "A-D, Connolly surface representations of structure.", "A, unligated AChE (6); B, TFK+ conjugated with AChE; note partial exposure of the white molecule TFK+, at the base of the gorge (33); C, fasciculin 2 bound AChE at the mouth of the gorge (5); D, fasciculin 2 complex with AChE, rotated 90°.", "Acrylodan conjugated to E84C is shown in yellow; and acrylodan conjugated to E81C is shown in green.", "(Note in D the proximity between arginine 11 on Fas 2 and the acrylodan side chain at position 84).", "FIG.", "2 shows a scheme showing combining of substrate at two discrete sites to form two binary complexes, ES and SE (where S is substrate; E is enzyme; and P is product).", "Only ES results in substrate hydrolysis.", "For simplicity, S is assumed to combine equally well with E and ES.", "The efficiency of substrate hydrolysis of the ternary complex SES, as compared with ES, is reflected in the value of the parameter, b, the relative catalytic turnover of the ternary complex (26).", "FIGS.", "3A-B show fluorescence emission spectra of acrylodan-labeled Y124C(A) and E84C (B) AChE free in solution (dashed line) and complexed with fasciculin (solid line).", "A, for acrylodan-labeled Y124C, fasciculin produces a hypsochromic shift and enhancement of fluorescence quantum yield.", "The large shift for Y124C reveals a clear isoemissive point indicative of the two (free and fasciculin bound) species.", "Equivalent concentrations of enzyme (215 nM) were present for all conditions.", "The concentration of fasciculin was 215 nM.", "B, for acrylodan-labeled E84C, fasciculin produces a bathochromic shift and reduction of fluorescence quantum yield.", "Equivalent concentrations of enzyme (270 nM) were present for all conditions.", "The concentration of fasciculin was 800 nM.", "FIGS.", "4A-B show association of TFK+ with acrylodan-modified E84C AChE.", "A, fluorescence emission spectra of acrylodan-labeled E84C AChE following addition of excess TFK+.", "TFK+ produces a bathochromic shift and reduction of fluorescence quantum yield.", "The large chromic shift reveals a clear isoemissive point indicative of the two (free and TFK+ bound) species.", "Initial enzyme concentration was 130 nM.", "Excess TFK+ (1.25 μM) was added, and fluorescence spectra were recorded at the following times: 0, 1, 2.5, 4.3, 5.8, 7.4, 10.6 and 22 min.", "B, time course of the fluorescence changes.", "Initial acrylodan-modified E84C AChE concentration was 150 nM.", "Excess TFK+ was added, and the decrease in fluorescence signal at 477 nm was monitored using an ISA Jobin Yvon-Spec Fluoromax fluorometer.", "The three TFK+ concentrations were 1.25 (▾), 2.5 (◯), and 5.0 (Δ) μM.", "Control enzyme samples, to which buffer rather than TFK+ was added, did not show decreases in fluorescence signals over the time intervals measured.", "The inset shows rates plotted as a function of TFK+ concentration.", "kon for TFK+is calculated based on ratios of the hydrated and unhydrated ketone (21).", "FIGS.", "5A-B show association of BW284c51 with acrylodan-modified E84C AChE.", "A, fluorescence emission spectra of acrylodan-labeled E84C AChE following titration with BW284c51.BW284c51 produces a bathochromic shift and reduction of fluorescence quantum yield.", "Initial enzyme concentration was 70 nM.", "BW284c51 concentrations were 0, 0.01, 0.035, 0.06, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50, and 100 μM.", "B, the decrease in fluorescence emission curves is plotted as a function of BW284c51 concentration.", "Kd is determined by fitting the data with Equation 1 as outlined below (EXAMPLES, Materials and Methods).", "FIG.", "6 shows an illustration showing regions of homology between AChE and various enzyme species.", "DETAILED DESCRIPTION OF THE INVENTION Before the present compositions and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, and reagents described as these may vary.", "It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be described by the appended claims.", "It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.", "Thus, for example, reference to “a subject” includes a plurality of such subjects, reference to “an enzyme” includes one or more enzymes and equivalents thereof known to those skilled in the art, and so forth.", "Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.", "Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods, devices, and materials are now described.", "All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the proteins, compounds, and methodologies which are reported in the publications which might be used in connection with the invention.", "Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.", "As used herein acetylcholinesterase (AChE) means a serine hydroxylase in the α/β-fold hydrolase protein superfamily which terminates nerve signals by catalyzing the hydrolysis of the neurotransmitter acetylcholine.", "In one embodiment, AChE is from mouse (e.g., but not limited to, Accession Nos.", "IMAAA, IAAB, IMAAC, IMAAD and IMAHA).", "In a related aspect, the mutagenized residues of AChE as envisaged in the instant invention are denoted by the wild type residue, followed by the residue number (based on, for example the Accession Numbers, supra), with the mutated residue last (i.e., cysteine, “C”).", "For example, based on Accession No.", "IMAAA, conversion of residue 81 (i.e., Glu or E) to cysteine (or C) would be denoted as follows: E81C.", "Corresponding structures on equivalent AChE molecules may be determined by alignment of homologous regions (see, e.g., FIG.", "6 and Soreq et al., Trends Biochem Sci (1992) 17(9):353-8).", "Equivalents of AChE may include, but are not limited to, Candida lipase (Acc.", "No.", "CAD86495), Dictyostelium crystal protein (Acc.", "No.", "CAA36702), Dictyostelium D2 esterase (Acc.", "No.", "A60531), Drosophila neurotactin (Acc.", "No.", "S12005), Drosophila glutactin (Acc.", "No.", "S12519), Heliothis juvenile hormone esterase (Acc.", "No.", "P12992), Culex esterase BI (Acc.", "No.", "A35986), Drosophila esterase 6 (Acc.", "No.", "AAD39965), Drosophila esterase P (Acc.", "No.", "B34089), Drosophila cholinesterase (Acc.", "No.", "A25363), Torpedo acetylcholinesterase (ACC.", "No.", "ACRYE), mammalian acetylcholinesterase (e.g., homo sapiens, ACC.", "No.", "AAA53473), mammalian butyrylcholinesterase (e.g., homo sapiens, Acc.", "No.", "AAH18141), mammalian carboxylesterase (6.1) (e.g., rattus norvegicus, Acc.", "No.", "P16303), mammalian lysophospholipase (e.g., mus musculus, Acc.", "No.", "AAH13536), thyroglobulin (e.g., rattus norvegicus, Acc.No.", "NP—112250), mammalian neuroligin (e.g., homo sapiens, Acc.", "No.", "AAH34018), and Drosophila gliotactin (NP—723931).", "In a related aspect, derivatives, fragments and variants of these AChE equivalents are also envisaged for the methods, compositions and devices of the present invention.", "(See, also e.g., FIG.", "6 and Soreq et al., 1992).", "In one embodiment, mutants can be prepared by site-directed mutagenesis (or evolution methods, see e.g., Devlin et al., Science (1990) 249:404-406; and Scott & Smith, Science (1990) 249:386-390) using a conventional oligonucleotide directed in vitro mutagenesis system such as that described by Eckstein et al., Nucleic Acids Research (1985) 13:8749-8785.", "(See also, U.S. Pat.", "No.", "6,001,625).", "Other conventional PCR techniques known in the art may be used.", "In one embodiment, the selection of residues for replacement is based on a molecular model, using the crystal structure of AChE published by Sussman, et al.", "on an Evans and Sutherland PS390 platform with Biosym software.", "As used herein, “cognate partner,” including grammatical variations thereof, means a molecule that stereoselectively binds within the active site of AChE.", "For example, a ligand (e.g., acetylcholine), modulator (e.g., galantamine) or inhibitor (e.g., organophosphates) of AChE would be considered a cognate partner.", "The instant invention is a sensitive method for the detection of AChE ligands, modulators and inhibitors using fluorescent labeled forms of AChE.", "The AChE of the present invention is labeled at sites peripheral to the active center with a fluorophore whose emission signal shows a large Stokes' shift upon binding of ligands.", "The attached fluorophore does not interfere with ligand binding, so the high affinity for inhibitors, modifiers or ligands seen in the native enzyme is maintained.", "The labeled AChE of the present invention can be used to detect interaction with inhibitors, modifiers and ligands.", "This is particularly useful for the detection of organophosphates such that insecticides or nerve agents and provides a method to determine if the concentrations of a substance present in the atmosphere or ground are sufficient to inhibit AChE.", "After a dye molecule has been electronically excited due to absorption of light, it may almost instantaneously emit light.", "This very fast process occurring on the nanosecond time scale (10−9 sec) is called fluorescence.", "A fluorescence dye is characterized by its spectral characteristics (excitation and emission spectra, lex and lem respectively), its quantum yield (QY) and its fluorescence lifetime (t).", "The spectral characteristics of fluorescence dyes depend strongly on their molecular backbone.", "For absorption and emission in the UV, near the visible spectral range, at minimum, a system of conjugated double bonds is needed.", "The wavelengths of absorption and emission are simply stated as shifted towards red or longer wavelengths.", "Additional electron donating substituents are necessary for the spectral characteristics of the dye to finally occur in the visible spectrum of light (400-700 nm), which is called the bathochromic shift.", "The emitted light occurs normally at a longer wavelength with respect to the absorbed light due to a loss in energy while the dye molecules remain in their electronically excited state.", "This so-called Stokes' shift makes the maximum emission occur at a few or up to several tens of nanometers (10−9 m) red-shifted with respect to the absorption maximum.", "These spectral properties of a dye are widely used for the identification of differently labeled probes.", "In a related aspect, the fluorophores of the present invention exhibit large Stokes' shift (i.e., the difference in wavelength between the excitation wavelength and the emission wavelength).", "One of the most important properties of a fluorescence dye is the quantum yield (QY).", "The quantum yield is the ratio of emitted light to the light absorbed prior to the observed fluorescence.", "Hence the quantum yield is a measure of the efficiency of fluorescence, which is concurring with other so-called dark processes.", "The higher the quantum yield of a fluorescence dye the better it can be observed.", "Due to the quantum nature of fluorescence, the emission of light from each molecule occurs arbitrarily after its excitation.", "In other words, the dye molecules remain in the electronically excited state for a very short but random time span.", "Nevertheless the statistics of emission follows a known model, which in the simplest case is an exponential decay.", "This well known kinetic law contains a time constant called fluorescence lifetime, mostly abbreviated as t, which is characteristic for the given dye and may be used for its identification.", "Fluorescence lifetime and the closely related quantum yield may strongly depend on the molecular environment of the individual dye molecule, such as the surrounding solvent or the local pH.", "Therefore a change of these properties indicates a change of the local environment of the dye molecules.", "Close investigations of this phenomenon have revealed several well-defined processes, often referred to as quenching, which can be used for switching the dye properties, allowing for the development of intelligent probes (GenePin).", "As used herein, “hypsochromic,” including grammatical variations thereof, means a shift of a spectral band to higher frequency or shorter wavelength upon substitution or change in medium (e.g., solvent).", "It is informally referred to as a blue shift, and is opposite to bathochromic shift.", "Sensitivity of assays and devices envisaged by the present invention is high, because the method of evaluating the presence of a conjugate (e.g., a labeled AChE-inhibitory conjugate) is based on detection with high quantum yield fluorophores.", "In one embodiment, the label is acrylodan.", "Fluorescence emission of acrylodan is exquisitely sensitive to the dielectric constant of the solvent.", "In general, the fluorescence emission spectrum of acrylodan shifts toward the red (bathochromic), and the quantum yield decreases as the polarity of solvent increases (20, 40-42).", "This sensitivity to solvent polarity arises from the interaction of the excited state of acrylodan with its surrounding solvent.", "The excited state is more polar than the ground state and, as such, will interact with a polar solvent so as to align solvent dipoles.", "This alignment lowers the energy of the excited state and causes the red shift of the emission spectrum.", "Hence, an acrylodan-labeled enzyme with an emission maximum of 510-525 nm likely reflects exposure of the side chain to solvent (20, 42).", "On the other hand, acrylodan emission maxima in the range of 475-500 nm likely reflect solvent exclusion and a more hydrophobic environment surrounding the fluorophore.", "For example, the time course of TFK+ reaction with acrylodan-E84C (FIG.", "3) reveals a large spectral shift from 477 to 512 nm, indicating acrylodan conjugated at this position has moved to a more hydrophilic environment with TFK+ bound.", "The large spectral shift yields a clear isoemissive point (i.e., the wavelength at which the intensity of emission of a sample does not change during a chemical reaction or physical change.", "The term derives from the Greek word for ‘same luminescence’), which arises when only two distinct emitting species are present, in this case the free enzyme and the TFK+ conjugate.", "As used herein, “accumulation of conjugates,” including grammatical variations thereof, means continuous increase in the number of AChE molecules bound (i.e., combine by chemical action) by a ligand, modulator or inhibitor.", "As used herein, “conditions sufficient for binding of an inhibitor to the enzyme” includes, but is not limited to, contacting a labeled AChE (or equivalents thereof) comprising a carrier (e.g., bovine serum albumin) in an appropriate buffer (e.g., sodium phosphate buffer, pH 7.0) with various concentrations of cognate partners, for example between about 0.05 mins to about 0.1 mins, about 0.1 mins to about 0.5 mins, about 0.5 mins to about 1 min, about 1 min to about 2.5 mins, about 2.5 mins to about 3 mins, about 3 mins to about 4.5 mins, about 4.5 mins to about 5.5 mins, about 5.5 mins to about 7 mins, about 7 mins to about 10 mins, about 10 mins to about 15 mins, about 15 mins to about 20 mins, or about 20 mins to 25 mins, at about 15° C. to about 20° C., about 20° C. to about 25° C., about 25° C. to about 30° C. to about 32° C., about 32° C. to about 37° C., about 37° C. to about 40° C. or about 40° C. to about 45° C. As used herein, “biological sample,” including grammatical variations thereof, means materials obtained from living organisms.", "For example, such samples include, but are not limited to, an integumentary system sample, sputum, feces, blood, urine, plasma, lacrimal secretions, cerumen, and semen.", "As used herein, “non-biological sample,” including grammatical variations thereof means materials obtained from inanimate objects or non-living materials.", "For example, such samples include, but are not limited to, soil, water, air and environmental (e.g., walls, floors, furniture etc.)", "surfaces.", "Methods are described which distinguish between various classes of organophosphates by analyzing the fluorescence shift of labeled-AChE of the present invention.", "In a related aspect, the AChE may be labeled with various fluorophores, including but not limited to, dimethoxyphosphoryl and diethoxyphosphoryl labels.", "In a further related aspect, fluorophores yield different emission maxima.", "In one embodiment, the labeled AChE can be immobilized on a chip to detect cumulative AChE inhibition.", "In a related aspect, such a chip can serve as a remote sensor for inadvertent or planned contamination.", "In general, the simplest biosensor for compounds as envisaged for the present invention comprises a material having AChE immobilized upon or within a stationary phase, secured upon a carrier such as plastic, glass, cloth, nylon, rubber, etc.", "Detection of cognate partners may be qualitatively determined by separation of conjugated enzyme from unconjugated enzyme.", "To test for cognate partners, the biosensor is exposed to the sample to be tested.", "Since the accumulation of conjugated-immobilized enzyme is substantially similar to that observed for the soluble form, only a short duration of exposure to the sample is required.", "In a related aspect, a shift in fluorescence indicates the presence of a conjugate.", "In one embodiment, the biosensor may be washed to remove compounds and/or compositions which may cause interference since the immobilized enzyme does not leach and the cognate partner is irreversibly bound to the immobilized enzyme.", "An appropriate buffer may be applied to the material.", "In a related aspect, in the field of drugs and testing of samples, it will be preferred to apply detectable cognate partners providing optimal signal in an aqueous system.", "In one embodiment, biosensors may be disposed after a single use or may be reused.", "The biosensor may be regenerated using a reagent to displace the cognate partner, e.g.", "fluoride salts.", "Accuracy of the biosensor is assured if it is recalibrated prior to use.", "The present invention also provides kits.", "Such kits will contain a container means which contains the instant enzyme.", "The container means may be any suitable container, but will typically be a glass vial or jar, a plastic pack, etc.", "In one embodiment, the container means may be a foil or plastic pouch which contains the enzyme immobilized on a microtitre plate or a chip.", "For example, a method of immobilization may include, but is not limited to, the method as described in Taylor et al.", "(U.S. Pat.", "No.", "5,192,507).", "In other embodiments, the container means may be a plastic, glass, or metal tube which contains the enzyme, and the tube may possess an inlet means at one end and an outlet means at the other end; this type of container means may be used as a column in a flow biosensor and may itself be contained in a second container means.", "The kit may further comprise a negative control sample.", "Such a negative control sample will contain either no cognate partner or a very low amount of cognate partner.", "The kit may also comprise a positive control sample, which will comprise, typically, an amount of cognate partner which is equal to or greater than the amount of cognate partner which is considered a positive result.", "The kit may also contain chemicals, such as buffers or diluents, and sample handling means, such as pipettes, reaction vials, vessels, tubes, or filters.", "In addition, the kit may comprise written instructions on a separate paper, or any of the container means, or any other packaging.", "These instructions will usually set forth the conditions for carrying out the detection method, such as mixing ratios, amounts, incubation times, etc., and criteria for evaluating the results of the method, including spectra charts.", "The following examples are intended to illustrate but not limit the invention.", "EXAMPLES The following examples are to exemplify certain embodiments of the invention.", "Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.", "Materials and Methods Inhibitors and Substrates-Acetylthiocholine iodide, 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman's reagent), dithiothreitol, tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate), BW284c51, decamethonium, and edrophonium were purchased from Sigma.", "m-(N,N,N-trimethylammonio)trifluoromethylacetophenone (TFK+) and (−)huperzine A were purchased from Calbiochem.", "Acrylodan was obtained from Molecular Probes (Eugene, Oreg.).", "Fasciculin 2 (purified from the venom of Dendroaspis angusticeps) was a gift of Dr. Pascale Marchot (University of Marseille, France).", "Drs.", "Yacov Ashani and Bhupendra P. Doctor (Walter Reed Army Research Center, Washington, D.C.) kindly provided 7-((methylethoxy) phosphinyl)-oxyl)-1-methylquinolinium iodide (MEPQ) and procainamide-linked Sepharose CL-4B resin.", "m-tert-Butyl trifluoromethylacetophenone (TFK+) was synthesized as described (21) and kindly provided by Dr. Daniel Quinn, University of Iowa, Iowa City, Iowa.", "All other chemicals were of the highest grade commercially available.", "Expression, Mutagenesis, and Purification of mAChE-Mouse AChE was produced by transfection of an expression plasmid (pCDNA3, Invitrogen, San Diego, Calif.) containing an encoding cDNA where the AChE sequence was terminated at position 548.The plasmid was transfected into HEK293 cells.", "Cells were selected with G418 to obtain stable producing cell lines, and AChE was expressed as a secreted soluble enzyme in serum-free media (20).", "Mutant enzymes were generated by standard mutagenesis procedures, and cassettes containing the mutation were subcloned into pCDNA3 (20).", "Nucleotide sequences of the cassettes were confirmed by double-stranded sequencing to ensure that spurious mutations were not introduced into the coding sequence.", "Affinity chromatography using (m-aminophenyl)trimethylammonium linked through a long chain to Sepharose CL-4B resin (Sigma) permitted one-step purification of AChE.", "From 4 to 6 liters of media, mutant and wild type enzyme were purified in quantities ranging between 5 and 25 mg, as described previously (22-24).", "Purity was ascertained by SDS-PAGE and by measurements of specific activity.", "Assay of Catalytic Activity—The spectrophotometric method of Ellman was used (25), and kinetic constants for acetylthiocholine hydrolysis were determined by fitting the observed rates as described (26).", "Titration of active sites with known concentrations of the irreversible phosphorylating agent, MEPQ, was accomplished by the method of Levy and Ashani (27).", "Acrylodan Labeling—Mutant enzymes were pretreated with 0.25 mM dithiothreitol for 30 min at room temperature to ensure reduction of the introduced cysteine.", "Excess dithiothreitol was removed by use of a G-50 Sephadex spin column (Roche Molecular Biochemicals) equilibrated in 10 mM Tris, 100 mM NaCl, 40 mM MgCl2, pH 8.0.A volume of 1 μl of acrylodan at 100 times the enzyme concentration was slowly mixed with the enzyme to achieve a ˜5-fold molar excess of acrylodan to mutant enzyme.", "Labeling was allowed to proceed for at least 12 h at 4° C., and unreacted acrylodan was removed by size exclusion chromatography using Sephadex G-25 (Amersham Pharmacia Biotech) in 0.1 M sodium phosphate buffer, pH 7.Concentrations of acrylodan-labeled enzyme were determined from the maximal absorbance found between 360 and 380 nm (ε˜16,400 M−1 cm−1).", "Stoichiometry of labeling of the various preparations, estimated from a comparison of enzyme concentration by protein (280 nm) to acrylodan (360-380 nm) absorbance, ranged as follows: L76C, 0.7-0.8; E81C, 0.79-1.0; E84C, 0.77-1.0; Y124C, 0.79-1.0; A262C, 0.69-0.85; and H287C, 0.82-0.88.Specificity of labeling was assessed by comparison of areas under the fluorescence emission curves for acrylodan-treated mutant and wild type enzymes.", "Specific labeling for each mutant was as follows; L76C, 70-85%; E81C, 81-91%; E84C, 85-93%; Y124C, 83-90%, A262C, 80-90%; H287C, 70-76%.", "Trifluoroacetophenone Inhibition—Picomolar amounts of enzyme in 0.01% bovine serum albumin in 0.1 M sodium phosphate buffer, pH 7.0, were reacted with TFK+ in the absence of substrate.", "Inhibition was monitored by measuring residual enzyme activity by removal of aliquots during the course of the reaction.", "Bimolecular rate constants of inhibition were determined by nonlinear fit of the data (28).", "ΔF=ΔFmax(Et+It+Kd−/(Et+It+Kd)2−4 EtIt∫0.5)(2Et)−1 (Eq.", "1) ΔF and ΔFmax are the change and maximum change in fluorescence, respectively; E1 is the total enzyme concentration, and I1 is the total inhibitor concentration.", "Association of TFK+ with acrylodan-labeled E81C and E84C was assessed from the kinetics of decrease in fluorescence at 470 and 477 nm respectively, following addition of a stoichiometric excess TFK+ at several concentrations.", "Data were fitted to a single exponential approach to equilibrium.", "Association and dissociation rate constants of edrophonium and BW286c51 with E81C and E84C AChEs were determined from changes in the tryptophan fluorescence using a stopped-flow spectrophotometer as described previously (29).", "Time-dependent decreases in tryptophan fluorescence were observed upon excitation at 276 nm by means of a 305 nm emission cut-off filter.", "EXAMPLE 1 Characteristics of Substrate Hydrolysis and Fasciculin 2 Inhibition Crystallographic Structures and Solution Dynamics of the Acetylcholinesterase Complex—In the several crystal structures of AChE with conjugated or reversible bound ligands that have been studied, little evidence for change in enzyme conformation has been detected with a difference of less than a root mean square of 2 Å for the α-carbon backbone between the apoenzyme and the various complexes (4-6, 13, 14, 33-35, 44).", "Changes in side chain orientation occur most notably in the phenyl ring at position 337 for certain reversible complexes (34) and phenylalanine 297 when bulky organophosphates are conjugated to the active site serine (44).", "However, based on the multiple positions of the outer trimethylammonio moiety in decamethonium for mouse (6) and Torpedo crystal structures (34), some flexibility may exist particularly within the gorge itself.", "Brownian dynamics often require lining the radii of the attacking ligands or the residues lining the gorge in order to simulate the kinetics of diffusion-limited substrate access observed experimentally (45).", "Thus, all of crystal structures reported to date reveal a closed gorge with constrained dimension.", "The solution-based fluorescence studies reported herein provide the first physical evidence for localizing the ligand-induced conformational change to the Cys69-Cys96 Ω loop.", "This finding raises an interesting possibility that the unliganded enzyme exists in a rapidly converting conformational equilibrium between open and closed states, and both ligand binding and conditions of crystallization favor formation of a closed gorge state.", "In fact, analysis of the molecule dynamics of a solvated mouse AChE shows fluctuations yielding an average widening of the motions of the gorge, which may also be integral to the catalytic cycle of transacylation and deacylation during ester hydrolysis.", "The cysteine-substituted enzymes show kinetics of acetylthiocholine hydrolysis similar to wild type enzyme (Table I and FIG.", "2) suggesting that all mutant enzymes fold correctly despite the presence of the newly introduced cysteine.", "The Km value of E84C shows slightly less than a 4-fold increase, whereas the change in turnover rate, kcat, is minimal.", "Similar changes in kinetic constants were observed previously for E84Q mAChE (28).", "Since Km, in the diffusion-limited catalysis, depicts the initial encounter between substrate and enzyme, an increase in Km likely arises from the reduction of negative charge that electrostatically steers the cationic substrate into the active center gorge.", "Interestingly, a similar E81C mutation has little or no effect on substrate hydrolysis.", "Not all negatively charged residues around the active center appear to be involved equivalently in electrostatic steering.", "Association and dissociation rates of fasciculin with A262C, H287C, and Y124C mutant enzymes were also found to be close to the rates of wild type enzyme (20).", "Fasciculin, at low concentrations, is also capable of associating with the mutant enzymes after acrylodan conjugation (FIG.", "2).", "In addition, enzyme activity measurements of fasciculin-bound acrylodan conjugates show greater that 99% inhibition.", "EXAMPLE 2 Influence of Residue Modification of Inhibition by m-Trimethylammoniotrifluoromethylacetophenone TFK+ binding to cysteine-substituted enzymes, both free and modified with acrylodan, was also examined (Table II).", "For E81C and E84C, the association rate constants (kon) for TFK+ were obtained from measurements of enzyme activity.", "Although positions 81 and 84 are both spatially removed from the TFK+ binding site, kon for E84C is slightly slower than that for wild type enzyme.", "By contrast, E81C shows no difference in the kinetic constants.", "Conjugation of acrylodan, a neutral naphthalene derivative, with E84C reduces kon of TFK+ 7-fold compared with unconjugated E84C, whereas conjugation of E81C with acrylodan only reduces kon of TFK+ slightly.", "For acrylodan-labeled mutants, kon was measured from the time-dependent decrease of fluorescence signal (FIG.", "3).", "Influence of Residue Modification of Ligand Binding—The changes in emission spectra of acrylodan-labeled Ω loop residues 81 and 84 have been exploited to monitor ligand binding (Table II).", "Cysteine substitution and acrylodan conjugation at position 84, but not at position 81, affect ligand binding kinetics.", "Cysteine substitution at position 84 has little influence on catalytic parameters derived from steady-state catalysis (Table I).", "TABLE I Constants for acetylthiocholine hydrolysis by wild type and mutant mouse AChEs.", "* kcat/Km Enzyme Km (μM) KSS (mM) b kcat (105/min) (109/M-min) WT# 54 ± 16 14 ± 5 0.2 ± 0.07 1.6 ± 0.4 3.0 Y124C# 65 ± 17 20 ± 14 0.2 ± 0.09 1.4 ± 0.3 2.2 H287C# 58 ± 7 12 ± 6 0.2 ± 0.06 1.8 ± 0.2 3.1 A262C# 59 ± 4 11 ± 3 0.2 ± 0.04 1.6 ± 0.1 2.7 L76C 97 ± 19 17 ± 1 0.2 ± 0.03 1.8 ± 0.1 1.9 E81C 57 ± 6 11 ± 1 0.2 ± 0.03 1.6 ± 0.1 2.9 E84C 190 ± 9 26 ± 2 0.2 ± 0.05 1.9 ± 0.4 1.0 Data shown as means ± S.D.", "typically from three measurements.", "Data were fit to the Equation ν = (1 + b[S]/KSS)/Vmax/(1 + [S]/KSS)(1 + Km/[S]), where [S] is substrate concentration, KSS is the substrate inhibition or activation constant, and b is the relative catalytic turnover of the ternary complex (12).", "# Data are from Boyd et al., J Biol Chem (2000) 275: 22401-22408.The Km of E84C increases less than 4-fold compared with the wild type enzyme.", "By contrast, a similar substitution at position 81 has no effect of ATCh steady-state catalysis.", "Precise quantitation of these catalytic parameters for the acrylodan-conjugated enzyme is complicated by incomplete modification by acrylodan.", "However inhibitor association can be measured using the change in fluorescence signal (Table II).", "TABLE II Kinetic and equilibrium constants for reaction of enzymes with TFK+, edrophonium, and BW284c51 in the presence of fluorescent (acrylodan) cysteine labeling compound.", " TFK+ Edrophonium BW284c51 Enzyme kon109M−1min−1 k on ⁢ ⁢ WT k on ⁢ ⁢ mutant KdnM K d ⁢ ⁢ mutant K d ⁢ ⁢ WT KdnM K d ⁢ ⁢ mutant K d ⁢ ⁢ WT Wild Type 150 250a 2.0a E81C 150 1 260b 1 2.6b 1.3 E81C- 94 1.6 640 2.6 6.9 3.5 acrylodan E84C 93 1.6 550b 2.2 35b 18 E84C- 13 11 6300 25 130 65 acrylodan Data are shown as means from two to three measurements.", "Individual determinations are within 35% of the mean.", "Rates for TFK+ are calculated based on ratios of the hydrated and unhydrated ketone (21).", "aData are from Radic et al.", "J Biol Chem (2001) 276: 4622-4633.bEquilibrium dissociation constants are derived from the ratio of koff/kon using stopped-flow measurement of tryptophan quenching.", "Reductions in binding kinetics were observed for several ligands (Table II) ranging between 1 or 2 orders of magnitude at position 84 but very little change at position 81.Although a portion of the reduction at position 84 is due to the cysteine substitution, acrylodan conjugation has a small, but significant (3-10-fold), influence on ligand binding.", "Even though both the 81 and 84 residues reside on the enzyme surface removed from the active center gorge, modification only at position 84 appreciably affects the energetics of ligand binding.", "The acrylodan moiety, whose dimension is slightly larger than the indole moiety of tryptophan, may impart steric restrictions to the region around the 84 site contributing to the energy cost in ligand binding.", "A small alteration in ligand binding energy (1.5-3.0 kcal/mol) is not unexpected if the conformation of the Ω loop plays a role in ligand binding.", "Velan et al.", "(18) examined steady-state kinetics for a large number of Ω loop substitutions and truncations.", "Modification of Glu84 and its neighboring residues was found to have limited effect on steady-state kinetics.", "Faerman et al.", "(19) inserted a cysteine at position 82 to pair with a second cysteine residing proximally in the body of the enzyme.", "Although it could not be firmly established that a disulfide bond formed, little change in kinetic parameters (Km and kcat) was observed.", "Because of compensating contributions of the component primary constants, it is often difficult to correlate changes in steady-state kinetic parameters with structural perturbations.", "Our site-directed fluorophore labeling provides a physical assessment of the localized conformational change in the Ω loop.", "In cases where the fluorophore makes direct contact with the ligand, as the acrylodan-labeled Y124C and H287C with fasciculin, the energetic perturbations from substitution are larger, since complementarity of the binding site may be altered through the insertion of acrylodan side chain at the interface between the ligand and its binding site (20).", "Acrylodan Modification at a Site Distal to the Active Center Core—The A262C modification was selected as a positional reference for a site distal to the active center.", "This residue is also located at the tip of the disulfide loop but is located ˜30 Å away from the rim of the active center gorge.", "Crystallographic studies show this region to have a high temperature coefficient (B factor), indicative of substantial molecular motion of this surface residue.", "In fact, the position of this residue and its immediate neighbors is the only secured in crystal forms where proximity of the symmetry-related AChE molecule limits its movement in the crystal structure (6).", "Acrylodan substitutions at this position show a long wavelength emission (λmax=517 nm) indicative of exposure to a hydrophilic environment (Table III).", "TABLE III Fluorescence emission parameters of mouse AChE mutants labeled with acrylodan in the presence of fasciculin.", " Acrylodan Emission Maxima (nm) Saturating Chromic Shift Relative Enzyme No Fasciculin Fasciculin (nm) Quantum Yield L76C 505 505 0 1.40 E81C 489 510 21 1.16 E84C 477 512 35 0.47 aY124C 500 477 −23 1.78 aA262C 517 517 0 0.97 aH287C 524 507 −17 5.0 Data are shown as mean values of at least three determinations.", "Relative quantum yields were determined by comparison of areas of the fluorescence emission curves.", "aData are from Boyd et al., J Biol Chem (2000) 275: 22401-22408.Moreover, none of the ligands studied, whether they are covalently attached to the active center (TFK or alkylphosphates), reversibly bound to the active center (edrophonium), span between the active center and peripheral site (decamethonium and BW286c51), or bind only to peripheral site (fasciculin), affect the spectroscopic properties of acrylodan conjugated at site 262 (Tables III-VI).", "TABLE IV Fluorescence emission parameters of mouse AChE mutants labeled with acrylodan in the presence of covalent active site inhibitors.", " Acrylodan Emission Maxima (nm) Relative Quantum Enzyme Conjugated TFK0 Chromic Shift (nm) Yield L76C 509 4 0.87 E81C 510 21 0.89 E84C 507 30 0.59 Y124C 478 −22 1.15 A262C 517 0 0.97 H287C 524 0 0.90 Acrylodan Emission Maxima (nm) Relative Quantum Enzyme Conjugated TFK+ Chromic Shift (nm) Yield L76C 511 6 0.92 E81C 510 21 0.89 E84C 512 35 0.52 Y124C 503 3 0.70 A262C 517 0 0.97 H287C 524 0 1.07 Acrylodan Emission Maxima (nm) Relative Quantum Enzyme Conjugated DDVP Chromic Shift (nm) Yield L76C 503 −2 1.27 E81C 510 21 0.19 E84C 496 19 0.39 Y124C 496 −4 1.27 A262C 517 0 0.97 H287C 524 0 1.03 Data are shown as mean values of at least three determinations.", "Relative quantum yields were determined by comparison of the areas of the fluorescence emission curves.", "Data for the unconjugated enzymes are found in Table III.", "TABLE V Fluorescence emission parameters of acrylodan-labeled mouse AChE mutants in the presence of reversible active site inhibitors.", " Acrylodan Emission Maxima (nm) Saturating Relative Quantum Enzyme Edrophonium Chromic Shift (nm) Yield L76C 509 4 0.92 E81C 510 21 0.91 E84C 510 33 0.60 Y124C 500 0 0.79 A262C 517 0 0.97 H287C 524 0 1.13 Acrylodan Emission Maxima (nm) Saturating Relative Quantum Enzyme Huperzine A Chromic Shift (nm) Yield L76C 511 6 0.86 E81C 510 21 0.88 E84C 510 33 0.55 Y124C 500 0 0.63 A262C 517 0 0.97 H287C 524 0 1.13 Acrylodan Emission Maxima (nm) Relative Quantum Enzyme Saturating Tacrine Chromic Shift (nm) Yield L76C 509 4 0.87 E81C 510 21 0.91 E84C 510 33 0.45 Y124C 497 −3 0.51 A262C 517 0 0.97 H287C 524 0 1.13 Data are shown as mean values of at least three determinations.", "Relative quantum yields were determined by comparison of the areas of the fluorescence emission curves.", "Data for the unliganded enzymes are found in Table III.", "TABLE VI Fluorescence emission parameters of acrylodan-labeled mouse AChE mutants in the presence of bisquaternary ligands.", " Acrylodan Emission Maxima (nm) Saturating Relative Quantum Enzyme BW284c51 Chromic Shift (nm) Yield L76C 508 3 1.13 E81C 510 21 0.98 E84C 512 35 0.47 Y124C 487 −13 1.05 A262C 517 0 0.97 H287C 510 −14 2.73 Acrylodan Emission Maxima (nm) Saturating Relative Quantum Enzyme Decamethonium Chromic Shift (nm) Yield L76C 508 3 1.05 E81C 510 21 0.94 E84C 505 28 0.59 Y124C 465 −35 1.85 A262C 517 0 0.97 H287C 517 −7 1.76 *Data are shown as mean values of at least three determinations.", "Relative quantum yields were determined by comparison of the areas of the fluorescence emission curves.", "Data for the unliganded enzymes are found in Table III.", "This pattern indicates a lack of global conformational change affecting residue environments in a disulfide loop well removed from the active center (FIG.", "1).", "Residues Residing on the Active Center Gorge in Apposition with the Ω Loop—Residues 124 and 287 lie in close proximity to the Ω loop with H287C at the rim of the gorge and Y124C, residing just below the rim in the gorge interior (FIG.", "1).", "The crystal structure of the complex shows fasciculin to “cap” these residues, hypsochromic shifts of acrylodan upon fasciculin binding (20).", "None of the reversibly bound active center ligands (edrophonium, huperzine, and tacrine) induce a spectral shift at position 124 or 287.However, modest quenching is observed at position 124 upon binding of these active center ligands.", "The bisquaternary ligands, which should approach or come in close apposition with these residues, cause significant hypsochromic shifts.", "The large shift for decamethonium at position 124 may reflect the ability of the cluster of aromatic residues to collapse around the methylene chain of decamethonium enlodged within the active center gorge.", "Crystallographic studies show one quaternary ammonium of decamethonium to be consistently positioned in the vicinity of Trp84; however, both the flexible side chain and the outermost quaternary group are found to assume multiple positions in the decamethonium-AChE complexes studied to date (6, 29).", "Covalent inhibition of cationic trifluoroacetophenone (TFK+) produces very little spectral shift of acrylodan at either position 124 or 287.This is consistent with the crystal structures where the trimethyl ammonio moiety of TFK+ forms a cation −π interconnection with Trp86, and the trifluoroacetophenone moiety forms a hemiketal bond with the active center serine 203 (33).", "However, the isosteric t-butyl congener (TFK0) shifts the environment of residue 124 to that resembling a hydrophobic state.", "This difference suggests that the orientation of this hemiketal conjugate differs where the t-butyl group extends toward the gorge exit.", "TFK0 inhibits the wild type enzyme 70-fold slower that TFK+, presumably due to lack of cation-π interaction and slightly different ligand orientation (21).", "Alkyl phosphorylation with small alkyl groups also has little influence on the environment at position 124 (Table IV).", "Ω Loop Substitutions—The residues modified, 76, 81 and 84, are all on the outer surface and do not form the inner gorge wall.", "Since residues 81 and 84 carry acidic side chains, they might be expected to show solvent exposure in the native enzyme and not be involved in the internal stabilization of the loop, as is evident in the crystal structure of the mouse enzyme (5, 6).", "In the absence of ligand, the spectra of the conjugated acrylodan moiety reveal different degrees of solvent exposure with the acrylodan at position 84 being the most protected in an hydrophobic environment, acrylodan at 81 being intermediate, and acrylodan at 76 being most exposed.", "Examination of the crystal structures of mouse enzyme revealed a surface cavity near the side chain of the 84 site (5, 6).", "The observed λmax likely reflects acrylodan buried in this surface cavity when conjugated to the 84 site (FIG.", "1.).", "The presence of fasciculin causes a large bathochromic shift of acrylodan fluorescence at both the 81 and 84 positions, as well as an increase in quantum yield of acrylodan at 76.The lack of shift in emission seen in quantum yield of acrylodan at the 76 position may simply reflect a balance between small environmental changes at 76 upon ligand binding by fasciculin.", "In the case of Glu84, the bathochromic shift likely reflects Arg11 of fasciculin loop I coming in van der Waals contact with the 84 side chain and displacing acrylodan into a more polar environment.", "However, an explanation of the bathochromic shift at position 81 requires a more involved analysis.", "Although 81 is removed from the fasciculin-binding site, fasciculin has a sufficient molecular dimension to restrict the Ω loop so that the entire loop freezes or closes upon fasciculin binding.", "Thus, fasciculin binding may confer strain on the α-carbon backbone structure of the Ω loop such that the acrylodan side chain at positions 81 and 84 becomes exposed to the hydrophilic environment.", "The fact that substitutions at both positions yielded acrylodan spectra with equivalent emission maxima after ligand binding suggests a conformational involvement of the entire loop.", "EXAMPLE 3 Influence of Residue Modification on Inhibition by Noncovalent Active Site Inhibitors A similar trend in inhibition kinetics was seen with noncovalent active site inhibitors such as edrophonium and BW286c51 (Table II).", "An increase over wild type Kd of 2-fold occurs for edrophonium binding to E84C, and an 18-fold increase in Kd is observed for BW286c51 binding.", "Similar increases in Kd of edrophonium and BW286c51 were seen E84Q human AChE (18).", "By comparison, E81C showed no alterations in ligand binding constants.", "For acrylodan-labeled mutants, Kd was measured from the fluorescence signals of an equilibrium titration (FIG.", "4).", "Acrylodan-labeled E84C shows Kd increases of 10-fold for edrophonium and 3-fold for BW286c51 as compared to unreacted E84C.", "For acrylodan-labeled E81C, only a slight increase in Kd is seen for both ligands.", "The high concentration of acrylodan-labeled E81C required for equilibrium titrations precludes an accurate estimate of Kd for high affinity ligands such as BW286c51.Similar to fasciculin, small ligands that bind to the active center produce a similar strain.", "All of the small ligands, whether reversibly bound or covalently attached, elicit marked changes in acrylodan emission with the largest spectral shift seen for E84C, an intermediate value seen for E81C, and only small change observed for L76C.", "In each case the conformational change induced by the ligand causes the acrylodan to move into a region of higher dielectric constant, presumably being more solvent-exposed.", "The pattern is remarkably consistent among the ligands, and only the small organophosphate when conjugated induces a shift of smaller magnitude.", "A likely explanation for the observed conformational changes is that ligand binding to the active center induces gorge closure, which is meditated throughout the Ω loop.", "The strain placed on the α-carbon backbone upon gorge closure causes the side chains to shift positions and become exposed to a hydrophilic environment.", "DeFari et al.", "(43) has noted that the peripheral site inhibitor, thioflavin T, when bound to AChE, shows a large enhancement of fluorescence.", "Simultaneous binding of an active center ligand and thioflavin T partially quenches the enhanced fluorescence of bound thioflavin T. Radic and Taylor (29) have observed that bound active center ligands cause a partial quenching of the native tryptophan fluorescence in AChE.", "Since these ligands lack the spectral overlap for fluorescence resonance energy transfer, the bound ligand is likely to influence the connectivity between aromatic residues present in the gorge, thereby influencing fluorescence quantum yields.", "Taken together, these studies suggest that ligands induce conformational changes in AChE giving rise to a gorge conformation collapsed around the bound ligand.", "The site-directed cysteine mutagenesis and fluorescence labeling studies herein suggest the involvement of particular resides on the Ω loop in this conformational change.", "EXAMPLE 4 Effect of Fasciculin on Acrylodan Fluorescence Emission The peptide toxin, fasciculin, inhibits AChE by tightly capping the mouth of the active center gorge (FIG.", "1) (11, 30-32).", "Table III shows changes in emission maxima of acrylodan-labeled AChE mutants in the presence of fasciculin.", "There is no discernible change in fluorescence emission of acrylodan-conjugated A262C (20), consistent with the position 262 being distal to the fasciculin-binding site.", "The large hypsochromic shifts seen at both the 124 and 287 positions reflect solvent exclusions and an increase in hydrophobicity experienced by the fluorophores in the gorge upon fasciculin binding (20).", "For the Ω loop mutant, L76C, fasciculin binding produces a 40% increase in quantum yield but no change in emission maximum.", "Bathochromic shifts are found at both the 81 and 84 positions, with position 84 producing a shift of larger magnitude (FIG.", "2 and Table III).", "EXAMPLE 5 Effect of Covalently Conjugated Active Site Inhibitors on Acrylodan Fluorescence Emission Changes in emission maxima of acrylodan-labeled AChE mutants in the presence of conjugating trifluoroacetophenones are shown in Table IV.", "The trifluoroacetophenones inhibit the enzyme by conjugating to form a hemiketal at the active site serine without dissociation of leaving group (33).", "Both the isosteric neutral and cationic trifluoroketones (TFKo and TFK+) produced no discernible changes in emission spectra of acrylodan conjugated at H287C and A262C, consistent with a fluorophore position distant from gorge base and hence not in direct contact with ligand.", "Remarkably, both TFKo and TFK+ produce a substantial bathochromic shift (at least 30 nm) with acrylodan-E84C.", "The trifluoroketones also produce a spectral shift of intermediate value (20 nm) for E81C and a much smaller change (4-6 nm) for L76C.", "Interestingly, neutral TFKo produces a large 22 nm of hypsochromic shift with the Y124C acrylodan conjugate.", "O,O-Dimethyl-O-(2,2-dichlorovinyl)phosphate, a smaller achiral organophosphonate, phosphorylates the active site serine of mAChE, with subsequent departure of the dichlorovinyloxy group (34, 35).", "The small and symmetrical dimethyl phosphoryl conjugate remaining at the active site serine might lead one to suspect very little perturbation, if any at all, in fluorescence spectra.", "Indeed, acrylodan conjugated at positions 124, 262, and 287 showed very little or no change in spectrum.", "However, bathochromic shifts at positions 81 and 84 were observed, although of smaller magnitude for E84C when compared with other ligands (Table IV).", "EXAMPLE 6 Effect of Noncovalent Active Site Inhibitors on Acrylodan Fluorescence Emission Noncovalent active site inhibitors, such as edrophonium, tacrine, and huperzine, associate primarily with the choline subsite at the base of the active site gorge.", "Crystal structures of inhibitors bound to Torpedo californica AChE revealed that these ligands should have no direct contact with the conjugated fluorophore at all six cysteine-substituted sites (36, 37).", "Upon edrophonium, tacrine, or huperzine association, alteration of acrylodan emission maxima is undetectable for positions 124, 287, and 262 (Table V).", "However, as seen for other ligands, acrylodan conjugated at E84C surprisingly shows a bathochromic shift of 33 nm (from 477 to 510 nm) upon inhibitor binding.", "A change of smaller magnitude is seen in the case of acrylodan-L76C (from 505 to 509 nm) and acrylodan-E81C (from 480 to 510 nm) with noncovalent active site inhibitors.", "Ligand binding results in a common emission maximum (λmax˜510 nm) for acrylodan at the three Ω loop positions.", "EXAMPLE 7 Effect of Bisquaternary Inhibitors on Acrylodan Emission Spectrum Extended bisquaternary inhibitors, such as BW286c51 and decamethonium, belong to a class of inhibitors that interact with two binding sites of AChE simultaneously (32, 38-39).", "The quaternary ammonium moiety on one end of the molecule associates with the Trp86 residue that characterizes the choline-binding site, whereas the other end resides near Trp286 at the active site gorge rim.", "Table VI shows changes in emission maxima of acrylodan-labeled AChE mutants in the presence of bisquaternary inhibitors.", "No changes are observed at position 262.By contrast, both decamethonium and BW284c51 caused a pronounced hypsochromic shift and increase in quantum yields with acrylodan conjugated at Y124C and H287C.", "Addition of decamethonium produced a hypsochromic shift of 35 nm at position 124, and a modest 7 nm shift at position 287.BW284c51 has a similar effect; for the Ω loop mutants, L76C, E81C, and E84C, bathochromic shifts of similar magnitude to the monoquaternary ligands were observed (Tables V and VI).", "EXAMPLE 8 Determination of the Presence of Potential Gaseous Cognate Partners in the Field Using a Portable Detection Device Containing Acrylodan-Labeled Mouse Acetylcholinesterase (Accession No.", "IMAAA) A chip is configured to comprise multiple compartments containing various forms of labeled, immobilized acetylcholinesterase (AChE).", "Acrylodan-labeled mouse AChE is generated by the method as outlined in Shi et al.", "(J Biol Chem (2001) 276(45):42196-42204 and see above in Materials and Methods)).", "Alternatively, the mouse AChE is mutagenized by the method as outlined in U.S. Pat.", "No.", "6,001,625 and labeled as outlined in Shi et al.", "(2001).", "Picomolar amounts of labeled AChE in 0.01% bovine serum albumin and 0.01 M phosphate buffer (pH 7.0) are immobilized on a solid phase by the method as described in U.S. Pat.", "No.", "6,406,876.The immobilized enzyme is then lyophilized as described in U.S. Pat.", "No.", "5,354,654 and sealed under a gas permeable membrane.", "Prior to use, the enzyme is reconstituted by rehydration with an appropriate aqueous buffer.", "Alternatively, the enzyme is immobilized directly on a gas permeable resin and affixed to the chip surface (see, e.g., U.S. Pat.", "No.", "4,619,897).", "In the latter system, the appropriate buffer system is added just prior to exposure to the deployment area.", "The chip is configured for both standard, right-angled fluorescence detection or for epifluoresence (i.e., comprising windows for incident and emission of excitation wavelengths at 90° relative to the incident [excitation] light source and/or detection of emitted wavelengths at 180°).", "The forms of immobilized AChE are as follows: acrylodan-labeled unconjugated AChE (comprising both single-site and multiple-site labeled enzymes, labeled at residues Leu76, Glu81, Glu84, Tyr124, Ala262 and/or His287), acrylodan-labeled conjugated AChE (comprising the labels as above and further comprising various organophosphates such as sarin, phosphine, soman, or tabun as positive controls), and unlabeled forms of both conjugated and unconjugated AChE.", "The chip is deployed in an area suspected of containing one or more airborne organophosphate agents.", "The chip remains in the area for a sufficient time (under ambient temperature conditions) to allow for diffusion of the surrounding gases across the gas permeable membrane and to contact/interact with the immobilized AChE.", "The chip is then removed from the area and placed in a device that can detect fluorescence (e.g., spectrofluorimeter).", "The fluorescence detected in compartments containing newly formed conjugates is compared with the fluorescence detected in the corresponding controls.", "Estimation of ratios of unconjugated to conjugated forms of the AChE is measured.", "Subsequent determination of an accumulation of conjugated AChE demonstrates the presence of one or more organophosphate agents in the deployment area.", "All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.", "While the compositions and methods of this invention have been described in terms of illustrative embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention.", "More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved.", "Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention.", "REFERENCES 1.Cygler et al., Protein Sci (1993) 2:366-382.2.Rosenberry T. 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[ [ "Information processing apparatus and method, and storage medium", "A secure connection between the main unit of a portable information device and a peripheral device via a wireless network is ensured by using an electronic seal that makes it possible to transmit an encryption key to the portable information terminal and the peripheral device thereof by an operation which is analogous to “seal affixing” by a user confirmed as an authorized user.", "For example, user confirmation is performed by an authentication technology using biometric information, such as “fingerprint authentication”.", "After the portable information terminal and the peripheral device thereof perform mutual recognition, they can perform secure mutual communication via a wireless network, etc., by using the encryption key provided via the electronic seal." ], [ "1.An information processing apparatus for outputting an encryption key to an authorized user, said information processing apparatus comprising: biometric information input means for inputting biometric information of a user; encryption key generation means for generating an encryption key in response to an input of biometric information; encryption key registration means for storing the encryption key so as to be associated with the biometric information of a user; and encryption key output means for permitting an output of the encryption key as a result of the input biometric information matching the registered biometric information of a user.", "2.An information processing apparatus according to claim 1, wherein said biometric information is a fingerprint of the user.", "3.An information processing apparatus according to claim 1, wherein said encryption key output means extracts the encryption key from said encryption key registration means and outputs the encryption key in response to a seal-affixing operation of contacting an output target device.", "4.An information processing apparatus according to claim 1, further comprising encryption key re-registration means for permitting generation of the encryption key and a re-registration thereof in said encryption key registration means.", "5.An information processing apparatus for securely performing data communication with another device, said information processing apparatus comprising: private key receiving means for receiving a private key authenticated based on biometric information of a user; mutual recognition means for performing mutual recognition of whether another device with which communication is performed possesses the same private key; and data communication means for performing data communication, which is encrypted using the mutually recognized private key.", "6.An information processing apparatus according to claim 5, wherein said private key receiving means receives the private key in response to a seal-affixing operation of contacting a device on a private key output side.", "7.An information processing apparatus according to claim 5, wherein said mutual recognition means performs mutual recognition by a technique of confirming the private key possessed by the other device without knowing the key itself.", "8.An information processing method for outputting an encryption key to an authorized user, said information processing method comprising: a biometric information input step of inputting biometric information of a user; an encryption key generation step of generating an encryption key in response to an input of biometric information; an encryption key registration step of storing the encryption key so as to be associated with the biometric information; and an encryption key output step of permitting output of the encryption key as a result of the input biometric information matching the registered biometric information.", "9.An information processing method according to claim 8, wherein said biometric information is a fingerprint of the user.", "10.An information processing method according to claim 8, wherein, in said encryption key output step, the encryption key stored in said encryption key registration step is output in response to a seal-affixing operation of contacting an output target device.", "11.An information processing method according to claim 8, further comprising an encryption key re-registration step of permitting generation of the encryption key and re-registration thereof as a result of the input biometric information matching the registered biometric information.", "12.An information processing method for securely performing data communication with another device, said information processing method comprising: a private key receiving step of receiving a private key authenticated based on biometric information of a user; a mutual recognition step of performing mutual recognition of whether another device with which communication is performed possesses the same private key; and a data communication step of performing data communication, which is encrypted using the mutually recognized private key and.", "13.An information processing method according to claim 12, wherein, in said private key receiving step, the private key is received in response to a seal-affixing operation of contacting a device on a private key output side.", "14.An information processing method according to claim 12, wherein, in said mutual recognition step, mutual recognition is performed by a technique of confirming the private key possessed by the other device without knowing the key itself.", "15.A storage medium having stored thereon in a computer-readable form, computer software described so as to execute processing for outputting an encryption key to an authorized user on a computer system, said computer software comprising: a biometric information input step of inputting biometric information of a user; an encryption key generation step of generating an encryption key in response to an input of biometric information; an encryption key registration step of storing the encryption key so as to be associated with the biometric information; and an encryption key output step of permitting output of the encryption key as a result of the input biometric information matching the registered biometric information.", "16.A storage medium having stored thereon in a computer-readable form, computer software described so as to execute processing for securely performing data communication with another device on a computer system, said computer software comprising: a private key receiving step of receiving a private key authenticated based on the biometric information of a user; a mutually recognizing step of performing mutual recognition of whether a device of a communication party possesses the same private key; and a data communication step of performing data communication, which is encrypted using the mutually recognized private key." ], [ "<SOH> BACKGROUND ART <EOH>Along with improvements in semiconductor manufacturing technology, electronic devices have become increasingly smaller, and various types of portable information devices, such as notebook computers, PDAs (Personal Digital Assistants), and cellular phones have appeared.", "These types of portable information device are driven by the supply of electricity from a battery incorporated in the main unit of the device, and are used in a mobile environment, that is, outdoors or at the location of the user.", "There has been an increasing demand for portable information devices to be equipped with various peripheral devices (for example, a position detecting device such as GPS (Global Positioning System), and user input/output devices such as a microphone, a speaker, a head set, and a keyboard) so as to expand the functions.", "Hitherto, it has been common practice to meet such needs for expanding the functionality of the main unit of the device by providing the main unit of the device with a space for housing and connecting peripheral devices, such as an expansion slot and a card slot.", "However, in order to maintain the portability, which is the most striking feature of portable information devices, the occupied volume, the weight, the power consumption, etc., of a device connected to the slot must be strictly limited.", "For this reason, the number of devices which can be connected to the main unit of the device is greatly limited, and thus it is not possible to satisfactorily meet the function expansion demanded by the user.", "In order to avoid such a limitation, recently, it has been proposed that the expansion of functions be realized by the main unit of the device communicating with a peripheral device via a wireless network.", "When devices are wirelessly connected to one another, there are secondary effects, for example, there is no need to use cables and therefore the desktop remains tidy, and there is no mechanical damage of connectors due to the mounting/removal of devices.", "When compared to a case in which connection among devices is made using cables, in a case where connection is made by a wireless network, the relationships regarding which portable information device corresponds to which peripheral device becomes difficult to keep track of.", "In particular, in a working environment where a plurality of portable information devices are clustered together, in order to maintain a normal operation in each information device even if a plurality of users come close to one another, a scheme whereby individual peripheral devices are capable of specifying the portable information device which is currently connected or the user thereof is necessary.", "Though the portable information device has a high economic value due to its high-function computing performance, since it can be easily carried, the risk of encountering loss or theft is high.", "Therefore, sufficient care must be paid so that the security of the entire system is tot degraded considerably.", "In a working environment in which devices are connected to one another by a wireless network, including the case in which the functions of an information device are expanded, such user specification is required often.", "For example, in order that the information device is used as part of a cellular phone system, various technologies having functions for specifying a user while eliminating various types of abuse, including wiretapping, have already been proposed; however, their development is still in progress.", "However, most existing techniques regarding the security of devices are presupposed on the intervention of special devices which provide network functions, for example, communication devices disposed in a base station.", "Due to such presupposed conditions, it is not possible to apply the above-described wireless network technology to security on a network which includes not only simple peripheral devices which simply operate in accordance with instructions from a portable information device, but also active devices capable of issuing instructions to another type of expansion device or portable information device.", "When data communication is performed between two or more information devices, encryption technology is generally used.", "That is, a device on the transmission side sends transmission data after encrypting the data, and a device on the receiving side decrypts the received data, and thereafter, uses the data for further processing.", "However, in order to use the encryption method, an encryption key must be shared between both devices.", "In a usage environment where the connection relationship between devices is fixed and stationary, secure data communication can be realized relatively easily by providing an encryption key to be shared, to both devices at the transmission and receiving sides and by providing the encryption key securely at a place which is protected by means of hardware.", "In contrast, the connection relationship between devices is not fixed, as with a portable information device and peripheral devices which expand the functions thereof, and for example, each time the user of the device moves, the connection relationship with the peripheral device varies dynamically.", "In such a usage environment where the connection relationship is dynamic and variable, the encryption key provided to the portable information device and the peripheral devices thereof (or the encryption key shared between the devices) is merely temporary, that is, it can be used only while the connection relationship continues, and when the next connection relationship is established, a new encryption key must be provided.", "In a case where the encryption key is made to be valid in any connection relationship between devices, it is difficult to crack down on re-use of the encryption key, and the encryption key can no longer function as an encryption key.", "Furthermore, if an unauthorized person can provide an encryption key to both a portable information device and a peripheral device for which a connection relationship is newly established, commonly called “posing” is permitted, and security on the wireless network is lost." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 is a block diagram schematically showing the functional configuration of an information processing apparatus 10 according to an embodiment of the present invention, which is capable of functioning as an “electronic seal”.", "FIG.", "2 is a block diagram schematically showing the functional configuration of a portable information device 30 for which seal affixing by an electronic seal is to be received, and a peripheral device 50 which expands the functions thereof.", "FIG.", "3 is a flowchart showing a series of processing procedures for registering a user of the electronic seal and for generating a key for the user.", "FIG.", "4 is a flowchart showing a series of processing procedures for re-registering a user of the electronic seal and for generating a key for the user.", "FIG.", "5 is a flowchart showing a series of processing procedures which is executed between the information processing apparatus 10 and another device (the portable information device 30 , the peripheral device 50 , etc.", "), for performing a seal-affixing procedure.", "FIG.", "6 is a flowchart showing a processing procedure for examining the matching of a private key by Fiat-Shamir recognition.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to an information processing apparatus and method for maintaining a normal operation among portable information devices and function expansion devices thereof, which are connected via a wireless network, and to a storage medium therefor and, in particular, it relates to an information processing apparatus and method for ensuring security among a plurality of devices which operate in cooperation via a wireless network, and to a storage medium therefor.", "More specifically, the present invention relates to an information processing apparatus and method for ensuring security by securely transmitting a private key to each device connected via a wireless network, and to a storage medium therefor and, in particular, it relates to an information processing apparatus and method for ensuring security on a wireless network by permitting only the transmission of a private key by an authorized user, and to a storage medium therefor.", "BACKGROUND ART Along with improvements in semiconductor manufacturing technology, electronic devices have become increasingly smaller, and various types of portable information devices, such as notebook computers, PDAs (Personal Digital Assistants), and cellular phones have appeared.", "These types of portable information device are driven by the supply of electricity from a battery incorporated in the main unit of the device, and are used in a mobile environment, that is, outdoors or at the location of the user.", "There has been an increasing demand for portable information devices to be equipped with various peripheral devices (for example, a position detecting device such as GPS (Global Positioning System), and user input/output devices such as a microphone, a speaker, a head set, and a keyboard) so as to expand the functions.", "Hitherto, it has been common practice to meet such needs for expanding the functionality of the main unit of the device by providing the main unit of the device with a space for housing and connecting peripheral devices, such as an expansion slot and a card slot.", "However, in order to maintain the portability, which is the most striking feature of portable information devices, the occupied volume, the weight, the power consumption, etc., of a device connected to the slot must be strictly limited.", "For this reason, the number of devices which can be connected to the main unit of the device is greatly limited, and thus it is not possible to satisfactorily meet the function expansion demanded by the user.", "In order to avoid such a limitation, recently, it has been proposed that the expansion of functions be realized by the main unit of the device communicating with a peripheral device via a wireless network.", "When devices are wirelessly connected to one another, there are secondary effects, for example, there is no need to use cables and therefore the desktop remains tidy, and there is no mechanical damage of connectors due to the mounting/removal of devices.", "When compared to a case in which connection among devices is made using cables, in a case where connection is made by a wireless network, the relationships regarding which portable information device corresponds to which peripheral device becomes difficult to keep track of.", "In particular, in a working environment where a plurality of portable information devices are clustered together, in order to maintain a normal operation in each information device even if a plurality of users come close to one another, a scheme whereby individual peripheral devices are capable of specifying the portable information device which is currently connected or the user thereof is necessary.", "Though the portable information device has a high economic value due to its high-function computing performance, since it can be easily carried, the risk of encountering loss or theft is high.", "Therefore, sufficient care must be paid so that the security of the entire system is tot degraded considerably.", "In a working environment in which devices are connected to one another by a wireless network, including the case in which the functions of an information device are expanded, such user specification is required often.", "For example, in order that the information device is used as part of a cellular phone system, various technologies having functions for specifying a user while eliminating various types of abuse, including wiretapping, have already been proposed; however, their development is still in progress.", "However, most existing techniques regarding the security of devices are presupposed on the intervention of special devices which provide network functions, for example, communication devices disposed in a base station.", "Due to such presupposed conditions, it is not possible to apply the above-described wireless network technology to security on a network which includes not only simple peripheral devices which simply operate in accordance with instructions from a portable information device, but also active devices capable of issuing instructions to another type of expansion device or portable information device.", "When data communication is performed between two or more information devices, encryption technology is generally used.", "That is, a device on the transmission side sends transmission data after encrypting the data, and a device on the receiving side decrypts the received data, and thereafter, uses the data for further processing.", "However, in order to use the encryption method, an encryption key must be shared between both devices.", "In a usage environment where the connection relationship between devices is fixed and stationary, secure data communication can be realized relatively easily by providing an encryption key to be shared, to both devices at the transmission and receiving sides and by providing the encryption key securely at a place which is protected by means of hardware.", "In contrast, the connection relationship between devices is not fixed, as with a portable information device and peripheral devices which expand the functions thereof, and for example, each time the user of the device moves, the connection relationship with the peripheral device varies dynamically.", "In such a usage environment where the connection relationship is dynamic and variable, the encryption key provided to the portable information device and the peripheral devices thereof (or the encryption key shared between the devices) is merely temporary, that is, it can be used only while the connection relationship continues, and when the next connection relationship is established, a new encryption key must be provided.", "In a case where the encryption key is made to be valid in any connection relationship between devices, it is difficult to crack down on re-use of the encryption key, and the encryption key can no longer function as an encryption key.", "Furthermore, if an unauthorized person can provide an encryption key to both a portable information device and a peripheral device for which a connection relationship is newly established, commonly called “posing” is permitted, and security on the wireless network is lost.", "DISCLOSURE OF THE INVENTION An object of the present invention is to provide a superior information processing apparatus and method which are capable of ensuring security among a plurality of devices which operate in cooperation via a wireless network, and a storage medium therefor.", "Another object of the present invention is to provide a superior information processing apparatus and method which are capable of ensuring security by securely transmitting a private key to each device connected via a wireless network, and a storage medium therefor.", "Another object of the present invention is to provide a superior information processing apparatus and method which are capable of ensuring security on a wireless network by permitting only the transmission of a private key by an authorized user, and a storage medium therefor.", "The present invention has been made in view of the above-described problems.", "In a first aspect, the present invention provides an information processing apparatus or method for outputting an encryption key by an authorized user, the information processing apparatus or method comprising: biometric information input means or step for inputting biometric information of a user; encryption key generation means or step for generating an encryption key in response to a new input of biometric information; encryption key registration means or step for storing the encryption key in such a manner as to be associated with the biometric information; and encryption key output means or step for permitting the output of the encryption key as a result of the input biometric information matching the registered biometric information.", "The biometric information referred to herein is, for example, the fingerprint of a user.", "Alternatively, other biometric information can be used from the human body of the user, such as the retina pattern, a voiceprint, or a pulse pattern.", "In the encryption key output means or step, the encryption key is extracted from the encryption key registration means or step in response to the seal-affixing operation of bringing into contact with an output target device, and this encryption key is then output.", "According to such a seal-affixing operation, the private key can be transmitted to an external device such that interception from peripheral device is difficult.", "According to the information processing apparatus or method in accordance with the first aspect of the present invention, the private key can be securely transmitted to an external device by a technique called an “electronic seal”.", "The electronic seal referred to herein makes it possible to transmit the encryption key to a portable information terminal and a peripheral device thereof by operation analogous to “seal affixing” by a user confirmed as an authorized user.", "User confirmation is performed by authentication technology using biometric information as in, for example, “fingerprint authentication”.", "After the portable information terminal and the peripheral device thereof perform mutual recognition, it is possible to perform secure mutual communication via a wireless network, etc., by using an encryption key provided via operation of affixing a seal by an electronic seal.", "The information processing apparatus or method in accordance with the first aspect of the present invention may further comprise an encryption key re-registration means or step for permitting the generation of the encryption key and the re-registration of the encryption key in the encryption key registration means or step.", "By permitting the registration of a fingerprint differing from the fingerprint authenticated in a previous step by the encryption key re-registration means or step, the electronic seal can be transferred to another user.", "In a second aspect, the present invention provides an information processing apparatus or method for securely performing data communication with another device, the information processing apparatus or method comprising: private key receiving means or step for receiving a private key authenticated on the basis of biometric information of a user; mutual recognition means or step for performing mutual recognition of whether the other device with which communication is performed, possesses the same private key; and data communication means or step for performing data communication, which is encrypted using the mutually recognized private-key.", "Here, in the private key receiving means or step, the private key may be transmitted among a plurality of devices in a state in which interception is impossible by receiving the private key in response to the seal-affixing operation of bringing into contact with the device on the private key output side.", "Since the private key can be shared securely among a plurality of devices by the seal-affixing operation, it is possible to perform secure data communication by a wireless network.", "The mutual recognition means may perform mutual recognition by a technique of confirming the private key possessed by the other device without knowing the key itself.", "In a third aspect, the present invention provides a storage medium having stored thereon in a computer-readable form, computer software described so as to execute processing for outputting an encryption key by an authorized user on a computer system, the computer software comprising: a biometric information input step of inputting biometric information of a user; an encryption key generation step of generating an encryption key in response to a new input of biometric information; an encryption key registration step of storing the encryption key in such a manner as to be associated with the biometric information; and an encryption key output step of permitting the output of the encryption key as a result of the input biometric information matching the registered biometric information.", "In a fourth aspect, the present invention provides a storage medium having stored thereon in a computer-readable form, computer software described so as to execute processing for securely performing data communication with another device on a computer system, the computer software comprising: a private key receiving step of receiving a private key authenticated on the basis of the biometric information of a user; a mutually recognizing step of performing mutual recognition of whether a device which is a communication party possesses the same private key; and a data communication step of performing data communication, which is encrypted using the mutually recognized private key.", "The storage medium according to the third or fourth aspect of the present invention is a medium for providing various codes in a computer-readable form to, for example, a general-purpose computer system capable of executing various program codes.", "Such a medium is a removable, portable storage medium, such as a CD (Compact Disc), an FD (Flexible Disk), or an MO (Magneto-Optical Disc).", "Alternatively, the provision of computer software to a specific computer system via a transmission medium such as a network (it does not matter whether the network is wireless or wired) is technically possible.", "Such a storage medium defines the structural or functional cooperative relationship between computer software and the storage medium for realizing the functions of predetermined computer software on a computer system.", "In other words, by installing predetermined computer software into a computer system via a storage medium according to the third or fourth aspect of the present invention, cooperative effects are exhibited on the computer system, and the same operational effects as those of the information processing apparatus and method in accordance with the first and second aspects of the present invention can be obtained.", "Further objects, features and advantages of the present invention will become apparent from the following detailed description of the embodiments of the present invention with reference to the attached drawings.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a block diagram schematically showing the functional configuration of an information processing apparatus 10 according to an embodiment of the present invention, which is capable of functioning as an “electronic seal”.", "FIG.", "2 is a block diagram schematically showing the functional configuration of a portable information device 30 for which seal affixing by an electronic seal is to be received, and a peripheral device 50 which expands the functions thereof.", "FIG.", "3 is a flowchart showing a series of processing procedures for registering a user of the electronic seal and for generating a key for the user.", "FIG.", "4 is a flowchart showing a series of processing procedures for re-registering a user of the electronic seal and for generating a key for the user.", "FIG.", "5 is a flowchart showing a series of processing procedures which is executed between the information processing apparatus 10 and another device (the portable information device 30, the peripheral device 50, etc.", "), for performing a seal-affixing procedure.", "FIG.", "6 is a flowchart showing a processing procedure for examining the matching of a private key by Fiat-Shamir recognition.", "BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will now be described below with reference to the drawings.", "A “seal” in the real world has the function of confirming personal identification or certifying one's identity to other persons through the action of affixing of a seal.", "In comparison, the information processing apparatus of the present invention confirms that a user is an authorized user and functions as an “electronic seal” which outputs an encryption key possessed by the confirmed authorized user.", "The operation of outputting the encryption key of the authorized user is analogous to the person himself/herself who possesses a seal affixing it.", "For this electronic seal, the encryption key can be transmitted to another device by an operation similar to “affixing of a seal” by the user confirmed as an authorized user.", "As technology for confirming an authorized user, authentication technology using biometric information, for example, “fingerprint authentication”, can be applied.", "Examples of devices to which the encryption key is transmitted include a portable information device and peripheral devices which expand the functions thereof.", "These devices further perform mutual authentication between themselves, and thereafter, can ensure security of mutual communication via a wireless network, etc., by using the encryption key provided by the authorized user via the electronic seal.", "The embodiments of the present invention will now be described below in detail with reference to the drawings.", "1.System Configuration FIG.", "1 is a block diagram schematically showing the functional configuration of an information processing apparatus 10 according to an embodiment of the present invention, which is capable of functioning as an “electronic seal”.", "As shown in FIG.", "1, the information processing apparatus 10 according to the embodiment of the present invention comprises a CPU (Central Processing Unit) 11, a RAM 12, a ROM 13, a fingerprint detector 14, and an encryption-key output device 15.The CPU 11 is a central controller for centrally controlling the operation of the entire information processing apparatus 10 as an electronic seal, and realizes various processes by executing program code stored in the ROM (Read Only Memory) 13.Examples of processes executed by the CPU 11 include the following: (1) Registration of user who uses the electronic seal, (2) Generation of key for authorized user, and (3) Affixing of seal using electronic seal.", "These will be described later.", "The RAM (Random Access Memory) 12 is a volatile memory through which reading and writing are possible, and is used to load program code executed by the CPU 11 and to temporarily store work data for an execution program.", "Examples of work data include fingerprints of persons for whom user registration is performed (or other biometric information used for an authentication process), and encryption keys generated for authorized users.", "The generated encryption key can be transmitted to the portable information device 30, the peripheral device 50, etc., by the action of “affixing a seal”.", "This point will be described later.", "The fingerprint detector 14 is, for example, a device for optically reading the fingerprint of the user of the electronic seal.", "The read fingerprint is used for user registration and for user confirmation.", "However, when biometric information other than the fingerprint is used for an authentication process, the fingerprint detector 14 may be replaced with another device.", "The encryption-key output device 15 is a device for outputting an encryption key when it, as an electronic seal, affixes a seal in another device (a portable information device and/or a peripheral device thereof).", "For example, the encryption key can be securely transferred among a plurality of devices by the “seal-affixing” operation of bringing an encryption-key input device (to be described later) into contact with the encryption-key output device 15 for the electronic seal by which user registration and key generation have been performed.", "FIG.", "2 schematically shows the functional configuration of the portable information device 30 for which affixing of a seal by an electronic seal is to be received, and the peripheral device 50 which is connected to this portable information device 30 via a network and which expands the functions thereof.", "The portable information device 30 comprises a CPU 31, a RAM 32, a ROM 33, a wireless network adapter 34, and an encryption-key input device 35.The CPU 31 is a central controller for centrally controlling the operation of the entire portable information device 30 under the control of the operating system (OS), and executes various processes by executing program codes stored in the ROM (Read Only Memory) 33, which is a read only memory, and another external storage device (not shown) such as a hard disk unit.", "Examples of processes executed by the CPU 31 include various application programs such as word processing or spreadsheet calculations, processes of connection with the peripheral device 50 via a wireless network or another communication medium, and application processes using the connected peripheral device 50.The RAM 32 is a volatile memory through which reading and writing are possible, and is used to load program code executed by the CPU 31 and to temporarily store work data for an execution program.", "Examples of work data stored in the RAM 32 include encryption keys (private keys) received via the encryption-key input device 35.The wireless network adapter 34 is a function module for exchanging data by a short-distance wireless data communication function to and from the peripheral device 50.As a result of the connection of the peripheral device 50, the function expansion of the portable information device 30 is realized.", "Of course, in the portable information device 30, means other than the wireless network may be used, for example, a peripheral device may be loaded into, for example, an expansion slot, a card slot, etc., or a peripheral device may be connected via a cable.", "The encryption-key input device 35 is a device for inputting an encryption key from the information processing apparatus 10 (described above) functioning as an electronic seal through the action of affixing a seal.", "The encryption key is ensured so as to be possessed by an authorized user for whom user registration has been performed, and can be used to perform secure data communication with the peripheral device 50 through the encryption of transmission data.", "Of course, the portable information device 30 may incorporate various devices (not shown).", "For example, user input/output devices such as a display, a keyboard, and a mouse, and external storage devices such as a hard disk unit and a CD-ROM drive, may be provided.", "On the other hand, the peripheral device 50 comprises a CPU 51, a RAM 52, a ROM 53, a wireless network adapter 54, an encryption-key input device 55, and an enhanced function module 56.The CPU 51 is a central controller for centrally controlling the operation of the entire portable information device 50 under the control of the operating system (OS), and realizes various processes by executing program codes stored in the ROM (Read Only Memory) 53, which is a read only memory.", "Examples of processes executed by the CPU 51 include processes of connection with the main unit of the portable information device 30 via a wireless network or another communication medium, and function expansion services for the portable information device 30 by controlling the driving of the enhanced function module 56.The RAM 52 is a volatile memory through which reading and writing are possible, and is used to load program code executed by the CPU 31 and to temporarily store work data for an execution program.", "Examples of work data stored in the RAM 52 include encryption keys (private keys) received via the encryption-key input device 55.The wireless network adapter 54 is a function module for exchanging data by a short-distance wireless data communication function to and from the main unit of the portable information device 30.As a result of the connection of the portable information device 30, the peripheral device 50 can provide expansion functions provided by the enhanced function module 56 to the portable information device 30.Of course, means other than the wireless network may be used, for example, the peripheral device 50 may be loaded into an expansion slot, a card slot, etc., of the main unit of the portable information device 30, or the peripheral device 50 may be connected to a portable information device via a cable.", "Examples of expansion functions provided by the enhanced function module 56 to the main unit of the portable information device 30 include an external storage device function such as expansion memory, a hard disk, and a CD-ROM; a user input function such as a mouse and a pad; and a mutual connection function to a computer network, such as a network interface card (NIC).", "However, since the function expansion itself for information devices are not directly related to the subject matter of the present invention, no further description is given here.", "2.User Registration and Key Generation In order to use this system, first, it is necessary for the user to register information for specifying the user himself/herself, such as the fingerprint (or other biometric information), in the information processing apparatus 10 which functions as an electronic seal.", "The information processing apparatus 10 as an electronic seal stores the user specification information in the internal RAM 12 via the fingerprint detector 14, and at the same time, generates a random number, and stores this random number as the private key of that user in the RAM 12.FIG.", "3 shows, in a flowchart, a series of processing procedures for registering a user of the electronic seal and for generating a key of the user.", "In practice, this processing procedure can be implemented in such a form that the CPU 11 inside the information processing apparatus 10 executes predetermined program code.", "Referring to this flowchart, the user registration and key generation processes are described below.", "Initially, it is determined whether or not the fingerprint has already been registered (step S1).", "When the fingerprint has already been registered, the user is prompted to input a fingerprint (step S2).", "Then, it is determined whether or not the fingerprint read via the fingerprint detector 14 matches the registered fingerprint (step S3).", "If they do not match, it is assumed that the user registration has failed, and the entire processing routine is terminated.", "On the other hand, if the fingerprint is not yet registered or if the input fingerprint matches the registered fingerprint, the user is further prompted to input a fingerprint (step S4), and this fingerprint is stored in the RAM 12 (step S5).", "Then, a random number, which serves as an encryption key, is.1generated, this is stored in such a manner as to be associated with the fingerprint (step S6), and the entire processing routine is terminated.", "In the information processing apparatus 10 according to this embodiment, it is possible to register a fingerprint and generate a key again after the fingerprint registration and the key generation are performed once.", "In this case, designing may be made so that the fingerprint is registered in the same processing procedure as that at the first time.", "However, by designing so as to register the fingerprint in the processing procedure described below, security can be improved further.", "Initially, whether or not the registrant is an authorized user is confirmed by fingerprint authentication (step S11).", "Next, fingerprint registration and key generation are performed (step S12).", "Here, designing may be made so that registration of a fingerprint differing from the fingerprint authenticated in the procedure which has already been described with reference to FIG.", "3 is permitted.", "By permitting this registration, it becomes possible to transfer the electronic seal to another person.", "Furthermore, by forming a plurality of pieces of user specification information in such a manner as to be stored in the information processing apparatus 10, it is also possible to realize an electronic seal which can be shared in a group.", "3.Affixing of Seal The information processing apparatus 10, in which user registration and key generation have been performed, is able to output an encryption key from the encryption-key output device 15 thereof.", "Each of the portable information device 30 and the peripheral device 50 becomes usable by bringing the encryption-key input device 35/55 into contact with the encryption-key output device 15 on the information processing apparatus 10 side.", "Such operation of exchanging an encryption key between the encryption-key output device 15 and the encryption-key input device 35/55 is called a “seal affixing” in this specification.", "FIG.", "5 shows, in a flowchart, a processing procedure, which is executed between the information processing apparatus 10 and another device (the portable information device 30, the peripheral device 50, etc.", "), for performing this seal-affixing procedure.", "The seal-affixing procedure will be described below with reference to this flowchart.", "In the information processing apparatus 10, initially, whether or not the current operator has been properly registered as a user, that is, whether or not the fingerprint has already been registered, is determined (step S21).", "If the fingerprint is not registered, assuming that the seal-affixing procedure has failed, the entire processing routine is terminated.", "When the fingerprint has already been registered, the operator is further prompted to input a fingerprint (step S22).", "Then, it is determined whether or not the fingerprint read via the fingerprint detector 14 matches the registered fingerprint (step S23).", "If they do not match, by assuming that the seal-affixing procedure has failed, the entire processing routine is terminated.", "On the other hand, when the input fingerprint matches the registered fingerprint, the encryption key corresponding to this fingerprint is extracted from the RAM 12, and this is output from the encryption-key output device 15 onto a device for which seal affixing is to be performed (step S24).", "In the device for which seal affixing is to be performed (for example, the portable information device 30 and the peripheral device 50), the seal-affixed encryption key is read from the encryption-key input device 35/55, and this is stored in the RAM 32/52 (step S25).", "The portable information device 30 and the peripheral device 50 are able to perform secure data communication via a wireless network by encrypting the transmission data using the encryption key obtained by such a seal-affixing process.", "In the manner described above, when affixing a seal, the information processing apparatus 10 as an electronic seal confirms that the person who is going to affix a seal is an authorized user by using technology such as fingerprint authentication.", "Then, the encryption key is transmitted to each device such as the portable information device 30 and the peripheral device 50.Here, the transmission of the encryption key must be performed by a method in which interception from another (unauthorized) device is difficult.", "For this purpose, a technique of transmitting the private key by bringing them into contact with each other rather than by wireless communication is preferred.", "However, as long as transmission in which interception is difficult is possible, of course, the encryption key may be transmitted by a method other than seal affixing, including wireless communication.", "The operation of “affixing a seal” is performed to transmit the private key to each device.", "However, in a particular device, when performing an operation which may cause a significant result, an embodiment may be conceived in which such a seal-affixing operation is used to confirm again that the user is an authorized user by making a request to the user.", "In such a case, there is no particular need to update the private key stored in the RAM 12 of the information processing apparatus 10 as the electronic seal.", "Furthermore, the information processing apparatus 10 may be configured so that a plurality of private keys can be registered.", "In this case, it is possible to add a new private key by affixing a seal of the owner of the device and by performing a user addition operation (a seal-affixing operation by a user other than the owner).", "4.Mutual Recognition After the seal-affixing operation is performed, when information is transmitted between devices such as the main unit of the portable information device 30 and the peripheral device 50, it is mutually confirmed that they possess the same private key.", "This confirmation is performed by using a recognition processing technique in which it is difficult to know the private key by intercepting communication of them as in a private key matching examination technique by challenge/response sequence and zero-knowledge interactive proof.", "FIG.", "6 shows, in a flowchart, a processing procedure for examining the matching of the private key.", "Here, the Fiat-Shamir recognition is applied.", "The two devices which are going to perform communication are able to confirm that another device possesses the private key by mutually performing the procedure shown in FIG.", "6 on the other device.", "Initially, on the authentication requesting device side, two prime numbers of p and q are generated (step S31), and the product n (=p×q) of them is computed (step S32).", "Furthermore, the remainder I produced by dividing the private key S squared with n is determined (step S33), and n and I are transmitted to the determination device (step S34).", "Then, the number of authentications is substituted in the counter value count (step S35).", "Next, a random number r is generated (step S36), and the remainder X produced by dividing the squared random number r by n is transmitted to the determination device (step S37).", "On the other hand, on the determination device side, when n and I transmitted in step S34 are received (step S51), the number of authentications is substituted in the counter value count (step S52).", "Furthermore, when X transmitted in step S37 is received (step S53), a random number eε [0, 1] is generated, and this is transmitted to the authentication requesting device (step S54).", "On the authentication requesting device side, when the random number e is received (step S38), the remainder Y produced by dividing the random number r multiplied by the encryption key raised to the e-th power by n, is determined, and this remainder Y is transmitted to the determination device side (step S39).", "In the determination device, when Y is received.", "(step S55), a check is made to determine whether or not Y squared is equal to the remainder produced when X multiplied by I raised to the e-th power is divided by n (step S56).", "If they are not equal, failure is reported to the authentication requesting device (step S60), and the authentication request is rejected.", "On the other hand, if Y squared is equal to the remainder produced when X multiplied by I raised to the e-th power is divided by n, the determination device reports success to the authentication requesting device (step S57).", "Then, the counter value count is decreased by 1 (step S58).", "If the count is still a positive value, the process returns to step S52, where processes similar to those described above are repeatedly performed.", "If the count reaches 0, the entire recognition processing routine is terminated.", "On the authentication requesting device side, when the authentication result is received in step S57 or S60 (step S40), it is determined whether or not this is a success report (step S41).", "When the authentication result is a failure report, by assuming that the authentication request has failed, the entire processing routine is terminated.", "On the other hand, when the authentication result is a success report, the counter value count is decreased by 1 (step S42).", "If the count is still a positive value, the process returns to step S36, where processes similar to those described above are repeatedly performed (step S43).", "If the count reaches 0, the entire recognition processing routine is terminated.", "5.Mutual Communication When the above-described mutual authentication process is successful, it is ensured that the two devices possess the common private key.", "Thereafter, by encrypting communication using this private key, security of data communication can be maintained.", "For this encryption, various shared key encryption methods, including the DES (Data Encryption Standard) encryption certified as standard encryption by the U.S. National Institute of Standards and Technology can be applied.", "Further Information In the foregoing, the present invention has been described in detail while referring to the specific embodiments.", "However, it is self-explanatory that a person skilled in the art can modify or substitute the embodiments without departing from the spirit and the scope of the invention.", "In this specification, a description has been given by using, as an example, a case in which the present invention is applied to a wireless network in which portable information devices and peripheral devices thereof are connected to one another; however, the subject matter of the present invention is not limited to this.", "For example, the present invention can exhibit the same operational effects even in a case where other types of information devices are connected to one another or devices are connected to one another by using a communication medium other than a wireless network.", "In addition to the embodiments shown as examples in this specification, an electronic seal can be implemented.", "For example, a central device such as a portable information device, and an electronic seal can be configured so as to be integrated.", "For example, when the master and slave relationship between devices is clear as a PDA (Personal Digital Assistant) and a peripheral device connected to the PDA, the convenience can be improved by incorporating the function of the above-described electronic seal in the PDA.", "Alternatively, a device having a specific function and an electronic seal can be configured so as to be integrated.", "Conversely, the function of an electronic seal can also be incorporated in a peripheral device.", "For example, a modification is conceived in which a retina pattern identification device is incorporated in a sensor device for tracking the line of sight, and this is made to have the function of an electronic seal.", "Furthermore, a modification in which a private key is transmitted by a technique other than contact with devices is conceived.", "That is, as long as it is a technique in which interception from an external source is difficult, the devices need not necessarily to be brought into contact with each other.", "For example, it is conceived that a private key is transmitted by using a human body as a transmission path.", "(The device itself in which a human body is used as a transmission path has already been realized.", "For example, the Japanese Unexamined Patent Application Publication No.", "7-170215 which has already been transferred to the present applicant discloses a configuration in which, in two mutually independent systems having electrodes, a very weak radio wave of such a degree as to be insufficient for communication just the way it is transmitted between systems, and as a result of the intervention of a human body between the systems, transfer of data between the systems is performed through the intervention of the human body).", "In summary, the present invention has been disclosed in an illustrative form, and is not intended to be construed as restrictive.", "In order to define the gist of the present invention, the section of CLAIMS noted at the beginning of the Description should be referred to.", "Industrial Applicability According to the present invention, it is possible to provide a superior information processing apparatus and method which are capable of ensuring security among a plurality of devices which operate in cooperation via a wireless network, and a storage medium therefor.", "According to the present invention, it is possible to provide a superior information processing apparatus and method which are capable of ensuring security by securely transmitting a private key to each device connected via a wireless network, and a storage medium therefor.", "According to the present invention, it is possible to provide a superior information processing apparatus and method which are capable of ensuring security on a wireless network by permitting only the transmission of a private key by an authorized user, and a storage medium therefor.", "By using the information processing apparatus according to the present invention as an “electronic seal” for transmitting an encryption key by an authorized user, it is possible to configure a group of devices in which a plurality of users use a wireless network independently of one another within a narrow range in which these are connected by, for example, short-distance wireless data communication.", "According to the present invention, since communication among devices can be encrypted using a private key which is provided securely, an unauthorized person cannot know the communication contents (that is, intercept) unless the private key is stolen.", "It is during “seal affixing” that this private key is transmitted among devices.", "Since only an authorized owner is ensured to affix a seal by applying the authentication technique using biometric information such as fingerprint, it is very difficult to transmit the private key to the device provided by an unauthorized person who tries to intercept, and security of data communication can be ensured.", "Even if the information processing apparatus according to the present invention functioning as an “electronic seal” is stolen, interception can easily be prevented by the authorized user performing fingerprint registration, key generation, and seal affixing.", "In the present invention, since a function for specifying an authorized user by using biometric information as in fingerprint authentication is provided, it is impossible to affix a seal to another device by using the stolen electronic seal.", "Furthermore, even if the stolen electronic seal is disassembled and the information stored in the memory can be analyzed, interception and unauthorized use can be easily prevented by the authorized user performing fingerprint registration, key generation, and seal affixing again.", "By using the information processing apparatus according to the present invention as an electronic seal, other devices such as portable information devices and peripheral devices need not to be provided with means for confirming that a user is an authorized user." ] ]	Patent_10469788
[ [ "Chain branching agent and polyamide composition containing the same", "The invention relates to a chain branching agent containing anhydride groups, which chain branching agent contains.", "(a) 5-75 mass % of a copolymer of at least an unsaturated dicarboxylic acid or a derivative thereof and a vinyl aromatic monomer; (b) 5-75 mass % of a copolymer of acrylonitrile and a vinyl aromatic monomer, (c) 0-80 mass % of an inert processing aid; and (d) 0-10 mass % customary additives; and in which (a) and (b) are miscible, the ratio (a)/(b) is from 1/3 to 3/1, and the total of (a)+(b)+(c)+(d) is 100%.", "Advantages of this chain branching agent in the preparation of, for instance, a polyamide composition with non-Newtonian melt flow behaviour include better reproducibility and strongly decreased formation of gel particles.", "The invention also relates to processes for the preparation of a chain branching agent and of a polyamide composition with non-Newtonian melt flow behaviour as well as to the use of such a polyamide composition for the manufacture of moulding articles." ], [ "1.Chain branching agent containing anhydride groups, characterized in that the chain branching agent consists of (a) 5-75 mass % of a copolymer of at least an unsaturated dicarboxylic acid or a derivative thereof and a vinyl aromatic monomer; (b) 5-75 mass % of a copolymer of acrylonitrile and a vinyl aromatic monomer; (c) 10-80 mass % of a homo- or copolymer of ethylene or propylene; and (d) 0-10 mass % customary additives; and in which (a) and (b) are miscible, the ratio (a)/(b) is from 1/3 to 3/1, and the total of (a)+(b)+(c)+(d) is 100%.", "2.Chain branching agent according to claim 1, in which (a) is a copolymer of maleic anhydride and styrene and (b) is a copolymer of acrylonitrile and styrene.", "3.Chain branching agent according to claim 1, which contains 40-80 mass % (c).", "4.Chain branching agent according to claim 1, which contains 10-30 mass % (a) and 10-30 mass % (b).", "5.Chain branching agent according to claim 1, in which (a) and (b) contain 10-35 mass % maleic anhydride and acrylonitrile, respectively.", "6.Chain branching agent according to claim 1, in which (a) and (b) contain 20-30 mass % maleic anhydride and acrylonitrile, respectively.", "7.Chain branching agent according to claim 1, in which the ratio (a)/(b) lies between 2/1 and 1/2.8.Chain branching agent according to claim 1, in which the inert processing aid is a thermoplastic polyolefin.", "9.Chain branching agent according to claim 8, in which the processing aid is a low-density polyethylene.", "10.Process for preparing a chain branching agent according to claim 1, in which the components (a)-(d) are melt mixed and then formed into a granulate.", "11.Process for preparing a polyamide composition with non-Newtonian melt flow behaviour, which comprises melt mixing a polyamide having a lower viscosity and substantially Newtonian melt flow behaviour is melt mixed with a chain branching agent containing anhydride groups according to claim 1, and optionally other additives.", "12.Process according to claim 11, wherein the polyamide is a PA 6, PA 66, PA 46 or a copolyamide thereof.", "13.Process according to claim 11, wherein the polyamide has an amine groups content of at least 20 meq/kg.", "14.Process according to claim 11, wherein an amount of chain branching agent is used such that the polyamide composition has a content of component (a) of 0.01-6 mass % (relative to the polyamide).", "15.Process according to claim 11, which further comprises melt-mixing at least 15-40 mass % glass fibre as additive.", "16.Polyamide composition obtainable with a process according to claim 11.17.Polyamide composition with non-Newtonian melt flow behaviour containing a polyamide, such an amount of chain branching agent according to claim 1 that the content of component (a) is 0.01-6 mass % (relative to the polyamide), and 0-60 mass % other additives.", "18.A method for the manufacture of a part or moulded article comprising injection moulding or extruding a polyamide composition according to claim 16.19.A method according to claim 18, which comprises forming said part by extrusion blow moulding said polyamide composition.", "20.Process for the preparation of a moulded article in which at least two parts are bonded together by means of a welding technique, wherein at least one of the parts substantially consists, at least at the location of a surface to be welded, of said polyamide composition.", "21.Process according to claim 20, in which all parts substantially consist of said polyamide composition.", "22.Process according to claim 20, wherein vibration welding is used as the welding technique.", "23.A molded article obtainable with the process according to claim 20.24.A molded article according to claim 23 for use in the automotive industry, such as a convoluted tube, a bellows, a liquid container, a component of the fuel system, an air-inlet manifold or an air duct." ], [ "The invention relates to a chain branching agent containing anhydride groups.", "The invention also relates to a process for preparing the chain branching agent.", "The invention also relates to a process for preparing a polyamide composition with non-Newtonian melt flow behaviour as well as a polyamide composition obtainable by the process and use thereof for the manufacture of a moulded article.", "Such a chain branching agent is known from EP-A-0495363.As chain branching agent said publication describes a copolymer containing anhydride groups, preferably a copolymer of maleic anhydride and styrene (SMA), which chain branching agent is used for the preparation of a polyamide composition with increased melt viscosity that exhibits non-Newtonian melt flow behaviour.", "Such a polyamide composition is prepared by melt mixing a low-viscosity polyamide with the chain branching agent.", "Non-Newtonian melt flow behaviour is here understood to mean rheological behaviour involving an increase in the melt viscosity of a molten polymer composition with decreasing shear rate.", "This phenomenon is also referred to as structural viscosity.", "Anhydride groups are here understood to mean anhydride groups or groups from which anhydride groups can be formed under processing conditions, for example a dicarboxylic acid group.", "A drawback of the known chain branching agent from EP-A-0495363 is that melt mixing of chain branching agent and polyamide involves a high risk of the formation of gel particles.", "Another drawback of the known chain branching agent is that the resulting polyamide composition exhibits viscosity values that are poorly reproducible, with differences in viscosity occurring not only between different lots but also between different granules within a lot.", "These differences greatly interfere with stable processing of the polyamide composition by means of an extrusion or injection moulding technique to form moulded articles.", "The object of the invention therefore is to provide a chain branching agent containing anhydride groups that can be used for the preparation of a polyamide composition with non-Newtonian melt flow behaviour and that does not exhibit said drawbacks or exhibits them to a substantially decreased extent.", "Surprisingly, this aim is achieved according to the invention with a chain branching agent that contains (a) 5-75 mass % of a copolymer of at least an unsaturated dicarboxylic acid or a derivative thereof and a vinyl aromatic monomer; (b) 5-75 mass % of a copolymer of acrylonitrile and a vinyl aromatic monomer; (c) 0-80 mass % of an inert processing aid; and (d) 0-10 mass % customary additives; and in which (a) and (b) are miscible, the ratio (a)/(b) is from 1/3 to 3/1, and the total of (a)+(b)+(c)+(d) is 100%.", "Use of the chain branching agent according to the invention yields a much better reproducibility when preparing a polyamide composition with non-Newtonian melt flow behaviour and allows much more accurate control of the viscosity, while in addition hardly any problems are caused by formation of gel particles.", "Another advantage is that larger amounts of chain branching agent can be added, so that there are fewer dosing problems in the production of a polyamide composition.", "Examples of suitable unsaturated dicarboxylic acids or derivatives thereof that can be used as monomer for component (a) are maleic acid or itaconic acid, or derivatives thereof, for example maleic anhydride (MA), N-phenyl maleinimide or itaconic anhydride.", "Dicarboxylic acid derivatives are here understood to be in particular anhydride or imide derivatives.", "Examples of suitable vinyl aromatic monomers are styrene, α-methylstyrene, or a styrene in which the aromatic ring contains a halogen or alkyl substituent.", "At least is here understood to mean that the copolymer (a) may also contain a minor amount of one or more other monomers.", "Preferably, however, (a) is a copolymer of maleic anhydride and styrene (SMA).", "The MA content of (a) generally lies between 5 and 40 mass %, preferably between 10 and 35, and more preferably between 20 and 30 mass %.", "The advantage of a high MA content is that a higher degree of branching can be achieved through reaction with a thermoplastic polymer containing amine groups, for example a polyamide.", "A further advantage of the chain branching agent according to the invention is that such a high MA content can be used without any undesirable degree of polymer chain crosslinking and formation of insoluble particles (gel particles) occurring Suitable vinyl aromatic monomers in component (b) are the same as described for (a), with (b) preferably being a copolymer of styrene and acrylonitrile (SAN).", "The acrylonitrile (AN) content of (b) generally lies between 5 and 40 mass %, preferably between 10 and 35, and more preferably between 20 and 30 mass %.", "The advantage of a higher AN content is a higher polarity of the copolymer, which improves the compatibility with other polar polymers such as a polyamide.", "As a rule, an SMA copolymer is miscible with a SAN copolymer if the ratio between the MA content and the AN content of the respective copolymers is roughly between 1.6 and 0.6.Preferably the chain branching agent according to the invention therefore contains SMA and SAN copolymers with a ratio between the MA content and the AN content of between 1.6 and 0.6, more preferably between 1.2 and 0.8.The ratio between the amounts of components (a) and (b) in the chain branching agent according to the invention may in principle vary between 3/1 and 1/3.Preferably the (a)/(b) ratio lies between 2/1 and 1/2.This has the advantage that a better dispersion of the chain branching agent according to the invention in polyamide is obtained.", "The chain branching agent according to the invention preferably contains 10-80 mass % of an inert processing aid.", "A thermoplastic polymer is an example of a suitable processing aid.", "This has as advantages a much simpler mixing process for the preparation of a chain branching agent according to the invention and in addition dosing of the chain branching agent is greatly improved and can be controlled much more accurately.", "This inert processing aid also serves as carrier material for the preparation of a chain branching agent in the form of a SMA and SAN concentrate.", "Inert is understood to mean that the processing aid or the carrier material does not react with the other components (a) and (b) and with a thermoplastic polymer containing amine groups to which the chain branching agent is later added, and neither does it interfere to any undesirable extent with the reaction between anhydride groups and amine groups.", "The inert processing aid is preferably a thermoplastic polyolefin.", "This is understood to be a polymer or copolymer of substantially at least one olefin, which may also incorporate minor amounts of other monomers.", "Suitable examples include various types of polyethylene, including low- and high-density polyethylene (LDPE, HDPE), ethylene/α-olefin copolymers such as plastomers, ethylene copolymers with a vinyl monomer or an alkyl(meth)acrylate, such as for example vinyl acetate or ethyl acrylate, and propylene homopolymer and copolymers.", "The chain branching agent according to the invention preferably contains an LDPE as inert processing aid in view of its good processability.", "In a special embodiment the chain branching agent according to the invention contains between 40 and 80 mass % of an inert processing aid.", "As additives (d) the chain branching agent according to the invention may contain the customary stabilizers, antioxidants and the like.", "The chain branching agent may also contain compounds that promote the desired reaction between anhydride groups of the chain branching agent and amine groups of a thermoplastic polymer.", "The invention also relates to a process for preparing a chain branching agent according to the invention in which the components (a), (b), (c) and (d) are melt mixed and then formed into a granulate.", "This granulate is preferably granular, with dimensions comparable to those of plastics granulate known to one skilled in the art, but may also be in powder form.", "The advantages of a granular granulate are the good dosing properties and good mixing with other granular components, such as plastics granulate.", "The invention also relates to the use of a chain branching agent according to the invention as additive to increase the viscosity of a thermoplastic polymer containing amine groups.", "Preferably this thermoplastic polymer containing amine groups is a polyamide.", "The invention also relates to a process for preparing a polyamide composition with non-Newtonian melt flow behaviour in which a polyamide having a lower viscosity and substantially Newtonian melt flow behaviour is melt mixed with a chain branching agent containing anhydride groups according to the invention and optionally other additives.", "Suitable polyamides are generally all thermoplastic polyamides, but preferably semi-crystalline polyamides in view of their favourable processing and mechanical properties.", "Examples include polyamide 6 (PA 6), PA 66, PA 46, PA 69, PA 610, PA 11, PA 12, PA MXD6, and copolymers and mixtures of such polyamides.", "Preferably the polyamide is a PA 6, PA 66, PA 46 or a copolyamide thereof.", "Preferably the polyamide used in the process according to the invention, in particular PA 6, has a relative solution viscosity (RSV) of 2.0 to 3.5 (measured on a 1 mass % solution in 90% formic acid at 25° C.).", "More preferably, the polyamide has an RSV of 2.2 to 2.8 1 and most preferably of 2.3 to 2.6.Suitable polyamides generally have 0.1 to 1 amine group as end groups per linear chain molecule, the amine groups content preferably being at least 20 meq/kg, more preferably 30 meq/kg and most preferably 40 meq/kg.", "The advantage of a higher amine groups content is a stronger increase in viscosity and more pronounced non-Newtonian melt flow behaviour as a result of reaction with anhydride groups in the chain branching agent.", "Preferably such an amount of chain branching agent is added in the process according to the invention that the content of component (a) in the polyamide composition is 0.01-6 mass % relative to the polyamide, more preferably 0.05-3, and most preferably 0.1-1.5 mass %, the higher contents relating to an SMA with a low anhydride groups content and the other way round.", "A larger amount of chain branching agent and/or a higher anhydride groups content in the chain branching agent generally results in a stronger increase in the viscosity and more pronounced non-Newtonian melt flow behaviour of the polyamide composition.", "The polyamide composition made using the process according to the invention may also contain 0-60 mass % other additives.", "Preferably these additives are chosen so that they do not significantly interfere with the desired reaction between amine groups and anhydride groups.", "Examples of additives are stabilizers, antioxidants, colourants, release agents and lubricants and the like, flame retardants, impact modifiers, and fillers and reinforcing agents.", "Preferably the polyamide composition contains at least a combination of a copper salt and an alkali halide, such as Cu/KI, as stabilizer.", "Examples of suitable impact modifiers are rubber-like polymers that not only contain a polar monomers such as olefins, but also polar or reactive monomers such as, among others, acrylates, epoxide, acid or anhydride containing monomers.", "Examples include a copolymer of ethylene with (meth)acrylic acid or an ethylene/propylene copolymer functionalized with anhydride groups.", "The advantage of such additives is that they do not only improve the impact strength of the polyamide composition but also contribute to an increase in viscosity.", "Suitable fillers and reinforcing agents are mineral fillers such as clay, mica, talc, glass spheres and fibrous reinforcing agents such as glass fibres.", "As reinforcing agent the polyamide composition preferably contains 5-50 mass % glass fibres, relative to the total composition, more preferably 10-45, and most preferably 15-40 mass % glass fibres.", "Suitable glass fibres generally have a diameter of 5-20 micron, preferably 8-15 micron, and are provided with a coating suitable for use in polyamide.", "The advantage of a polyamide composition with glass fibres is its increased strength and stiffness, particularly also at higher temperatures, which allows use at temperatures up to close to the melting point of the polyamide in the polyamide composition.", "This is important especially for use in the automotive sector, and particularly in or near the engine compartment.", "In a preferred embodiment of the process according to the invention, polyamide, chain branching agent according to the invention and other additives (components) are melt mixed using an extruder, preferably a twin-screw extruder, it being possible for components to be supplied both to the throat of the extruder and to the melt.", "The temperature at which the process is carried out depends on the melting point of the polyamide used and generally lies between 200 and 350° C. Preferably such a throughput rate is chosen that the residence time in the extruder, combined with the temperature, is sufficient for the reaction between anhydride groups of the chain branching agent and amine groups of the polyamide to take place virtually completely.", "The advantage of a complete reaction is that the polyamide composition will not undergo any strong changes in viscosity when it is later melt-processed using a shaping technique.", "Surprisingly, it has been found that this reaction proceeds virtually to completion with the chain branching agent according to the invention, whereas when use is made of a pure SMA as chain branching agent the reaction does not proceed to completion in the same available time, while moreover no or hardly any gel particles are formed with the chain branching agent according to the invention.", "After melt mixing and granulation followed by drying, the polyamide composition is suitable for further use.", "If desired the viscosity can be increased further by means of a solid-phase post-condensation reaction.", "This involves exposure of the polyamide composition under reduced pressure for, for example, 1-50 hours to a temperature up to at most about 10° C. below the melting point of the polyamide in the polyamide composition until the desired viscosity level is reached.", "Such a post-condensation is applied in particular for the preparation of a polyamide composition to be processed by means of an extrusion blow moulding technique, this yielding an even better melt strength.", "In another embodiment of the process according to the invention the components of a polyamide composition are first mixed in solid condition and melt mixing takes place during a shaping step, for example during a an extrusion or injection moulding technique.", "The advantage compared with a process in which first a polyamide composition is prepared, which is then shaped, is that one processing step is left out.", "This process is possible because the chain branching agent according to the invention is more readily and better dispersed in a polyamide and the chain branching reaction takes place in a more controlled way than when using a state of the art chain branching agent.", "In a special embodiment a polyamide composition as obtained in granular form using the process according to the invention is then mixed with granules of a conventional polyamide composition having a lower viscosity and optionally a different composition, and processed as such as a granular mixture in a shaping step.", "This has the advantage that various compositions whose rheological properties are suited to the demands of a particular application can be obtained in a flexible and economic manner.", "The invention also relates to a polyamide composition that can be obtained with the process according to the invention.", "Compositions containing PA6, SAN and SMA are also known inter alia from the publication in J. Polym.", "Sci., Part B: Polym.", "Phys.", "30(11), 1273-84 (1992), but these are mixtures of, for example, 75 mass % PA6 with SAN, to which a smaller amount of SMA is added as compatibilizer, for example about 2.5 mass %.", "It is the effects on the morphology of the mixture that are important here, not the Theological behaviour.", "The invention therefore also relates to a polyamide composition with non-Newtonian melt flow behaviour containing a polyamide, such an amount of chain branching agent according to the invention that the content of component (a) is 0.01-6, preferably 0.05-3, and most preferably 0.1-1.5 mass % (relative to the polyamide), and 0-60 mass % of other additives, of which preferably 1045 mass % glass fibres.", "The invention also relates to the use of a polyamide composition according to the invention for manufacturing a part or moulded article by means of an injection moulding or extrusion technique.", "The higher viscosity and the non-Newtonian rheological behaviour of the polyamide composition according to the invention is advantageous in particular when shaping takes place by means of extrusion, for example into a rod, profile or tube, and in particular when a (hollow) moulded article is manufactured using an extrusion blow moulding technique.", "Extrusion blow moulding generally involves the manufacture of a tubular, still molten preform, the melt strength of the polyamide composition having to be such that the preform does not stretch or change shape under its own weight.", "Examples of extrusion blow moulding techniques that are used in practice include coextrusion blow moulding, sequential extrusion blow moulding and 3D extrusion blow moulding.", "The polyamide composition according to the invention also offers advantages when processing takes place by means of art injection moulding technique, because the high viscosity at a low shear rate to a large extent prevents the formation of burs caused by the flow of the molten polyamide composition between mould parts.", "The invention also relates to a process for the preparation of a moulded article in which at least two parts, for instance obtained by means of an injection moulding or extrusion technique, are bonded together by means of a welding technique, with at least one of the parts substantially consisting, at least at the location of a surface to be welded, of a polyamide composition according to the invention.", "A special embodiment is a process in which all parts substantially consist of a polyamide composition according to the invention, so that an optimum weld seam strength is obtained.", "A welding technique is here understood to be a way of bonding several parts together in which the plastic is heated to above its melting point at the location of the contact areas to be bonded together, following which the parts are pressed together and cooled.", "Heating of the contact areas can be effected in several ways, for instance by contacting the contact areas with a heated metal plate, by heating the plastic using ultrasonic vibrations (ultrasonic welding), or by generating friction heat by rotating the contact areas against each other (rotation welding) or rubbing them together (vibration welding) at a high speed.", "Preferably vibration welding is used for joining parts as this makes it possible to produce complex moulded articles.", "It has been found that if at least one part is made from a polyamide composition according to the invention this results in a substantial improvement of the weld seam strength.", "The invention also relates to a moulded article obtainable by means of an injection moulding or extrusion technique and to a moulded article consisting of two or more parts bonded together by means of a welding technique, which moulded parts contain a polyamide composition according to the invention.", "In particular the invention relates to a moulded article for use in the automotive industry, such as a convoluted tube, a bellows, a liquid container, a component of the fuel system, an air-inlet manifold or an air duct.", "The invention will now be further elucidated on the basis of the following examples and comparative experiments.", "Materials Used: SMA a styrene-maleic anhydride copolymer with an MA content of 28 mass % and an Mw of approx.", "110,000 g/mol (type Stapron ® SZ28110, DSM, NL); SAN a styrene-acrylonitrile copolymer with an AN content of 28 mass %, MFI (220° C., 10 kg) 50 g/10 min (DSM, NL); LDPE a low-density polyethylene (type Lupolen ® 1810H, BASF, DE); EP Impact modifier, ethylene-propylene rubber modified with rubber approx.", "0.5 mass % MA, MFI (230° C., 2.16 kg) = 0.7 g/10 min, density 0.87 g/cm3 (type Tafmer ® MP0610, Mitsui, JP) PA 6 a polyamide 6 with RSV = 2.5 (1 mass % in formic acid, 25° C.), Mn approx.", "15,000 g/mol, Mw approx.", "29,000 g/mol, amine groups content 48 meq/kg (type Akulon ® C225, DSM, NL); GF standard polyamide glass fibres, fibre diameter 10 mm PA 6 a standard injection-moulding polyamide 6 type with 30 mass % GF30 glass fibres, heat-stabilized, coloured black (type Akulon ® K224-HG6, DSM, NL); EXAMPLE I A 50/50 (m/m) mixture of SMA and SAN was prepared by feeding the two components to a ZSK57 twin-screw extruder, with a temperature setting of 230° C., at a speed of 200 r.p.m.", "Only with great difficulty could the exiting, clear melt be processed into granules, the strand formed being very brittle.", "EXAMPLE II On a ZSK57, with a temperature setting of 230° C. and at a speed of 200 rpm, a mixture of SMA/SAN/LDPE was prepared, in a mass ratio of 25/25/50.The throughput rate was 110 kg/hour, with 85% torque as control value.", "The mixture was readily extruded and processed into granules with regular dimensions.", "EXAMPLES III AND IV In an analogous manner to Example II SMA/SAN/LDPE mixtures with mass ratios of 37.5137.5/25 and 12.5/12.5/75 were prepared.", "The processability of the sample with 25% LDPE was slightly lower than that of the other samples.", "EXAMPLE V AND COMPARATIVE EXPERIMENT A The compositions indicated in Table 1 were prepared by feeding PA 6, chain branching agent according to Example I or SMA, 0.3 mass % copper/iodide stabilizer and 0.5 mass % black colour concentrate to the throat of a ZSK57.Furthermore an impact modifier was added.", "This modifier consisted in part of a modified EP rubber and in part of LDPE.", "The temperature was set at 270° C., the throughput rate was about 50 kg/hour and the melt was degassed by applying a negative pressure.", "The extruded strands were chopped into granules and dried.", "Subsequently the materials were post-condensed in the solid phase at a granule temperature of 185° C. until the materials had an RSV of 5.1 (1 mass % in formic acid, 25° C.).", "The rheological behaviour of the compositions was measured by means of a Rheometrics RMS800 Dynamic-Mechanical Spectrometer (DMS) in the shear rate range of 0.1-100 rad/s at a temperature of 260° C. (parallel plate geometry, diameter=25 mm).", "The results are summarized in Table 1.From the higher melt viscosity found it can be deduced that a chain branching agent according to the invention is more effective than pure SMA.", "This is also apparent from the higher viscosity ratio at low and high shear rates, respectively.", "In addition, a melt drawing test (melt temperature=260° C.) was conducted.", "The force needed to draw the strand in the melt is a measure of the melt strength of the material.", "The melt drawability is sensitive to inhomogeneities in the melt such as gels.", "The higher melt drawing force is indicative of the increased effectiveness of the branching reaction and the higher melt drawability indicates that the number of gel particles is reduced in comparison with the comparative experiment.", "TABLE 1 unit Example V Comp.", "Exp.", "A Composition PA 6 mass % 88.5 88.9 Example I mass % 0.72 0 SMA mass % 0 0.36 EP rubber mass % 4.0 4.0 LDPE mass % 6.0 6.0 rheological properties RSV 5.1 5.1 η (0.1 rad/s) Pa · s 22,400 17,800 η (100 rad/s) Pa · s 1600 1550 η (0.1)/η (100) 14.0 11.5 Melt drawability 38 24 Melt drawing force cN 8.5 7.9 EXAMPLES VI-VII AND COMPARATIVE EXPERIMENTS B-C The compositions indicated in Table 2 were prepared by feeding PA 6, chain branching agent according to Example II or SMA, 0.25 mass % copper/iodide stabilizer, 0.3 mass % release agent and 2.0 mass % black colour concentrate to the throat of a ZSK30 twin-screw extruder and 30 mass % glass fibre via a side feed to the melt.", "The temperature was set at 270° C., the throughput rate was about 10 kg/hour and the melt was degassed by applying a reduced pressure.", "The extruded strands were chopped into granules and dried.", "In comparative experiment A a commercial PA6 GF30 was used as reference material.", "The rheological behaviour of the compositions was measured by means of a Rheometrics RMS800 Dynamic-Mechanical Spectrometer (DMS) in the shear rate range of 0.1-100 rad/s at a temperature of 260° C. (parallel plate geometry, diameter=25 mm) The compositions were processed into test bars on an Engel 80e injection moulding machine, with a temperature setting of 260° C. (melt temperature).", "The tensile properties were determined in accordance with ISO 527-1, the impact strength was measured in accordance with ISO 179/1eU and ISO179/1eA.", "The results are summarized in Table 2.From the higher melt viscosity found it can be deduced that a chain branching agent according to the invention is more effective than pure SMA.", "In addition, during the injection moulding of Examples VI and VII a more stable process was observed, with smaller pressure variations with time, than in comparative experiment C. The increased viscosity is not found to have any adverse effect on the injection moulding behaviour as such, supporting a non-Newtonian flow behaviour.", "The higher viscosity does result in higher toughness of the polyamide composition, judging from the somewhat higher elongation at break and (unnotched) impact strength and the slight decrease in tensile strength.", "TABLE 2 Comp.", "Comp.", "unit Ex.", "VI Ex.", "VII Exp.", "B Exp.", "C composition PA 6 mass % 66.3 65.7 PA 6 GF30 67.0 GF mass % 30.0 30.0 30 Example II mass % 1.2 1.8 0 0 SMA mass % 0 0 0 0.45 tensile properties E-modulus MPa 9830 9690 9680 9440 Tensile MPa 160 155 169 170 strength Elongation at % 4.1 4.3 3.7 4.0 break impact strength Charpy kJ/m2 16 15 15 14 notched, 23° C. Ch.", "kJ/m2 92 86 82 99 unnotched, 23° C. Charpy kJ/m2 9 9 9 9 notched, −30° C. Ch.", "kJ/m2 75 73 68 78 unnotched, −30° C. rheological properties RSV 3.35 3.88 2.4 η (0.1 rad/s) Pa.s 7450 15150 940 8900 η (100 rad/s) Pa.s 2230 2610 850 2150 η(0.1)/η(100) 3.3 5.8 1.1 4.1 EXAMPLE VIII Analogously to Examples III and IV a larger amount of a polyamide composition with 1.1 mass % of the chain branching agent from Example II was prepared using a ZSK58 extruder, temperature setting 250° C. and throughput rate about 150 kg/hour at a speed of 250 rpm.", "The product had an RSV of 3.43.By means of DMS 1 (0.1 rad/s) was measured to be 7415 and η (100 rad/s)=1990, so that η(0.1)/η(100) was 3.4.This polyamide composition and the reference material PA 6 GF30 (comparative experiment B) were injection moulded into two articles which were subsequently joined by means of vibration welding to form an air-inlet manifold.", "In general, the resulting weld seam is the weakest point of such a part.", "Its strength was determined by means of bursting pressure tests under different conditions.", "The results obtained are presented in Table 3.It can be concluded that the polyamide composition according to the invention results in an appreciably higher weld seam strength than the reference material, while in addition it meets the required minimum values for this moulded article.", "TABLE 3 Unit requirement Example VIII Comp.", "Exp.", "B Bursting pressure MPa ≧0.55 0.66 0.40 at 23° C. Bursting pressure MPa ≧0.45 0.60 0.34 at −30° C. Bursting pressure MPa ≧0.50 0.50 0.42 at 120° C." ] ]	Patent_10469797
[ [ "Mobile advertising methods and improvements", "Mobile advertising methods and improvements, for instance, the adding of global positioning satellite tracking devices to mobile advertising platforms for purposes of verification of location, travel routes, mileage, etc.", "Incorporation of the Internet, Intranet, peer-to-peer and/or other networking systems and associated software for improving business methods, account access, and the interface between the advertiser or client, the ad agency or administration, and those persons or entities associated with the mobile platforms.", "Providing wireless communication systems for mobile advertising platforms for enabling real-time messages and for real-time interaction and/or for connecting central or distributed service operations.", "Providing improved compensation particularity to those persons or entities associated most closely with the mobile platforms.", "Incorporation of video and/or still e-cameras and/or audio equipment into the mobile advertising platform." ], [ "1.Improved method of advertising via non-common carrier automobiles by providing adequate compensation to the persons or entities associated with the vehicles, whereby for example only, ideally a person or person and car, etc., in a desired target market, which also conforms with possible other client/sponsor or agency desired parameters, might list him/herself as available, might request, be requested, or might even bid to contract to provide advertising via their vehicle, and in exchange receive adequate compensation in one or more forms.", "2.Improved method of advertising via automobiles by providing an onboard or portable location device, such as a GPS device, whereby for example only, a global positioning satellite system device (“GPS” device) might either communicate various related data in real-time and/or record such data for down loading periodically.", "3.Improved method of advertising via automobiles by providing one or more onboard or portable wireless communication systems or devices for improved up-dating of the ad message, spot location advertising, and for other communication purposes, whereby for example only, such wireless device may be used to update, on a real-time, live or down loadable basis, the advertising media or other information in order to for instance change to a new ad as the vehicle approaches a client's store, or for instance to issue a warning to other drivers of an accident ahead, and such communication system may be integrated or not integrated or associated with the GPS system.", "4.Improved method of advertising via automobiles by providing an Internet, Intranet, peer-to-peer, or other networking and software systems, as an significant communication and business interface between the ad agency and the persons associated with the vehicles, whereby for example only, a comprehensive business tool consisting of computer hardware, mobile and stationary devices and appropriate software so that, for instance persons interested in signing up as potential ad drivers may review criteria, contracts and other information, potential clients may review advertising plans and rates, and data from various sources may be entered, such as when the car is fueled and the GPS data, payments, etc., and established clients and ad drivers may access their accounts.", "5.Improved method of advertising via automobiles by providing one or more onboard or portable video or still cameras, with or without an audio pickup, whereby for example only, such camera captured data may be recorded and stored and/or be transmitted in real-time or by streaming video for various purposes, for instance traffic updates, accidents, and route, parking, delivery/location verification, special events, etc.", "6.A comprehensive method of advertising via automobiles, the method comprising the following steps: a) providing adequate compensation to the person associated with the vehicle; b) providing an onboard or portable vehicle location device.", "7.A comprehensive method of advertising via automobiles, the method comprising the following steps: a) providing adequate compensation to the person associated with the vehicle; b) providing the Internet, Intranet, or other such networking system as a communication and/or business interface between the ad agency and persons.", "8.A comprehensive system regarding the business methods of providing advertising via automobiles, the methods comprising the following steps: a) providing the Internet, Intranet, or other networking system as a communication and/or business interface between the ad agency and persons.", "b) providing adequate compensation to the person associated with the vehicle.", "9.Improved method of advertising via mobile platforms by providing adequate compensation to the person associated with the platform, whereby for example only, ideally a person or person carried platform, etc., in a desired target market, whom/which also conforms with other client/sponsor or agency desired parameters, might list him/herself as available, might request, be requested, or might even bid to contract to provide advertising via their body or personally carried platform, and in exchange receive adequate compensation in one or more forms.", "10.Improved method of advertising via mobile platforms, by providing an onboard or portable location device, whereby for example only, said location device, such as a global positioning satellite system device (“GPS” device) might either communicate various related data in real-time and/or record such data for down-loading periodically.", "11.Improved method of advertising via mobile platforms by providing one or more wireless communication systems or devices, whereby for example only, such wireless device may be used to update, on a real-time, streaming or down loadable basis, the advertising media or other information in order to, for instance, change ad copy/messages as the mobile platform approaches a client's store, or for instance, to issue a warning to others of some hazard, and such communication system may be integrated or not integrated or associated with other systems.", "12.Improved method of advertising via mobile platforms, by providing the Internet, Intranet, peer-to-peer or other networking and software systems as an significant communication and business interface between the ad agency and persons, whereby for example only, a comprehensive business tool consisting of computer hardware, mobile and stationary devices and appropriate software so that, for instance, persons interested in signing up as potential mobile platform carriers, etc., may review criteria, contracts and other information, potential clients may review advertising plans and rates, and data from various sources may be entered, such as when the platform is delivering its message, GPS data, payments, etc., and established clients and ad persons may gain access to their private access account information, etc.", "13.Improved method of advertising via mobile platforms, by providing one or more onboard or video and/or still cameras, with or without an audio pickup, whereby for example only, such camera captured data may be recorded and stored and/or be transmitted in real-time or by streaming video for various purposes, for foot traffic updates, store coverage, and routes, delivery and location verification, special events, etc.", "14.A comprehensive method of advertising via mobile platforms, the method comprising the following steps: a) providing adequate compensation to the person associated with the platform; b) providing an onboard or portable platform location device, such as a GPS device.", "15.A comprehensive method of advertising via mobile platforms, the method comprising the following steps: a) providing adequate compensation to the person associated with the platform; b) providing the Internet, Intranet, or other networking system as a communication and/or business interface between the ad agency, etc.", "and platform persons.", "16.A comprehensive system regarding the business methods of providing advertising media via mobile platforms, the methods comprising the following steps: a) providing the Internet, Intranet, peer-to-peer or other networking system as a communication and/or business interface between the ad agency and/or other administrative organization and the platform persons; b) providing adequate compensation to the persons associated with the platforms." ], [ "<SOH> BACKGROUND <EOH>1.Field of Invention This invention relates generally to new or improved methods relating to advertising on mobile platform mobile advertising and mobile information business and methods (together, herein meaning “advertising”, “advertisements”, “ads” or “adverts”).", "The word “method” herein will generally be used in place of one or more of the following terms: advertising services, business services, business concepts, mobile advertising processes, business method, business model, etc.", "These improvements and new methods are accomplished through the inventive integration and/or mixing and matching of various new and existing mobile platforms, the Internet, software, wireless devices, incentives, compensation, communication systems, location technology such as GPS, and methods and/or by the inventive overcoming of serious limitations related to the prior art of mobile advertising.", "Although this application will primarily apply to mobile advertising platforms, other platforms and methods are included when the inventive combinations and/or new methods herein proposed are applicable.", "2.Description of Prior Art Although not so limited, this invention generally relates to mobile platforms used for advertising, such as automobiles (herein, the word “car” may refer to vehicles, trucks, motorcycles, scooters, etc.)", "boats, planes, bikes, skateboards, signs, phones, PDAs, game and gaming platforms, appliances, computers, living beings, articles of clothing, toys, etc.", "(these and other related mobile platforms, herein may be referred to as simply “mobile platforms”).", "Examples of prior art are taxi and bus ads, signs on company owned cars, airplanes pulling ad banners, bumper stickers, license plate frames, dealership logos, race and other cars with sponsorship ads, hats, clothing, etc.", "These examples represent three common financial models of mobile platform advertising: a. volunteer, i.e., wearing your favorite baseball team's cap, branded shoes, a civic ad (“give blood”, etc.)", "on a tee shirt, etc.", "; b. sponsored ads, i.e., race cars, etc.", "; c. paid mobile platform advertising, i.e., taxi ads, moving billboards, etc.", "A paid example that will be used herein, is commuter cars whose owners contract with advertising firms, etc.", "to use their car as a mobile advertising platform in exchange for significant valuable compensation.", "The general concept of actually paying money to car owners for placing advertisements on their cars may not be new.", "Reportedly, starting in 1972, a company named Beetleboards paid VW bug car owners about $20 USD per month to place advertising on their Volkswagen cars.", "In addition to the low monetary compensation, the VW bug owners apparently also benefited from an exclusive “bug club” or Beetleboard membership effect that encouraged them to sport the third partly advertising arranged by the Beetleboard’ company.", "Apparently, the belonging to the bug ad club effect did not spread to other makes of cars.", "As the popularity of owning VW bugs died off so did the Beetleboard advertising business.", "Herein, the “prior art” means, the prior art, prior to the inventor's press publications, related articles, Website and his provisional patent filing.", "Although not intended to list all the disadvantages related to the prior art, generally the prior art advertising methods and/or platforms present one or more of the following disadvantages or limitations: a) low or too little monetary or other compensation to the platform owner, i.e., “free” bumper stickers, printed tee shirts, antenna balls, hats, etc.", "; b) no tracking of the amount of public exposure (herein may mean the target audience, etc.)", "i.e., a bumper sticker could be removed within hours or days, and a tee shirt may never be worn, and a taxi could be in the shop for repairs, etc.", "; c) no location tracking for mobile platforms, i.e., lack of GPS, etc., enabled mobile platforms; d) limited real-time interactive advertising; e) no or limited automatic inventory and/or automatic and/or interactive replacement ordering platforms or systems; f) no or limited live or real-time updates and live or down loadable changes to the ad copy; g) mostly visual ads; h) limited video and streaming video; i) a very limited variety of platforms; j) no interactive shopping handheld or basket attached PDAs to provide in store or in mall or in car specials, ads, suggestions, recipes, reminders for additional related items, etc.", "; k) no or limited use of RF and IR technologies; l) no physical interaction technology; m) no or limited artificial intelligence (“AI”) technology used; n) little or no instant public or target audience feedback to the advertising message; o) limited to the use of two of the five human senses." ], [ "<SOH> SUMMARY <EOH>In accordance with the present invention, improvements, various methods and various combinations of apparatus and/or technology for mobile advertising are disclosed.", "Herein, the proposed improvements, combination of improvements, various methods, models, concepts and combinations of such and combinations of apparatus and/or technology, may be referred to simply as “methods”.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "CROSS-REFERENCES TO RELATED APPLICATIONS This application claims the benefit of U.S.", "Provisional Patent Application Ser.", "No.", "60/170,088, filed 9 Dec. 99, on behalf of Craig L. Linden and titled AD-DRIVE.", "Additionally, U.S. Patent Office Disclosure Documents Nos.", "467551 and 471830, were respectively mailed on Jan. 1, 2000 and Mar.", "24, 2000, and they now carry the respective USPTO date stamp markings of Jan. 11, 2000, and Apr.", "3, 2000.BACKGROUND 1.Field of Invention This invention relates generally to new or improved methods relating to advertising on mobile platform mobile advertising and mobile information business and methods (together, herein meaning “advertising”, “advertisements”, “ads” or “adverts”).", "The word “method” herein will generally be used in place of one or more of the following terms: advertising services, business services, business concepts, mobile advertising processes, business method, business model, etc.", "These improvements and new methods are accomplished through the inventive integration and/or mixing and matching of various new and existing mobile platforms, the Internet, software, wireless devices, incentives, compensation, communication systems, location technology such as GPS, and methods and/or by the inventive overcoming of serious limitations related to the prior art of mobile advertising.", "Although this application will primarily apply to mobile advertising platforms, other platforms and methods are included when the inventive combinations and/or new methods herein proposed are applicable.", "2.Description of Prior Art Although not so limited, this invention generally relates to mobile platforms used for advertising, such as automobiles (herein, the word “car” may refer to vehicles, trucks, motorcycles, scooters, etc.)", "boats, planes, bikes, skateboards, signs, phones, PDAs, game and gaming platforms, appliances, computers, living beings, articles of clothing, toys, etc.", "(these and other related mobile platforms, herein may be referred to as simply “mobile platforms”).", "Examples of prior art are taxi and bus ads, signs on company owned cars, airplanes pulling ad banners, bumper stickers, license plate frames, dealership logos, race and other cars with sponsorship ads, hats, clothing, etc.", "These examples represent three common financial models of mobile platform advertising: a. volunteer, i.e., wearing your favorite baseball team's cap, branded shoes, a civic ad (“give blood”, etc.)", "on a tee shirt, etc.", "; b. sponsored ads, i.e., race cars, etc.", "; c. paid mobile platform advertising, i.e., taxi ads, moving billboards, etc.", "A paid example that will be used herein, is commuter cars whose owners contract with advertising firms, etc.", "to use their car as a mobile advertising platform in exchange for significant valuable compensation.", "The general concept of actually paying money to car owners for placing advertisements on their cars may not be new.", "Reportedly, starting in 1972, a company named Beetleboards paid VW bug car owners about $20 USD per month to place advertising on their Volkswagen cars.", "In addition to the low monetary compensation, the VW bug owners apparently also benefited from an exclusive “bug club” or Beetleboard membership effect that encouraged them to sport the third partly advertising arranged by the Beetleboard’ company.", "Apparently, the belonging to the bug ad club effect did not spread to other makes of cars.", "As the popularity of owning VW bugs died off so did the Beetleboard advertising business.", "Herein, the “prior art” means, the prior art, prior to the inventor's press publications, related articles, Website and his provisional patent filing.", "Although not intended to list all the disadvantages related to the prior art, generally the prior art advertising methods and/or platforms present one or more of the following disadvantages or limitations: a) low or too little monetary or other compensation to the platform owner, i.e., “free” bumper stickers, printed tee shirts, antenna balls, hats, etc.", "; b) no tracking of the amount of public exposure (herein may mean the target audience, etc.)", "i.e., a bumper sticker could be removed within hours or days, and a tee shirt may never be worn, and a taxi could be in the shop for repairs, etc.", "; c) no location tracking for mobile platforms, i.e., lack of GPS, etc., enabled mobile platforms; d) limited real-time interactive advertising; e) no or limited automatic inventory and/or automatic and/or interactive replacement ordering platforms or systems; f) no or limited live or real-time updates and live or down loadable changes to the ad copy; g) mostly visual ads; h) limited video and streaming video; i) a very limited variety of platforms; j) no interactive shopping handheld or basket attached PDAs to provide in store or in mall or in car specials, ads, suggestions, recipes, reminders for additional related items, etc.", "; k) no or limited use of RF and IR technologies; l) no physical interaction technology; m) no or limited artificial intelligence (“AI”) technology used; n) little or no instant public or target audience feedback to the advertising message; o) limited to the use of two of the five human senses.", "SUMMARY In accordance with the present invention, improvements, various methods and various combinations of apparatus and/or technology for mobile advertising are disclosed.", "Herein, the proposed improvements, combination of improvements, various methods, models, concepts and combinations of such and combinations of apparatus and/or technology, may be referred to simply as “methods”.", "OBJECTS AND ADVANTAGES Although not so limited the general object of the present invention is to propose new and improved methods related to the mobile advertising business.", "The proposed methods herein may be applied to a large variety of platforms.", "It is accordingly another object of the present invention to provide GPS or other available technology for tracking the movement, location, speed, and/or other parameters of the associated mobile platform.", "For example only, an advertising car equipped with a GPS device, which may or may not provide for real-time data to a remote location, and/or which may or may not have an onboard data recorder.", "GPS data may be used in any number of ways (on an update or real-time basis), such as verification of routes, mileage driven, times when driven, parking, platform and/or personal security, providing location specific ads and/or coupons (e-coupons or printed), traffic updates, emergency or police vehicle safety warnings, family location updates, spot weather, alternate routes, accidents, matchmaking or similar interest information regarding other mobile or stationary persons, etc.", "Note that the ad car (herein, it should not be necessarily assumed that an ad car, or any other mobile platform, has a visual display, i.e., a platform may be instead or also be equipped with streaming or providing down loadable data related to one or more of the other senses) platform illustrates only one of many mobile platforms that may be equipped with such tracking devices, e.g., even a human ad platform may be so equipped.", "Another object of the present invention is to provide one or multiple interactive methods and devices for presenting advertising messages to a plurality of persons.", "One example of such interactive messaging would be an ad car equipped with one or more radio-type (“RF”) or other transceiving device(s) so that other drivers (or other persons) for instance, may request additional and/or basic information from the ad car.", "The ad car or other platform may contain a network server computer or such server may be a convention Internet server, etc.", "Herein “media” refers to various types of advertising media, which may include one or more of the known and even future media, such as audio, visual, taste, smell, touch, electronic stimulation, etc.", "Another object is to provide mobile platforms with live streaming or real-time up-datable media displays by equipping the platforms with at least one-way receivers or two-way data, etc.", "transceivers.", "Of course any combination of technologies may be applied to any appropriate platform, i.e., GPS and digital or analog receivers or transceivers (herein, “receivers” and “transceivers” may be referred to, as simply “transceivers” or “RF”).", "And herein, “RF” may act as a catchall for various types of communication technologies when such are more appropriate, such as infrared “IR” or other technologies.", "Please note that herein, “displays” mean whatever single or multiple technologies and devices necessary to deliver the desired type of media.", "Therefore, herein “display” (the word “herein” is used throughout this application to mean something mentioned within this application, i.e., not within just the associated paragraph) may refer to one or more or a combination or integration of devices/technologies, for example only, which may be represented by any type of electronic visual display, audio display, smell, taste, interactive touch devices and other technologies.", "Another object is to provide a comprehensive mobile advertising method that combines the advantages of the stationary and/or mobile versions Internet, Intranet, peer-to-peer networking and/or other such systems with the one or more of the platforms, devices and/or technologies set forth herein.", "Yet another object is to provide new and/or improved and/or otherwise fully adequate compensation to the persons or entities (herein, referred to as “persons”, regardless of the type of entity) to ensure sufficient interest from a sufficient number of persons, in any and all desired target areas, to produce a reasonable profit by the mobile advertising organization, etc.", "As noted above, lack of proper levels of compensation (herein, “compensation” may be any single thing or combination of such things as, for example only, money, cars, insurance, fuel, mortgage or other payments, food, entertainment, college tuition/expenses, travel, lodging, homes, psychological interest, etc.)", "is one reason the prior art VW bug based advertising venture failed.", "Another object is providing methods for making sure that the above mentioned compensation properly fit the degree of performance and level of platform advertising.", "This of course also goes to the methods herein mentioned regarding verification of various parameters.", "The value of such compensation might be based upon one or more of many different factors for example only: when the ad is displayed on a private automobile, the level of compensation could be related to the number of miles, the demographics of the area and its population, traffic counts, where and how long the vehicle is parked, the time of day, the status of the owner, the type, size, age, value and/or appearance of the vehicle and associated media and other characteristics of the ads/signs/sounds/motions/displays, etc.", "The verification techniques are also very important to assure the client (advertiser) that their money is being well spent.", "The overall object is to provide a mix of devices, platforms, compensation or compensation packages, technologies, communication systems, electronic or other networking, etc.", "from which a business person may select the appropriate ingredients necessary for producing a mobile platform advertising method success in any desired market for any desired product or service, etc.", "The long-term success of a citywide, national or globally replicable method must offer appropriate levels of compensation to the platform persons, reasonable profit levels to the mobile advertising agency or entity, while providing verifiable results, proof of advertising coverage, and/or appropriate impression data to the client or sponsor.", "While another object is to provide, when desired, methods for mobile tag advertising.", "For example only, an ad car may on one hand be covered or otherwise present a client's message, while also carrying or presenting, via any desired media, a tag ad for the ad agency or perhaps just an URL for signing up possible new ad car or other platform persons.", "It is still further an object of the present invention to provide, when desired (herein, one or more improvements, objects, methods, etc.", "may be selected and used singularly or in combination with other improvements, etc., and/or with other mobile or stationary advertising methods not mentioned herein) instant and/or delayed methods for providing information, input from and/or other interaction and/or ordering of products or services by the target or otherwise end user.", "For an example, the platform may be equipped with either physical, RF or other input/our means adaptable to serve the above mentioned purposes.", "Another object is to provide new space for advertising, i.e., at the time the inventor invented these methods, etc., he was unaware of any compensated advertising programs particularly for mobile platforms controlled, used, driven, owned, rented or leased by private persons, let alone any which included the methods set forth herein.", "Another object is to provide part or full time employment and/or independent contractor, etc.", "opportunities for literally millions of persons, who otherwise would not have the chance to enter into one or more functional, creative, administrative areas of the advertising field.", "Yet further object is to provide a lower cost method of advertising.", "For example companies currently paid for taxi, bus, billboard, raid, TV, computer, or even purchase special vehicles and provide paid drivers, insurance, fuel, etc.", "to display their ads.", "The purchase or dedication of owned or leased vehicles or the lease of space on taxies, buses, benches, etc.", "is relatively expensive as compared to the new methods described above.", "Another object is the optional use/incorporation of the mobile and/or stationary point-of-sale and/or business sign-up and/or information connectivity terminals, making the advertising more effective by allowing the subjects more instance access (while the ads are in their thoughts and/or sight, etc.).", "Another object or advantage is that some of these methods bring advertising closer to the person-to-person level or word-of-mouth effect.", "For example, when one sees a private citizen driving their car with an ad, one may assume that person believes in the product or service being advertised (i.e., more than believing in an ad on a taxi or bus, for instance).", "Alternatively, or additionally, people could purchase a franchise or otherwise be provided a business license for one or more of these business methods—or they may just decide to contract to add an ad to their car, scooter, hat, etc.", "Another object and/or advantage is to provide more advertising opportunities in situations and areas that have little coverage currently.", "For example in stop and go traffic one is limited to how many taxis or bus ads one is exposed.", "In a shopping mall in the near future one will see more persons sporting compensated advertising of various wearable media.", "These as well as additional methods, objects and advantages will become more fully apparent from consideration of the claims.", "DESCRIPTION OF THE DRAWING FIG.", "1 is a combination pictorial and block diagram representing the wireless exchange of information.", "FIG.", "1 represents an advertising vehicle that may carry, display and/or transmit various forms of advertising media.", "In one type of preferred embodiment, the vehicle might be fully equipped with an onboard computer, wireless communication system(s), video and/or still camera(s), GPS device, live or up dateable displays (and/or fixed signage of some type) and even a short distance radio transmitter for opt-in audio ads delivered to other motorists, etc.", "FIG.", "2 represents a GPS satellite, from which the vehicle in FIG.", "1 calculates its location and other information.", "FIG.", "3 represents, for example only, a cellular type communication system and the Internet.", "FIG.", "4 represents the server, monitor and workstation at the regional mobile advertising agency.", "FIG.", "5 represents one or more desktop computers or handheld computers that might be in use at any time by the public to access sales and sign up information, by the advertiser or client for account information, and/or by the persons in control of the mobile platform, i.e., the car in FIG.", "1.The foregoing descriptions of specific and suggested embodiments of the present invention have been presented for purposes of illustration and description only.", "The embodiments were chosen, drawn and described in order to best explain and illustrate, in an economical manner, the basic improvements of the invention and at least one of its practical applications.", "Many known and common platforms may be adapted or integrated with the present invention.", "Illustrations of common mobile advertising platforms without the added methods, technology, and devices discussed herein, are available elsewhere therefore, the preparation of additional drawings showing more examples has been avoided.", "This document is meant to be read and understood as a whole, thereby if some descriptive point or other information is presented in one area but not another, such information and descriptions are to be applied generally as appropriate to other discussions and descriptions.", "The necessary components, materials and software to enable any and all of the proposed methods are available.", "Thereby, with the such available building blocks, along with wise and experienced electronics, mobile communications systems, GPS, advertising, marketing, finance, appropriate business knowledge, and the descriptions herein, enabling those skilled in these arts to best utilize the inventive methods and various embodiments with various modifications as are suited to the particular platform use, area and markets contemplated.", "Accordingly, the scope of the invention is defined by the claims appended hereto and their equivalents." ] ]	Patent_10469800
[ [ "Peptide-chelate conjugates", "A peptide-chelate with affinity for the ST receptor is disclosed, wherein the chelate is tetradentate.", "The peptide-chelate conjugate of the invention may be labelled with a radiometal to provide a metal complex.", "A radiopharmaccutical composition comprising the metal complex is provided, which is suitable for the diagnostic imaging of colorectal cancer.", "Also provided for in the invention is a kit for the preparation of the radiopharmaceutical preparation." ], [ "1) A peptide-chelate conjugate comprising a peptide having affinity for the ST receptor conjugated to a tetradentate chelating agent, wherein the peptide having affinity for the ST receptor comprises 10 to 25 amino acids.", "2) The peptide-chelate conjugate of claim 1 wherein the peptide having affinity for the ST receptor comprises 13 to 19 amino acids.", "3) The peptide-chelate conjugate of claim 1 where the tetradentate chelating agent is chosen from diaminedioximes, N3S ligands, N4 ligands, diaminediphenols and N2S2 ligands.", "4) The peptide-chelate conjugate of claim 1 where the tetradentate chelating agent is chosen from diaminedioximes and N3S ligands.", "5) The peptide-chelate conjugate of claim 1 where the tetradentate chelating agent comprises ligands which deprotonate at basic pH to allow binding of 99mTc.", "6) The peptide-chelate conjugate of claim 1 where the tetradentate chelating agent is of formula A: where R1-R6 are all CH3 and Q is —(CH2)2NR(CH2)2— and wherein R is an R group as defined for Formula (A) in claim 1.7) The peptide-chelate conjugate of claim 1 where the tetradentate chelating agent is of formula B: where R1 and R2 are both H and X is a thiol protecting group.", "8) The peptide-chelate conjugate of claim 1 where the peptide is chosen from SEQ ID Nos.", "1 to 6 and FF—(CH2)5-SEQ ID No.", "7.9) The peptide-chelate conjugate of claim 1 where the conjugation is either at the N-terminus or at the C-terminus of the peptide.", "10) The peptide-chelate conjugate of claim 1 which further comprises a linker group between the peptide and the tetradentate chelating agent.", "11) The peptide-chelate conjugate of claim 10 where the linker group comprises a peptide sequence of 5 to 9 amino acids.", "12) The peptide-chelate conjugate of claim 10 where the linker group is selected from -poly-Lys-, -poly-Glu-, -(Gly)2-Glu-(Lys)3-, (Gly)2-Glu-Lys-Glu-Lys-, (Phe)2-(CH2)5-, (Lys)6-Gly-, -(Gly)3-(DGlu)3- and -(Gly)3-(aminocaproic acid)2-.", "13) A metal complex which comprises a radiometal complexed to the tetradentate chelating agent of the peptide-chelate conjugate of claim 1.14) The metal complex of claim 13 where the radiometal is chosen from 64Cu, 65Cu and 99mTc.", "15) The metal complex of claim 13 where the radiometal is 99mTc.", "16) A radiopharmaceutical composition in a form suitable for human administration, which comprises the metal complex of claim 13.17) The radiopharmaceutical composition of claim 16, where the radiometal is 99mTc.", "18) Use of the radiopharmaceutical composition of claim 16 for imaging cancer of colorectal origin.", "19) A kit for the preparation of the radiopharmaceutical composition of claim 16 which comprises: i) the peptide-chelate conjugate of claim 1 ii) a reducing agent.", "20) The kit of claim 19 where the reducing agent is a stannous salt.", "21) The kit of claim 19 further comprising one or more of stabilisers; antioxidants; bulking agents for lyophilisation;" ], [ "<SOH> BACKGROUND ART <EOH>Colorectal carcinoma (CRC) is the fourth most common malignancy worldwide following cancers of the lung, breast and prostate.", "Metastases of CRC origin are the main cause of death in patients diagnosed with colorectal primary tumours.", "Approximately 150,000 to 200,000 new cases are diagnosed annually in the USA and around 50,000 deaths are attributed to this disease.", "Many patients die due to the metastatic spread of the disease with 60-80% of cases developing liver lesions during the illness.", "The positive identification of liver metastasis is therefore a clear indication of latent disease.", "Although much less common, other possible sites of spread include lung, brain and occasionally bone.", "Due to the strong correlation between extent of liver involvement (number and location of metastasis) and resectability, the appropriate evaluation of patients regarding suitability for surgery is becoming more important (Liver Metastasis: Biology, diagnosis and treatment.", "Garden O. J., Gereghty J. G., Nagorney D. M. eds.", "1998).", "The need for an agent capable of detecting metastasis of small size (<1 cm) will have a profound impact on the treatment and management of CRC patients.", "There is a need to specifically detect small (<1 cm) metastatic lesions in the liver.", "The early detection of number and location of metastatic lesions is critical.", "There is also a need to characterise and specifically identify the origin of the tumour with no interference from other possible lesions (e.g.", "cysts, benign lesions, non-treatable tumours).", "A low-molecular weight heat-stable toxin is produced by enterotoxic strains of E. coli.", "This toxin, known as ST peptide, mediates acute diarrheal disease by binding to its receptor on colorectal cells and stimulating guanylate cyclase.", "Synthetic ST peptides that bind to the ST receptor without mediating acute diarrheal disease are disclosed in U.S. Pat.", "No.", "4,545,931 and U.S. Pat.", "No.", "4,886,663.These synthetically produced peptides are suitable for human administration for therapeutic and diagnostic purposes.", "Targeted ligands directed towards receptors that are expressed selectively on tumour cells of colorectal origin are a means to specifically detect the presence of cancers of colorectal origin.", "Thus, the ST receptor is a potential target mechanism.", "Gastrointestinal mucosal cells specifically express the ST receptor and the expression persists after colonic and rectal mucosal cells undergo malignant neoplasic transformation.", "No ST peptide has been found in any other extra-intestinal tissues, therefore specificity of ligands to tissue of gastrointestinal origin is maintained.", "Similar levels of expression have been found in human primary and metastatic colorectal tissues with different grades of differentiation and location.", "A specific ligand for the ST receptor will only bind to metastatic disease, as access to the apical side of intestinal cells will be avoided if the compound is injected intravenously.", "Radiolabelled ST peptides for CRC imaging and diagnosis have been previously documented.", "U.S. Pat.", "No.", "5,518,888 claims radiodiagnostic agents based on ST peptides.", "In one embodiment of that invention, the peptides may be linked to a radioactive imaging agent, such as radioactive iodine, 111 In or 99m Tc.", "99m Tc is chelated by DTPA, which is converted to an anhydride and reacted with an ST peptide.", "No other chelates are disclosed in U.S. Pat.", "No.", "5,518,888.Radiolabelling thus renders the peptides suitable for use in radioimaging metastasized colorectal cancers.", "U.S. Pat.", "No.", "6,060,037 discloses a method of radioimaging metastatic CRC using such radiolabelled ST peptides.", "The detection of localised accumulation or aggregation of radioactivity following administration of radiolabelled ST peptide is indicative of the presence of cells with ST receptors.", "WO 99/21587 and WO 99/39748 also disclose radiolabelled ST peptides for diagnostic imaging.", "In WO 99/21587 the preferred classes of radiometal complexing agents are terpyridines and phenanthrolines.", "In WO 99/39748 the complexing agents comprise a macrocyclic oligo-2,6-pyridine-containing ring which is a derivative of a terpyridine, quaterpyridine, quinpyridine, or sexipyridine." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 shows the effect of pH 8.0, 8.5 and 9.0 in the radiolabelling reaction on the formation of Species 2 (CA-ST 5-18 ).", "FIG.", "2 shows the effect of varying the temperature of the radiolabelling reaction on the formation of Species 2 (CA-ST 5-18 ).", "The temperatures evaluated were 40° C., 60° C. and 70° C. FIG.", "3 shows the effect of compound mass on the percent formation of Species 2 (CA-ST 5-18 ), tested at 12.5 μg, 25 μg, 50 μg and 100 μg compound.", "FIG.", "4 shows SPECT images of mice bearing T84 tumours subcutaneously.", "The images are planar posterior static images with LEUHR collimator using BPLC purified 99m Tc-labelled CA1-ST 5-18 at 20 MBq per animal.", "Tumour to liver ratios were; 1.1 at 15 minutes, 2 at 60 minutes and 4 at 120 minutes.", "Image quality was comparable when crude preparations were used.", "FIG.", "5 shows the relative retention of CA1-ST 5-18 versus the negative control, CA1-ST 5-18(cys-ala) .", "FIG.", "6 shows a comparison of HPLC purified 99m Tc-labelled CA1-ST 5-18 and CA1-ST 5-18(cys-ala) uptake in CD-1 nude mice bearing subcutaneous T84 tumours.", "Planar posterior static imaging with LEUHR collimator using HPLC purified CA1-ST 5-18 at 20 MBq per animal.", "FIG.", "7 shows the biodistribution of purified CA1-ST 5-18 at 2 MBq per animal in RNU/mu nude rats and mice bearing T84 liver tumours.", "The data is expressed in as relative retention (RR) and ratios of tumour to liver and tumour to muscle.", "FIG.", "8 shows an image of an RNU/rnu nude rat bearing T84 liver tumours.", "It is a planar posterior static image at 120 minutes post-injection (p.i.)", "using HPLC purified CA1-ST 5-18 at 20 MBq with LEUHR collimator.", "The image is a whole body image with background counts removed and kidneys masked.", "Image tumour:liver ratio was typically 2.5.FIG.", "9 shows an image of RNU/rnu nude mice bearing T84 liver tumours in the presence and absence of excess peptide.", "The images are planar posterior static images 120 minutes p.i.", "of BPLC purified CA1-ST 5-18 at 20 MBq per animal with LEUHR collimator.", "FIG.", "10 shows the receptor density (RD) of two human and four xenograft human cell line CRC tumours (nd=receptor density data not detected).", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION This invention relates to radiopharmaceuticals, in particular the invention relates to a peptide-chelate conjugate, comprising a peptide with affinity for the ST receptor.", "The compound of the invention is suitable for diagnostic imaging of colorectal cancer in a mammal.", "The invention additionally relates to a radiolabelled peptide-chelate conjugate with affinity for the ST receptor, wherein radiolabelling of the peptide-chelate conjugate does not interfere with the affinity of the peptide for the ST receptor.", "In another aspect of the invention, use of such a compound for imaging of cancer of colorectal origin is provided.", "A kit for the production of a radiolabelled peptide-chelate conjugate for imaging colorectal cancer is also disclosed.", "BACKGROUND ART Colorectal carcinoma (CRC) is the fourth most common malignancy worldwide following cancers of the lung, breast and prostate.", "Metastases of CRC origin are the main cause of death in patients diagnosed with colorectal primary tumours.", "Approximately 150,000 to 200,000 new cases are diagnosed annually in the USA and around 50,000 deaths are attributed to this disease.", "Many patients die due to the metastatic spread of the disease with 60-80% of cases developing liver lesions during the illness.", "The positive identification of liver metastasis is therefore a clear indication of latent disease.", "Although much less common, other possible sites of spread include lung, brain and occasionally bone.", "Due to the strong correlation between extent of liver involvement (number and location of metastasis) and resectability, the appropriate evaluation of patients regarding suitability for surgery is becoming more important (Liver Metastasis: Biology, diagnosis and treatment.", "Garden O. J., Gereghty J. G., Nagorney D. M. eds.", "1998).", "The need for an agent capable of detecting metastasis of small size (<1 cm) will have a profound impact on the treatment and management of CRC patients.", "There is a need to specifically detect small (<1 cm) metastatic lesions in the liver.", "The early detection of number and location of metastatic lesions is critical.", "There is also a need to characterise and specifically identify the origin of the tumour with no interference from other possible lesions (e.g.", "cysts, benign lesions, non-treatable tumours).", "A low-molecular weight heat-stable toxin is produced by enterotoxic strains of E. coli.", "This toxin, known as ST peptide, mediates acute diarrheal disease by binding to its receptor on colorectal cells and stimulating guanylate cyclase.", "Synthetic ST peptides that bind to the ST receptor without mediating acute diarrheal disease are disclosed in U.S. Pat.", "No.", "4,545,931 and U.S. Pat.", "No.", "4,886,663.These synthetically produced peptides are suitable for human administration for therapeutic and diagnostic purposes.", "Targeted ligands directed towards receptors that are expressed selectively on tumour cells of colorectal origin are a means to specifically detect the presence of cancers of colorectal origin.", "Thus, the ST receptor is a potential target mechanism.", "Gastrointestinal mucosal cells specifically express the ST receptor and the expression persists after colonic and rectal mucosal cells undergo malignant neoplasic transformation.", "No ST peptide has been found in any other extra-intestinal tissues, therefore specificity of ligands to tissue of gastrointestinal origin is maintained.", "Similar levels of expression have been found in human primary and metastatic colorectal tissues with different grades of differentiation and location.", "A specific ligand for the ST receptor will only bind to metastatic disease, as access to the apical side of intestinal cells will be avoided if the compound is injected intravenously.", "Radiolabelled ST peptides for CRC imaging and diagnosis have been previously documented.", "U.S. Pat.", "No.", "5,518,888 claims radiodiagnostic agents based on ST peptides.", "In one embodiment of that invention, the peptides may be linked to a radioactive imaging agent, such as radioactive iodine, 111In or 99mTc.", "99mTc is chelated by DTPA, which is converted to an anhydride and reacted with an ST peptide.", "No other chelates are disclosed in U.S. Pat.", "No.", "5,518,888.Radiolabelling thus renders the peptides suitable for use in radioimaging metastasized colorectal cancers.", "U.S. Pat.", "No.", "6,060,037 discloses a method of radioimaging metastatic CRC using such radiolabelled ST peptides.", "The detection of localised accumulation or aggregation of radioactivity following administration of radiolabelled ST peptide is indicative of the presence of cells with ST receptors.", "WO 99/21587 and WO 99/39748 also disclose radiolabelled ST peptides for diagnostic imaging.", "In WO 99/21587 the preferred classes of radiometal complexing agents are terpyridines and phenanthrolines.", "In WO 99/39748 the complexing agents comprise a macrocyclic oligo-2,6-pyridine-containing ring which is a derivative of a terpyridine, quaterpyridine, quinpyridine, or sexipyridine.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 shows the effect of pH 8.0, 8.5 and 9.0 in the radiolabelling reaction on the formation of Species 2 (CA-ST5-18).", "FIG.", "2 shows the effect of varying the temperature of the radiolabelling reaction on the formation of Species 2 (CA-ST5-18).", "The temperatures evaluated were 40° C., 60° C. and 70° C. FIG.", "3 shows the effect of compound mass on the percent formation of Species 2 (CA-ST5-18), tested at 12.5 μg, 25 μg, 50 μg and 100 μg compound.", "FIG.", "4 shows SPECT images of mice bearing T84 tumours subcutaneously.", "The images are planar posterior static images with LEUHR collimator using BPLC purified 99mTc-labelled CA1-ST5-18 at 20 MBq per animal.", "Tumour to liver ratios were; 1.1 at 15 minutes, 2 at 60 minutes and 4 at 120 minutes.", "Image quality was comparable when crude preparations were used.", "FIG.", "5 shows the relative retention of CA1-ST5-18 versus the negative control, CA1-ST5-18(cys-ala).", "FIG.", "6 shows a comparison of HPLC purified 99mTc-labelled CA1-ST5-18 and CA1-ST5-18(cys-ala) uptake in CD-1 nude mice bearing subcutaneous T84 tumours.", "Planar posterior static imaging with LEUHR collimator using HPLC purified CA1-ST5-18 at 20 MBq per animal.", "FIG.", "7 shows the biodistribution of purified CA1-ST5-18 at 2 MBq per animal in RNU/mu nude rats and mice bearing T84 liver tumours.", "The data is expressed in as relative retention (RR) and ratios of tumour to liver and tumour to muscle.", "FIG.", "8 shows an image of an RNU/rnu nude rat bearing T84 liver tumours.", "It is a planar posterior static image at 120 minutes post-injection (p.i.)", "using HPLC purified CA1-ST5-18 at 20 MBq with LEUHR collimator.", "The image is a whole body image with background counts removed and kidneys masked.", "Image tumour:liver ratio was typically 2.5.FIG.", "9 shows an image of RNU/rnu nude mice bearing T84 liver tumours in the presence and absence of excess peptide.", "The images are planar posterior static images 120 minutes p.i.", "of BPLC purified CA1-ST5-18 at 20 MBq per animal with LEUHR collimator.", "FIG.", "10 shows the receptor density (RD) of two human and four xenograft human cell line CRC tumours (nd=receptor density data not detected).", "DESCRIPTION OF THE INVENTION ST peptides are cyclic peptides containing three disulphide bonds.", "It has been found that these ST peptides are unstable under the basic pH conditions required for some 99mTc chelator radiolabelling, giving in cleavage of the disulphide bridges and unacceptable loss of potency.", "This instability may rule out a large number of 99mTc chelates that require pH ca.", "8-9 for efficient labelling.", "Consequently, peptide-chelate conjugates would have to be labelled under acidic conditions to maintain high potency.", "However, such chelates need complicated labelling protocols and can generate 99mTc-conjugates with poor efficacy, e.g., elevated hepatobiliary uptake and fast blood clearance.", "The present invention provides peptide-chelate conjugates that can be labelled under basic conditions without loss of potency of the ST peptide, to provide effective imaging agents.", "In a first aspect, the present invention provides a peptide-chelate conjugate comprising a peptide having affinity for the ST receptor conjugated to a tetradentate chelating agent, wherein the peptide having affinity for the ST receptor comprises 10 to 25 amino acids.", "“A peptide having affinity for the ST receptor” is a biologically active peptide which is suitably between 10 and 25 amino acids, and preferably between 13 and 19 amino acids, derived from the ST peptide sequence, that binds the ST receptor with high affinity.", "The peptides of the present invention may be of naturally occurring or synthetic origin, but are preferably synthetic.", "These peptides may be conjugated directly to the chelate, or by means of a “linker group”.", "“Linker groups” may comprise a peptide sequence of about 5 to 9 amino acids, with or without the inclusion of other groups such as aliphatic chains of up to 5 carbons in length.", "Preferred linker groups are poly-Lys, poly-Glu, (Gly)3-(DGlU)3 and (Gly)3-(aminocaproic acid)2.Labelled with a suitable radiometal, ST peptides of the invention are suitable for use as imaging agents to detect cancers of colorectal origin.", "Preferred peptides of the present invention are sequences 1-7.Especially preferred peptides having affinity for the ST receptor are SEQ ID NOs 1 to 5, presented in the included sequence listing.", "The negative control used in the experiments of the present invention is SEQ ID NO 8, i.e., SEQ ID NO 1 with all of the cysteines replaced with alanines and therefore lacking the disulphide bridges required for binding.", "As used herein the term “tetradentate chelating agents” are chelates that are suitable for the formation of the peptide-chelate conjugates of the present invention, in which the radiometal is coordinated by the four metal donor atoms of the tetradentate chelating agent.", "A suitable radioactive metal ion is complexed by a tetradentate chelating agent by means of a donor set consisting of four metal donors which form at least one 5- or 6-membered chelate ring with the radiometal.", "Preferably, the tetradentate chelating agent forms 2 or more such 5- or 6-membered chelate rings with the radiometal.", "These ligands are particularly suitable for the complexation of technetium (99mTc), but may also be used for other radiometals.", "The tetradentate chelating agent is present in the conjugate in order to produce a radiolabelled form of the peptide, suitable for diagnostic imaging.", "A suitable radioactive metal ion may be incorporated into the peptide-chelate conjugate by means of complexation with the tetradentate chelating agent.", "It is an important feature of the present invention that the potency of the ST peptide is not compromised by the process of radiolabelling.", "Tetradentate chelating agents suitable for the present invention include but are not limited to the following: (i) diaminedioximes of formula A: where RA1-RA6 are each independently an R group; where R is H or C1-10 alkyl, alkylaryl, alkoxyalkyl, hydroxyalkyl, fluoroalkyl, aminoalkyl or carboxyalkyl; and Q is a bridging group of formula —(W)n—; where n is 3, 4 or 5 and each W is independently —O—, —NR— or —CR2— provided that (W)n contains a maximum of one W group which is —O— or —NR—.", "Preferred diaminedioximes have RA1 to RA6=C1-3 alkyl, alkylaryl alkoxyalkyl, hydroxyalkyl, fluoroalkyl or aminoalkyl.", "Most preferred diaminedioximes have RA1 to RA6=CH3.", "(ii) N3S ligands of formula B: where X is a thiol protecting group such as benzoyl, acetyl or ethoxyethyl that is cleaved before or during the labelling process, and; RB1 and RB2 may be H or the side chain of any amino acid, (iii) N4 ligands of formula C: where RC1-RC4 may be H, alkyl, aryl or combinations thereof and where one of RC1-RC4 must be a functional group such as alkylamine, alkyl sulphide, alkoxy, alkyl carboxylate, alkyl arylamine or aryl sulphide.", "Ligands of this type include those with C═O amide linkages.", "Macrocyclic versions of formula C are also included, such as: where RC5 is as defined for RC1 to RC4, above.", "(iv) Diaminediphenols of formula D: where Y is either C or N; RD1 and RD2 may be either H, alkyl or aryl and where one of RD1 and RD2 must be a functional group such as alkylamine, alkyl sulphide, alkoxy, alkyl carboxylate, alkyl arylamine or aryl sulphide, and; m=n=1 or 2.v) N2S2 ligands of formula E: where Z is a thiol protecting group such as benzoyl, acetyl or ethoxyethyl that is cleaved before or during the labelling process, and; RE1-RE12 may be each chosen from H, an aryl group or an alkyl group and where one of RE1-RE12 must be a functional group such as alkylamine, alkyl sulphide, alkoxy, alkyl carboxylate, alkyl arylamine or aryl sulphide, and; one or more of the pairs RE3/RE4, RE5/RE6, RE7/RE8, RE9/RE10 may represent a C═O bond.", "A preferred chelating agent of the present invention is a diaminedioxime of formula A, where RA1-RA6 are all CH3, and Q is —(CH2)2NR(CH2)2—, where R is an R group as defined for Formula (A).", "A most preferred chelating agent is where R of —(CH2)2NR(CH2)2— is aminoalkyl, especially R═—(CH2)2NH—.", "The latter will be referred to in the remainder of this document as “chelating agent 1⇄ (CA1).", "Another preferred chelating agent is an N3S ligand of formula B where RB1 and RB2 are both H and X is acetyl.", "This will be referred to as “chelating agent 2” (CA2) for the remainder of the document.", "“Chelating agent 3” (CA3) is an N3S ligand of formula B where RB1 is CH2CH2CH2C═O, RB2 is H and X is ethoxyethyl.", "The synthetic peptides of the present invention can be synthesised by solid phase methodology, as is well known in the art.", "A representative synthesis of ST5-18 is given in Example 2.The peptide-chelate conjugates of the present invention can be prepared using bifunctional chelators, i.e., compounds in which the tetradentate chelator bears a pendant fictional group.", "Preferred functional groups are amino or carboxyl functional groups, which permit facile coupling via amide bonds to the amine or carboxyl groups on the peptides of interest, especially the amino- or carboxyl-terminus of the peptide.", "N3S bifunctional chelators can be prepared by the method of Sudhaker et al [Bioconjugate Chem., Vol.", "9, 108-117(1998)].", "The synthesis of diaminedioxime bifunctional chelators is described in Example 3.Diaminediphenol compounds can be prepared by the method of Pillai et al [Nucl.", "Med.", "Biol., Vol.", "20, 211-216(1993)].", "Bisamidedithiol compounds can be prepared by the method of Kung et al [Tetr.", "Lett., Vol 30, 4069-4072 (1989].", "Monoamidemonoaminebisthiol compounds can be prepared by the method of Hansen et al [Inorg.", "Chem., Vol 38, 5351-5358 (1999)].", "Functional tetraamines can be prepared by the method of Simon et al [J.", "Am.", "Chem.", "Soc., Vol 102, 7247 (1980)].", "The critical processes in the synthesis of the CA1-ST5-18 conjugate can be summarised as shown in Scheme 1 on the following page.", "Conjugation of the peptide to the chelate is carried out prior to the labelling reaction.", "The direct method of conjugation is exemplified in the compound CA1-ST5-18 (or CA1-SEQ ID No 1).", "Conjugation via a linker molecule is exemplified in the compound CA1-(Gly)3-(D-Glu)3-ST5-18 (or CA1-SEQ ID No 5).", "Conjugations may be carried out via either the N-terminus or the C-terminus of the peptide molecule.", "Refer to Table I for these structures and structures of other suitable compounds of the present invention.", "The compounds of the present invention have reproducibly high radiochemical purity (RCP), advantageous pharmacokinetics such as reduced gastrointestinal uptake, and imaging efficacy in tumoured animals.", "The compounds offer a distinct advantage over previously disclosed CRC-targeting compounds since they have improved pharmacokinetics and specific retention in tumour tissue compared to background tissues.", "In a second aspect, the present invention provides a metal complex comprising a radiometal complexed to the tetradentate chelating agent of the peptide-chelate conjugate.", "Preferred chelating agents for the metal complex are chosen from diaminedioximes of formula A where RA1-RA6 are all CH3 and Q is —(CH2)2NR(CH2)2—.", "An especially preferred diaminedioxime of formula A is CA1, as defined above.", "Other preferred chelating agents for the metal complex are the N3S ligands CA2 and CA3, defined above.", "A suitable radiometal may be chosen from positron emitters such as 64Cu and 65Cu, or from gamma emitters such as 99mTc.", "A preferred embodiment of the invention is a peptide-chelate conjugate radiolabelled with 99mTc.", "A peptide-chelate conjugate radiolabelled with 99mTc, where the radiolabelling reaction is carried out at basic pH is a most preferred embodiment of the present invention.", "Radiolabelling as defined by the present invention is the chemical attachment of a radiometal, such as those described above.", "Suitable radiolabelled compounds of the invention have affinity for the ST receptor to enable diagnostic imaging of cancers of colorectal origin.", "Preferred radiolabelled compounds are thus cleanly labelled with the minimum production of by-products.", "An example of such a by-product is reduced hydrolysed technetium (RHT) in the case of 99mTc labelling.", "In a preferred embodiment of the invention, the peptide-chelate conjugate is labelled with 99mTc maintaining high RCP and low RHT levels.", "The results of various studies described in the Examples show that the preferred compounds of the present invention are stable to the labelling conditions and maintain their biological efficacy following radiolabelling.", "The RHT levels are of particular importance in the case of 99mTc-labelled conjugates of the present invention as one of the main biological targets is in the liver.", "RHT localises in the liver and thus if allowed to remain, will therefore increase background tissue activity, decrease target to background ratios and lead to an overall decrease in image quality.", "The amount of RHT is relatively low for all the preparations (Example 10).", "There is little difference between the labelling characteristics of CA1-ST5-18 at conjugate levels of 12.5-100 μg, in terms of the rate of formation of species 2.However, there was an increase in RHT at reduced conjugate levels (Example 10).", "Compared to the non-HPLC purified unfiltered preparation, both filtering and addition of methylene diphosphonate (MDP, which is known to reduce RHT levels) had a similar effect in reducing liver uptake (Example 24).", "Neither method reduced background tissue counts to the level seen with HPLC purified preparations but high quality images of liver metastases are still possible with non-HPLC purified preparations.", "Radiolabelling of the peptide-chelate conjugate may be carried out in a suitable buffering system such as carbonate, borate, triethanolamine hydrochloride-NaOH, dimethylleucylglycine, tris(hydroxymethyl)aminomethane, 2-amino-2-methyl-1:3-propanediol-HCl, diethanolamine-HCl, Clark and Lubs solutions (Bower and Bates, J. Res.", "Nata.", "Bur.", "Stand.", "55, 191 (1957), Glycine, Glycylglycine or TAPS®.", "The labelling reaction is preferably carried out at a basic pH, i.e., between pH 8.0 and pH 10, most preferably at a pH of around 8.5.Examples 5 and 6 describe conducting the radiolabelling reaction using either sodium hydroxide or borate buffer in order to maintain a suitable pH.", "A preferred method of the present invention is the use of a borate buffering system in the radiolabelling reaction.", "The competition binding experiments carried out (as described in Example 15) investigated the relative potency of various ST peptide compounds.", "Ki values in the nM range suggest good binding of competing ligand.", "These data show firstly that variations outside the pharmacophore in the ST peptide make little difference to binding (ST1-17 vs. ST5-18, Ki 0.6 and 0.4 nM, respectively).", "The presence of CA3 on the ST peptide results in a small increase in Ki, i.e., a small resulting decrease in binding.", "The presence of CA1 has a greater effect on binding to reduce further the Ki value to 2.8 nM, almost a ten fold reduction in binding efficacy.", "Binding affinity in the nanomolar range is considered acceptably high and the resulting differences in reduced binding are not observed in vivo.", "The negative control demonstrated no binding (up to 500 μM).", "The preferred compounds of the present invention demonstrated Ki values of less than 100 nM, indicating high affinity for the receptor (see Example 17).", "Of all the compounds tested, CA1-ST5-18, CA3-ST5-18 and CA1-gly3-Dglu3-ST5-18 showed the greatest potency in this assay, with Ki values of 2.8 nM, 0.8 nM and 1.0 nM, respectively.", "The in vivo efficacy of these screened compounds is discussed below.", "The data obtained in Example 15, and the high binding (Ki) of mock-labelled peptide (peptide subjected to the radiolabelling protocol in the absence of radiometal), supports the fact that the radiolabelled material is still bioactive.", "In a third aspect, the invention also encompasses radiopharmaceutical preparations.", "By the term “radiopharmaceutical preparation” is meant a composition comprising the radiometal complex of the invention in a form suitable for human administration.", "For human administration a radiopharamceutical preparation must be sterile.", "It is preferably in an injectable form, e.g., for 99mTc, reconstitution of the peptide-chelate conjugate with sterile pertechnetate in saline.", "The radiopharmaceutical preparation of the present invention may also be provided in a unit dose form ready for human injection and could for example be supplied in a pre-filled sterile syringe.", "The syringe containing the unit dose would also be supplied within a syringe shield (to protect the operator from potential radioactive dose).", "A fourth aspect of the present invention is the use of the radiopharmaceutical preparation of the invention for the imaging of cancer of colorectal origin.", "Imaging using the radiopharmaceutical preparation of the present invention may be carried out by means of PET or SPECT imaging, depending on the nature of the radiometal.", "In a fifth aspect, the present invention provides kits for the production of the radiopharmaceutical preparation of the invention.", "Suitable kits are designed to give sterile radiopharmaceutical products suitable for human administration, e.g.", "via injection into the bloodstream.", "When the radiometal is 99mTc, the kit comprises a vial containing the peptide-chelate conjugate for the metal together with a pharmaceutically acceptable reducing agent.", "Suitable such reducing agents are: sodium dithionite, sodium bisulphite, ascorbic acid, formaridine sulphinic acid, stannous ion, Fe(II) or Cu(l).", "The pharmaceutically acceptable reducing agent is preferably a stannous salt such as stannous chloride or stannous tartrate.", "Alternatively, the kit may comprise a metal complex which, upon addition of the radiometal, undergoes transmetallation (i.e., ligand exchange) giving the desired product.", "For 99mTc, the kit is preferably lyophilised and is designed to be reconstituted with sterile 99mTc-pertechnetate (TcO4−) from a 99mTc radioisotope generator to give a radiopharmaceutical solution suitable for human administration without further manipulation.", "The peptide-chelate conjugate is preferably present in the kit in a form amenable to transport and storage over relatively long time-periods.", "Reconstitution with eluate from a 99mTc generator under the preferred radiolabelling conditions results in a 99mTc-labelled peptide with specificity for the ST receptor.", "It is an important feature of the present invention that in such a kit, the radiolabelling reaction does not alter the affinity of the peptide for the ST receptor.", "99mTc-labelled peptides of the present invention have been shown to be stable for up to 4 hrs at room temperature and in the presence of activity levels of up to 1 GBq.", "The above kits or pre-filled syringes may optionally contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g.", "cyclodextrins or surfactants such as Pluronic™, Tween™ or phospholipids); pharmaceutically acceptable stabilisers, radioprotectants or antioxidants (such as ascorbic acid, gentisic acid or para-aminobenzoic acid) or bulking agents for lyophilisation (such as sodium chloride or mannitol).", "It is a feature of the invention that the metal complexes have maintained potency for the ST receptor as well as suitable phannacokinetics.", "Data obtained in Example 16 indicate that positive control, 125I-ST-1-17 has the highest binding of 20% of added radioactive material.", "The negative control, CA3-T5-18(Cys-Ala), showed no specific binding (binding at NSB level, <1%).", "99mTc-labelled CA1-STs5-18 shows reduced binding (6% of total added) compared to the positive control but is still significantly higher than that of the negative control.", "The compounds of the present invention also have optimal blood clearance, fast liver clearance and fast background tissue clearance.", "Liver clearance is a particularly significant feature of the present invention due to the high incidence of CRC metastases in that organ.", "Furthermore, the compounds of the present invention have been demonstrated to permit imaging of colorectal cancer metastases which are less than 1 cm in diameter.", "This is a feature which additionally permits distinction to be drawn between focal and diffuse metastases.", "It has not been previously possible to achieve these features in an agent directed towards the ST receptor.", "This is therefore a surprising development over the prior art.", "Metal complexes of the present invention are cleared rapidly via the urine (>90% at 60 min p.i.).", "An additional feature of the present invention is low gastrointestinal uptake, which reduces the risk of unwanted side effects.", "Low uptake in background tissues such as liver, which may interfere with image quality, is an advantage of the metal complexes of the present invention.", "For CA1-ST5-18 the liver uptake was 0.93% of the injected dose per gram at 2 hours p.i.", "(Example 17).", "CA1-ST5-18 shows the greatest tumour to background tissue ratio, the greatest uptake (% ID/g) and relative retention of all the compounds assessed.", "This shows that of all the compounds examined, CA1-ST5-18 showed the best pharmacokinetic qualities for a CRC imaging agent (Example 17).", "The preferred mode of imaging of the present invention is SPECT imaging.", "It was demonstrated to be possible to image tumours of between 0.5-1 cm in diameter by SPECT imaging using the subcutaneous model (described in Example 17).", "The images acquired in Example 18 show that imaging is possible from 15 min p.i., and by 120 min p.i.", "only the tumour and kidneys are visible.", "The tumour size imaged was of the region expected in the target population.", "The images have been acquired in a planar mode, with a relatively low radioactive dose of 20 MBq/animal.", "Spatial resolution between organs is known to be better in humans such that the images obtained would be expected to be at least as good as seen in the animal models.", "As shown in Examples 18, 19, 21 and 23, the preferred compounds of the invention display superior imaging properties not previously reported in the art.", "Specific in vivo tumour uptake has been demonstrated in two different animal models.", "These models are described in Examples 17 and 20.Imaging of both subcutaneous tumours and liver metastases was achieved at <2 hr p.i.", "in mice.", "It was possible to acquire images in mice from 15 minutes p.i.", "This will support the ideal clinical imaging window in humans of between 1-6 hours p.i.", "The images produced in Example 18 (FIG.", "4) have consistently shown clear identification of tumours from 15 min p.i.", "with good ratios for tumour to muscle (not shown) and tumour to liver, which improves over time so that by 120 min p.i.", "only the tumour and kidneys are visible.", "The data obtained in Example 19 demonstrate good uptake for 99mTc-labelled CA1-ST5-18 and poor uptake for the negative control (FIG.", "5).", "Planar posterior static images of the same animals acquired at 120-min p.i.", "show a similar pattern; good image of 99mTc-labelled CA1-ST5-18 and poor image of negative control (FIG.", "6).", "In the image produced in Example 21 (FIG.", "8), metastases are clearly visible, with a tumour:liver ratio of 1.5-3.0 based on ROI analysis.", "Images were acquired consistently and without major manipulation, with both of the formulations: HPLC purified and crude preparations.", "Imaging experiments as described in Example 21 demonstrate specific retention of 99mTc-labelled CA1-ST5-18 into CRC tumours.", "These data support the specificity of 99mTc-labelled CA1-ST5-18 for ST-receptors, and highlight the importance of the conformational structure of ST peptides.", "When mice bearing sub-cutaneous Lewis lung tumours were injected with CA1-ST5-18, as described in Example 22, significantly higher uptake of CA1-ST5-18 into colorectal tumours than into those of non-colonic origin was demonstrated.", "Literature studies indicate little variation in receptor expression between tumour samples, and the reported data (80-120 fmol/mg protein) compared well with the average expression found in this study.", "Although a large differential in receptor expression has been seen between the two human donors (Example 25) In biodistribution studies on a range of xenograft tumours with different ST receptor expression, highest expression was shown in T84 cells but similar to the expression in human tumours (Example 25).", "The relative expression in a number of different cell lines was analysed and was found to be in the following order: T84>CaCO2>LS180>HT29>SW480.The data show comparatively higher retention of CA1-ST5-18 in high ST receptor-expressing tumours, CaCO2 and T84, compared to the rest.", "Images of tumours expressing receptor density lower than that found in human tumours (LS180) was achieved, as a result CA1-ST5-18 will be able to detect a wide range of human tumours.", "Expected values from previous studies suggest little loss in expression with either origin of tumour or decrease in grade.", "Therefore, a population of patients with low ST receptor-expressing metastases is unlikely.", "More importantly, the expression of receptors in human CRC tumours lies directly in between that of two imageable CRC tumours, suggesting that expression of the receptor in Human tumours is sufficient for imaging with 99mTc-labelled CA1-ST5-18.Low retention in tumours was observed with the negative control, CA3-ST5-18(cys-ala).", "The data also show that despite very low receptor density there is still specific retention of 99mTc-labelled CA1-ST5-18 in those tumours.", "The tumour uptake in tumours from T84 and LS180 cells, grown subcutaneously or in the liver of immunocompromised animals, is sufficient for imaging, as demonstrated in both the subcutaneous model and in the liver metastases model.", "By doing so it has been shown that it is possible to image small tumours in the region of expression levels of ST receptor found in human tumours.", "The compounds of the present invention satisfy all major requirements identified for an improved CRC imaging agent, in particular successfully imaging tumours in an animal model thought to represent the more challenging aspects of the clinical disease.", "Exceptional efficacy has been demonstrated in the critical efficacy model of liver metastases.", "The preferred embodiments allow imaging of tumours that represent the range of receptor densities found in human liver metastases from CRC.", "The efficacy is maintained in the presence of excess peptide, at levels expected in a potential clinical formulation hanging studies have shown that preferred compounds are likely to be more sensitive and specific than X-ray CT.", "The compounds of the present invention could thus be used in the initial diagnosis of disease, and more importantly in the appropriate staging or determination of the extent of the disease, measuring response to therapy and in the follow up of patients treated for primary CRC.", "The compounds of the present invention are therefore superior to any other reported CRC imaging compound.", "They are retained specifically in tumours of CRC origin.", "Imaging potential was demonstrated in a clinically acceptable time frame in a desired target tumour size.", "Surprising uptake, retention, pharmacokinetics, and efficacy have been demonstrated in view of reported instability of the vector at basic pH.", "EXAMPLES Example 1 Peptide-Chelate Conjugates The compounds used are given in Table I. Conjugates were prepared as described in Example 2 as 100 μg aliquots in plastic vials, and stored at −18° C. The conjugates were allowed to warm to ambient temperature before use.", "TABLE I Compounds of the invention CA3-ST5-18 CA3-ST5-18 (cys-ala) CA1-ST5-18 CA2-(Gly)2-Glu-(Lys)3-ST5-18 CA2-(Gly)2-Glu-Lys-Glu-Lys-ST5-18 ST5-17 C terminus amide CA2-(Phe)2-(CH2)5-ST5-18 CA1-(Glu)6-ST5-18 CA1-(Lys)6-Gly-ST5-18 CA1-(Gly)3-(D-Glu)3-ST5-18 CA1-(Gly)3-(aminocaproic acid)2-ST5-18 Example 2 Synthesis of CA1-ST5-18 2.1 Solid-Phase Synthesis The solid-phase assembly of the peptide sequence was performed on the support Fmoc-Tyr(tBu)-SASRIN (10 g polymer; 0.6 mmol/g loading) using an Advanced Chemtech 90 automated peptide synthesiser.", "The amino acids were all of the L-configuration with the α-amino function blocked with the 9-Fluorenylmethoxycarbonyl (Fmoc) protecting group.", "The reactive side-chain functional groups were bearing the following protecting groups: Trityl (Trt)—for cysteines 6 and 14 and asparagine, Acetamidomethyl (Acm)—for cysteines 5 and 10, 4Methoxybenzyl (Mob)—for cysteines 9 and 17, tert-Butyl ester—for glutamic acid tert-Butyl ether—for tyrosine 2.2 Coupling and Deprotection Reactions Double coupling cycles were carried out using a 5-fold excess of activated Fmoc-amino acid derivatives dissolved in N-methylpyrrolidone (NMP).", "The first coupling was carried out with Fmoc amino acid/DIC/HOBt activation, and the second coupling with Fmoc amino acid/HBTU/HOBt/DIEA activation.", "Excess reagents were removed from the polymer by washing three times with NMP, three times with methanol and three times with NMP before removal of the Fmoc group in 20% piperidine/NMP (5 minute and 20 minute cycles performed).", "Once assembled the peptide resin was washed three times with NMP, three times with DCM and three times with methanol before drying overnight in a vacuum oven at room temperature.", "Weight of final resin 20.76 g (yield 84.2%).", "2.3 Cleavage of Partially Protected Peptide from the Solid Support To the dry peptide resin in the nitrogen bubbler was added TFA (25 ml) containing 5% TIS and 5% water and the mixture bubbled under nitrogen for 30 minutes.", "The peptide solution was then drained into toluene (200 ml) and evaporated in vacuo at RT.", "Cold diethyl ether was added to the oily residue in order to precipitate out the product as a solid.", "Following trituration with diethyl ether and drying, 600 mg of the crude dithiol peptide was obtained.", "The HPLC purity of the crude product was shown to be around 70% with several side-products identified due to modifications during the TFA cleavage step.", "The most significant impurity (10-15%) was caused by the cleavage of one of the CA1 oxime side arms.", "2.4 Cyclisation 1: Oxidation of the Crude Dithiol Peptide: 6,14 Disulphide Crude dithiol peptide from 2.3 above (0.132 mmol, 300 mg) was dissolved portionwise in 600 mL of 0.1M NH4HCO3 (pH 8) in water/acetonitrile (80:20) containing potassium ferricyanate (0.3 mmol, 100 mg).", "The reaction was left stirring under nitrogen for 16 hours then HPLC analysis was used to confirm complete conversion of starting dithiol to oxidised product.", "The peptide solution was then acidified to pH 2 with TFA, filtered through a 0.45 micron filter (Millipore) then pumped directly onto the preparative HPLC column and gradient elution initiated.", "Fractions of >80% purity were combined and freeze-dried yielding 140 mg of the desired product.", "2.5 Cyclisation 2: Acm Group Deprotection and Cyclisation 6,14: 5,10 The mono-disulphide product from 2.4 above (0.14 g, 0.062 mmol) was dissolved in 150 ml of acetic acid/DMSO (1:1) containing 0.1 mL anisole and iodine (0.1 g, 0.39 mmol) added.", "The reaction mixture was stirred in the dark for 30 minutes before dilution to a total volume of 450 ml with distilled water.", "The peptide solution was then extracted twice with diethyl ether in a separating funnel and the aqueous phase analysed by HPLC.", "BPLC analysis revealed the presence of two new products present in a 4:1 ratio.", "Both products had the desired molecular weight as evidenced by MS analysis and corresponded to two conformational isomers.", "The aqueous layer was once again filtered through a 0.45 micron filter (Millipore) before pumping directly onto the preparative BPLC column and gradient elution initiated.", "Fractions of >80% purity were combined and freeze-dried yielding 40 mg of the desired product.", "2.6 Cyclisation 3: Mob Group Deprotection and Cyclisation To the bis-disulphide product from 2.5 above (40 mg, 0.019 mmol) was added a solution of 10% DMSO/TFA (50 ml) containing 0.1 ml of anisole and the mixture gently shaken (20 min.).", "The TFA was evaporated in vacuo at room temperature and the product precipitated by the addition of diethyl ether.", "Following trituration with diethyl ether 25 mg of the fully folded product was recovered.", "Preparative HPLC of crude product and freeze-drying yielded 16 mg of the desired product (purity >98%).", "MS analysis by LCQ; found (M+H)+=1873, expected (M+H)+=1873.2.7: Salt Exchange The pure TFA salt from above was dissolved in water containing 0.1% ammonium acetate.", "Gradient elution on a C18 reverse phase column using a 10 to 60% B gradient over 40 minutes where buffer A=0.1% ammonium acetate/H2O; B=0.1% ammonium acetate in 80% acetonitrile/H2O) was performed.", "The acetate salt (15 mg) was recovered following freeze-drying.", "Example 3 Synthesis of Chelating Agent 1 (CA1) To a solution of tris-(2-aminoethyl) amine (Aldrich; 2 ml, 13 mmol) in acetonitrile (20 ml) was added sodium bicarbonate (2.2 g, 26 mmol).", "A solution of 3-chloro-3-methyl-2-nitrosobutane (1.8 g, 13 mmol) in dry acetonitrile (10 ml) was added slowly at 0° C. The reaction mixture was left to stir at room temperature for 4 hours, and then filtered.", "The filtrant was washed with acetonitrile and the filtrate evaporated.", "The crude product was dissolved in acetonitrile and purified by HPLC (Hamilton PRP-1; A: 2% aqueous NH3, B: MeCN; 0-100% B in 20 min; 3 ml/min) to afford CA1.Yield: 0.88 g, 19%.", "Example 4 Conjugation of Peptide to Chelating Agent 4.1 Coupling of Succinic Acid Linker An aliquot (1 g, ca 0.3 mmol) of the peptide resin from Example 2 was transferred to a nitrogen bubbler where a further set of washes in DMF (3×15 ml) were carried out.", "A solution containing 1 mmol of succinic anhydride dissolved in DMF (20 ml) was then added and the mixture bubbled under nitrogen for 30 minutes.", "The polymer was washed with 5×15 ml quantities of DMF and a resin sample taken for analysis by Kaiser Test to confirm the absence of free amino groups.", "4.2 Coupling of Chelating Agent 1 (CA1) The resin bound acid functionality was then pre-activated in situ by the addition of a solution in DMF (15 ml) containing PyAOP (0.52 g, 1 mmol), HOAt (0.14 g, 1 mmol) and NMM (0.2 ml, 2 mmol).", "On-resin activation was carried out for 10 minutes followed by addition of a solution in DMF (10 ml) of CA1 (0.4 g, 1.1 mmol).", "The mixture was bubbled for 3 hours then excess reagents removed by filtration followed by washing with DMF (5×15 ml), DCM (3×15 ml) and ether (3×15 ml).", "The peptide-resin was then dried in a stream of nitrogen.", "Example 5 Radiolabelling: Sodium Hydroxide Method An aliquot of conjugate (100 μg) was dissolved in nitrogen-purged saline (900 μl) and added to a silanised P6 vial using silanised pipette tips.", "The pH of the solution was adjusted to pH 8.5 with 0.01M sodium hydroxide solution and the vial capped.", "To the solution was added Na99mTcO4 (1 ml, 1 GBq/ml) from a freshly eluted generator (eluate less than 2 hours old).", "The pH of the solution was again adjusted to pH 8.5 with 0.01M sodium hydroxide solution.", "Freshly prepared nitrogen-purged SnCl2 solution (0.1 ml, 10 μmg/100 μml saline) was then added to the solution.", "The pH of the solution was again adjusted to pH 8.5 with 0.01M sodium hydroxide solution.", "The vial was shaken after each addition to ensure mixing.", "The preparation was left to stand at room temperature.", "Example 6 Radiolabelling: Borate Buffer Method 12.5 mM borate buffer (1 ml) was added to an aliquot of conjugate and sonicated for 30 seconds.", "The resulting solution was added to a silanised P6 vial, using silanised pipette tips, and the vial capped.", "To the solution was added Na99mTcO4 (1 ml, 1 GBq/ml) from a freshly eluted generator (eluate less than 2 hours old).", "Freshly prepared nitrogen-purged SnCl2 solution (0.1 ml, 10 mg/100 ml saline) was then added to the solution.", "The vial was shaken after each addition to ensure mixing.", "Example 7 Labelling Analysis Investigation of the labelling characteristics of the CA1 conjugates was performed by labelling CA1-ST5-18 using the sodium hydroxide method outlined in Example 5.Simultaneous BPLC and ITLC analysis was performed at approximately 15, 60 and 120 minutes post reconstitution.", "In addition, preparative HPLC was performed to obtain pure samples of the two 99mTc species.", "ITLC analysis was performed on each purified sample to confirm agreement between the two analytical techniques.", "Further studies were performed on alternative CA1 conjugates to confirm the observed labelling characteristics.", "The results of the initial investigation of the labelling characteristics showed a single major 99mTc species at early time points, converting almost completely to a second species over a number of hours.", "Good agreement between ITLC and BPLC analysis was observed.", "Two 99mTc-conjugate species were resolved by both BPLC and ITLC and the relative quantities of each species correlated well.", "For further confirmation, preparative “PLC was performed to obtain pure samples of the two species.", "Example 8 Effect of pH on Radiolabelling The effect of pH on the labelling characteristics was investigated by comparing the labelling of CA1-ST5-18 (100 μg), using the borate buffer method outlined in Example 6, at pH 8.1, 8.5 and 9.0.ITLC analysis was performed at 15, 30, 60, 90, 120 and 180 minutes post-reconstitution.", "A relatively narrow pH range was studied due to the known instability of the ST5-18 vector to high pH, caused by the presence of three disulphide bridges.", "The results of the comparison of the labelling of CA1-ST5-18 at pH 8.1, 8.5 and 9.0 are shown (FIG.", "1).", "The results are expressed as % species 2 formed, as seen by ITLC (Gelman ITLC/SG paper, 70:30 saline: acetonitrile eluent) analysis.", "Example 9 Effect of Temperature on Radiolabelling The effect of temperature on the labelling characteristics was investigated by comparing the labelling of CA1-ST5-18 (100 μg), using the borate buffer (pH 9.0) method outlined above, followed by heating at 40, 60 and 75° C. Each preparation was allowed to stand at room temperature for 10 minutes post reconstitution then heated for 20 minutes.", "ITLC analysis was performed prior to heating (10 minutes post reconstitution), 10 minutes post heating (40 minutes post reconstitution) and 60 minutes post heating (100 minutes post reconstitution).", "The results of the comparison of the labelling of CA1-ST5-18 at 40, 60 and 70° C. are shown (FIG.", "2).", "The results are again expressed as % Peak 2 formed, as measured by ITLC (Gelman ITLC/SG paper, 70:30 saline: acetonitrile eluent) analysis.", "Example 10 Effect of Conjugate Level on Radiolabelling The effect of conjugate level on the labelling characteristics was investigated by comparing the labelling of various amounts of CA1-ST5-18 (12.5, 25, 50 and 100 μg) using the borate buffer (pH 9.0) method outlined above.", "ITLC analysis was performed at 15, 30, 60, 90, 120 and 180 minutes post reconstitution.", "The results of the labelling CA1-ST5-18 at varying levels (12.5, 25, 50 and 100 μg, equivalent to 6.6×10−9, 1.3×10−8, 2.7×10−8 and 5.3×10−8 moles) are shown (FIG.", "3).", "The results are expressed as the percentage of species 2 formed, as seen by ITLC (Gelman ITLC/SG paper, 70:30 saline: acetonitrile eluent) analysis.", "RHT data has been shown (Table II).", "TABLE II RHT levels in radiolabelled preparations.", "100 μg 50 μg 25 μg 12.5 μg RHT (%) 0.6 1.5 1.9 2.7 Example 11 Stability of Conjugate to Labelling Conditions To establish the stability of the starting material to the labelling conditions, an inactive, “blank” labelling experiment was performed.", "A “blank” labelling involved the substitution of saline for Na99mTcO4.CA1-ST5-18 (100 μg) was subjected to the borate buffer labelling method (pH 9.0, 60° C., 20 minutes).", "The starting material was then HPLC purified and a competition binding assay performed to determine whether there was any loss of affinity for the receptor compared with before the labelling procedure.", "HPLC analysis was performed to detect any degradation of the compound.", "No degradation of the compound was observed by HPLC and potency (measured by competition binding assay) did not significantly alter.", "Example 12 Competition Between CA1-benzamide and ST5-18 To investigate co-ordination of 99mTc to the peptide conjugate, a competition study was performed.", "Firstly, CA1-benzamide (which was chosen to mimic the CA1 portion of CA1-ST5-18) and ST5-18 were labelled independently using the borate buffer method (pH9.0, 60° C. for 20 minutes) and characterised by BPLC and ITLC.", "For the competition study, equimolar quantities of CA1-benzamide (31 μg, 6.92×10−8) and ST5-18 (100 μg, 6.92×10−8) were labelled together in the same pot using the borate buffer method.", "ITLC and HPLC analyses, as outlined in Examples 13 and 14, determined where the technetium had coordinated.", "Example 13 ITLC Gelman ITLC/SG paper eluted with saline Gelman ITLC/SG paper eluted with methylethyl ketone (MEK, 2-butanone) Gelman ITLC/SG paper eluted with a 70:30 v/v mixture of saline and acetonitrile Whatman No.", "1 (W1) paper eluted with a 50:50 v/v mixture of water and acetonitrile Example 14 HPLC All analysis was carried out using Gilson hardware and Unipoint software.", "Column: Phenomenex Luna C-18, 4.6×250 mm, 5 μm Eluent A:0.1% acetic acid (pH raised to pH 5.0 with ammonia) in 90% water/10% acetonitrile Eluent B:0.1% acetic acid (pH raised to pH 5.0 with ammonia) in 10% water/90% acetonitrile Flow:1 ml/min UV Detector:)λ=230 mn Similar gradients were used for the different conjugates.", "A typical gradient was: T0:0% B T5:15% B T20:15% B T21:0% B T30:0% B Example 15 Competition Binding Assay Compounds were assessed for their potency by radioligand binding assay to determine inhibition constant (Ki) as a measure of potency of binding.", "Methods are described elsewhere, but briefly, [125]]ST-1-17 was competed with cold ST compounds (0.0001-50 μM) in 96 well plates containing ca.", "1 μg rat intestinal membrane and incubated 1 hr, 37° C. Samples were filtered (GF/B filters, Whatman) to retrieve bound radiolabelled peptide and counted to determine % Bound/Free.", "Potency of compounds was determined by inhibition constant (Ki) for their ability to compete with radioligand, [125I]ST1-17.Potencies of screened compounds are shown in Table III.", "TABLE III Ki assay data for CRC compounds.", "Ki values shown are means of triplicate values of assay wells.", "Non-specific binding accounts for less than 0.1% in all cases and total binding (no competing peptide) is no greater than 20%.", "Compound Mean Ki value (nM) Comments ST1-17 0.6 ST peptide ST5-18 0.4 Selected Core Vector CA3-ST5-18 0.8 Labelled CA3-ST5-18(cyc-ala) none Negative Control CA1-ST5-18 2.8 Unlabelled CA-ST5-18 3.5 Mock-labelled Example 16 Radioactive Binding Assay To determine whether radiolabelling affected the binding of the compound, labelled compounds were tested for their ability to bind the receptor.", "Briefly HPLC purified radiolabelled compounds (ca.", "0.05 nM) were incubated with 6 μg/150 μl final volume of rat intestinal membrane (one hour), in the presence and absence of competing peptide (50 μM ST5-18).", "Samples were filtered (GF/B filters, Whatman) to retrieve bound radiolabelled peptide and counted to determine % Bound/Free.", "Non-specific binding accounted for less than 1% in all cases.", "In order to ensure that potency was maintained post-labelling, an assay measuring binding of labelled compounds was developed.", "Example 17 Biodistribution in a Subcutaneous Tumour Model The sub-cutaneous model was used as an appropriate initial in vivo screen.", "Briefly, mice (female nude CD-1, ca.", "20 g; Charles River) were injected sub-cutaneously into the neck with T84 human colon carcinoma cells (0.1 ml, 1×108 cells/ml in medium) through a 23-gauge needle and allowed to develop tumours over 6-8 weeks.", "At least three animals per test point were studied.", "Results are expressed as % injected dose per gram (% ID/g) of tissue.", "Due to the fast clearance of 99mTc-labelled CA1-ST5-18 (greater than 90% excreted via urine 60 mins p.i.)", "tumour uptake is also expressed as relative retention (RR).", "Compounds were screened for their uptake into sub-cutaneous T84 tumours in the mouse (Table M).", "This model allows assessment of the imageability of CA1-ST5-18 by allowing comparison between tumour and background tissue uptake.", "TABLE IV Screening data for compounds of the invention.", "Screen CA3-ST5-18(cys-ala) CA3-ST5-18 CA1-ST5-18 CA1-lys6-gly-ST5-18 CA1-gly3-Dglu3-ST5-18 Ki value (nM) none 0.8 2.8 72 1 HBS 26 5 1.0 1.48 0.71 (% id at 2 hours p.i.)", "Urinary (% id at 2 32 90 >90 76 63 hours p.i.)", "Liver (% id at 2 hours p.i.)", "0.93 0.5 0.1 4.9 0.46 Tumour uptake & ratios % id/g at 2 hours p.i.)", "0.21 0.8 0.94 1.0 0.26 RC at 2 hours p.i.", "0.1 0.24 0.32 0.3 0.08 RR at 2 hours p.i.", "0.17 2.5 6.4 1.6 1.9 Tumour/liver 0.08 2 12.4 0.23 0.63 Retention over 2 hours 8 30 20 34 5.5 Example 18 Imaging in the Subcutaneous Tumour Model Tumours were grown as in Example 17.Animals were monitored for tumour growth for 8 to 10 weeks, until tumours were 0.5-1 cm in size.", "After this time, animals were injected with test article (0.1 ml, 20 MBq/ml or 200 MBq/ml) as an intravenous bolus via the tail vein.", "At various times p.i.", "animals were euthanased and whole body planar images acquired between 5 minutes and 24 hours p.i.", "using a gamma camera (Park Medical Isocam-1).", "Image acquisition times were typically 15-30 min or 150-250K counts (whole body, LEUHR collimator).", "Example 19 Uptake of 99mTc-Labelled CA1-ST5-18 and CA3-ST5-18(cys-ala) (Negative Control) into CRC Tumours CA3-ST5-18(cys-ala) has the same peptide sequence as 99mTc-labelled CA1-ST5-18, except all cysteines have been replaced by alanine amino acids, removing the three disulphide bridges required for binding.", "CA3-ST5-18(cys-ala) also contains the CA3 as opposed to CA1 and so was labelled using a suitable protocol for this chelate.", "To examine the uptake and retention of CA3-ST5-18(cys-ala) and 99mTc-labelled CA1-ST5-18, CD-1 nude mice bearing sub-cutaneous T84 tumours were injected, euthanased and dissected at various times p.i.", "The data is presented in FIGS.", "5 and 6.Example 20 Biodistribution in Liver Metastases Model The liver metastases model was used in both mice and rats.", "As for the subcutaneous tumour model except in establishing tumours, cells were injected via the spleen and allowed to transfer to the liver via the circulation.", "Two minutes after injection of cells, a splenectomy was performed.", "Animals were allowed to generate tumours over 6-8 weeks.", "Animals were monitored for tumour growth for 8 to 10 weeks, until tumours were 0.5-1 cm in size.", "After this time, animals were injected with test article (0.1 ml, 20 MBq/ml or 200 MBq/ml) as an intravenous bolus via the tail vein.", "At various times p.i.", "animals were euthanased.", "Muscle, kidneys, urine, lung, liver, stomach, small intestine, large intestine, thyroid and tumour were dissected and a blood sample taken.", "Dissected tissues, blood samples and standards were weighed and counted (Wallac Wizard).", "At least three animals per test point were studied.", "Results are expressed as % ID/g of tissue.", "Due to the fast clearance of 99mTc-labelled CA1-ST5-18 (greater than 90% excreted via urine 60 mins p.i.)", "tumour uptake is also expressed as RR (FIG.", "7).", "Example 21 Imaging in Liver Metastases Model Tumours were grown in animal as described in Example 20.Animals were monitored for tumour growth for 8 to 10 weeks, until tumours were 0.5-1 cm in size.", "After this time, animals were injected with test article (0.1 ml, 200 MBq/ml) as an intravenous bolus via the tail vein.", "At various times p.i.", "animals were euthanased and whole body planar images acquired between 5 minutes and 24 hours p.i.", "using a gamma camera (Park Medical Isocam-1).", "Image acquisition times were typically 15-30 min or 150-250K counts (whole body, LEUHR collimator).", "The uptake of 99mTc-labelled CA1-ST5-18 in the rat liver metastases model is shown (FIG.", "8).", "The image has the kidneys masked to improve the resolution of the imaged metastases.", "Example 22 Uptake of CA1-ST5-18 into a Non Colonic Tumour To examine the specificity of the compounds of the present invention for colonic carcinomas, C57BL/6 mice bearing sub-cutaneous Lewis lung tumours were injected with CA1-ST5-18 and dissected at various times p.i.", "Lewis lung is carcinoma that originally spontaneously arose in the lung of C57BL/6 mice and therefore is not of colorectal origin and is not thought to express the ST receptor.", "Example 23 ST Receptor Mediated Uptake of CAT-ST5-18 To further examine the specificity of the compounds of the present invention for 5 colonic carcinomas, nude mice bearing T84 tumours as described in Example 17 were imaged.", "Co-injection of 100 μg/mouse of ST peptide injected with CA1-ST5-18 reduced the uptake by 40%.", "(FIG.", "9).", "Example 24 Effect of Impurities It was observed that there was an increase in background tissue activity observed with non-HPLC purified preparations; this could be potentially due to several factors: presence of excess peptide, presence of 99mTc labelled impurities (e.g.", "RHT or others).", "Further studies were carried out to investigate the other possible causes for the increased background seen with non-HPLC purified preparations, different approaches to reduce impurities such us RHT were tested.", "Preparations were either filtered (0.2 μm filter size) or had MDP added, known to reduce RHT levels.", "Example 25 Comparison of Tumour Model used with Human Tissue T84 receptor density data show good comparison with those of human liver metastases (FIG.", "10).", "Direct comparison of human liver metastases and xenograft tumour receptor density (Bmax) data clearly show that mean receptor density of human tumours lies in between that seen for T84 and LS180 tumours.", "In order to determine whether uptake varied with differences in receptor density, biodistribution studies were undertaken in a range of xenograft tumours with different ST receptor expressions." ] ]	Patent_10469801
[ [ "Telecommunications networks", "A first multi protocol label switching (MPLS) enabled Internet Protocol (IP) data network is able to transmit data to a second MPLS enabled IP network via a legacy optical network, which would not otherwise be able to handle the user network interface (UNI) protocols required to be used within an MPLS network environment, by means of configuring the legacy optical network and its traditional network management system (TNMS) so that they simulate or emulate an MPLS enabled optical network.", "The simulation/emulation of an MPLS network is performed as follows: when a first legacy network element (NE) receives a connection request (a UNI request) from the MPLS network under a UNI protocol, the UNI request is passed to the TNMS, which then sets the required connection across the legacy network via a second edge NE to an NE of the second IP network.", "Once the connection has been set, the TNMS instructs the edge NE to send a return signal to the requesting network indicating that the connection has been successfully set.", "Data packets may then be transmitted across the network." ], [ "1-12.", "(Canceled) 13.A method of operating a connection-oriented first communications network having a plurality of first network elements in which connections across the first network elements are established by a network management system, the first network being connectable to a second communications network by an edge network element, the second network having a plurality of second network elements each operative for making connections or routing data across the second network in accordance with a connection request received by the edge network element, the connection request being in accordance with a predetermined protocol, the method being performed to establish a connection across the connection-oriented first network in response to the connection request from the second network, and comprising the steps of: a) upon the edge network element receiving the connection request from the second network, sending to the network management system information relating to the connection request; b) sending signals by the network management system to set the connection in response to said information received from the edge network element; and c) causing the edge network element by the network management system to send a return signal according to the predetermined protocol to the second network indicating a status of a setting of the connection.", "14: The method according to claim 13, wherein the connection-oriented first network is further connectable to a third communications network by a further edge network element, the third network having a plurality of third network elements each operative for making connections or routing data across the third network in accordance with a connection request received by the further edge network element, the method further comprising the step of: causing the further edge network element by the network management system to send a connection request according to the predetermined protocol to the third network, thereby enabling a connection of the second and third networks via the connection-oriented first network.", "15.The method according to claim 13, and comprising the step of operating the connection-oriented first network such that, in use, topology information relating to the first network is not made available outside the first network.", "16: The method according to claim 13, wherein the predetermined protocol is a user network interface (UNI) protocol.", "17: A connection-oriented first communications network, comprising: a plurality of first network elements in which connections across the first network elements are established by a network management system, the first communications network being interoperable with a second communications network by an edge network element, the second network having a plurality of second network elements each operative for making connections or routing data across the second network in accordance with a connection request received by the edge network element, the connection request being in accordance with a predetermined protocol, the edge network element upon receiving the connection request from the second network, being operative for sending to the network management system information relating to the connection request, the network management system being operative for sending signals to the first network elements to set a connection in response to said information received from the edge network element, and the network management system being operative for causing the edge network element to send a return signal according to the predetermined protocol to the second network indicating a status of a setting of the connection.", "18.The connection-oriented first communications network according to claim 17, and further comprising a further edge network element for connecting the first network to a third communications network having a plurality of third network elements each operative for making connections or routing data across the third network in accordance with a connection request received by the further edge network element, the network management system being configured to cause the further edge network element to send the connection request according to the predetermined protocol to the third network, thereby enabling the connection of the second and third networks via the first network.", "19.The connection-oriented first communications network according to claim 17, wherein the first network is operated such that, in use, topology information relating to the first network is not made available outside the first network.", "20: The connection-oriented first communications network according to claim 17, wherein the predetermined protocol is a user network interface (UNI) protocol.", "21.The connection-oriented first communications network according to claim 17, wherein the first network is an optical communications network.", "22.The connection-oriented first communications network according to claim 17, wherein the second network elements of the second network are operative for establishing connections or routing data in accordance with multi-protocol label switching (MPLS).", "23.An edge network element for use in a connection-oriented first communications network according to claim 17.24.A network management system for use in a connection-oriented first communications network according to claim 17." ], [ "The present invention relates to a method of communicating across a telecommunications network and associated apparatus.", "In particular, the invention relates to a method of communicating across a telecommunications network, to a telecommunications network, to a network management system for setting connections in a network and to a network element of such networks.", "Telecommunications networks, particularly optical networks, have in the past routed data across the network by setting routes by means of a manually operated network management system.", "When changes are required to be made to the route or routes set in the network, response times can be very long in comparison to the rate of transmission of data.", "Significant improvements in routing of data have been made in recent years in the context of electrical networks.", "One such improvement is the ability of network elements of the network to route data packets without reverting to a separate network management system.", "Recently, the use of Multi Protocol Label Switching (MPLS), which is currently used in both IP and ATM networks, has been recognised as being particularly advantageous.", "One of the benefits of MPLS is that network elements of the network are able to route a given data packet quickly, by reference to a label in the data packet.", "Furthermore, since the routing of data packets does not require the exchange of data with a network management system the use of MPIS has a major advantage in that it facilitates dynamic network control without the delays often associated with networks controlled by a network management system.", "It has been proposed, so as to facilitate dynamic network control, to introduce MPLS, in the form of a Generalised Multi-protocol Label Switching (GMPLS) method, into optical networks.", "However, incorporating GMPLS into an optical network is not straightforward.", "Two proposals have been made for implementing GMPLS in an optical network as will now be described.", "The first proposal may be referred to as the “Peer-to-Peer Model” and is illustrated by FIG.", "1 of the accompanying drawings.", "With reference to FIG.", "1, a first IP network 1 is connected via an optical network 2 to a second IP network 3.The optical network 2 is required to make available to the IP networks 1, 3 topological information (in the form of IP information) so that data packets can be routed from the first IP network 1 to the second IP network 3 via the optical network 2 by means of IP data in the data packet.", "If the optical network 2 is privately owned, making such topological information publicly available may however be undesirable.", "For example, such information may be considered to be commercially sensitive and it may be desired to keep such information confidential.", "The second proposal, which may be referred to as the “Client-Server Model”, does not require the optical network to make public such topological information.", "In this second proposal, which is described with reference to FIG.", "2 of the accompanying drawings, a first IP network 1 is connected via an optical network 2 to a second IP network 3 in a manner similar to that of the first proposal.", "However, in this proposal the interfaces between the first and second IP networks 1, 3 (the clients) and the optical network 2 (the server) each include a user network interface 4 (UNI).", "Thus the first IP network 1, via a first UNI 4a, effectively request a connection across the optical network 2 by means of IP data in a data packet.", "Topology information relating to the optical network 2 is however not made available outside the optical network 2.Both of the proposals described above suffer from a significant disadvantage.", "In order for the optical network to operate in a GMPLS environment it is necessary, in the proposals made, for the network elements of the optical network to process and handle network topology information and to set up and tear down network connections.", "In order for the individual network elements to be able to perform such tasks the network elements each require significant processing capability and access to significant amounts of memory.", "Whilst such requirements can be met when installing new optical networks, many existing optical network elements are not able to perform at the required level.", "Replacing such existing optical networks (often referred to as legacy networks) would be costly and is therefore undesirable.", "It is therefore an object of the present invention to provide a method of communicating across a telecommunications network which allows a given protocol, for example a protocol used in an MPLS environment, to be used in combination with other networks, for example optical networks, and which mitigates one or more of the problems associated with the above-mentioned proposals.", "The present invention also seeks to provide suitable means or apparatus for performing such a method or aspects of such a method.", "According to a first aspect of the invention there is provided a method of operating a connection-oriented communications network, the communications network comprising a plurality of network elements in which connections across the network elements are established by a network management system; the communications network being connectable to a second communications network by an edge network element, the second communications network comprising a plurality of network elements each of which is capable of making connections or routing data across the second network in accordance with a connection request received by the element; the connection request being in accordance with a predetermined protocol, the method being for establishing a connection across the connection-oriented network in response to a connection request from the second network and characterised by: the edge network element upon receiving a connection request from the second network, sending to the network management system information relating to the connection request; the network management system sending signals to set a connection across the network in response to said information received from the edge network element; and the network management system causing the edge network element to send a return signal according to the predetermined protocol to the second network indicating the status of the setting of a connection.", "The method of the present invention enables a connection-oriented network to establish connections in response to connection requests, which it would otherwise be unable to handle, by the steps of passing the connection request from a suitably configured edge network element to a suitably configured network management system, which is able to process the request, make the connection in accordance with the request and respond via the edge network element in accordance with the given protocol.", "As far as the second network is concerned the connection-oriented communications network is able to communicate with it under the given protocol.", "In the context of the method of the invention, the manner in which connection requests are made can be considered as being a client-server arrangement, in which the connection-oriented network is the server network and the second network is the client network.", "The return signal indicating the status of the setting of the connection may for example indicate either that the connection has been successfully made or that the connection could not be made.", "After the second network receives a return signal indicating that the connection has been successfully made, data can then be transmitted from the second network across the communications network.", "Advantageously the connection-oriented communications network is further connectable to a third communications network by a further edge network element, the third network comprising a plurality of network elements each of which is capable of making connections or touting data across the third network in accordance with a connection request received by the element, and the method further comprises: the network management system causing the further edge network element to send a connection request according to the predetermined protocol to the third network thereby enabling connection of the second and third networks via the connection-oriented network.", "Preferably the connection-oriented network is operated such that, in use, topology information relating to the network is not made available outside the network to for example the second and/or third networks.", "Of course, information concerning the possible connections to and from network elements within, but at the edge of the network, may be made available to network elements outside the communications network and accordingly such information may be considered as not relating to topology information relating to the communications network.", "Advantageously the predetermined protocol is a user network interface (UNI protocol The UNI protocol may be such that topology information is not revealed over the interface between the communications networks.", "The user network interface protocol used may, for example, be in accordance with the standards laid down by the Optical Interface Forum (OIF).", "An appropriate standard is described in document number OIF 2000.125 available from the Optical Interface Forum.", "In the case where the communications network is connected to MPLS enabled networks, the arrangement may be such that the MPLS network elements require a connection request in order to make a connection and transmit data.", "Such MPLS network elements may also be arranged automatically to send appropriate connection requests.", "Thus, when setting a connection from the communications network to the third network that is MPLS enabled, the network management system advantageously causes the further edge network element to send an appropriate connection request, for example, a U request.", "The method may be such that other protocols are used when further connection requests ar made between network elements of the network.", "For example, a network network interface (or NNI) protocol may be used An NNI protocol may be especially convenient when connection requests are made between MPLS enabled network elements.", "The NNI protocol may be such that topology information is revealed over the interface between the relevant network elements.", "According to a second aspect of the invention there is provided a connection-oriented communications network comprising a plurality of network elements in which connections across the network elements are established by a network management system; the communications network being adapted to be interoperable with a second communications network by an edge network element, the second network comprising a plurality of network elements each of which is capable of making connections or routing data across the second network in accordance with a connection request received by the element; the connection request being in accordance with a predetermined protocol, the connection-oriented network being characterised by the edge network element upon receiving connection request from the second network, sending to the network management system information relating to the connection request; the network management system sending signals to the network elements to set a connection across the network in response to said information received from the edge network element; and the network management system causing the edge network element to send a return signal according to the predetermined protocol to the second network indicating the status of the setting of a connection.", "The present invention finds particular application to legacy connection-oriented networks that comprise network elements unable to make connections or route data in accordance with connection requests.", "The step in which the edge network element sends to the network management system information relating to the connection request can comprise relaying, or repeating, the connection request.", "The edge network element may therefore be required to perform little or preferably no processing of the connection request, Conveniently the edge network element can comprise a legacy edge network element that has been appropriately adapted.", "Conversion of the legacy network element may include a step of programming the network element with appropriate updated software.", "The requirements of such computer software will be apparent to the relevant persons skilled in the art and, as such, further details of such software are not provided here.", "Alternatively, the conversion could be made by means of extra hardware in addition to, or instead of, providing such software.", "The network management system advantageously comprises the legacy network management system that has been appropriately adapted.", "The conversion of such a legacy network management system may include a step of programming the network management system with appropriate updated software.", "Again the requirements of such computer software would be apparent to those skilled in the art, when presented with the details of the present invention.", "Alternatively, the conversion could be made by means of extra hardware in addition to, or instead of, providing such software.", "Advantageously the communications network further comprises a further edge network element for connecting the communications network to a third communications network, the third network comprising a plurality of network elements each of which is capable of making connections or routing data across the third network in accordance with a connection request received by the element, the network management system being configured to cause the further edge network element to send a connection request according to the predetermined protocol to the third network thereby enabling connection of the second and third networks via the communications network.", "Such a network is advantageous where the third network requires a connection request according to the predetermined protocol to be received before a connection can be made.", "Since the network management system causes the further edge network to send the connection request this eliminates the need for the further edge network element to be able to itself generate the connection request Preferably the communications network is operated such that, in use, topology information relating to the network is not made available outside the network.", "Advantageously the predetermined protocol is a user network interface (TM protocol.", "The network management system may also be able to handle that some UNI protocol insofar as is necessary to enable it to cause the edge network element to send the return signal under the same UNI protocol.", "The relevant edge network elements of the network may simply be programmed with appropriate software to enable them to handle the same UNI protocol.", "Preferably the second and/or third networks are packet based networks, such as Internet Protocol (OP) networks in which data packets are routed by the network elements in dependence upon the connection request within the packets.", "Alternatively the second and/or third networks can comprise Asynchronous Transfer Mode (ATM networks, and alike, in which connections are established by the network elements in dependence upon connection requests.", "The invention finds particular application for connection to networks able to handle Multi-Protocol Label Switching (MPLS).", "It will be understood that multi-protocol label switching may take many forms, any of which could be used in the context of the present invention.", "For example, a generalised form of MPLS (GMPLS) may be used The form of multi-protocol label switching used may however be conveniently chosen to be in accordance with an accepted standard, for example a standard set by the Internet Engineering Task Force.", "According to a farther aspect of the invention there is provided an edge network element for use in a communications network in accordance with the second aspect of the invention.", "According to a yet further aspect of the invention there is provided a network management system for use in a communications network in accordance with the second aspect of the invention.", "Embodiments of the present invention will now be described by way of example only with reference to the accompanying schematic drawings, of which: FIGS.", "1 and 2 show prior art proposals for a telecommunications network; FIG.", "3 illustrates a telecommunications network in accordance with a first embodiment of the invention; and FIG.", "4 illustrates a telecommunications network in accordance, with a second embodiment of the invention.", "FIGS.", "1 and 2 relate to prior art proposal and are described above.", "A telecommunications network according to the first embodiment of the invention is shown in FIG.", "3.With reference to FIG.", "3 the network includes a first MPLS enabled IP network 11 connectable to a second MPLS enabled IP network 13 via a legacy optical network 12.The legacy optical network 12 includes a plurality of internal network elements 15 (only one of which is shown in FIG.", "3 for the sake of clarity) and a plurality of edge network elements 14a, 14b (only two of which are shown).", "The legacy optical network 12 is also connected to a traditional network management system 16.At the interface between the first IP network 11 and the optical network 12, signals may be received and sent by an edge network element 14a of the optical network 12.Similarly, at the interface between the second IP network 13 and the optical network 12, signals may be received and sent by an edge network element 14b of the optical network 12.Network access and connection requests may be made through the edge network elements of the optical network.", "The network may thus be considered as forming a client/server system with interfaces, between the server (the optical network 12) and the client (the first or second IP networks 11, 13).", "The protocol used at such interfaces, in relation to the provision of network access and connection requests, is a UNI (user network interface) protocol.", "A UNI protocol is also used throughout the first and second IP networks 11, 13 and connection requests in the form of UNI requests are, in the IP networks, processed by the local network elements, connections being established by use of the topology protocols running across the network.", "The topology protocols used may for example be an OSPF (Open Shortest Path First) protocol.", "However, the network elements within the legacy optical network 12 are not able to process such requests locally.", "The making of a connection across the optical network 12, including the handling of UNI requests made to edge network elements of the optical network 12, will now be described.", "A UNI request is sent to a first edge network element 14a of the optical network 12 from a network element (not shown separately) of the first IP network 11, the request effectively requesting a connection to a network element of the second WP network 13.The request is sent (arrow A) directly to the network management system 16.The network management system 16 then processes the request and determines an appropriate connection across the optical network 12.The network management system 16 then sends signals (arrows B) instructing the edge network element 14a, the relevant internal network elements 15 and a second network element 14b of the optical network to establish the required connection.", "The network management system 16 then formulates a suitable response for sending to the network element of the first network 11 that sent the UNI request.", "The network management system 16 then sends (arrow C) a signal to the edge network element 14a originally receiving the UNI request, causing that edge network element 14a to send such a suitable response to that network element of the first network 11.The response sent from the edge network element 14a originally receiving the UNI request to the network element of the first network 11 indicates either that a connection has been successfully made or that the connection failed, as appropriate.", "If the connection is successfully made the network element of the first network 11 is then able to send data via the optical network 12 to the appropriate network element of the second IP network 13.The IP networks 11, 13 are not able to discover the topology of the optical network 12, as such information is not made available outside of the optical network 12.Thus it will be appreciated that such an arrangement enables a client/server UNI network to be established without the need for network elements of the server network (the legacy optical network 12) to run locally any topology protocols.", "This is especially advantageous in legacy networks, such as the optical network 12 illustrated in FIG.", "3, where the network elements do not have the required memory and/or processing power to handle such protocols.", "A telecommunications network according to a second embodiment of the invention is illustrated with reference to FIG.", "4.In this second embodiment, MPLS enabled network elements are combined with legacy network elements in a common transport network.", "The MPLS enabled parts of the network can operate with full topology protocols, whereas the legacy part of the network effectively emulates a UNI interface to enable connections across the legacy part of the network to be provided automatically on request.", "With reference to FIG.", "4, the network includes a first MPLS enabled IP network 21a connectable to a second MPLS enabled IP network 23a via an optical network 20.The optical part of the network comprises a legacy optical network 22 connectable between first and second MPLS enabled optical networks 21b, 23b.", "The legacy optical network 22 includes a plurality of internal network elements (not shown) and a plurality of edge network elements 24a, 24b and is connected to a traditional network management system 26, in a manner similar to that of the legacy optical network 12 shown in FIG.", "3.In FIG.", "4, the first IP network 21a has a UNI interface to the optical network 20.Topology information relating to the first MPLS optical network 21b, the second MPLS optical network 23b and the legacy optical network 22 is not made available outside each network, respectively.", "As far as the first IP network 21a is concerned the optical network 20 may, or may not, be separated into MPLS enabled and legacy optical networks.", "The making of a connection from the first IP network 21a to the second IP network 23a will now be described.", "The first IP network 21a sends a UNI request to the first MPLS enabled optical network 211b of the optical network 20, the UNI request effectively, requesting a connection to a network element (not shown separately) of the second IP network 23a via the optical network 20.The UNI request is processed locally within the first MPLS optical network 21b, and the requested connection made to the edge of that network, where it meets another UNI interface (the interface between the first MPLS optical network 21b and the legacy optical network 22).", "A new UNI request is therefore sent from an edge network element of the first MPLS optical network 21b to a first edge network element 24a of the legacy optical network 22.This UNI request effectively requests a connection from the interface between the MPLS optical network 21b and the legacy optical network 22 to the destination network element of the second IP network 23a.", "Since the network receiving the UNI request is a legacy optical network 22, the request cannot be processed locally by the network elements of the network 22.In a manner similar to that described above with reference to the first embodiment, the request is sent (arrow A) directly to the traditional network management system 26.The network management system 26 then processes the request and determines an appropriate connection across the optical network 22.The network management system 26 then sends signals (arrow B) instructing the edge network element 24a, the relevant internal network elements and a second network element 24b of the optical network to establish the required connection.", "In this embodiment, however, the legacy optical network 22 interfaces to another optical network, which is MPLS enabled (i.e.", "the second MPLS optical network 23b).", "The second MPLS optical network 23b requires a UNI request to cause it to make the connection to the second IP network 23a.", "Thus the network management system 26 sends (arrow X) to the second edge network element 24b of the legacy network 22 a signal causing the second edge network element 24b to send a UNI request to an edge network element of the second MPLS optical network 23b.", "This UNI request again effectively requests a connection to be made to the destination network element of the second IP network 23a.", "The UNI request is processed locally within the second MPLS optical network 23b, and the requested connection made across that network to the destination network element of the second IP network 23a.", "The edge network element of the second MPLS optical network 23b receiving the UNI request from the second edge network element 24b of the legacy network 22, sends a return signal under the UNI protocol to the second edge network element 24b, the return signal indicating that the requested connection has been successfully made.", "The return signal is sent (arrow Y) directly to the network management system 26.On receipt of that return signal the network management system 26 formulates a suitable response for sending to the network element of the first MPLS optical network 21b that sent the UNI request to the legacy network 22.The network management system 26 then sends (arrow C) a signal to the edge network element 24a originally receiving the UNI request from the first MPLS optical network 21b, causing that edge network element 24a to send such a suitable response under the UNI protocol to the relevant edge network element of the first MPLS optical network 21b.", "The indication of the successful connection is then finally relayed to the first IP network 21a by the first MPLS optical network 21b to complete the connection process.", "The network element of the first IP network 21a is then able to send data via the optical, network 20 to the appropriate network element of the second IP network 23a.", "Should the process fail at any stage, then a failure response is sent back to the requesting network.", "The network is arranged such that on such a failure any intermediate connections that have been made in relation to the given connection request are cleared down.", "As will be appreciated, various modifications may be made to the above described embodiments.", "For example, two IP networks could be connected via an optical network, such that a first IP network is connected to a single MPLS optical network, which is connected to a single legacy network, which is connected to a second IP network.", "In such a case, the receipt from the first network of a UNI request requesting connection to the second IP network would be handled in a similar manner to that described with reference to the first embodiment.", "The UNI request passed on by the MPLS optical network would be passed from an ingress legacy network element to the network management system of the legacy network, which would set a connection across the legacy network to the second IP network, and then cause the ingress legacy network element to send an appropriate response to the requesting IP network, data thereafter being transmitted from the first IP network across the optical network and to the second IP network.", "Also, two IP networks could be connected via an optical network, such that a first IP network is connected to a single legacy network, which is connected to a single MPLS optical network, which is connected to a second IP network.", "In such a case, the receipt from the first network of a UNI request requesting connection to the second AP network would be handled in a similar manner to that described with reference to the second embodiment.", "The UNI request would be passed from an ingress legacy network element to the network management system of the legacy network, which would set a connection, send a UNI request via an egress legacy network element to the MPLS optical network, receive an appropriate response via the egress legacy network element from the MPLS optical network, and then cause the ingress legacy network element to send an appropriate response to the requesting IP network, data thereafter being transmitted from the first IP network across the optical network and to the second IP network.", "Other permutations of legacy optical, optical MPLS, and IP networks will of course be apparent to those skilled in the art.", "Whilst MPLS optical networks have been referred to above, the embodiments described would of course also be of use if the MPLS optical networks were in the form of GMPLS (generalised MPLS) optical networks.", "The IP based networks described above need not be IP data networks and could alternatively be ATM data networks, since such networks may also be used with MPLS and user network interfaces (UNI) The topology protocols used across the MPLS networks need not be OSPF.", "For example, the topology protocol used may alternatively be an IS/IS (Intermediate System to Intermediate System Routing Exchange) protocol from the OSI (Open Systems Interconnection)." ] ]	Patent_10469802
[ [ "Communications network", "A communications network determines for itself its own network topology, that is, the identity and interconnection of nodes comprising the network.", "The network comprises a plurality of nodes each having at least one port.", "The ports are interconnected in accordance with the network topology in which communication traffic is conveyed over the network via the interconnected ports.", "Each port is arranged to transmit first information within the communication traffic including the identity of the port (Section trace identity) from which the communication traffic originates.", "Second information is transmitted between nodes identifying which first information identity relates to which node and which port.", "A processor is operative for determining for each node from the first and second information the identity of adjacent nodes and the identity of the ports to which its ports are connected." ], [ "1-16.(Canceled).", "17.A communications network, comprising: a plurality of nodes each having at least one port, the ports being interconnected in accordance with a network topology in which communication traffic is conveyed over the network via the interconnected ports, each port having associated with it a unique port identifier, each node having associated with it a unique node identifier, each port being operative for transmitting within the communication traffic its respective port identifier identifying the port from which the communication traffic originates, means for transmitting to each node second information, including for each node ofthe network, the node identifier and the port identifier for each port associated with the node, and processing means for determining for each node from the received port identifier and the second information, an identity of an adjacent node and an identity of the port to which its ports are connected.", "18.The communications network according to claim 17, in which the processing means is operative for determining the topology of the network from the interconnection of the adjacent nodes.", "19.The communications network according to claim 17, in which the second information is transmitted to the nodes over connections which are physically separate to those interconnecting the nodes and carrying the communication traffic.", "20.The communications network according to claim 19, in which the second information is transmitted as part of control signaling information.", "21.The communications network according to claim 17, in which the processing means is distributed among the nodes of the network.", "22.The communications network according to claim 17, in which the nodes include storage means for storing pre-loaded check information, the check information including the port identifiers that can be expected to be received on respective ports of the node, and means for comparing the port identifiers received at its ports with the check information for validating an integrity of the connection of the ports of the nodes.", "23.The communications network according to claim 17, in which the port identifier is transmitted as a part of an overhead part of the communication traffic.", "24.The communications network according to claim 17, in which the second information includes a modified internet protocol.", "25.The communications network according to claim 17, in which the network is an optical communications network in which the communication traffic is transmitted between the nodes as optical radiation modulated with the communication traffic, and in which the radiation is conveyed by radiation guiding means interconnecting the nodes.", "26.The communications network according to claim 17, in which the network comprises a synchronous digital hierarchy (SDH) network.", "27.The communications network according to claim 17, in which the network comprises a synchronous optical network (SONET).", "28.The communications network according to claim 26, in which the port identifier comprises a section trace identifier which is carried within section overhead.", "29.The communications network according to claim 26, in which the second information is transmitted within a data communications channel (DCC).", "30.The communications network according to claim 17, in which the network comprises a wavelength division multiplex (WDM) network.", "31.The communications network according to claim 30, in which the second information is transmitted within an optical supervisory channel (OSC).", "32.A method of operating a communications network comprising a plurality of nodes each having at least one port, the ports being interconnected in accordance with a network topology in which communication traffic is conveyed over the network via the interconnected nodes, each port having associated with it a unique port identifier, each node having associated with it a unique node identifier, each port transmitting its respective port identifier within the communication traffic identifying the port from which the communication traffic originates, the method being performed for determining the network topology and comprising the steps of: a) transmitting to each node second information which includes for each node of the network the node identifier and the port identifier for each port associated with the node; and b) determining for each node from the received port identifier and the second information, an identity of an adjacent node and an identity of the port interconnecting the adjacent nodes." ], [ "The invention relates to communications network and in particular to a network that can determine for itself its own network topology, that is the identity and interconnection of nodes comprising the network.", "As is known communications network comprise a plurality of interconnected nodes.", "At each node there is provided a node unit having a plurality of ports.", "Ports of the respective node units are interconnected to one another in accordance with the network topology, e.g.", "a ring configuration, mesh configuration etc.", "Communication traffic (information/data) is conveyed between node units by means of the interconnected ports.", "Communication between node units can be electrical in which case the ports of the node units are generally connected by electrical conductors and information is conveyed between nodes as modulated electrical signals or optical in which case communication between ports is by means of modulated optical radiation which is conveyed by optical waveguides, typically optical fibres.", "In the management and operation (routing of communication traffic etc) of communications networks it is a requirement that the network topology be known that is the identity and interconnection of node units and in particular which port of each unit is connected to which port of which other node unit.", "It is particularly advantageous for the network to be capable of determining for itself its own network topology.", "An example of a known communications network that is capable of determining its network topology is an ATM (asynchronous transfer mode) or IP (internet protocol) network.", "Such networks use Multi Protocol Label Switching (MPLS) to provide a label addressing, quality of service together with an associated capability of determining their own network topology.", "An example of such a network is illustrated in FIG.", "1.The network comprises five nodes A to E which are interconnected to one another by respective ports labelled A1, A2 .", ".", ".", "to E1.As is known communication traffic (customer data/information) is communicated across the network by selective routing between ports in dependence up signalling information which, it is to be noted, is carried over the same physical interconnection between ports, that is in-band signalling.", "In the context of the present patent application control signalling information refers to information such as routing/management/quality of service etc which is used for control/operation of the network.", "In contrast communication traffic, e.g.", "data, voice, video etc is the information to be communicated across the system.", "For the network to determine its topology, each node needs initially to determine the identity of adjacent node/s to which it is connected and how its ports are physically connected to which ports of the/each adjacent node/s.", "In the ATM network illustrated each node determines this information by transmitting control signalling information over each port including the identities (ID) of the node and the port.", "This information is transmitted in accordance with the MPLS protocol Thus each port of each node will receive the identity of each node and port it is physically connected to.", "In this manner each node can determine a map of adjacent nodes to which it is connected.", "Once each node has determined a map of adjacent nodes, each node then broadcasts to all other nodes this information thereby enabling each node to determine an overall map of the network topology.", "Whilst such a method of determining network topology is adequate in ATM/IP networks the inventor has appreciated that a problem exists in determining network topology in optical communications networks especially those operating using synchronous digital hierarchy (SDH) or synchronous optical networks (SONET).", "In such networks, unlike ATM and IP, there is no direct access to the in-band traffic and therefore limited capability to carry in-band signalling and communication of control signalling information between nodes is carried over paths which are physically separate to those carrying the communication traffic.", "As a result the receiving node cannot assume a direct association (physical interconnection) of ports and consequently a problems arises in each node determining a mapping of adjacent nodes.", "EP 1026916 discloses an automatic telecommunications link identification system, in which a link comprises interconnected ports of adjacent nodes of the network.", "Each port has a unique identifier, and is configured to transmit over the link an identification message which includes its own identifier and the presumed identifier (remote identifier) of the port at the receiving end of the link.", "Similarly the port at the receiving end sends an identifier message which includes its own identifier together a remote identifier.", "If the identification message from the ports at opposite ends of a link agree, that is, if the port identifiers are reciprocals of each other, the link is identified and the interconnectivity of the ports determined.", "If however, the identification messages do not agree one port will update, in response to the received port identifier, its remote identifier and re-send an identification message as an acknowledgement of the updated remote port identification.", "The present invention arose in an endeavour to provide a communications network that is capable of determining its own topology which in part, at least, overcomes the limitations of the known arrangements.", "According to a first aspect of the invention there is provided a communications network comprising a plurality of nodes each having at least one port, the port being interconnected in accordance with the network topology and in which communication traffic is conveyed over the network via the interconnected ports, wherein each port has associated with it a unique port identifier and each node has associated with it a unique node identifier, and each port is arranged to transmit within the communication traffic its respective port identifier identifying the port from which the communication traffic originates, the network characterised by: comprising means arranged to transmit to each node second information, including for each node of the network, the node identifier and port identifier/s for each port associated with the node; and processing means arranged for determining for each node from received port identifier/s and second information, the identity of adjacent node/s and the identity of the port/s to which its ports are connected.", "Advantageously the processing means is arranged to further determine the network topology, that is the interconnection of all ports of all nodes, from the interconnection of adjacent nodes.", "The present invention finds particular application for communications network, such as synchronous systems, in which the second information is transmitted between nodes over connections which are physically separate to those interconnecting the nodes and which carry the communication traffic.", "Preferably in such a network the second information is transmitted as part of the control signalling information.", "Preferably the processing means is distributed among the nodes of the network.", "In a preferred arrangement each node includes processing means capable at least of determining the identity of adjacent node/s and the identity of the respective ports that each of its ports is interconnected.", "Preferably, the nodes include storage means for storing pre-loaded check information, the check information including the port identifiers that can be expected to be received on respective ports of the node, and means for comparing the port identifiers received at its ports with the check information for validating the integrity of the connection of the nodes ports.", "Preferably the port identifier is carried within an overhead part of the communication traffic.", "Advantageously the second information is transmitted in the form of a modified Internet Protcol.", "Preferably, the network is an optical communications network in which communications traffic is transmitted between nodes in the form of optical radiation modulated with the communication traffic said radiation being is conveyed by radiation guiding means, preferably optical fibres.", "In one arrangement, the network comprises a Synchronous Digital Hierarchy (SDH) network.", "Alternatively the network comprises a Synchronous Optical Network (SONET).", "In such communication networks the port identifier comprises a section trace identifier which is advantageously carried within section overhead of the synchronous traffic frame.", "Advantageously the second information is transmitted within a data communications channel (DCC).", "Advantageously the communications network comprises a wavelength division multiplex (WDM) network, preferably a dense WDM network.", "For such a network the second information is advantageously transmitted in an Optical Supervisory Channel (OSC).", "According to a second aspect of the invention there is provided a method of operating a communications network of a type comprising a plurality of nodes each having at least one port, the ports being interconnected in accordance with the network topology and in which communication traffic is conveyed over the network via the interconnected nodes, wherein each port has associated with it a unique port identifier and each node has associated with it a unique port identifier; and each port transmits its respective port identifier within the communication traffic identifying the port from which the communication traffic originates, the method being for determining the network topology and characterised by: transmitting to each node second information which includes for each node of the network, the node identifier and port identifier/s for each port associated with the node; and determining for each node from the received port identifier/s and second information, the identity of adjacent node/s and the identity of the ports interconnecting the adjacent nodes.", "A communications network in accordance with the invention will now be described by way of example only with reference to the accompanying drawings in which: FIG.", "1 is a schematic representation of a known ATM communications network as described above; and FIG.", "2 schematic representation of an optical communications network in accordance with the invention.", "Referring to the accompanying drawing, there is shown an optical communications network comprising five node 1, 2, 3, 4 and 5 respectively identified as A to E in the FIG.", "2.Node 1 has six optical ports identified as A1 to A6 respectively.", "Node 2 has three optical ports identified B1 to B3 respectively; node 3 has two optical ports C1 and C2, node 4 has two optical ports D1 and D2 and node 5 a single optical port labelled E1.The optical ports of the nodes are connected to each other by optical fibres 7 to 13 in accordance with the network topology and enable the transmission of communication traffic between the nodes.", "In the example embodiment being described the communications traffic is conveyed between the nodes in the form of modulated optical radiation in accordance with a synchronous digital hierarchy (SDH) or synchronous optical network (SONET).", "The interconnection of the optical ports of the nodes 1 to 5 will now be described.", "The optical ports A1 to A3 of the node are connected to the optical ports B1 to B3 of the node 2 by optical fibres 7 to 9 respectively.", "The optical port A4 of node 1 is connected to the optical port C1 of node 3 by the optical fibre 10.The optical ports A5 and A6 of node 1 are respectively connected to the optical ports D1 and D2 of node 4 by the optical fibres 11 and 12 respectively.", "Finally the optical port C2 of node 3 is connected to the optical port E1 of node 5 by the optical fibre 13.The network being described is a simplified network topology for the purposes of elucidating the invention.", "In practical communications networks the topology would likely comprise many tens or even hundreds of nodes connected in ring, mesh or other network topologies.", "The network further comprises a management source 6 for controlling operation of the network.", "The source 6 is connected to each node 1 to 5 by an independently routed signal control communications link 14.The communications link 14 accesses the node 1 to 5 via ports which are unlabelled in FIG.", "2 and are independent of the ports of the node used for conveying communication traffic.", "The management source 6 represents an information source, which may belong to the network management system.", "The communications link 14 allows bi-directional communication from the source 6 to each node and also bi-directional communication between the nodes.", "As is known in SDH networks each node transmits within the communications traffic a first piece of information which is unique to the port from which the communications traffic originates.", "This first information termed a section trace identifier, is typically sixteen bytes long, is carried within the section overhead of the synchronous traffic frame.", "More specifically the section trace identifier is carried within sixteen consecutive J0 bytes of the SDH frames.", "On initial configuration of the network the unique section trace identifier associated with each physical port is allocated by the management source 6 and communicated to the respective node via the communications link 14.A section refers to the physical link between two ports.", "Thus each section is uniquely defines by two section trace identifiers.", "For example port A1 of node 1 includes a section trace identifier indicated as h in FIG.", "2.Conversely port B1 of node 2 which is connected to node A1 and thus represents the same physical connection (section) is arranged to transmit trace information indicated as i.", "Similarly ports A2, B2, A3, B3, A4, C1, A5, D1, A6, D2, C2, E1 are arranged to respectively transmit section trace identifiers indicated as j to u.", "As well as communicating the respective section trace identifiers to each node the source 6 also on initial configuration communicates to each port the section trace identifiers that can be expected to be received on respective ports.", "Each node is configured to examine the section trace identifier received at its ports and compare theses information with the expected value and thereby validate the integrity of section (physical connection).", "In the event that received and expected section trace identifiers do not correspond the node generates an error flag which it communicates to the management source 6.The use of section trace identifiers are well known and documented and the network described thus far is of a known configuration.", "It will be appreciated that the section trace identifiers alone only provides a means of checking the correct interconnection of nodes.", "They do not, however, enable each node to determine for itself the identities of adjacent node to which it is connected nor to which port of the node it is attached.", "In accordance with the communications network of the present invention each node is arranged to communicate with every other node second information via the communications link 14.This information is communicated between nodes using a modified internet protocol (IP).", "This second information comprises the identity of the node, the identity of its physical ports and the section trace identity for each port.", "As an example the information that node 1 communicates is given in table 1 below.", "TABLE 1 Node Port Section Trace identifier A A1 h A2 j A3 l A4 n A5 p A6 r In this way each node ends up with information for the node identity, port identity and corresponding section trace identity for each and every node.", "This information is set out in table 2.TABLE 2 Node Port Section Trace identifier A A1 h A2 j A3 l A4 n A5 p A6 r B B1 i B2 k B3 m C C1 o C2 t D D1 q D2 s E E1 u In operation each node combines the section trace identity extracted from the communications traffic received at its respective ports with the further information (table 2) to determine the node identity and port identity to which each of its ports is connected.", "For example node 1 identifies that communication traffic received at its first port A1 has a section trace identity i.", "From the information in table 2 processing means in the node deduces that this identity corresponds to the port B1 of node 2 and thereby determines that its port A1 is physically connected to port B1 of node 2.In a like manner each node determines a mapping of adjacent nodes to which its ports are directly connected.", "Once each node has determined the mapping of adjacent nodes it transmits this information to all other nodes via the communications link 14, enabling each node to build up a map of the layout of the network as a whole.", "In this way each node is able to determine for itself the identity and interconnection of all nodes comprising the network i.e.", "each is able to determine the network topology.", "Although in the above description the determination of network topology is described as being determined at initialisation of the network it is envisaged that this process is continued during operation of the network as the SDH network topology can vary dynamically during operation.", "A particular benefit of the network of the present invention is that the network can determine its own topology even though communication of control signalling information between nodes takes place over physically separate communications paths to those of the communication traffic.", "A particular advantage of the network described is the utilisation of the section trace identity that is inherently present in SDH/SONET networks to carry the first information.", "The network of the present invention is thus readily realisable by the provision of processing means within the nodes and modification of the existing internet protocol used to communicate between nodes.", "It will be appreciated that modifications can be made to the network describe which fall within the scope of the invention.", "For example in an alternative configuration it is envisaged to communicate the second information between nodes using the Data Communications Channel (DCC).", "Although this is an in-band communication channel it operates independently of communications traffic channels.", "Furthermore the communications network can comprise a wavelength division multiplex (WDM) network or dense WDM network in which communications traffic is conveyed in the form of modulated radiation of a specified wavelength often termed a wavelength channel.", "In such networks one of the wavelength channels is reserved for purposes other than carrying communications traffic and is termed an Optical Supervisory Channel (OSC).", "Preferably in such a network the section trace identities (first information) of the node ports are carried by the OSC.", "Although the above network is an optical network, it will be understood by those skilled in the art that the invention is applicable to non-optical networks in which it cannot be guaranteed that communication of signal control information will take place over the same physical paths as the communication of communications traffic.", "It is the division of the information into two parts as described above that enables the network of the present invention to determine its own network topology." ] ]	Patent_10469804
[ [ "Evolutionary programming of configurable logic devices", "A configurable hardware logic device is configured to implement a complete evolutionary algorithm in hardware.", "The configured hardware logic device produces an initial population of individuals, carries out fitness tests on the initial population of individuals, selects an individual or individuals of the initial population on the basis of results of the fitness tests, breeds a further population of individuals from the selected individual or individuals, and provides a single preferred selected individual from the further population of individuals." ], [ "1-24.", "(Canceled) 25: An uncommitted configurable hardware logic device when configured to implement a complete evolutionary algorithm in hardware, the configured hardware logic device comprising: a) population producing means having an output, and operative for producing an initial population of individuals at the output; b) fitness evaluating means having an input and first and second ouputs, the output of the population producing means being connected to the input of the fitness evaluating means, and the fitness evaluating means being operative for carrying out fitness tests on the initial population of individuals, and for providing results of the fitness tests and the respective individuals at the first output of the fitness evaluating means; c) selector means having an input and an output, and operative for selecting at least one individual of the initial population on the basis of the results of the fitness tests and the respective individuals connected from the output of the fitness evaluating means to the input of the selector means, and for providing the selected at least one individual at the output of the selector means; d) breeding means having an input connected to the output of the selector means, and operative for breeding a further population of individuals from the selected at least one individual, and having an output connecting the further population of individuals to the input of the fitness evaluating means; and e) the fitness evaluating means being further operative for providing a single preferred selected individual from the further population of individuals at the second output of the fitness evaluating means.", "26: The uncommitted configurable hardware logic device when configured as claimed in claim 25, wherein the evolutionary algorithm is genetic programming.", "27: The uncommitted configurable hardware logic device when configured as claimed in claim 25, wherein the uncommitted configurable hardware logic device is a field programmable gate array (FPGA).", "28: The uncommitted configurable hardware logic device when configured as claimed in claim 25, which is configured to perform a selected step or steps each in a plurality of parallel steps.", "29: The uncommitted configurable hardware logic device when configured as claimed in claim 25, wherein a configuration is by means of a high-level language to hardware compilation system.", "30: The uncommitted configurable hardware logic device when configured as claimed in claim 29, wherein the high-level language to hardware compilation system is Handel-C. 31: The uncommitted configurable hardware logic device when configured as claimed in claim 25, wherein the uncommitted configurable hardware logic device is reconfigurable.", "32: The uncommitted configurable hardware logic device when configured as claimed in claim 25, and a random access memory included or connected to the device.", "33: The uncommitted configurable hardware logic device when configured as claimed in claim 25, and a random number generator configured from a part of the device.", "34: The uncommitted configurable hardware logic device when configured as claimed in claim 25, when configured to operate on scalar data.", "35: The uncommitted configurable hardware logic device when configured as claimed in claim 25, when configured to operate on vector data.", "36: The uncommitted configurable hardware logic device when configured as claimed in claim 25, when configured to operate on sub-machine-code.", "37: A method of implementing a complete evolutionary algorithm in hardware by configuring an uncommitted configurable hardware logic device to carry out the steps of: a) creating an initial population of individuals; b) evaluating a fitness of the initial population of individuals; c) selecting at least one individual from the initial population of individuals; d) breeding a further population of individuals from the selected at least one individual; e) evaluating a fitness of the further population of individuals; and f) selecting a preferred one of the further population of individuals based on the fitness of the further population of individuals.", "38: The method of claim 37, wherein the evolutionary algorithm is genetic programming.", "39: The method of claim 37, wherein the uncommitted hardware logic device is a field programmable gate array (FPGA).", "40: The method of claim 37, wherein at least one of the steps is performed in a plurality of parallel steps.", "41: The method of claim 37, wherein the configuring is performed by a high-level language to hardware compilation system 42: The method of claim 41, wherein the high-level language to hardware compilation system is Handel-C. 43: The method of claim 37, wherein the uncommitted configurable hardware logic device is reconfigurable.", "44: The method of claim 37, and the step of storing data in a random access memory.", "45: The method of claim 37, and the step of configuring a random number generator.", "46: The method of claim 37, and the step of operating on scalar data.", "47: The method of claim 37, and the step of operating on vector data.", "48: The method of claim 37, and the step of operating on sub-machine code." ], [ "Genetic Programming (GP) systems are generally realized as programs running on general purpose computers.", "This work was motivated by the observation that as problems get harder, the performance of traditional computers can be severely stretched.", "This is despite the continuing increase in performance of modern CPUs, and the use of multiple processors to exploit the fact that GP can be parallelised.", "By implementing a GP system directly in hardware the aim is to increase the performance by a sufficiently large factor so as to make it possible to tackle harder problems.", "The present invention shows how a GP system can be implemented in hardware using a hardware compilation technique.", "Initially there is a description of the hardware and the hardware compilation language.", "This is followed by a description of the GP system in general and a discussion of the design decisions that had to be made in order to successfully fit a GP system into a Field Programmable Gate Array (FPGA).", "This is followed by some example problems chosen to exercise the implementation.", "The results of running the system and comparisons with a traditional implementation follow.", "The following section briefly describes FPGAs in general.", "This is followed by a description of the high level language to hardware compilation system.", "This is not intended to be a full description of the tool, but it describes the most important features, especially those that influence the design decisions described later.", "This introduction is intended to make some of the following discussions easier to understand for those with little or no experience of FPGAs.", "More comprehensive data can be located at various manufacturers web sites.", "FPGAs are a class of programmable hardware devices, consisting of an array of Configurable Logic Blocks (CLBs) and configurable interconnects.", "A general model of an FPGA is shown in FIG.", "1 .", "Each CLB consists of two function generators (F & G) and two flip-flops (FF).", "Internal Carry and Control logic (H) connects the function generators and the flip-flops.", "A general model of a CLB is shown in FIG.", "2.The configuration of these devices is achieved by loading configuration bit patterns.", "In the Xlinx XCV4000 FPGA, which is the example device used herein, the bit patterns are programmed into static RAM on the chip.", "This has to be done each time the chip is re-powered.", "The configuration bit patterns are generated using software tools that take a high level description of the configuration information.", "Handel-C is a high-level language to hardware translation system, designed to compile programs written in a C-like language into synchronous hardware.", "The output from Handel-C is a file that can be used to create the configuration data for a FPGA.", "Handel-C is produced by Celoxica [www.celoxica.com].", "Handel-C has a C-like syntax.", "This makes the tool appealing for software engineers with no experience of hardware, in that they can quickly translate a software algorithm into hardware, without having to learn about FPGAs in detail, or VHDL.", "VHDL is a standard hardware design language.", "It stands for VHSIC Hardware Design Language.", "VHSIC itself stands for Very High Speed Integrated Circuit.", "One of the advantages of using hardware is the ability to exploit parallelism directly.", "This is in contrast to the simulated software parallelism that is found on single CPU computers achieved using time-slicing.", "Handel-C has additional constructs to support the parallelisation of code.", "The block par{ a=10; b=20; } would generate hardware to assign the value 10 to a and 20 to b in a single clock cycle.", "Using arrays of functions or by generating inline code, large blocks of functionality can be generated that execute in parallel.", "Hardware can be replicated using the construct par (i=0;i<10;i++) { a[i] = b[i]; } which would result in 10 parallel assignment operations.", "To make efficient use of the hardware, Handel-C requires the programmer to declare the width of all data, for example, int 5 count; is a signed integer that is 5 bits wide, and so will be able to represent the values between −15 and +15.Handel-C supports a single Integer data type.", "Communication between the hardware and the outside world is performed using interfaces.", "These may be specified as input or output, and as with assignment a write to or a read from an interface will take one clock cycle.", "The language allows the designer to target particular hardware, assign input and output pins, specify the timing of signals, and generally control the low level hardware interfacing details.", "Macros are available to help target particular devices.", "According to the Handel-C documentation, the simple rule about timing of statements is that “assignment takes 1 clock cycle, the rest is free”.", "This means that expressions are constructed using combinatorial logic, and data is clocked only when an assignment is performed.", "For example, Handel-C would generate hardware for the following statement that executed in a single clock cycle.", "y=((xx)+3x); This feature makes it easy to predict the performance in terms of clock cycles.", "However, there is a penalty in that the more complex the expression, the deeper the logic required to implement the expression.", "This in turn limits the maximum clock rate at which the design can be run because of the propagation delays associated with deep logic.", "In practice this means that the designer needs to trade clock cycles against clock rate, and this is typically an iterative process.", "Because Handel-C targets hardware, there are some programming restrictions when compared to a traditional ANSI-C compiler.", "These need to be taken into consideration when designing code that can be compiled by Handel-C.", "Some of these restrictions particularly affect the building of a GP system.", "Firstly, there is no stack available, so recursive functions cannot be directly supported by the language.", "This in turn means that standard GP, which relies heavily on recursion, cannot be implemented without some modification.", "Secondly, there is a limit on the size of memory that can be implemented using standard logic cells on an FPGA, because implementing memory is expensive in terms of silicon real estate.", "However, some FPGAs have internal ram that can be used by Handel-C. For example the Xlinx Virtex and Spartan series support internal memory that Handel-C allows the user to declare as RAM or ROM.", "For example the definition ram int 8 mem[128]; declares a RAM block of 128 cells, each 8 bits wide, which can be accessed as a normal array.", "A limitation of using RAM or ROM is that it cannot be accessed more than once per clock cycle, so restricting the potential for parallel execution of code that accesses it.", "Thirdly, expressions are not allowed to have side effects, since this would break the single cycle assignment rule.", "Therefore code such as a=++b; is not allowed and needs to be re-written as: b=b+1; a=b; Handel-C supports two targets.", "The first is a simulator target that allows development and testing of code without the need to use any hardware.", "This is supported by a debugger and other tools.", "The second target is the synthesis of a netlist to input to place and route tools.", "A netlist is a formal description of the gates and their connections.", "Place and route is the process of translating a netlist into a hardware layout.", "This allows the design to be translated into configuration data for particular chips.", "An overview of the process is shown in FIG.", "3.Analysis of cycle count is available from the simulator, and an estimate of gate count is generated by the Handel-C compiler.", "To get definitive timing information and actual hardware usage, the place and route tools need to be invoked.", "FPGAs have been used to implement parts of a GP system.", "The system described in “Register-based Genetic Programming on FPGA computing platforms” (M I Heywood and A N Zincir-Heywood; Genetic Programming, Proceedings of Euro GP 2000 Volume 1802 of LNCS, pages 44-59, Edinburgh 15-16 April 2000; Springer-Verlag) was simulated using more traditional FPGA tools.", "The proposal in his work was to use the FPGA only for evaluating the individuals and performing mutation and crossover.", "Koza et al used an FPGA to speed up the evaluation of fitness of a sorting network “Evolving sorting networks using genetic programming and the rapidly configurable Xilinx 6216 field-programmable gate array” (John R Koza et al; Proceedings of the 31st Asilomar Conference on Signals, Systems and Computers; IEEE Press, 1997).", "In this work the FPGA was used solely to perform the fitness evaluation.", "The initial population was created by a host computer, and then individuals were downloaded to the FPGA and the FPGA instructed to evaluate the fitness of the individual.", "Subsequent selection and breeding were again performed by the host computer.", "The next section describes the general design decisions taken to implement GP in hardware.", "The aim was to realise a complete GP system in hardware.", "That is initial population generation, fitness evaluation, breeding and the delivery of the final result.", "This is in contrast to all other examples of using FPGAs with Genetic Programming.", "This high level aim guided many of the following design decisions.", "The lack of a built-in stack when using Handel-C makes the use of recursive functions difficult.", "Although there are well known methods of removing recursion from algorithms, a stack of some form is still required to store intermediate results.", "An alternative to the standard tree representation is the linear GP system.", "The details of the internal representation depend on the word size, number of functions, and number of terminals used, and these are dependent on the problem being tackled.", "For the present work, a register like machine was chosen for its simplicity.", "A general layout of an instruction is shown in table 1.Each program is an array of instructions together with some control information.", "To simplify the manipulation of programs, each program is a fixed maximum size, with a maximum active length stored against it.", "The representation of a program is shown in table 2.TABLE 1 General layout of an instruction Field Comments Opcode The operation being encoded.", "Effective Address 1 The primary source operand and the destination address.", "Always a register.", "Effective Address 2 The secondary operand.", "Can be a register, a new Program counter or an index into a table of constants.", "TABLE 2 General layout of an individual Field Comments Length The active length of the program.", "Raw The raw fitness of the program fitness Instructions An array of instructions According to the present invention there is provided an uncommitted configurable hardware logic device when configured to implement a complete evolutionary algorithm in hardware, the configured hardware logic device comprising; (a) population producing means having an output and arranged to produce an initial population of individuals at the output; (b) fitness evaluating means having an input and first and second outputs, the output of the population producing means being connected to the input of the fitness evaluating means and the fitness evaluating means being arranged to carry out fitness tests on the initial population of individuals and to provide the results of the fitness tests and the respective individuals at the first output of the fitness evaluating means; (c) selector means having an input and an output and arranged to select an individual or individuals of the initial population on the basis of results of the fitness tests and the respective individuals connected from the output of the fitness evaluating means to the input of the selector means and to provide the selected individual or individuals at the output of the selector means; (d) breeding means having an input connected to the output of the selector means and arranged to breed a further population of individuals from the selected individual or individuals and having an output connecting the further population of individuals to the input of the fitness evaluating means; the fitness evaluating means being further arranged to provide a single preferred selected individual from the further population of individuals at the second output thereof.", "There is further provided a method of implementing a complete evolutionary algorithm in hardware comprising configuring an uncommitted configurable hardware logic device so as to carry out the steps of; (e) creating an initial population of individuals; (f) evaluating the fitness of the initial population of individuals; (g) selecting one or more individuals from the initial population thereof; (h) breeding a further population of individuals from the selected one or more individuals; (i) evaluating the fitness of the further population of individuals; (j) selecting a preferred one of the further population of individuals based on the fitness of the further population of individuals.", "The present invention will now be described by way of example, with reference to the accompanying drawings, in which: FIG.", "1 shows a general model of an FGPA; FIG.", "2 shows general model of a CLB; FIG.", "3 shows an overview of the process of translating code into hardware; FIG.", "4 shows a diagram of a Linear Feedback Shift Register Random Number Generator; FIG.", "5 illustrates the number of cycles to carry out one fitness function evaluation for the population; FIG.", "6 illustrates the total number of cycles for the problem; and FIG.", "7 illustrates an estimated NAND gate count as the number of parallel fitness evaluations is increased.", "A key difference between the present work and others, is that it is not constrained to a function set that a microprocessor designer sees fit to implement.", "That is to say the functions can be designed to have a higher level of abstraction than machine instructions.", "An example of a problem where the function set is expressed at a high level of abstraction is the function and terminal set require several steps to be performed.", "If implemented using a RISC or CISC architecture, each step would require several RISC instructions to be executed.", "With Handel-C the functions could be encoded efficiently and compactly.", "One of the key advantages of hardware is that it is possible to design truly parallel machines.", "In order to make the most efficient use of the available silicon, choosing which parts to make parallel needs some care.", "The three main phases of GP are the initial population creation, the fitness evaluation of all individuals, and the breeding of new generations.", "Since the first is only performed once, there is little advantage in consuming excessive silicon resources to speed this phase up.", "The evaluation of fitness is traditionally cited as the most expensive in terms of processing resources, so this is a prime candidate for making a parallel phase.", "The breeding phase normally accounts for significantly less than the fitness evaluation, so there may be some advantage in making this parallel only in critical sections.", "The design used in this work exploits parallel execution of all simple statements where possible.", "This is done regardless of the phase of GP since there is no penalty in executing two assignments in parallel; the hardware will be generated for each assignment anyway.", "This is especially useful when initialising variables at the beginning of a function.", "The fitness evaluation phase exploits the parallel capabilities by executing a number of evaluations at the same time.", "If the problem has a total population size of P then a subpopulation of size S=P/n individuals are evaluated in parallel.", "To make this as efficient as possible, and to make the maximum use of the hardware, all parameter settings are powers of 2.So if P=16 and n=4 then S=4.Parallelisation of the evaluation is implemented by using the inline keyword in Handel-C.", "This causes as many copies of the hardware to be generated as required.", "A random number generator (RNG) is used in two of the major steps in GP.", "Firstly, during initial population creation, and secondly, during the breeding phase.", "When using Handel-C, the use of the standard multiply and divide instructions are inefficient in terms of silicon because of the deep logic generated.", "Because of this the usual linear congruential generators normally found were rejected.", "Instead, a linear feedback shift register (LFSR) design was used.", "A word size of 64 was chosen, as this could be implemented efficiently on a standard modern CPU, and so the LFSR can be ported easily to ANSI-C.", "It is important to choose a good polynomial to make sure that the RNG can generate a maximal sequence of 2n−1 random numbers, while keeping the number of taps to a minimum for efficiency.", "For a 64 bit word the polynomial x64+x4+x3 +x2+x1+x0 was used (FIG.", "4 shows 4 terms from a polynomial; 5 terms from a 64 bit word; x0 is always 1 and so is ignored).", "The block diagram of the LFSR is shown in FIG.", "4.The RNG is designed so that a random number is generated in one cycle.", "The required number of bits are then read from the 64 bit register, starting at bit 1 to give a random number.", "For example if the system has 8 instructions, then 3 bits are needed to encode the instruction.", "During initial program creation the random selection of an instruction uses the bottom 3 bits.", "Handel-C allows efficient bit operations, and the code to select the 3 bits is: unsigned int 3 instruction; instruction=randReg[2:0]; where randReg is the shift register variable.", "Seeding of the RNG is done by reading a 64 bit port during the initialisation phase.", "This allows the RNG to be seeded from an external source, such as a time of day clock, or other source of noise.", "It also allows the RNG to be preset to a known seed for reproducing particular results.", "A limitation of the current implementation of the generator is that it is not possible to allow parallel execution blocks to generate a random number because this would violate a condition of Handel-C that says a single instance of a function may not be called from a parallel block.", "This means that the initial program generation and breeding phases cannot be parallelised.", "While this can be overcome by implementing multiple generators, it was thought more important to dedicate resources to the fitness evaluation phase.", "To conserve memory, a steady state breeding policy is used.", "For a population size of P, every generation P individuals are selected at random and an operator applied to that individual.", "If the operator is crossover or copy, then this is counted as two individuals having been processed.", "Tournament selection is used with a tournament size of two.", "Larger tournament selection makes little sense with very small populations.", "The operators were selected using the following probabilities.", "Mutation 10%, Crossover 70%, copy 20%.", "The mutation operator works by reusing the function that generates a program node during initial program creation.", "This is done primarily to economise on hardware.", "The result is that a mutation can change zero, one or more of the instruction details.", "Crossover for a linear program representation causes some problems in that it is generally wanted to avoid performing large block memory moves.", "The present work maintains a fixed maximum program size, and copies segments from one program to another.", "By exploiting the parallel nature of hardware, the effects of performing block memory copies can be reduced to an acceptable level.", "Again by exploiting the parallel nature of the hardware, a copy of an individual of length l requires l+k clock cycles, where k represents the small overhead to set up the copy.", "The next section describes the problems chosen to test the implementation of GP using Handel-C and the environment used for the experiments.", "There were four aims of running experiments.", "1.To determine if a limited GP system could solve the problems chosen 2.To determine whether the system could be implemented using Handel-C and to verify that the design would fit on an FPGA 3.To obtain some indicative performance comparisons between a traditional C implementation and a hardware implementation 4.To determine whether the design was realisable as hardware and to implement the design in hardware Two problems demonstrate the general concept of GP in hardware using Handel-C.", "The two problems chosen were a regression problem and a boolean logic problem.", "The problem chosen is x=a+2b.", "The boolean logic problem is the 2 bit XOR function x=a⊕b.", "In both problems the raw fitness was arranged to be zero for a 100% correct program, thereby reducing the amount of logic required to test for fitness.", "In both problems, the run was terminated if a 100% correct program was found, or if the maximum number of generations was evaluated.", "To meet the above aims the problems were run using five different environments.", "Firstly, as a standard C application running under Linux.", "This was to prove the initial program operation, and enable the application to be debugged using standard GNU tools.", "The program was compiled using gcc v2.95.2 and executed on a 200 MHz AMD K6 PC running Linux.", "Secondly, the program was compiled using Handel-C, and optimisations made to the code to reduce logic depth, reduce gate count, and increase parallelism.", "Thirdly, the Handel-C implementation was run using the Handel-C simulator.", "This gave the number of clock cycles needed to execute the program.", "Fourthly, the C code was compiled using a cross compiler and executed on an instruction simulator for the Motorola Power-PC architecture.", "This was performed to get a count of instruction and memory cycles needed for a modem processor.", "The choice of the PowerPC for this work was made on the basis of a readily available simulator for the PowerPC.", "The Power-PC simulation was performed by using gcc 2.95.2 configured as a Power-PC cross compiler using the “eabi” calling convention.", "This version of the program was optimised so as to have a minimal start-up overhead and to not use any I/O.", "It is therefore as close to the FPGA program as is possible, allowing a meaningful comparison of performance to be made.", "The simulator itself was psim which is built into the GNU debugger (gdb).", "Psim can also be run as a stand-alone application.", "Lastly the output from Handel-C was used to generate a hardware layout for the place and route tools which gave the maximum clock frequency the design could be run at, and an indication of the FPGA resources required.", "For the Handel-C simulation and hardware implementation, the code was compiled using Handel-C v3.0 using maximum optimisation.", "The final FPGA configuration data was produced using Xlinx Design Manager version 3.1i for a Xlinx Virtex XCV1000-6 chip hosted on a Celoxica RC1000 development board.", "The FPGA design wrote its output to a 16 bit output port as a sequence of key, data pairs.", "This data was read and saved to a disk file for later analysis.", "A disassembler was written to decode the output data for analysis.", "When measuring the clock counts of both the Handel-C simulation and the Power-PC simulation, the code was modified to run to the maximum number of generations.", "They also both used the same random number seed.", "This was done to ensure that comparisons were made using identical conditions.", "Eight instructions were chosen for the regression problem, requiring three bits.", "Each instruction can specify up to two registers, and there are four registers available, requiring 2 bits each.", "Therefore each instruction requires 7 bits of storage.", "The instructions for this problem are: add(Rn,Rm) adds the contents of Rm to the contents of Rn and places the result back into Rn.", "sub(Rn,R m) subtracts the value in Rm from the value in Rn and places the result back to Rn.", "shl(Rn) shifts the contents of Rn left by one bit, leaving the result in Rn.", "shr behaves in a similar fashion, but shifting right by one bit.", "nop is a no-operation function.", "This was included to make the number of instructions a power of 2.halt causes the evaluation to finish, returning the value in Rn.", "ldim(Rn,Kn) causes the constant Kn to be placed into Rn.", "jmpifz(Rn, Rm)tests the value in Rn If the value is zero, then jumps to the location in Rm modulo program size.", "Program termination occurs on the following conditions: 1.a halt instruction is encountered 2.the last instruction in the program is executed 3.a jmpifz instruction has caused a loop to be created, and a predetermined number of loops have been executed.", "The machine that implements these instructions can execute one instruction every two clock cycles, including instruction fetch, decode, operand address evaluation and operand read/write.", "To speed this up even further it would be possible to build a pipeline, reducing the cycle count to one per instruction.", "Four random constants are made available to each individual.", "These are created once during the construction of individuals.", "Most examples of regression in the literature include the multiply and divide functions.", "Since these two functions generate very deep logic using the default Handel-C operators, these were replaced with single bit shift left and shift right operators, generating much shallower and therefore faster logic, and multiplying by two and dividing by two respectively.", "The full set of parameters for the regression problem are given in Table 3.The input values a and b were placed in registers R0 and R1 before the fitness evaluation, and the result x read from register R0 if the program was terminated at the end, or the value in Rn if terminated by a Halt instruction.", "The fitness data was pre-computed once at the start of the program and made available to all copies of the fitness evaluation.", "TABLE 3 Parameters for the regression problem Parameter Value Population Size 16 Functions Add(Rn, Rm), sub(Rn, Rm), shl(Rn), shr(Rn), nop, halt(Rn), Idim(Rn, Kn), jmpifz(Rn, Rm) Terminals 4 registers, R0 .", ".", ".", "R3 Word Size 8 bits Max Program 8 Size Generations 511 Fitness Cases 4 pairs of values of a and b. Subpopulation 4 Size The XOR function uses the four basic logic primitives AND, OR, NOR and NAND.", "Each of these functions takes two registers, Rn and Rm.", "The result is placed into Rn.", "These have been shown to be sufficient to solve the boolean XOR problem.", "Execution is terminated when the last instruction in the program has been executed.", "The two inputs a and b were written to registers R0 and R1 before the fitness evaluation, and the result x read from register R0 after the fitness evaluation.", "The full set of parameters is given in Table 4.With only four functions for this problem, each instruction requires six bits.", "TABLE 4 Parameters for the XOR problem Parameter Value Population Size 16 Functions AND(Rn, Rm), OR(Rn, Rm), NOR(Rn, Rm), NAND(Rn, Rm) Terminals 4 registers, R0 .", ".", ".", "R3 Word Size 1 bit Max Program Size 16 Generations 511 Fitness Cases 4 pairs of values of a and b. Subpopulation Size 4 Raw fitness The number of fitness cases that failed to yield the expected result.", "The results from the simulator for the regression problem are given in Table 5.The figures for the PowerPC were taken from the output of running psim directly from the command line, and specifying the -Iswitch to get the total number of cpu cycles.", "The cycles column takes into account the pipeline stalls and branch prediction failure, but does not take into account any memory sub-system delays.", "TABLE 5 Results of running the regression problem PPC Measurement Handel-C Simulation cycles 332,368 16,612,624 Clock 67 MHz 200 frequency(MHz) Estimated Gates tbd n/a Number of CLBs tbd n/a Speedupcycles 1 49 Speeduptime 1 16.7 The speed-up factors are given for two conditions, the raw cycle counts and the actual time taken to execute the programs.", "The first is a comparison made in terms of raw clock cycles.", "This treats the two implementations as though they were operating at the same clock frequency.", "The second is a comparison made using a typical clock rate for the PPC and the fastest frequency the FPGA could be clocked as reported by the place and route tools.", "The speed-up factor Speedupcycles=Cyclesppc/Cyclesfpga and the speed-up factor for time Speeduptime=SpeedupcyclesFreqppc/Freqfpga.", "An (annotated) example program from this problem found in generation 16 of one run is: shl(r1) //r1=b2 add(r1,r2) //nop add(r0,r1) //r0=a+(b*2) halt(r0) //Return the result in r0 The XOR problem was executed using the same environments as the regression problem.", "The results are presented in Table 6.TABLE 6 Results of running the XOR problem PPC Measurement Handel-C Simulation cycles 814,301 47,885,760 Clock 67 MHz 200 frequency(MHz) Estimated Gates Tbd N/a Number of CLBs Tbd N/a Speedupcycles 1 58 Speeduptime 1 19.7 An (annotated) example program from this problem found in generation 86 of one run is: or(r3, r1) // r3 = b or(r3, r0) // r3 = a + b or(r2, r1) // nop nand(r0, r1) // r0 = {overscore (ab)} and(r0, r3) // r0 = (a + b){overscore (ab)} The effect of using parallelisation was measured by implementing the XOR problem using different subpopulation sizes, and running the problem in the Handel-C simulator.", "A total population size of 8 was chosen, together with a maximum of 4 nodes per individual.", "These low numbers were chosen to allow the programs to be compiled in a reasonable amount of time.", "The number of subpopulations was modified each time, using the values 1, 2, 4 and 8.Data was collected for the number of cycles to perform the initial population creation, the number of cycles to evaluate the first generation, and the number of cycles to perform the breeding operators on the first generation.", "These are shown in tabular form in Table 7.TABLE 7 Cycle counts and gate estimates for various stages of the GP and different subpopulation sizes.", "Sub- population Initial Evaluation Breeding Total cycles Gate size population of of generation for run estimate 1 214 324 123 6517 35,666 2 214 180 123 4669 43,314 4 214 108 123 3549 58,588 8 214 60 123 2877 89,136 FIG.", "5 shows the effect on the number of cycles for one fitness evaluation with different sub population sizes.", "It can be seen from this graph that as the number of parallel fitness evaluations increases, so the benefit tails off.", "This is due to the constant overhead associated with setting up the fitness evaluations.", "This effect would not be so marked if the population size was greater.", "The total number of cycles for the problem is shown in FIG.", "6.The program was run for 16 generations.", "Here the effect of the breeding phase can be seen.", "The benefit gained from doubling the number of parallel fitness evaluations from four to eight only reduces the cycles required by 18%.", "The contribution of the initial population is about 7.5% of the total cycles when 8 parallel evaluations were performed.", "Lastly, the effect of increasing the number of parallel fitness evaluations on the hardware resources required was measured.", "This is shown in FIG.", "7.This shows a linear increase in the size of the hardware resources needed.", "The XOR program discussed in the previous section was also run using the PPC simulator.", "The Handel-C version used a subpopulation size of 8.The results are shown in table 8.The maximum frequency for the FPGA was 67 MHz.", "TABLE 8 Comparison of a fully parallel Handel-C program and the PPC simulator FPGA PPC cycles Cycles Speedupcycles Speeduptime 2877 299,775 104 34 The two problems presented here, though trivial when compared to many problems that have been solved using GP, have proved the general concept of using, Handel-C to produce GP systems that can be run on FPGAs.", "The use of a C-like language has some valuable properties, the most important being that a GP algorithm can be developed and tested using traditional software tools.", "This is an important consideration for software engineering, in that there is no need to become proficient in hardware design.", "The results shown above show clearly that using the current implementation, the benefits of increasing the number of parallel fitness evaluations falls off above 4.This is due to the breeding phase taking a significant portion of the cycles when compared to the fitness evaluation.", "With regard to GP, the speed increase (Speeduptime ) of 19 for the XOR problem is a valuable contribution to tackling harder GP problems.", "As faster FPGAs become available, this factor can be exploited even further.", "Currently Xilinx is offering its Virtex II series that has a maximum clock rate of 450 MHz.", "While the speed up in terms of clock cycles is worthwhile, it must be remembered that this work has in effect implemented traditional instructions that operate on scalar data.", "Greater gains can be made using an FPGA by operating on vector data, for example an image.", "Using vectors of data the function set can be tuned to exploit the parallelism of the hardware to a far greater extent than the problems described in this paper.", "When considering problems than can expressed as boolean logic problems, the technique of sub-machine-code GP has been shown to give an improvement in performance of 2 orders of magnitude.", "Using an FPGA, the possible word size is only limited by available CLBs, therefore using sub-machine-code GP and very long length words opens up the possibility of even greater performance gains.", "Input and output can be directly encoded into the function set, embedding the GP system and having it directly control hardware while evaluating the fitness of the programs.", "An example of this would be a robotic control that reads sensor inputs directly using some of the I/O pins on the FPGA and generates control signals directly to the robot.", "Since FPGAs do not need a lot of support circuitry such a controller could be embedded directly into even the smallest robot.", "The two example problems presented so far (the regression problem and the XOR problem) represent small problems that can be solved using this system.", "To solve larger problems one or more of the following parameters needs to be increased: 1.the number of generations in a run 2.the size of the population 3.the number of nodes in an individual The system so far described can accommodate increases in the number of generations without any changes, but to increase the number of individuals in the population and/or the size of an individual, some changes need to be made.", "There are three ways of realising random access memory in an FPGA system.", "Firstly, memory can be synthesised by using the single bit storage elements present in each CLB.", "This results in fast memory that can be accessed by parallel hardware.", "Secondly, some FPGAs have on-chip RAM available that can be used by a Handel-C program.", "This results in fast memory, but can only be accessed once per clock cycle thereby limiting its usefulness when accessing memory in parallel machines.", "Lastly, external off-chip RAM can be interfaced to the FPGA using some of the input/output pins available.", "This gives access to potentially many Gbytes of RAM, but at the expense of access speed.", "Extending the system to handle larger population sizes and larger individuals requires that the population is divided into an active sub-population that is held in fast synthesised memory and an inactive sub-population that is held in the slower on-chip RAM or off-chip RAM.", "Individuals are moved between the slow inactive sub-population and the fast active sub-population.", "By arranging the movement of individuals to be performed in parallel with the main evaluation and breeding phases, the delay imposed by accessing the slow inactive sub-population can be minimised." ] ]	Patent_10469808
[ [ "Method of modulating the proliferation of medullary thyroid carcinoma cells", "The present invention is directed to a method of decreasing the rate of proliferation of medullary thyroid carcinoma cells which comprises contacting medullary thyroid carcinoma cells with one or more SSTR2 agonist." ], [ "1.A method of modulating the rate of proliferation of medullary thyroid carcinoma cells which comprises contacting MTC cells with one or more SSTR2 agonist and one or more SSTR5 agonist, wherein said SSTR2 agonist reduces the rate of proliferation of the MTC cells and said SSTR5 agonist attenuates the SSTR-2 agonist-induced reduction in cellular proliferation rate.", "2.A method according to claim 1 wherein said SSTR-5 agonist is D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2 (SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof.", "3.A method of decreasing the rate of proliferation of medullary thyroid carcinoma cells which comprises contacting said medullary thyroid carcinoma cells with one or more SSTR2 agonist or a pharmaceutically acceptable salt thereof.", "4.A method according to claim 3 wherein said SSTR-2 agonist is a SSTR-2 selective agonist or a pharmaceutically acceptable salt thereof.", "5.A method according to claim 3 wherein said SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value for SSTR-5 that is at least 2 times higher than it has for SSTR-2.6.A method according to claim 3 wherein said SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value for SSTR-5 that is at least 5 times higher than it has for SSTR-2.7.A method according to claim 3 wherein said SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value for SSTR-5 that is at least 10 times higher than it has for SSTR-2.8.A method according to claim 3 wherein said SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value of less than 5.9.A method according to claim 3 wherein said SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value of less than 1.10.A method according to claim 3 wherein said SSTR-2 selective agonist is a compound selected from the list consisting of: D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 (SEQ ID NO:2); cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe] (SEQ ID NO:3); 4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2 (SEQ ID NO:4); and 4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2 (SEQ ID NO:5); or a pharmaceutically acceptable salt thereof.", "11.A method of treating medullary thyroid carcinoma which comprises administering to a patient in need thereof an effective amount of a SSTR2 agonist or a pharmaceutically acceptable salt thereof.", "12.A method according to claim 11 wherein said SSTR2 agonist comprises an SSTR2 selective agonist, or a pharmaceutically acceptable salt thereof.", "18.A method according to claim 11 wherein said SSTR-2 agonist or pharmaceutically acceptable salt thereof is a compound selected from the list consisting of: D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 (SEQ ID NO:2); cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe] (SEQ ID NO:3); 4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2 (SEQ ID NO:4); and 4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2 (SEQ ID NO:5); or a pharmaceutically acceptable salt thereof.", "19.A method according to claim 11, wherein said SSTR-2 agonist or pharmaceutically acceptable salt thereof comprises a Tyr(I) residue, wherein the iodine atom of said Tyr(I) residue comprises a radioactive iodine isotope.", "20.A method according to claim 11 or claim 19, wherein said medullary thyroid carcinoma cells have formed metastases outside the thyroid.", "21.A method according to claim 20, wherein said metastases are present in the lymph, the lung, the liver, the brain, or in bone.", "22.A method of claim 21, wherein said iodine is 125I, 127 I or 131I." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Somatostatin (SS), a tetradecapeptide discovered by Brazeau et al., has been shown to have potent inhibitory effects on various secretory processes in tissues such as pituitary, pancreas and gastrointestinal tract.", "SS also acts as a neuromodulator in the central nervous system.", "These biological effects of SS, all inhibitory in nature, are elicited through a series of G protein coupled receptors, of which five different subtypes have been characterized (SSTR1-SSTR5) (Reubi J C, et al., Cancer Res 47: 551-558, Reisine T, et al., Endocrine Review 16: 427-442, Lamberts S W, et al., Endocr Rev 12: 450-482, 4 Patel Y C, 1999 Front Neuroendocrinology 20: 157-198).", "These five subtypes have similar affinities for the endogenous SS ligands but have differing distribution in various tissues.", "Somatostatin binds to the five distinct receptor (SSTR) subtypes with relatively high and equal affinity for each subtype.", "There is evidence that SS regulates cell proliferation by arresting cell growth via SSTR1, 2, 4, and 5 subtypes (Buscail L, et al., 1995 Proc Natl Acad Sci USA 92: 1580-1584; Buscail L, et al., 1994 Proc Natl Acad Sci USA 91: 2315-2319; Florio T, et al., 1999 Mol Endocrinol 13: 24-37; Sharma K, et al., 1999 Mol Endocrinol 13: 82-90), or by inducing apoptosis via SSTR3 subtype (Sharma K, et al., 1996 Mol Endocrinol 10: 1688-1696).", "SS and various analogues have been shown to inhibit normal and neoplastic cell proliferation in vitro and vivo (Lamberts S W, et al., Endocr Rev 12: 450-482) via specific SS receptors (SSTR's) (Patel Y C, 1999 Front Neuroendocrinology 20: 157-198) and possibly different postreceptor actions (Weckbecker G, et al., Pharmacol Ther 60: 245-264; Bell G I, Reisine T 1993 Trends Neurosci 16: 34-38; Patel Y C, et al., Biochem Biophys Res Commun 198: 605-612; Law S F, et al., Cell Signal 7:1-8).", "In addition, there is evidence that distinct SSTR subtypes are expressed in normal and neoplastic human tissues (Virgolini I, et al., Eur J Clin Invest 27: 645-647), conferring different tissue affinities for various SS analogues and variable clinical response to their therapeutic effects.", "Binding to the different types of somatostatin receptor subtypes has been associated with the treatment of various conditions and/or diseases.", "For example, the inhibition of growth hormone has been attributed to the somatostatin type-2 receptor (“SSTR2”) (Raynor, et al., Molecular Pharmacol.", "43:838 (1993); Lloyd, et al., Am.", "J. Physiol.", "268: G 102 (1995)) while the inhibition of insulin has been attributed to the somatostatin type-5 receptor (“SSTR5”) (Coy, et al.", "197:366-371 (1993)).", "Activation of types 2 and 5 have been associated with growth hormone suppression and more particularly GH secreting adenomas (Acromegaly) and TSH secreting adenomas.", "Activation of type 2 but not type 5 has been associated with treating prolactin secreting adenomas.", "Other indications associated with activation of the somatostatin receptor subtypes include inhibition of insulin and/or glucagon for treating diabetes mellitus, angiopathy, proliferative retinopathy, dawn phenomenon and nephropathy; inhibition of gastric acid secretion and more particularly peptic ulcers, enterocutaneous and pancreaticocutaneous fistula, irritable bowel syndrome, Dumping syndrome, watery diarrhea syndrome, AIDS related diarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitis and gastrointestinal hormone secreting tumors; treatment of cancer such as hepatoma; inhibition of angiogenesis; treatment of inflammatory disorders such as arthritis; retinopathy; chronic allograft rejection; angioplasty; preventing graft vessel and gastrointestinal bleeding.", "It is preferred to have an analog which is selective for the specific somatostatin receptor subtype or subtypes responsible for the desired biological response, thus, reducing interaction with other receptor subtypes which could lead to undesirable side effects.", "Somatostatin (SS) and its receptors (SSTR1 to SSTR5) are expressed in normal human parafollicular C cells and medullary thyroid carcinoma (MTC) cells.", "MTC is a tumor originating from thyroid parafollicular C cells that produces calcitonin (CT), somatostatin, as well as several other peptides (Moreau J P, et al., Metabolism 45 (8 Suppl 1): 24-26).", "Recently, Mato et al.", "showed that SS and SSTR's are expressed in human MTC (Mato E, et al., J Clin Endocrinol Metab 83: 2417-2420).", "It has been documented that SS and its analogues induce a decrease in plasma CT levels and a symptomatic improvement in MTC patients.", "However, until now the antiproliferative activity of SS analogues on tumor cells had not been clearly demonstrated (Mahler C, et al., Clin Endocrinol 33: 261-9; Lupoli G, et al., Cancer 78: 1114-8; Smid W M, et al., Neth J Med 40: 240-243).", "Thus, development and assessment of SSTR subtype analogues selective on MTC cell growth provides a useful tool for clinical application.", "Until now, no data concerning specific SSTR subtype involvement in MTC cell growth regulation have been reported.", "The present invention relates to the discovery that the human MTC cell line TT, which displays MTC cell characteristics (Zabel M, et al., 1992 Histochemistry 102: 323-327, 2 Gagel R F, et al., 1986 Endocrinology 118: 1643-1651, Liu J L, et al., 1995 Endocrinology 136: 2389-2396) and which stably expresses all the SSTR subtypes, responds to SSTR2 and SSTR5 activation by subtype selective agonists with two different patterns in terms of [ 3 H]thy incorporation and cell number.", "SSTR2 preferential agonists significantly suppress [ 3 H]thy incorporation, i.e., inhibit DNA synthesis, and reduce cell proliferation.", "SSTR5 selective agonists significantly increase [ 3 H]thy incorporation in TT cells, i.e., increase DNA synthesis, but alone fail to influence cell proliferation.", "Further, SSTR2 antagonists counteract the action of SSTR2 preferential agonists on TT cells.", "Further still, increasing concentrations of an SSTR5 selective agonist dose-dependently prevents the suppression of TT cell [ 3 H]thy incorporation and proliferation produced by an SSTR2 preferential agonist, and vice versa, showing an antagonism between such agonists.", "Hetero- and homodimeric interactions between subtypes of the opiate (Jordan B A, et al., 1999 Nature 399:697-700.)", "and SS (Rocheville M, et al., 2000 J. Biol.", "Chem.", "275:7862-7869) receptor families have been recently demonstrated.", "Studies in cultured pituitary adenoma cells have demonstrated that SSTR subtype 2 and 5 act synergistically in the suppression of growth hormone and prolactin secretion (Shimon I, et al., 1997 J.", "Clinical Invest.", "100:2386-2392, Jaquet P, et al., 2000 J Clin Endocrinol Metab.", "85:781-792).", "The finding that SSTR5 activation reduces the antiproliferative activity mediated by SSTR2 differs from results in other tissues (Patel Y C, 1999 Front Neuroendocrinology 20: 157-198, Buscail L, et al., 1995 Proc Natl Acad Sci USA 92: 1580-1584, Buscail L, et al., 1994 Proc Natl Acad Sci USA 91: 2315-2319, Sharma K, et al., 1996 Mol Endocrinol 10: 1688-1696).", "This is the first demonstration that SSTR subtypes 2 and 5 can act antagonistically in regulating cell growth.", "Thus, SSTR2 and SSTR5 preferential agonists exert differential effects on proliferation of human medullary thyroid TT cell line in vitro, according to their specific SSTR selectivity.", "Proliferation of the TT cell line can be reduced by SSTR2 selective agonists, but not by SSTR5 agonists, and an SSTR5 agonist can prevent SSTR2 mediated antiproliferative effects.", "The key inhibitory role of SSTR2 on MTC cell proliferation demonstrates that analogues with enhanced SSTR2 affinity and selectivity versus SSTR5 would be useful as antiproliferative agents in MTC treatment." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based on the discovery that somatostatin agonists selective for SSTR-2 are effective in reducing the rate of proliferation of medullary thyroid carcinoma cells, and that somatostatin agonists selective for SSTR-5 are effective in attenuating this SSTR-2 agonist-induced reduction in rate of proliferation.", "In one aspect, the present invention is directed to a method of modulating the rate of proliferation of MTC cells which comprises contacting MTC cells with one or more SSTR2 agonist and one or more SSTR5 agonist, wherein said SSTR2 agonist serves to reduce the rate of proliferation of the MTC cells and said SSTR5 agonist serves to attenuate the SSTR-2 agonist-induced reduction in proliferation rate.", "In one embodiment, the invention is directed to the immediately foregoing method wherein said SSTR-5 agonist is D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH 2 or a pharmaceutically acceptable salt thereof.", "In another embodiment the invention is directed to a method of decreasing the rate of proliferation of medullary thyroid carcinoma cells which comprises contacting medullary thyroid carcinoma cells with one or more SSTR2 agonist or a pharmaceutically acceptable salt thereof.", "In a preferred example of the immediately foregoing embodiment the SSTR-2 agonist is a SSTR-2 selective agonist.", "In a more preferred example, the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value for SSTR-5 that is at least 2 times higher than it has for SSTR-2, more preferably at least 5 times higher than it has for SSTR-2, more preferably still at least 10 times higher than it has for SSTR-2.In another preferred example of the foregoing embodiment the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value of less than 5 nM, more preferably less than 1 nM.", "In another preferred example of the foregoing embodiment, the SSTR-2 selective agonist is a compound selected from the list consisting of D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH 2 , cyclo [Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH 2 , and 4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH 2 ; or a pharmaceutically acceptable salt thereof, wherein “4-(2-Hydroxyethyl)-1-piperazinylacetyl” denotes the structure: and “4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-” denotes the structure: In a third embodiment, the invention is directed to a method of treating medullary thyroid carcinoma which comprises administering to a patient in need thereof an effective amount of a SSTR2 agonist.", "In a preferred example of the third embodiment the SSTR-2 agonist is a SSTR-2 selective agonist.", "In a more preferred example, the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value for SSTR-5 that is at least 2 times higher than it has for SSTR-2, more preferably at least 5 times higher than it has for SSTR-2, more preferably still at least 10 times higher than it has for SSTR-2.In another preferred example of the third embodiment the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value of less than 5 nM, more preferably less than 1 nM.", "In yet another preferred example of the third embodiment, the SSTR-2 selective agonist is a compound selected from the list consisting of D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH 2 , cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH 2 , and 4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH 2 ; or a pharmaceutically acceptable salt thereof, wherein “4-(2-Hydroxyethyl)-1-piperazinylacetyl” and “4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-” are as previously defined.", "Importantly, as is well known in the art, standard radioactive iodine therapy, e.g., administration of a radioactive iodine salt to a patient, is not available for the treatment of medullary thyroid carcinoma since parafollicular cells do not take up iodine.", "Thus in another aspect the invention provides a method of treating medullary thyroid carcinoma patient comprising administering to a patient in need thereof an effective amount of a SSTR2 agonist or a pharmaceutically acceptable salt thereof, wherein said SSTR-2 agonist or pharmaceutically acceptable salt thereof comprises a Tyr(I) residue, wherein the iodine atom of said Tyr(I) residue comprises a radioactive iodine isotope.", "Preferably said iodine isotope comprises 125 I, 127 I or 131 I.", "In one embodiment of said medullary thyroid carcinoma cells have formed metastases outside the thyroid.", "In a further embodiment said metastases are present in the lymph, the lung, the liver, the brain, or in bone." ], [ "BACKGROUND OF THE INVENTION Somatostatin (SS), a tetradecapeptide discovered by Brazeau et al., has been shown to have potent inhibitory effects on various secretory processes in tissues such as pituitary, pancreas and gastrointestinal tract.", "SS also acts as a neuromodulator in the central nervous system.", "These biological effects of SS, all inhibitory in nature, are elicited through a series of G protein coupled receptors, of which five different subtypes have been characterized (SSTR1-SSTR5) (Reubi J C, et al., Cancer Res 47: 551-558, Reisine T, et al., Endocrine Review 16: 427-442, Lamberts S W, et al., Endocr Rev 12: 450-482, 4 Patel Y C, 1999 Front Neuroendocrinology 20: 157-198).", "These five subtypes have similar affinities for the endogenous SS ligands but have differing distribution in various tissues.", "Somatostatin binds to the five distinct receptor (SSTR) subtypes with relatively high and equal affinity for each subtype.", "There is evidence that SS regulates cell proliferation by arresting cell growth via SSTR1, 2, 4, and 5 subtypes (Buscail L, et al., 1995 Proc Natl Acad Sci USA 92: 1580-1584; Buscail L, et al., 1994 Proc Natl Acad Sci USA 91: 2315-2319; Florio T, et al., 1999 Mol Endocrinol 13: 24-37; Sharma K, et al., 1999 Mol Endocrinol 13: 82-90), or by inducing apoptosis via SSTR3 subtype (Sharma K, et al., 1996 Mol Endocrinol 10: 1688-1696).", "SS and various analogues have been shown to inhibit normal and neoplastic cell proliferation in vitro and vivo (Lamberts S W, et al., Endocr Rev 12: 450-482) via specific SS receptors (SSTR's) (Patel Y C, 1999 Front Neuroendocrinology 20: 157-198) and possibly different postreceptor actions (Weckbecker G, et al., Pharmacol Ther 60: 245-264; Bell G I, Reisine T 1993 Trends Neurosci 16: 34-38; Patel Y C, et al., Biochem Biophys Res Commun 198: 605-612; Law S F, et al., Cell Signal 7:1-8).", "In addition, there is evidence that distinct SSTR subtypes are expressed in normal and neoplastic human tissues (Virgolini I, et al., Eur J Clin Invest 27: 645-647), conferring different tissue affinities for various SS analogues and variable clinical response to their therapeutic effects.", "Binding to the different types of somatostatin receptor subtypes has been associated with the treatment of various conditions and/or diseases.", "For example, the inhibition of growth hormone has been attributed to the somatostatin type-2 receptor (“SSTR2”) (Raynor, et al., Molecular Pharmacol.", "43:838 (1993); Lloyd, et al., Am.", "J. Physiol.", "268: G 102 (1995)) while the inhibition of insulin has been attributed to the somatostatin type-5 receptor (“SSTR5”) (Coy, et al.", "197:366-371 (1993)).", "Activation of types 2 and 5 have been associated with growth hormone suppression and more particularly GH secreting adenomas (Acromegaly) and TSH secreting adenomas.", "Activation of type 2 but not type 5 has been associated with treating prolactin secreting adenomas.", "Other indications associated with activation of the somatostatin receptor subtypes include inhibition of insulin and/or glucagon for treating diabetes mellitus, angiopathy, proliferative retinopathy, dawn phenomenon and nephropathy; inhibition of gastric acid secretion and more particularly peptic ulcers, enterocutaneous and pancreaticocutaneous fistula, irritable bowel syndrome, Dumping syndrome, watery diarrhea syndrome, AIDS related diarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitis and gastrointestinal hormone secreting tumors; treatment of cancer such as hepatoma; inhibition of angiogenesis; treatment of inflammatory disorders such as arthritis; retinopathy; chronic allograft rejection; angioplasty; preventing graft vessel and gastrointestinal bleeding.", "It is preferred to have an analog which is selective for the specific somatostatin receptor subtype or subtypes responsible for the desired biological response, thus, reducing interaction with other receptor subtypes which could lead to undesirable side effects.", "Somatostatin (SS) and its receptors (SSTR1 to SSTR5) are expressed in normal human parafollicular C cells and medullary thyroid carcinoma (MTC) cells.", "MTC is a tumor originating from thyroid parafollicular C cells that produces calcitonin (CT), somatostatin, as well as several other peptides (Moreau J P, et al., Metabolism 45 (8 Suppl 1): 24-26).", "Recently, Mato et al.", "showed that SS and SSTR's are expressed in human MTC (Mato E, et al., J Clin Endocrinol Metab 83: 2417-2420).", "It has been documented that SS and its analogues induce a decrease in plasma CT levels and a symptomatic improvement in MTC patients.", "However, until now the antiproliferative activity of SS analogues on tumor cells had not been clearly demonstrated (Mahler C, et al., Clin Endocrinol 33: 261-9; Lupoli G, et al., Cancer 78: 1114-8; Smid W M, et al., Neth J Med 40: 240-243).", "Thus, development and assessment of SSTR subtype analogues selective on MTC cell growth provides a useful tool for clinical application.", "Until now, no data concerning specific SSTR subtype involvement in MTC cell growth regulation have been reported.", "The present invention relates to the discovery that the human MTC cell line TT, which displays MTC cell characteristics (Zabel M, et al., 1992 Histochemistry 102: 323-327, 2 Gagel R F, et al., 1986 Endocrinology 118: 1643-1651, Liu J L, et al., 1995 Endocrinology 136: 2389-2396) and which stably expresses all the SSTR subtypes, responds to SSTR2 and SSTR5 activation by subtype selective agonists with two different patterns in terms of [3H]thy incorporation and cell number.", "SSTR2 preferential agonists significantly suppress [3H]thy incorporation, i.e., inhibit DNA synthesis, and reduce cell proliferation.", "SSTR5 selective agonists significantly increase [3H]thy incorporation in TT cells, i.e., increase DNA synthesis, but alone fail to influence cell proliferation.", "Further, SSTR2 antagonists counteract the action of SSTR2 preferential agonists on TT cells.", "Further still, increasing concentrations of an SSTR5 selective agonist dose-dependently prevents the suppression of TT cell [3H]thy incorporation and proliferation produced by an SSTR2 preferential agonist, and vice versa, showing an antagonism between such agonists.", "Hetero- and homodimeric interactions between subtypes of the opiate (Jordan B A, et al., 1999 Nature 399:697-700.)", "and SS (Rocheville M, et al., 2000 J. Biol.", "Chem.", "275:7862-7869) receptor families have been recently demonstrated.", "Studies in cultured pituitary adenoma cells have demonstrated that SSTR subtype 2 and 5 act synergistically in the suppression of growth hormone and prolactin secretion (Shimon I, et al., 1997 J.", "Clinical Invest.", "100:2386-2392, Jaquet P, et al., 2000 J Clin Endocrinol Metab.", "85:781-792).", "The finding that SSTR5 activation reduces the antiproliferative activity mediated by SSTR2 differs from results in other tissues (Patel Y C, 1999 Front Neuroendocrinology 20: 157-198, Buscail L, et al., 1995 Proc Natl Acad Sci USA 92: 1580-1584, Buscail L, et al., 1994 Proc Natl Acad Sci USA 91: 2315-2319, Sharma K, et al., 1996 Mol Endocrinol 10: 1688-1696).", "This is the first demonstration that SSTR subtypes 2 and 5 can act antagonistically in regulating cell growth.", "Thus, SSTR2 and SSTR5 preferential agonists exert differential effects on proliferation of human medullary thyroid TT cell line in vitro, according to their specific SSTR selectivity.", "Proliferation of the TT cell line can be reduced by SSTR2 selective agonists, but not by SSTR5 agonists, and an SSTR5 agonist can prevent SSTR2 mediated antiproliferative effects.", "The key inhibitory role of SSTR2 on MTC cell proliferation demonstrates that analogues with enhanced SSTR2 affinity and selectivity versus SSTR5 would be useful as antiproliferative agents in MTC treatment.", "SUMMARY OF THE INVENTION The present invention is based on the discovery that somatostatin agonists selective for SSTR-2 are effective in reducing the rate of proliferation of medullary thyroid carcinoma cells, and that somatostatin agonists selective for SSTR-5 are effective in attenuating this SSTR-2 agonist-induced reduction in rate of proliferation.", "In one aspect, the present invention is directed to a method of modulating the rate of proliferation of MTC cells which comprises contacting MTC cells with one or more SSTR2 agonist and one or more SSTR5 agonist, wherein said SSTR2 agonist serves to reduce the rate of proliferation of the MTC cells and said SSTR5 agonist serves to attenuate the SSTR-2 agonist-induced reduction in proliferation rate.", "In one embodiment, the invention is directed to the immediately foregoing method wherein said SSTR-5 agonist is D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2 or a pharmaceutically acceptable salt thereof.", "In another embodiment the invention is directed to a method of decreasing the rate of proliferation of medullary thyroid carcinoma cells which comprises contacting medullary thyroid carcinoma cells with one or more SSTR2 agonist or a pharmaceutically acceptable salt thereof.", "In a preferred example of the immediately foregoing embodiment the SSTR-2 agonist is a SSTR-2 selective agonist.", "In a more preferred example, the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value for SSTR-5 that is at least 2 times higher than it has for SSTR-2, more preferably at least 5 times higher than it has for SSTR-2, more preferably still at least 10 times higher than it has for SSTR-2.In another preferred example of the foregoing embodiment the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value of less than 5 nM, more preferably less than 1 nM.", "In another preferred example of the foregoing embodiment, the SSTR-2 selective agonist is a compound selected from the list consisting of D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, cyclo [Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2, and 4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2; or a pharmaceutically acceptable salt thereof, wherein “4-(2-Hydroxyethyl)-1-piperazinylacetyl” denotes the structure: and “4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-” denotes the structure: In a third embodiment, the invention is directed to a method of treating medullary thyroid carcinoma which comprises administering to a patient in need thereof an effective amount of a SSTR2 agonist.", "In a preferred example of the third embodiment the SSTR-2 agonist is a SSTR-2 selective agonist.", "In a more preferred example, the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value for SSTR-5 that is at least 2 times higher than it has for SSTR-2, more preferably at least 5 times higher than it has for SSTR-2, more preferably still at least 10 times higher than it has for SSTR-2.In another preferred example of the third embodiment the SSTR-2 agonist or pharmaceutically acceptable salt thereof has a Ki value of less than 5 nM, more preferably less than 1 nM.", "In yet another preferred example of the third embodiment, the SSTR-2 selective agonist is a compound selected from the list consisting of D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2, and 4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2; or a pharmaceutically acceptable salt thereof, wherein “4-(2-Hydroxyethyl)-1-piperazinylacetyl” and “4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-” are as previously defined.", "Importantly, as is well known in the art, standard radioactive iodine therapy, e.g., administration of a radioactive iodine salt to a patient, is not available for the treatment of medullary thyroid carcinoma since parafollicular cells do not take up iodine.", "Thus in another aspect the invention provides a method of treating medullary thyroid carcinoma patient comprising administering to a patient in need thereof an effective amount of a SSTR2 agonist or a pharmaceutically acceptable salt thereof, wherein said SSTR-2 agonist or pharmaceutically acceptable salt thereof comprises a Tyr(I) residue, wherein the iodine atom of said Tyr(I) residue comprises a radioactive iodine isotope.", "Preferably said iodine isotope comprises 125I, 127I or 131I.", "In one embodiment of said medullary thyroid carcinoma cells have formed metastases outside the thyroid.", "In a further embodiment said metastases are present in the lymph, the lung, the liver, the brain, or in bone.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1: In vitro SSTR2 mediated intracellular calcium mobilization CHO-K 1 cells, expressing the human SSTR2, were harvested as described in Material and Methods and then the SS analogues (10−7-10−6 M) were added for measurement of intracellular Ca2+ mobilization, expressed as the ratio between the intracellular calcium concentration measured after the addition of SS analogues and the value observed at basal level.", "The excitation and emission wavelengths were 340 and 510 nm, respectively.", "The data are represented as mean±SEM.", "FIG.", "2: In vitro SSTR5 mediated intracellular calcium mobilization CHO-K1 cells, expressing the human SSTR5, were harvested as described in Material and Methods and then the SS analogues (10−7-10−6 M) were added for measurement of intracellular Ca2+ mobilization, expressed as the ratio between the intracellular calcium concentration measured after the addition of SS analogues (Compound 1, Compound 5 and Compound 6) and the value observed at basal level.", "The excitation and emission wavelengths were 340 and 510 nm, respectively.", "The data are represented as mean±SEM.", "The structures of the compounds appearing in FIG.", "2 are as follows: Compound 1: D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2; Compound 2: cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe]; Compound 3: 4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2; Compound 4: 4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2; Compound 5: D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2; and Compound 6: Cpa-cyclo(D-Cys-4-Pal-D-Trp-Lys-Thr-Cys)-Nal-NH2 FIG.", "3: In vitro inhibition of SS-stimulated intracellular calcium mobilization by SSTR2 antagonist CHO-K1 cells, expressing the human SSTR2, were harvested as described in Material and Methods, and then Compound 6 (10−9-10−6 M) and SS (10 nM) were added for measurement of the effect of Compound 6 on SS (10−8 M)-stimulated intracellular calcium mobilization, and expressed as the percentage vs. SS alone.", "The excitation and emission wavelengths were 340 and 510 nm, respectively.", "The data are represented as mean±SEM.", "FIG.", "4: Somatostatin receptors mRNA expression in TT cells.", "Extracted RNA (1 μg/reaction) was treated with deoxyribonuclease and subjected to reverse transcription using Oligo(dT) as primer.", "Samples incubated without RT enzyme served as control.", "Aliquots from the generated cDNA and the negative controls were subjected to subsequent PCR amplification of SSTR's, using the primers indicated in Table 1.PCR products were resolved on a 2% agarose gel.", "The expected PCR products of SSTR 1-5 are depicted in A (lane M, PCR Marker; G, PCR product of GAPDH amplification).", "FIG.", "5: Effect of SS analogues on [3H]thy incorporation in TT cells.", "Cells were incubated in 24-well plates for 48 hours in a culture medium supplemented with SS analogues at various concentrations (10−9, 10−8, 10−7, and 10 −6 M).", "Control wells were treated with vehicle solution and [3H]thy incorporation was measured as radioactivity in TCA-precipitated material.", "Data from six individual experiments evaluated independently in quadruplicate are expressed as the mean±SEM percent [3H]thy incorporation inhibition versus untreated control cells P<0.05 and P<0.01 vs. control.", "FIG.", "6: Effect of SS analogues on TT cells proliferation.", "Cells were incubated in 96-well plates for 48 hours in a culture medium supplemented with SS analogues at various concentrations (10−9, 10−8, 10−7, and 10 −6 M).", "Control wells were treated with vehicle solution.", "TT cell proliferation was measured as absorbance at 490 nM of each well.", "Data from six individual experiments were evaluated independently with eight replicates expressed as the mean±SEM percent cell proliferation inhibition versus untreated control cells P<0.05 and *P<0.01 vs. control.", "FIG.", "7: Effect of SSTR2 selective antagonist on T[cell [3H]thy incorporation and cell proliferation during treatment with SSTR2 agonist.", "Upper panel: Cells were incubated in 24-well plates for 48 hours in a culture medium supplemented with 100 nM Compound 1 or Compound 2, with or without Compound 6 (10−7 M).", "Control wells were treated with vehicle solution.", "[3H]thy incorporation was measured as radioactivity in TCA-precipitated material.", "Data from six individual experiments were evaluated independently with four replicates expressed as the mean±SEM percent [3H]thy incorporation inhibition versus untreated control cells P<0.05 and *P<0.01 vs. control.", "Lower panel: Cells were incubated in 96-well plates for 48 hours in a culture medium supplemented with 10−7 M Compound 1 or Compound 2, with or without Compound 6 (10−7 M).", "Control wells were treated with vehicle solution.", "TT cell proliferation was measured as absorbance at 490 nM of each well.", "Data from six individual experiments were evaluated independently with eight replicates expressed as the mean±SEM percent cell proliferation inhibition versus untreated control cells P<0.05 and *P<0.01 vs. control.", "FIG.", "8: Effect of SSTR5 selective agonist on TT cell [3H]thy incorporation and cell proliferation during treatment with SSTR2 selective agonist.", "Upper panel: Cells were incubated in 24-well plates for 48 hours in a culture medium supplemented with Compound 2 (10−7 M) without or with increasing concentrations of Compound 5 (10−9, 10−8, 10−7, and 10−6 M), or with Compound 5 (10−7 M) with or without decreasing concentrations of Compound 2 (10−6, 10−7, 10 −8, and 10−9 M).", "Control wells were treated with vehicle solution.", "[3H]thy incorporation was measured as radioactivity in TCA-precipitated material.", "Data from six individual experiments were evaluated independently with four replicates expressed as the mean±SEM percent [3H]thy incorporation inhibition versus untreated control cells P<0.05 and *P<0.01 vs. control.", "Lower panel: Cells were incubated in 96-well plates for 48 hours in a culture medium supplemented with Compound 2 (10−7 M) without or with increasing concentrations of Compound 5 (10−9, 10−8, 10−7, and 10−6 M), or with Compound 5 (10−7 M) with or without decreasing concentrations of Compound 2 (10−6, 10−7, 10−8, and 10−9 M).", "Control wells were treated with vehicle solution and TT cell proliferation was measured as absorbance at 490 nM of each well.", "Data from six individual experiments were evaluated independently with eight replicates expressed as the mean±SEM percent cell proliferation inhibition versus untreated control cells P<0.05 and **P<0.01 vs. control.", "DETAILED DESCRIPTION OF THE INVENTION It is believed that one skilled in the art can, based on the description herein, utilise the present invention to its fullest extent.", "The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.", "Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.", "Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference, each in its entirety.", "Various somatostatin receptors (SSTR's) have been isolated, e.g., SSTR-1, SSTR-2, SSTR-3, SSTR-4, and SSTR-5.Thus, a somatostatin agonist may be one or more of an SSTR-1 agonist, SSTR-2 agonist, SSTR-3 agonist, SSTR-4 agonist or a SSTR-5 agonist.", "What is meant by a somatostatin type-2 receptor agonist (i.e., SSTR-2 agonist) is a compound which (1) has a high binding affinity (e.g., Ki of less than 100 nM or preferably less than 10 nm or less than 1 nM) for SSTR-2 (e.g., as defined by the receptor binding assay described below) and (2) decreases the rate of proliferation of medullary thyroid carcinoma cells (e.g., as shown by the biological assay described below).", "What is meant by a somatostatin type-2 receptor selective agonist is a somatostatin type-2 receptor agonist which has a higher binding affinity (i.e., lower Ki) for SSTR-2 than for SSTR-5.What is meant by a somatostatin type-5 receptor agonist is a somatostatin agonist which (I) has a high binding affinity (e.g., Ki of less than 100 nM or preferably less than 10 nm or less than 1 nM) for SSTR-5 (e.g., as defined by the receptor binding assay described below) and (2) attenuates the SSTR-2 agonist-induced decrease in the rate of proliferation of medullary thyroid carcinoma cells (e.g., as shown by the biological assay described below).", "What is meant by a somatostatin type-5 receptor selective agonist is a somatostatin type-5 receptor agonist which has a higher binding affinity (i.e., lower Ki) for SSTR-5 than for SSTR-2.In one embodiment, the SSTR-2 agonist is also a SSTR-2 selective agonist.", "In another embodiment, the SSTR-2 selective agonist has a Ki value for SSTR-5 that is at least 2 times (e.g., at least 5 times or at least 10 times) higher than it has for the SSTR-2 receptor (e.g., as defined by the receptor binding assay described below).", "Examples of SSTR-2 agonists which may be used to practice the present invention include, but are not limited to: D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, (Compound 1), cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe], (Compound 2), 4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2, (Compound 3), and 4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2, (Compound 4).", "An example of SSTR-5 agonist which may be used to practice the present invention includes, but is not limited to: D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2.", "(Compound 5).", "Further examples of somatostatin agonists are those covered by formulae or those specifically recited in the publications set forth below, all of which are hereby incorporated by reference.", "EP Application No.", "P5 164 EU (Inventor: G. Keri); Van Binst, G. et al.", "Peptide Research 5:8 (1992); Horvath, A. et al.", "Abstract, “Conformations of Somatostatin Analogs Having Antitumor Activity”, 22nd European peptide Symposium, Sep. 13-19, 1992, Interlaken, Switzerland; PCT Application No.", "WO 91/09056 (1991); EP Application No.", "0 363 589 A2 (1990); U.S. Pat.", "No.", "4,904,642 (1990); U.S. Pat.", "No.", "4,871,717 (1989); U.S. Pat.", "No.", "4,853,371 (1989); U.S. Pat.", "No.", "4,725,577 (1988); U.S. Pat.", "No.", "4,684,620 (1987); U.S. Pat.", "No.", "4,650,787 (1987); U.S. Pat.", "No.", "4,603,120 (1986); U.S. Pat.", "No.", "4,585,755 (1986); EP Application No.", "0 203 031 A2 (1986); U.S. Pat.", "No.", "4,522,813 (1985); U.S. Pat.", "No.", "4,486,415 (1984); U.S. Pat.", "No.", "4,485,101 (1984); U.S. Pat.", "No.", "4,435,385 (1984); U.S. Pat.", "No.", "4,395,403 (1983); U.S. Pat.", "No.", "4,369,179 (1983); U.S. Pat.", "No.", "4,360,516 (1982); U.S. Pat.", "No.", "4,358,439 (1982); U.S. Pat.", "No.", "4,328,214 (1982); U.S. Pat.", "No.", "4,316,890 (1982); U.S. Pat.", "No.", "4,310,518 (1982); U.S. Pat.", "No.", "4,291,022 (1981); U.S. Pat.", "No.", "4,238,481 (1980); U.S. Pat.", "No.", "4,235,886 (1980); U.S. Pat.", "No.", "4,224,199 (1980); U.S. Pat.", "No.", "4,211,693 (1980); U.S. Pat.", "No.", "4,190,648 (1980); U.S. Pat.", "No.", "4,146,612 (1979); U.S. Pat.", "No.", "4,133,782 (1979); U.S. Pat.", "No.", "5,506,339 (1996); U.S. Pat.", "No.", "4,261,885 (1981); U.S. Pat.", "No.", "4,728,638 (1988); U.S. Pat.", "No.", "4,282,143 (1981); U.S. Pat.", "No.", "4,215,039 (1980); U.S. Pat.", "No.", "4,209,426 (1980); U.S. Pat.", "No.", "4,190,575 (1980); EP Patent No.", "0 389 180 (1990); EP Application No.", "0 505 680 (1982); EP Application No.", "0 083 305 (1982); EP Application No.", "0 030 920 (1980); PCT Application No.", "WO 88/05052 (1988); PCT Application No.", "WO 90/12811 (1990); PCT Application No.", "WO 97/01579 (1997); PCT Application No.", "WO 91/18016 (1991); U.K.", "Application No.", "GB 2,095,261 (1981); and French Application No.", "FR 2,522,655 (1983).", "Note that for all somatostatin agonists described herein, each amino acid residue represents the structure of —NH—C(R)H—CO—, in which R is the side chain (e.g., CH3 for Ala).", "Lines between amino acid residues represent peptide bonds which join the amino acids.", "Also, where the amino acid residue is optically active, it is the L-form configuration that is intended unless D-form is expressly designated.", "For clarity, disulfide bonds (e.g., disulfide bridge) which exist between two free thiols of Cys residues are not shown.", "Abbreviations of the common amino acids are in accordance with IUPAC-IUB recommendations.", "Synthesis of Somatostatin Agonists The methods for synthesizing somatostatin agonists is well documented and are within the ability of a person of ordinary skill in the art.", "Synthesis of short amino acid sequences is well established in the peptide art.", "For example, synthesis of H-D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2, described above, can be achieved by following the protocol set forth in Example I of European Patent Application 0 395 417 A1.The synthesis of somatostatin agonists with a substituted N-terminus can be achieved, for example, by following the protocol set forth in WO 88/02756, European Patent Application No.", "0 329 295, and PCT Publication No.", "WO 94/04752.Some of the compounds of the instant invention can have at least one asymmetric center.", "Additional asymmetric centers may be present on the molecule depending upon the nature of the various substituents on the molecule.", "Each such asymmetric center will produce two optical isomers and it is intended that all such optical isomers, as separated, pure or partially purified optical isomers, racemic mixtures or diastereomeric mixtures thereof, are included within the scope of the instant invention.", "The compounds of the instant invention generally can be isolated in the form of their pharmaceutically acceptable acid addition salts, such as the salts derived from using inorganic and organic acids.", "Examples of such acids are hydrochloric, nitric, sulfuric, phosphoric, formic, acetic, trifluoroacetic, propionic, maleic, succinic, D-tartaric, L-tartaric, malonic, methane sulfonic and the like.", "In addition, certain compounds containing an acidic function such as a carboxy can be isolated in the form of their inorganic salt in which the counter-ion can be selected from sodium, potassium, lithium, calcium, magnesium and the like, as well as from organic bases.", "The pharmaceutically acceptable salts can be formed by taking about 1 equivalent of a SSTR-2 agonist, e.g., compound 1, and contacting it with about 1 equivalent or more of the appropriate corresponding acid of the salt which is desired.", "Work-up and isolation of the resulting salt is well-known to those of ordinary skill in the art.", "The compounds of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual or topical routes of administration and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.", "Accordingly, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one SSTR-2 agonist in association with a pharmaceutically acceptable carrier.", "Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.", "In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch.", "Such dosage forms can also comprise, as is normal practice, additional substances other than such inert diluents, e.g., lubricating agents such as magnesium stearate.", "In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.", "Tablets and pills can additionally be prepared with enteric coatings.", "Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water.", "Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.", "Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.", "Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.", "Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents.", "They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.", "They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.", "Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as coca butter or a suppository wax.", "Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.", "In general, an effective dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained.", "The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment, all of which are within the realm of knowledge of one of ordinary skill in the art.", "Generally, dosage levels of between 0.0001 to 100 mg/kg of body weight daily are administered to humans and other animals, e.g., mammals.", "A preferred dosage range is 0.01 to 10.0 mg/kg of body weight daily, which can be administered as a single dose or divided into multiple doses.", "Materials and Methods RT-PCR analysis was used to demonstrate that all five SSTR subtype mRNA's are expressed in a human MTC cell line, TT.", "The ability of SS analogues with differing affinity and specificity for SSTR2 and 5 subtypes to influence TT cell proliferative activity may be assessed by considering [3H]thy incorporation, considered an indirect measure of DNA synthetic activity, and number of viable cells.", "All SSTR2 preferential agonists were able to significantly suppress TT cell number at concentrations ranging from 10−9 M to 10−6 M. Compound 3 and Compound 4 significantly (p<0.05) reduced [3H]thy incorporation at 10−9 M but not at 10−8 M and 10−7 M, when their maximal inhibitory effect on cell number was apparent.", "Each SSTR2 compound tested showed a trend for decreased efficacy with increasing concentration, however, bell-shaped response curves are common for SS.", "The inhibition of [3H]thy incorporation and TT cell number by Compound 1 and Compound 2 at 10−7 M was not associated with any cytotoxic action, as demonstrated by Trypan Blue staining.", "Moreover, this effect was completely counteracted by cotreatment of TT cells with Compound 6, a selective SSTR2 antagonist.", "Taken together, these results indicate that SS analogues with preferential selectivity for SSTR2 inhibit TT cell proliferation by specifically interacting with SSTR2.TT Cell Line Culture The TT cell line was obtained from the American Type Culture Collection (ATCC, Manassas, Va., USA).", "The TT cell line consists of aneuploid transformed CT-producing parafollicular cells which are characterised by the presence of a TGC to TGG mutation (Cys to Trp) at exon 11 codon 634 in the RET protooncogene (Cooley L D, et al., 1995 Cancer Genet Cytogenet 80: 138-149), a characteristic that we confirmed in the cell line we worked with.", "Moreover, TT cells display an impaired expression of the tumor suppressor gene p53 (Velasco J A, et al., 1997 Int J Cancer 73: 449-455).", "Immunohistochemistry studies demonstrated that TT cells express CT and CT receptor (Frendo J L, et al., 1994 FEBS Lett.", "342: 214-216), carcino-embrionic antigen (CEA), SS, neurotensin, gastrin-releasing peptide (GRP), Leu- and Met-enkephalin, parathyroid hormone releasing peptide (PTHrp), Chromogranin A, SP-I, Synaptophysin, Neuron-specific enolase (NSE), 1,25-dihydroxyvitamin D3 receptor, Thyrosin hydroxylase, α-Tubulin, and Cytocheratin (Zabel M, et al., 1995 Histochemical J.", "27: 859-868).", "TT cells secrete a significant amount of CT and respond to changes in ionised calcium levels (Zabel M, et al., 1992 Histochemistry 102: 323-327).", "Thus the TT cell line is suitable for studies on parafollicular function and responses to endocrine and pharmacological stimuli.", "Cells were maintained in Ham's Nutrient Mixture F12 with Glutamine (EuroClone Ltd, Torquay, UK), supplemented with 10% fetal bovine serum (FBS, Life Technologies, Milano, Italy), 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 100 μg/mL amphotericin (EuroClone Ltd, Torquay, UK) at 37° C. in a humidified atmosphere of 5% CO2 and 95% air.", "Isolation of RNA Total RNA was extracted from subconfluent TT cells by using TRIZOL (Life Technologies, Milano, Italy).", "The TRIZOL protocol is a modification of the guanidinium/phenyl extraction.", "Briefly, the cultured cell media was aspirated and the cells washed with 1×PBS.", "The TRIZOL reagent was added and cells lysed at room temperature for 10 min.", "Chloroform was added to the TRIZOL/cell lysate mixture, and left to stand for 2-3 min, and then centrifuged 12000×g for 15 min.", "The aqueous layer was removed from the centrifuged mixture.", "Isopropanol was added to precipitate the RNA, the pellet collected, washed with 75% ethanol and dried in air.", "Total RNA was resuspended in diethylpyrocarbonate-treated (DEPC) water and quantified using UV spectrophotometry at 260 nM.", "To prevent DNA contamination, RNA was treated with ribonuclease-free deoxyribonuclease (Promega, Milano, Italy).", "RT-PCR Using a first strand complementary DNA (cDNA) synthesis kit (SuperScript Preamplification System for First Strand cDNA Synthesis, Life Technologies, Milano, Italy), 1 μg total RNA was reverse transcribed according to the manufacturer's protocol.", "RT mix in PCR tubes was covered with 50 μl light white mineral oil (Sigma-Aldrich Corp. Milano, Italy); the RT was carried out in the Minicycler (MJ Research Inc., Watertown, Mass., USA) using a program with the following parameters: 10 min at 70° C., 1 min at 4° C., 5 min at 4° C. After supplementing with SuperScript II, the reaction was completed at 42° C. for 50 min then at 70° for 15 min.", "Samples were digested with RNAse H (Promega, Milano, Italy) at 37° C. for 20 min, and then stored at −20° C. until the first PCR.", "The cDNA (I μl of RT reaction) was then amplified by PCR with 1 U Taq DNA polymerase (Life Technologies, Milano, Italy), in the conditions recommended by suppliers in a 50-μl reaction mixture.", "After initial denaturation at 95° C. for 5 min, PCR reactions were carried out using the oligonucleotide primers and the conditions listed in Table 1, describing the size of expected fragments.", "PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide (ETB) staining.", "To assure that no contamination occurred during the course of the RT-PCR procedure, two kinds of negative control were prepared.", "The first negative control was made by omitting the total RNA in the RT.", "The second was prepared by replacing the cDNA mix with water in the PCR reaction.", "The PCR was considered useful only if no band was observed in the negative control lanes on a 2% agarose gel.", "Each PCR product was subjected to restriction enzyme digestion and analysed on 2% agarose gel to further confirm the correct identification of the amplicons.", "SSTR Selective Agonists and Antagonists SS analogues used in this study and their respective affinities to the different SSTR's are listed in Table 2.Each compound, provided by Biomeasure Incorporated (Milford, Mass., USA), was resuspended in 0.01 N acetic acid containing 0.1% bovine serum albumin (BSA) in order to provide uniform solubility and prevent non-specific binding to the various preparation surfaces.", "Specificity and selectivity of the analogues were determined by Radioligand Binding Assay on CHO-K1 cells stably transfected with each of the SSTR subtypes, as follows.", "The complete coding sequences of genomic fragments of the SSTR 1, 2, 3, and 4 genes and a cDNA clone for SSTR 5 were subcloned into the mammalian expression vector pCMV (Life Technologies, Milano, Italy).", "Clonal cell lines stably expressing SSTR's 1-5 were obtained by transfection into CHO-K1 cells (ATCC, Manassas, Va., USA) using the calcium phosphate co-precipitation method (Davis L, et al., 1994 In: Basic methods in Molecular Biology, 2nd edition, Appleton & Lange, Norwalk, Conn., USA: 611-646).", "The plasmid pRSV-neo (ATCC) was included as a selectable marker.", "Clonal cell lines were selected in RPMI 1640 media containing 0.5 mg/ml of G418 (Life Technologies, Milano, Italy), ring cloned, and expanded into culture.", "Membranes for in vitro receptor binding assays were obtained by homogenizing the CHO-K1 cells expressing the SSTR's subtypes in ice-cold 50 mM Tris-HCl and centrifuging twice at 39000 g (10 min), with an intermediate resuspension in fresh buffer.", "The final pellets were resuspended in 10 mM Tris-HCl for assay.", "For the SSTR 1, 3, 4, and 5 assays, aliquots of the membrane preparations were incubated 90 min.", "at 25° C. with 0.05 nM [125I-Tyr11]SS-14 in 50 mM HEPES (pH 7.4) containing 10 mg/ml BSA, 5 mM MgCl2, 200 KIU/ml Trasylol, 0.02 mg/ml bacitracin, and 0.02 mg/ml pbenylmethylsuphonyl fluoride.", "The final assay volume was 0.3 ml.", "For the SSTR 2 assay, 0.05 nM [125I]MK-678 was employed as the radioligand and the incubation time was 90 min at 25° C. The incubations were terminated by rapid filtration through GF/C filters (pre-soaked in 0.3% polyethylenimine) using a Brandel filtration manifold.", "Each tube and filter were then washed three times with 5 ml aliquots of ice-cold buffer.", "Specific binding was defined as the total radioligand bound minus that bound in the presence of 1000 nM SS-14 for SSTR 1, 3, 4, and 5, or 1000 nM MK-678 for SSTR2.Biological Activity Evaluation Biological activity of SSTR selective agonists and antagonists was evaluated by the calcium mobilization assay in CHO-K1 cells expressing the human SSTR2 or SSTR5.The cells were harvested by incubating in a 0.3% EDTA/phosphate buffered saline solution (25° C.), and washed twice by centrifugation.", "The washed cells were resuspended in Hank's-buffered saline solution (HBSS) for loading of the fluorescent Ca2+ indicator Fura-2AM.", "Cell suspensions (approximately 106 cells/ml) were incubated with 2 mM Fura-2AM for 30 min at 25° C. Unloaded Fura-2AM was removed by centrifugation twice in HBBS, and the final suspensions were transferred to a spectrofluorometer (Hitachi F-2000) equipped with a magnetic stirring mechanism and a temperature-regulated cuvette holder.", "After equilibration to 37° C., the SS analogues were added for measurement of intracellular Ca2+ mobilization.", "The excitation and emission wavelengths were 340 and 510 nm, respectively.", "In the SSTR2 expressing cells (FIG.", "1), Compound 2 and Compound 1 stimulated significant intracellular Ca2+ mobilization (indicated as the ratio between stimulated and basal value), whereas Compound 6 did not, at the concentrations tested.", "In addition, Compound 4 and Compound 3 were also highly potent in stimulating Ca mobilization.", "In the SSTR5 expressing cells (FIG.", "2), Compound 5 and Compound 1 stimulated significant intracellular Ca2+ mobilization, whereas Compound 6 displayed slight agonist activity in the range of 300 to 1000 nM.", "In the SSTR2 expressing cells (FIG.", "3), Compound 6 inhibited SS-induced intracellular Ca2+ mobilization in SSTR2 expressing cells in a dose dependent manner with complete suppression of SS action at about 10−7 M. Therefore the evaluation of intracellular Ca2+ mobilization demonstrated that the biological activity of each of the various analogues was in keeping with its receptor binding profile.", "DNA Synthesis The effects of SSTR selective agonists and antagonists on TT cell DNA synthesis were assessed by determining the rate of [3H]thymidine ([3H]thy) incorporation, as previously described (Davis L, et al., 1994 In: Basic methods in Molecular Biology, 2nd edition, Appleton & Lange, Norwalk, Conn., USA: 611-646, degli Uberti E C, et al., 1991 J Clin Endocrinol Metab 72: 1364-1371).", "TT cells were plated in 24-multiwell plates (105 cells/well) and incubated for 48 hours in a medium supplemented with 10% FBS in the presence of [3H]thy (1.5 μCi/mL; 87 Ci/mmol) with or without each SS analogue at concentrations ranging from 10−6 to 10−9 M. Treatments were renewed by adding fresh analogues to the wells after the first 24 h of incubation, without removing the medium.", "After incubation, the cells were washed three times with ice-cold PBS and twice with 10% ice-cold trichloroacetic acid (TCA).", "TCA-precipitated material was solubilized in 500 μL 0.2 mol/L sodium hydroxide and 0.1% SDS.", "Cell-associated radioactivity was then counted in a scintillation spectrometer.", "Results (counts per min per well) were obtained by determining the mean value of at least six experiments in quadruplicate.", "The viability of TT cells in control and treated cultures was evaluated by Trypan blue staining both after 24 and 48 hours, and the number of viable cells was always 85-95%.", "Cell Proliferation The effects of SSTR selective agonists and antagonists on TT cell proliferation were assessed by the CELLTITER 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Milano, Italy), a colorimetric method for determining the number of viable cells in proliferation assays.", "The assay contains solutions of a tetrazolium compound (Owen's reagent; MTS) and an electron coupling reagent (phenazine methosulphate; PMS).", "MTS is bioreduced by cells into a formazan that is soluble in tissue culture medium.", "The absorbance of the formazan at 490 nm can be measured directly from 96 well assay plates (Zatelli M C, et al., 2000 J Clin Endocrinol Metab 85: 847-852; Cory A H, et al., 1991 Cancer Coniniun 3: 207-212).", "The conversion of MTS into the aqueous soluble formazan is accomplished by dehydrogenase enzymes found in metabolically active cells.", "The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.", "Briefly, TT cells were plated in 96-multiwell plates (2×104 cells/well) and incubated for 48 hours in a medium supplemented with 10% FBS in the presence or absence of each SS analogue at concentrations ranging from 10−6 to 10−9 M. Treatments were renewed by adding fresh analogues to the wells after the first 24 hours of incubation.", "At the end of the incubation period, 20 μl of a combined MTS/PMS solution were added to each well with a repeating pipette, and the plates were incubated for an additional 4 hours at 37° C. in a humidified 5% CO2 atmosphere.", "The absorbance at 490 nm was then recorded using an ELISA plate reader (EASIA Reader, Medgenix, Camarillo, Calif.).", "Results (absorbance at 490 nm) were obtained by determining the mean value of at least six experiments in eight replicates.", "Results SSTR Expression in the Human MTC Cell Line TT To understand the individual role of SSTR2 and SSTR5 subtypes in controlling parafollicular C cell proliferation, we evaluated whether TT cells express SSTR's that could mediate a potential response to selective compounds for individual SSTR subtypes.", "To address this question, we isolated total RNA from cultured TT cells and performed RT-PCR reactions in the conditions described in Material and Methods.", "Integrity of cDNA was assured by the presence of the GAPDH signal.", "The absence of genomic DNA contamination in the cDNA samples was assessed by the lack of any amplification in a PCR reaction using non-reverse transcribed samples.", "Positive amplification of SSTR1, 2, 3, 4, and 5 was found in the examined cell line (FIG.", "4), demonstrating that these receptors are expressed in human MTC cell-line TT.", "The demonstration that the TT cell line stably expresses SSTR subtypes made this cellular model system suitable for evaluating the action of receptor-selective SS analogues.", "Effect of Selective SS Analogues on TT cell [3H]thy Incorporation [3H]Thy incorporation values obtained with 10−9 to 10−6 M concentrations of SSTR2 preferential agonists (Compound 1, Compound 2, Compound 3, and Compound 4), SSTR5 preferential agonist (Compound 5) and SSTR2 preferential antagonist (Compound 6) are presented in FIG.", "5.As indicated, Compound 2 significantly suppressed [3H]thy incorporation by 58-23% at concentrations ranging from 10−9 to 10−7 M. Compound 1 significantly suppressed [3H]thy incorporation by 41-21% at concentrations ranging from 10−9 to 10−6 M. [3H]thy incorporation was also significantly reduced by Compound 4 (−13%, p<0.05) and Compound 3 (−17%, p<0.05) at 10−9 M. In contrast, Compound 5 significantly increased [3H]thy incorporation in TT cells by 80-175%.", "The SSTR2 selective antagonist, Compound 6, did not alter TT cell [3H]thy incorporation compared with untreated control cells.", "Effect of Selective SS Analogues on TT Cell Proliferation To examine in more detail the activity of SS-analogues on TT cell growth, their effect on viable cell number was also analyzed.", "The effects of SSTR2 preferential agonists, an SSTR5 preferential agonist and an SSTR2 preferential antagonist on viable TT cell number at concentrations ranging from 10−9 to 10−6 M are represented in FIG.", "6.As indicated, all SSTR2 preferential compounds significantly inhibited cell proliferation when compared with untreated control cells at each concentration tested.", "The selective SSTR5 agonist, Compound 5, produced a slight increase of TT cell proliferation-(up to 11% at 10−8 M), however this did not represent a statistical difference from the untreated control cells.", "The selective SSTR2 antagonist, Compound 6, did not appear to affect TT cell growth at the concentrations tested.", "Selective SSTR2 Antagonist Counteracts the Effects of SSTR2-Preferential Agonist To further clarify whether SSTR2 is specifically involved in mediating the antiproliferative activity of SSTR2 preferential agonists, [3H]thy incorporation and cell growth were evaluated in TT cells exposed for 48 hours to Compound 1 and Compound 2, each either alone (at 10−7 M) or in combination with Compound 6, a selective SSTR2 antagonist at equimolar concentration (10−7 M).", "The inhibition of [3H]thy incorporation induced by both Compound 1 and Compound 2 was suppressed by cotreatment of TT cells with Compound 6 (FIG.", "7, upper panel).", "TT cell proliferation inhibition induced by Compound 1 was significantly reduced from 46% to 10% by cotreatment with Compound 6.Further, Compound 6 appeared to block completely the antiproliferative activity of Compound 2 (FIG.", "7, lower panel).", "Thus, the specific involvement of SSTR2 in mediating the inhibitory effect of an SSTR2 agonist on TT cell proliferation is clearly demonstrated.", "Effect of Combination of a Preferential SSTR2 Agonist with a Preferential SSTR5 Agonist on [3H]Thy Incorporation and Cell Proliferation In order to analyze the effects of an SSTR2 and an SSTR5 agonist in combination, TT cell [3H]thy incorporation and proliferation were examined testing each of Compound 2 and Compound 5 at 10−7 M in combination with increasing doses (from 10−9 M to 10−6 M) of the other compound.", "The results are summarized in FIG.", "8.Increasing concentrations of the SSTR5 agonist (10−9 M to 10=6 M) dose-dependently prevented the suppression of TT cell [3H]thy incorporation (FIG.", "8, upper panel) and proliferation (FIG.", "8, lower panel) produced by the SSTR2 agonist (10−7 M).", "These data demonstrate an antagonism between SSTR5 and SSTR2 mediated effects on proliferation.", "It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention defined by the appended claims.", "Other aspects, advantages, and modifications are within the claims.", "TABLE 1 Primers and PCR conditions for SSRs amplification Expected SSR Primers Denaturation Annealing Extension Cycles fragment (bp) 1 For: 5′-AGCCGGTTGACTATTACGCC-3′ 95° C., 30″ 60° C., 1′ 72° C., 2′ 45 334 Rev: 5′-GCTCTCACTTCTACCATTGTC-3′ 2 For: 5′-GGTGAAGTCCTCTGGAATCC-3′ 95° C., 30″ 63° C., 1′ 72° C., 2′ 45 461 Rev: 5′-CCATTGCCAGTAGACAGAGC-3′ 3 For: 5′-TCATCTGCCTCTGCTACCTG-3′ 95° C., 30″ 65° C., 1′ 72° C., 2′ 45 221 Rev: 5′-GAGCCCAAAGAAGGCAGGCT-3′ 4 For: 5′-CGGCAGTCTTCGTGGTCTAC-3′ 94° C., 30″ 63° C., 1′ 72° C., 2′ 45 247 Rev: 5′-GCATCAAGGTCGGTCACGAC-3′ 5 For: 5′-AACACGCTGGTCATCTACGTGGT-3′ 94° C., 1′ 60° C., 1′ 72° C., 1′15″ 40 211 Rev: 5′-AGACACTGGTGAACTGGTTGAC-3′ GAPDH For: 5′-ATGACCCCTTCATTGACCTC-3′ 95° C., 30″ 60° C., 1′ 72° C., 2″ 40 820 Rev: 5′-AAGTGGTCGTTGAGGGCAAT-3′ TABLE 2 Human somatostatin receptor subtype specificity (IC50, nM) Receptor Subtype Compound 1 2 3 4 5 1 2129 0.75 98 1826 12.7 2 1000 0.34 412 1000 213.5 3 5210 0.35 215 7537 11.2 4 6016 0.19 26.8 3897 9.8 5 1152 166 1000 1618 2.4 6 2757 6.4 44 423 86.5 (antagonist) Subtype affinity was determined by radioligand membrane receptor binding assays in Chinese hamster ovary cells expressing human SSR2 gene or SSR5 cDNA" ] ]	Patent_10469835
[ [ "Projector", "Projector (1) enabling a remarkable flexibility of use and a marked reduction of the dimensions thereof, comprising: a main body (2) housing light generating means and heat disposal means; and a secondary body (3), removably associable to said main body (2), housing means (40) for holding and sliding a film (F), means (30) for cooling said film (F) and an optical projection group (4)," ], [ "1.A projector (1), comprising: a main body (2), housing light generating means and heat disposal means; and a secondary body (3), removably associable to said main body (2), housing means (40) for holding and sliding a film (F) and an optical projection group (4), characterised in that said secondary body houses means (30) for cooling said film (F) having a fan (12), located at the bottom edge of the film (F), so as to generate a cooling flow tangential to both the surfaces of said film (f).", "2.The projector (1) according to claim 1, wherein said means (40) for holding and sliding a film (F) comprises a film wind shaft (41) about which a film (F) initially wound on a start spool (7a) is wound, and a film rewind shaft (42), about which a take-up spool (7b) gradually winds up as the projection goes on, each shaft (41, 42) having a length such as to house films of different sizes.", "3.The projector (1) according to claim 1, wherein said shafts (41, 42) comprise, at a respective end, presser members 43, each kept pressed onto the top edge of the film (F) by a respective spring (44), the presser members keeping the film (F) in the projection position.", "4.The projector (1) according to claim 2, wherein the means (40) for holding and sliding comprises auxiliary motor drives (8) connected to said spools (7a, 7b) for the rewinding and the unwinding of the film.", "5.The projector (1) according to claim 2, wherein the means (40) for holding and sliding comprises a step-by-step motor drive (9) connected to a motor wheel (10) cooperating with an idle wheel (17), both gripping the film (F) for the sliding thereof, the step-by-step motor drive (9) being apt to determine the shifting rate and accuracy of the film (F).", "6.The projector (1) according to claim 1, comprising a first plate (13) constrainable to the secondary body (3) located parallel to the film section at a projection area (6) and orthogonal to an axis of projection (A-A) so as to form, when said first plate (13) is constrained to said secondary body (3), a wall thereof facing the projection optics (4) and so as to face a projection lamp inserted in the main body (2) when the secondary body (3) is constrained to said main body; said first plate being apt, when constrained to the secondary body (3), to keep the film (F) pressed between two idle wheels (15) and the corresponding wheels (10) and (17) of the secondary body (3) and it being apt, when released from said secondary body, to ease the mounting and the dismounting of the film (F) therefrom according to a direction substantially corresponding to the axis of projection (A-A).", "7.The projector (1) according to claim 1, wherein to the secondary body (3) a first plate (13), interchangeable with a second plate (20) bearing a device (21) for applying marks (33), is constrainable.", "8.Projector (1) according to claim 6, wherein the first plate (13) has an opening (14) which, when said first plate is constrained to the secondary body (3), is centered with respect to the axis of projection (A-A) and has dimensions not smaller than those of the frames impressed onto the film (F); onto the surface of said first plate (13) facing the inside of the secondary body (3) the at least two idle wheels (15) and a film pressure frame (18) are constrained.", "9.The projector (1) according to claim 8, wherein said idle wheels (15) have the respective axes of rotation mutually parallel and positioned orthogonal to the sliding direction of the film (F); the idle wheels (15) are constrained to the first plate (13) in a position such as to be capable of cooperating, when said first plate is constrained to the secondary body (3), with the corresponding wheels (10, 17); the idle wheels (15) have supports (16) thereof constrained to the first plate (13) in an elastically yielding way, said elastic compliance being apt to ensure steadiness of pressure between said idle wheels (15) and the wheels (10, 17), said pressure being apt to ensure the normal unwinding of the film (6) between the idle wheels (15) and the wheels (10, 17); all the wheels (15, 17, 10) are coated with friction material, e.g.", "gummy material.", "10.The projector (1) according to claim 9, wherein the film pressure frame (18) is substantially made of a frame enclosing an opening (19), true with respect to the axis of projection (A-A) and having dimensions not smaller than those of the frames impressed on the film (F); said frame (18) being constrained to the first plate (13) by elastic supports apt to give to said film pressure frame (18) a second compliance, it also of elastic type.", "11.The projector (1) according to claim 10, wherein the edges of the frame making the film pressure frame (18) have the surface thereof facing the inside of the secondary body (3) provided with a plurality of inserts (35) of a non-stick material, e.g.", "PTFE.", "12.The projector (1) according to claim 11, wherein the bottom edge of the film pressure frame (18) is provided with a plurality of nicks (31) oriented as the axes of rotation of the idle wheels (15); jointly to the scanty thickness of the film pressure frame (18), said nicks being apt to ease the air flow for cooling the film (6) outletted from the fan (12); the reduced thickness of the film pressure frame (18) being also apt to offer the least drag to the cooling air flow outletted from said fan.", "13.The projector (1) according to claim 6, wherein the first plate (13) is provided with fastening members (28) apt to enable the mere upturning thereof with respect to the sliding plane of the film (F) as well as the complete releasing of said first plate from the secondary body (3).", "14.The projector (1) according to claim 7, wherein said second plate (20) has shapes and dimensions identical to those of the first plate (13) and it is provided with identical idle wheels (15) and identical fastening means (28); the device (21) for applying marks (33) is apt to the applying, onto the film (6), of a plurality of said marks, said applying being viable solely when the second plate (20) is constrained to the secondary body (3) in lieu of the first plate (13); said marks being apt to form finder members aimed at restoring the synchronism of the frames in the case wherein the auxiliary drive motors, the motor drive (9) and/or the wheels (15, 17, 10) cause deviations in the sliding of the film (F).", "15.The projector (1) according to claim 14, wherein the applying device (21) comprises a shelf (29) to which there are constrained: one idle spool (22) housing a strip (23) which supports a plurality of the marks (33); a plurality of gripping members (25) apt to grip and to shift the strip (23); shifting means (26) apt to shift said gripping members, said shifting being connected to at least one of the idle wheels (15), said connection being apt, when the second plate (20) is constrained to the secondary body (3), to drive the motion of the motor wheel (10) to the strip (23) eliminating the need of a dedicated motor drive.", "16.The projector (1) according to claim 14, wherein onto the second plate (20), in lieu of the film pressure frame (18), there is constrained a plate (27) of the film (F) lacking in any opening; said pressure plate being constrained to the second plate (20) in an elastically yielding way; said pressure plate having a surface destined to contact said film coated with a layer of a non-stick material, e.g.", "PTFE." ], [ "The present invention refers to a projector, in particular of the type apt to project images, slides and films onto large-sized surfaces, and optionally in Multivision, i.e.", "with an array of projectors synchronised thereamong.", "The use of specific image projectors onto large-sized surfaces in environments destined to entertainment like, e.g., discotheques and theatres has been widespread for quite some time.", "The surfaces onto which the images are projected do not necessarily require a specific preparation, optionally being the common walls of the projection premises, and hence exhibiting also all the uneven spots typical of a common wall: e.g.", "recesses, juts, openings, doors and windows, etc.", "Of late years, this specific type of projections also involves outdoor environments, like fronts of monuments and palaces.", "The effect resulting from these projections is particularly enthralling and spectacular, capable of engendering/creating peculiar and evocative atmospheres.", "This type of projections is best used during great multimedia shows and events involving vast spaces with monumental architectural surfaces, like e.g., the front of a castle or of a cathedral.", "Another putative field of use of said projections is that of scenic performances.", "Such surfaces constitute an exceptional screen for the projected images, requiring however projectors of ever-increasing candlepower.", "The art has already studied and implemented projection units gradually improved with regard to the image rendering, the candlepower and, accordingly, the width of the outdoor surfaces to be used as projection screens.", "However, in the projectors used to date the employ of strong brilliances, i.e.", "of lamps characterised by an elevated candlepower and a greater concentration of the light beam, results in a disproportionate increase of the dimensions, of the weight and of the overall complexity thereof.", "This increase in turn causes non-negligible drawbacks.", "In fact, considerable dimensions and weight, of up and beyond the 100 kg, can limit the use and the applications of such known-type projectors, which further require complex maintenance, lamp replacement and film loading interventions.", "Apart from this major drawback, the known projectors are affected by further problems limiting the diffusion thereof.", "In fact, the known-type projectors generally employ special and large-sized (up to 24×24 cm) films, which are hard to find on the market and to expose and develop, as few laboratories are adequately equipped therefor.", "Above all in the case of the multivision, often the use of non-standard projectors, hence hardly found on the market, is required.", "Another drawback of the known-type projectors lies in a possible imperfect positioning of the film due to slippage and deviations of the rotation rate of the take-up wheels with respect thereto.", "The imperfect positioning of the film is particularly serious when, due to specific requirements, a multivision with the concomitant use of a plurality of projectors is carried out.", "In these cases, the required overlapping and/or concomitance of the images is compromised, with the decaying or the undoing of the desired visual effects.", "To date, the attempts to obviate this drawback resulted in the adoption of punched strips or the like onto the film edges.", "This punching entails high costs and requires the employ of skilled personnel.", "Another drawback in the known-type projectors lies in the difficult cooling of the film subjected to ever-increasing candlepower, with temperatures onto the film that can even exceed the 200° C. U.S. Pat.", "No.", "3,712,725 A (Eckerdt) discloses an adapter for enabling a slide tray projector to project film strip but intended to allow the view of images in proximity of the projector, thus no requiring special features for cooling the film strip.", "DE 421,372 C (Rohde) discloses a film projector wherein an air flow is driven through the assembly supporting the film but not directly on the film, thus only allowing the heat removal from said assembly which is directly illuminated by the light source.", "In both cases, means for holding the film are positioned at least in part outside the adapter or the assembly as well, to maintain the film (when not illuminated) in a cooler environment.", "The technical problem underlying the present invention is that of providing a projector allowing to obviate the problem of the dimensions and of the excessive weight, allowing a greater effectiveness and an effective adaptation to elevated candlepowers, at the same time providing a substantial portability of the film to be projected in a specific housing to be directly mounted on the projector body.", "This problem is solved by a projector, comprising: a main body, housing light generating means and heat disposal means; and a secondary body, removably associable to said main body, housing means for holding and sliding a film and an optical projection group, characterised in that said secondary body houses means for cooling said film having a fan, located at the bottom edge of the film, so as to generate a cooling flow tangential to both the surfaces of said film.", "The main advantage of the projector according to the present invention lies in subdividing the componentry so as to make more flexible and practical any function associated thereto, concomitantly remarkably reducing the bulks and the weights thereof.", "As it will be apparent hereinafter, preferred embodiments of the projector enable to satisfy further needs.", "In particular, a first object of the present projector is to implement a projector, employable in multivision, provided with high candlepower and yet being highly compact and weight-light, capable of using standard-type films, e.g.", "multisize 6×7 cm and 6×6 cm, or 70 mm and 24×36 ones with a simple adapter, with no need to add perforations to an unpunched film.", "Other objects of the present invention are to manufacture a projector having a quick and reliable system for loading the film and also to provide the former with an effective forced cooling of the latter.", "A further object of the present invention is to install an active frame synchronization system with a limited maximum error, e.g.", "of +/−0.05 mm per 100 frames in the 6×7 cm format.", "The present invention will hereinafter be described according to a preferred embodiment thereof, given by way of example and without limitative purposes, making reference to the attached drawings, wherein: FIG.", "1 is a perspective view of a projector according to the invention, comprising a main body and a secondary body thereof; FIG.", "2 is a perspective view of a detail associated to said secondary body; FIG.", "3 is a perspective view of said secondary body incorporating the detail of FIG.", "2; FIG.", "4 is a perspective view of a specific attachment associated to said secondary body; and FIG.", "5 is a perspective view of said secondary body incorporating the specific attachment of FIG.", "4.With reference to FIG.", "1, the reference number 1 generally indicates a projector of the high candlepower type, i.e.", "destined to project onto wide surfaces, and apt to operate in multivision, which comprises a reciprocally detachable main body 2 and a secondary body 3.In particular, the main body 2 is boxed and parallelepiped-shaped, having a substantially horizontal development with a rear end, and a front end to which said body 3 is removably associated.", "The main body 2 houses light generating means, i.e.", "lamps having the candlepower required for the projection over wide surfaces, a primary optical group which concentrates the light onto the film to be projected, and heat disposal means, i.e.", "suitable fans for forcedly cool the inside thereof, with vents and fins formed thereon.", "The body 2 will be made of a material having an elevated thermal conductivity, like anodised aluminium.", "The secondary body 3 (FIG.", "3) can be mounted onto the main body 2 by lever couplings 28.The former substantially forms the driving device of the film to be projected and it comprises an optical projection group 4, a control button strip 5 and means, generally indicated with 40, for holding and sliding a film.", "Such means 40 hold a film F stretched on a projection area 6, the primary optical group of the main body 2 and the optical projection group 4 resting thereon.", "At each side of this area 6, the holding and sliding means 40 comprises on the one hand a wind shaft 41, onto which a film, initially wound on a start spool 7a, is mounted, and on the other hand a rewind shaft 42, about which a take-up spool 7b gradually winds up as the projection goes on.", "Each shaft 41, 42 has a length such as to house films of different sizes, and moreover comprises, at a respective end, presser members 43, each kept pressed onto the top edge of the film F by a respective spring 44.The presser members keep the film F in the projection position.", "Said means 40 further comprises electrical and mechanical members apt to shift the film F stretched between the two spools 7a, 7b.", "These members are power-supplied via a dedicated outlet 32.Among said electrical and mechanical members there are indicated two ratiomotors 8, each dedicated to said two spools 7a, 7b, a first motor drive 9 apt to generate a step-by-step motion, connected to a motor wheel 10 which is located adjacent to the rewind shaft 42.The secondary body 3 further comprises means 30 for cooling the film, having a second motor drive 11 that actuates a radial-flow fan 12.In particular, the fan 12 is located at the bottom edge of the film in said projection area 6 and it generates a cooling flow tangential to both the surfaces of the film F, as well as substantially perpendicular to the path thereof.", "At the back of the secondary body 3, i.e.", "at the portion thereof destined to contact the main body 2, there is located a first supporting plate 13.The latter, when it is completely constrained to the former, is parallel to the sliding direction of the film F and orthogonal to an axis of projection A-A.", "Said plate 13 has an opening 14 centered with respect to the axis of projection A-A.", "Said opening has dimensions not smaller than those of the frames impressed onto the film F. With reference to FIGS.", "2 and 3, onto the surface of the first plate 13 facing the inside of the secondary body 3, two idle wheels 15 are constrained whose axis of rotation is orthogonal to the motion plane of the film.", "Each of said idle wheels is located in substantial correspondence to the vertical edges of the opening 14 and is constrained to a support 16, which in turn is elastically constrained to the first plate 13.Each of the two idle wheels 15, when said first plate is constrained to the secondary body 3, corresponds to and is into contact with two wheels constrained to the secondary body 3.The first wheel 17 is idle as well, whereas the second wheel 10 has already been defined as motor wheel, connected to the first motor drive 9.Onto the surface of the first supporting plate 13 facing the inside of the secondary body 3 also a frame-shaped film pressure frame 18 is elastically constrained.", "The latter encloses an opening 19 having dimensions not smaller than those of the frames impressed onto the film F. The edges of said film pressure frame 18, which face said film F when the first plate 13 is constrained to the secondary body 3, are provided with a plurality of inserts of a non-stick material, e.g., PTFE.", "Moreover, the bottom edge thereof is also provided with a plurality of nicks 31 oriented as the axes of rotation of the idle wheels 15.The radial-flow fan 12 is integrally constrained to the secondary body 3 and it is positioned below the film 6 with the air flow outlet mouth substantially centered with respect to the thickness of the latter, in order to ensure the tangentiality of the cooling flow.", "A second supporting plate 20 (FIG.", "4), identical in shape and dimensions to the first supporting plate 13, is interchangeable with the latter onto the secondary body 3.Said second plate is identical to the first plate also with regard to the idle wheels 15 and the related elastically constrained supports 16.The second plate 20 bears a device 21 for applying marks 33 made of a shelf 29 which supports one idle spool 22.About the latter, a strip 23 is wound which supports a plurality of said marks.", "The latter, of the label type, have dimensions of about 3×10 mm.", "An infrared sensor 34 is fork-shaped in order to embrace the edge of the film 6 along which the marks 33 are applied.", "Said strip, in the unwinding thereof outside of said idle spool, is first inserted into an application station 24, and then into gripping members 25 made of three mutually cooperating and substantially cylinder-shaped members.", "The axis of rotation of one of such gripping members is connected to shifting means 26 which are cooperating with one of the idle wheels 15, said shifting means made, in the illustrated device, of a pair of pulleys.", "To the shelf 29 also said gripping and shifting members are constrained.", "The second plate 20 bears, onto the surface thereof facing the inside of the secondary body 3, a presser plate 27 made of a frame which, unlike the film pressure frame 18, is blind, having no opening.", "The presser plate 27 is elastically constrained to the second plate 20, analogously to the constraint of the film pressure frame 18 to the first plate 13.The application station 24 is constrained to the presser plate 27.Both the first plate 13 and the second plate 20 are provided with identical fastening members 28, i.e.", "the said lever couplings, to the secondary body 3.Hereinafter, the operation of the projector according to the present embodiment will be described, with particular reference to the secondary body 3.When a projection is to be carried out, the secondary body 3 is parted from the main body 2 in order to insert the film F, and the first plate 13 is released from said secondary body making use of the fastening members 28.Instead of completely releasing the first plate 13 from the secondary body 3, the former can even be only partially released, overturning it sideways as it is shown in FIG.", "3.Thus, the insertion of the film is particularly easy, taking place assembling the start spool 7a at the wind shaft 41 and hooking the free end of the film F to the rewind shaft 42.Upon installing the film F, said first plate 13 is reapplied to the secondary body 3.Thus, the section of the former stretched onto the projection area 6 between the two spools 7a, 7b, is imprisoned between the two idle wheels 15 and the corresponding wheels 17, idle, and 10, motor.", "By virtue of the elastic constraint provided to the supports 16 of the idle wheels 15, the latter always exert a steady pressure onto the corresponding rolls 17 and 10, ensuring at all times a steady pressure onto said film.", "To the rear surface of the film F the film pressure frame 18 adheres, by virtue of its constraint elastically yielding with respect to the first plate 13, factually concurring to keep the former perfectly planar at the axis of projection A-A.", "Then, the secondary body 3 can again be constrained to the main body 2 and, acting onto the control button strip 5, the film F is made to slide.", "Thus, the selecting of all the desired positions or frames and the storing thereof by means of the electronic management logic incorporated in the secondary body 3 of the projector 1 can be carried out.", "Such storing is apt to involve even more than 100 positions.", "Then, the projection can be started and, at any command given by the control button strip 5 or even by a remote computer, the take-up device subject matter of the present invention will exactly position the film F onto the pre-stored spot.", "In order to attain the total safety and accuracy for said film, and all the more so when plural projectors 1 are used, prior to the projection the following is carried out.", "Upon mounting the film F into the secondary body 3 as abovedescribed, and releasing the main body 2 from the secondary body 3, the first plate 13 is removed from the latter.", "The film F is installed in the latter, and, in lieu of said first plate 13, the second plate 20 is constrained thereto, always making use of the fastening members 28.By acting onto the control button strip 5, the film F will be set into motion and the marks 33 onto the strip 23 will quickly and automatically be applied by the applying device 21 over the entire length of said film at the rate of about one mark per each frame.", "Said applying device places said marks onto the 3 mm edge of the frame, thereby without interfering in any way with the projection and without weakening the film F. The rotary motion of the motor wheel 10 is contact-driven to the corresponding idle wheel 15, and therefrom, by the shifting members 26 and the gripping members 25, to the strip 23 unwinding from the idle spool 22.Passing the strip 23 into the application station 24, the desired applying of the marks 33 onto the film F is attained.", "Apparently, the applying of said marks could abide to a laxer rule, also generating rather uneven spacing between the marks, entailing no drawbacks.", "In fact, the electronic management logic incorporated in the secondary body 3 will automatically provide synchronicity during the projection.", "The marks 33 applied onto the film F are detected by the infrared sensor 34.Said sensor 34 is apt to operate by dimming, i.e.", "each mark dims the infrared beam, whereas the film F is transparent thereto.", "The reading by a suitable sensor (not shown) of a single mark positioned onto the film edge facing that onto which the plurality of said marks is applied provides the reference for the starting of the wheel and the synchronising of the film F. Thus, the restoring of the position register after having turned off the projector, and even in the case of a blackout, is ensured.", "The applying of the marks 33 can also take place with the secondary body 3 released from the main body 2, as said secondary body is provided with its own outlet 32.Upon having applied said marks onto the film F, the second plate 20 is removed from the secondary body 3, in its place the first plate 13 is installed, and the projector 1 is ready to be used.", "The sole step-by-step motor drive 9 is the main motor drive, and it determines the shifting rate and accuracy of the film F, whereas the two ratiomotors 8 serve as auxiliary motor drives and control the rewinding and the unwinding of the spools 7.An advantage provided by the structure subject-matter of the present invention lies in the certainty that the sliding of the film F is free from position deviations even at the hundredth frame.", "The synchronism thereby attained compensates not merely the physiological mutual film/wheels sliding, but also any sliding due to mechanical problems, wear, dust, and film aging; The active synchronisation system of the present invention positions the frames with a maximum error of +/−0.05 mm/100 (6×7 cm) frames.", "Another advantage lies in the optimal cooling of the film whose two sides are concomitantly enveloped by the air flow outletted from the fan 12 positioned quite near and bottomwise to the film, in a position substantially true to the thickness thereof.", "There ensues the viability of a lamp exhibiting, frame size being equal, a higher candlepower, and, hence, of the projector.", "Said advantage fosters other advantages, like the greater power and useful projection size, the higher brightness of the projected image and lower risks of heat-induced film deterioration, though with an extremely compact and handy projector.", "A further advantage of the projector subject-matter of the present invention lies in the optional applying, automatically and within a few seconds, marks apt to synchronise the frames, with a wholly variable spacing between the same marks, as detectable by the electronic management logic incorporated into the secondary body.", "This applying is attained using the second plate 20 interchangeable with the first plate 13.The presence of said marks onto the film F ensures, during the projection, a total accuracy in the shifting of said film regardless of any factor apt to alter the normal unwinding thereof.", "To the abovedescribed projector a person skilled in the art, in order to satisfy further and contingent needs, may effect several further modifications and variants, all however falling within the protective scope of the present invention, as defined by the appended claims." ] ]	Patent_10469885
[ [ "Methods and devices for treating and processing data", "A data processing unit (VPU) is described, having a field of clocked logic cells (PAEs) which is operable in different configuration states and a clock preselecting means for preselecting logic cell clocking.", "It is provided here that the clock preselecting means is designed in such a way that, depending on the state, a first clock is preselected at least at a first cell (PAE) and an additional clock is preselected at least at an additional cell." ], [ "1.A data processing unit (VPU) comprising a field of clocked logic cells (PAES) which is operable in different configuration states and a clock preselecting means for preselecting logic cell clocking, wherein the clock preselecting means is designed in such a way that, depending on the state, a first clock is preselected at least at a first cell (PAE) and an additional clock is preselected at least at an additional cell.", "2.The data processing unit as recited in the preceding claim, wherein the clock preselecting means is designed in such a way that it receives the setpoint clock for at least one first cell from a unit preselecting configuration states.", "3.The data processing unit as recited in the preceding claim, wherein the unit preselecting the configuration states includes a compiling unit and/or a cell configuration preselecting unit.", "4.The data processing unit as recited in one of the preceding claims, wherein the clock preselecting means is designed in such a way that it receives the setpoint clock from a logic cell.", "5.The data processing unit as recited in one of the preceding claims, wherein the clock preselecting means includes at least one central clock preselecting unit and at least one local clock generating unit for generating the local clock from the preselected central clock, in particular one time synchronizing clock generating unit per cell.", "6.The data processing unit as recited in one of the preceding claims, wherein at least a portion of the logic cells includes at last one ALU and/or is formed by such.", "7.The data processing unit as recited in one of the preceding claims, wherein at least one memory and/or register is assigned to at least a portion of the logic cells.", "8.The data processing unit as recited in one of the preceding claims, wherein a plurality of identical logic cells is provided.", "9.The data processing unit as recited in one of the preceding claims, wherein all logic cells are identical.", "10.A method for operating a field of clocked logic cells which are settable into different configuration states, wherein a first state is determined, at least temporarily, for at least one first cell, a clock which is to be assigned to the first cell being determined dependent on the first state and the cell being operated using this clock; a second state is determined for at least one additional cell, a second clock which is to be assigned to the second cell being determined dependent on the second state and the second cell being operated using this second clock, which differs from the first clock.", "11.The method as recited in the preceding claim, wherein the clock is preselected for at least one first cell, either together with or determined by its configuration.", "12.The method as recited in one of the preceding method claims, where a group of cells is jointly configured for executing algebraic and/or other operations which require a different number of clock cycles and where at least one cell, executing an operation which requires fewer clock cells than that operation requiring the most clock cycles within the group, is clocked slower than at least one other cell.", "13.The method as recited in one of the preceding method claims, wherein cells of at least one group are configured for sequential data processing.", "14.The method as recited in one of the preceding method claims, wherein the field in at least two cell groups is configured for executing at least two different tasks which are assigned different priorities, and the cell group appointed for executing the task having the lower priority is clocked using a lower clock frequency.", "15.The method as recited in one of the preceding method claims, wherein the condition of a voltage supply source and/or a temperature is determined and the cell clock is determined as a function of the voltage and/or temperature condition thus determined.", "16.A method for operating a system of reconfigurable logic elements which are operable in different configurations, wherein a still permissible frequency, in particular the still executable maximum frequency, is determined for a plurality of possible configurations, in particular such that are simultaneously configured into the field, a plurality of cells being operated using this frequency; the plurality of cells is larger than the plurality which is assigned for executing this so-called slowest configuration and the plurality is able, in particular, to include the entire field of the configurable elements.", "17.The method for operating a system of reconfigurable logic elements which are operable in different configurations, wherein configurations are selected such that, by taking signal transmissions via bus lines into account, maximum frequencies are maintained during transmissions via bus systems." ], [ "The present invention relates to the definition of the species in the main claim and its object is to achieve an optimization of the hardware used in data processing.", "Data processing requires the optimization of the available resources, as well as the power consumption of the circuits involved in data processing.", "This is the case in particular when reconfigurable processors are used.", "Reconfigurable architecture is defined herein as modules (VPU) having a configurable function and/or interconnection, in particular integrated modules having a plurality of unidimensionally or multidimensionally positioned arithmetic and/or logic and/or analog and/or storage and/or internally/externally interconnecting modules, which are connected to one another either directly or via a bus system.", "These generic modules include in particular systolic arrays, neural networks, multiprocessor systems, processors having a plurality of arithmetic units and/or logic cells and/or communication/peripheral cells (IO), interconnecting and networking modules such as crossbar switches, as well as known modules of the type FPGA, DPGA, Chameleon, XPUTER, etc.", "Reference is also made in particular in this context to the following patents and patent applications of the same applicant: P 44 16 881.0-53, DE 197 81 412.3, DE 197 81 483.2, DE 196 54 846.2-53, DE 196 54 593.5-53, DE 197 04 044.6-53, DE 198 80 129.7, DE 198 61 088.2-53, DE 199 80 312.9, PCT/DE 00/01869, DE 100 36 627.9-33, DE 100 28 397.7, DE 101 10 530.4, DE 101 11 014.6, PCT/EP 00/10516, EP 01 102 674.7, PCT/DE 97/02949(PACT02/PCT), PCT/DE 97/02998 (PACT04/PCT), PCT/DE 97/02999 (PACT05/PCT), PCT/DE 98/00334 (PACT08/PCT), PCT/DE 99/00504 (PACT10b/PCT), PCT/DE 99/00505 (PACT10c/PCT), DE 101 39 170.6 (PACT11), DE 101 42 903.7 (PACT11a), DE 101 44 732.9 (PACT11b), DE 101 45 792.8, (PACT11c), DE 101 54 260.7 (PACT11d), DE 102 07 225.6 (PACT11e), PCT/DE 00/01869 (PACT13/PCT), DE 101 42 904.5 (PACT21), DE 101 44 733.7 (PACT21a), DE 101 54 259.3 (PACT21b), DE 102 07 226.4 (PACT21c), PCT/DE 00/01869 (PACT13/PCT), DE 101 10 530.4 (PACT18), DE 101 11 014.6 (PACT18a), DE 101 46 132.1 (PACT18II), DE 102 02 044.2 (PACT19), DE 102 02 175.9 (PACT19a), DE 101 35 210.7 (PACT25), DE 101 35 211.5 (PACT25a), DE 101 42 231.8 (PACT25II), (PACT25b).", "The entire contents of these documents are hereby included for the purpose of disclosure.", "The above-mentioned architecture is used as an example to illustrate the invention and is referred to hereinafter as VPU.", "The architecture includes an arbitrary number of arithmetic, logic (including memory) and/or memory cells and/or networking cells and/or communication/peripheral (IO) cells (PAEs—Processing Array Elements) which may be positioned to form a unidimensional or multidimensional matrix (PA); the matrix may have different cells of any desired configuration.", "Bus systems are also understood here as cells.", "A configuration unit (CT) which affects the interconnection and function of the PA through configuration is assigned to the entire matrix or parts thereof.", "The configuration of a VPU is determined by writing configuration words into configuration registers.", "Each configuration word determines a subfunction.", "PAEs may require a plurality of configuration words for their configuration, e.g., one/or more words for the interconnection of the PAE, one/or more words for the clock determination and one/or more words for the selection of an ALU function, etc.", "It is known that a processor which is operated at a higher clock frequency requires more power.", "Thus, the cooling requirements in modern processors increase substantially as the clock frequency increases.", "Moreover, additional power must be supplied which is critical in mobile applications in particular.", "To determine the clock frequency for a microprocessor based on the state is known.", "Such technologies are known from the area of mobile computers.", "However, problems arise in the overall speed with which certain applications are carried out.", "The object of the present invention is to provide a novel method for commercial application.", "The achievement of the object is claimed independently.", "The present invention thus shows how the power consumption may be reduced and/or optimized in VPU technology.", "As far as different methods are addressed in the following, it should be pointed out that they provide advantages, either individually or in combination.", "In a data processing unit (VPU) according to a first essential aspect of the present invention, by using a field of clocked logic cells (PAEs)-which is operable in different configuration states and a clock preselecting means for preselecting logic cell clocking, the clock preselecting means is designed in such a way that, depending on the state, a first clock is preselected at least at a first cell (PAE) and an additional clock is preselected at least at an additional cell (PAE).", "It is therefore suggested to operate different cells using different clocking.", "As a rule, the additional clock corresponds to the first clock; the former is thus situated in a defined phase angle to the latter.", "In order to achieve optimum data processing results, in particular with regard to the required data processing time, as well as the power consumption of the entire data processing unit, it is suggested that clocking takes place depending on the state, which means that no clock is preselected jointly for all cells based on a certain state, but rather an appropriate clock is assigned to each cell based on the state.", "Furthermore, it is suggested that the clocking be designed to be totally configurable, so that one calibration (configuration) mutually influences the clocking of the total number of cells.", "It is possible and desired that the clock preselecting means is designed in such a way that it receives the setpoint clock for at least one first cell from a unit which preselects configuration states.", "This makes it possible to select the clocking of the cell based on its configuration as soon as this configuration is determined.", "This has the advantage that configuration may take place free of problems.", "The unit preselecting configuration states may be a compiling unit, which means that required or desired clocking of the cell is already determined during the compiling of the program.", "If the compiling unit preselects the configuration states, then the cell configuration preselecting unit may convey clocking for cell configuration to a cell to be configured.", "This is advantageous since it is possible to merely add clock-determining information to the configuration word or the configuration instruction with which the configuration of a cell is determined, without additional measures being required such as the implementation of clock-assigning buses which separately transmit the clock-determining signals, or the like; it should be noted that this is possible in principle.", "It may also be provided that the clock preselecting means is designed in such a way that it receives the setpoint clock or a clock-influencing signal from one of the other logic cells, in particular a configurable logic cell.", "This is particularly advantageous if a first logic cell awaits an input signal from an external unit and not until arrival of such signals are the cells to be activated which process subsequently arriving signals.", "This makes it possible to implement a logic field sleeping mode in which only one or a plurality of cells are activated, if necessary, on a very low level, i.e., very slow clocking, and the remaining field is clocked extremely slowly.", "The clock frequencies required in the remaining field are dependent on physically necessary clocking which is required for the preservation of memory contents or the like.", "It is also advantageous to receive a clock-influencing signal from another logic cell if, using one logic cell, one or a series of a plurality of different arithmetic and/or logical operations may be executed which, at least in part, require a different number of clock cycles, but this may not be determined in advance by the compiling unit.", "Also in such a case, the subsequent cells do not need to be operated at a high clock frequency if they are appropriately clocked down by corresponding signals which indicate the state of the cell participating in a processing sequence.", "In a preferred variant, the clock preselecting means includes a central clock preselecting unit, e.g., a central clock generator, whose clock is transmitted to the individual cells via a clock line, as well as a local clock-generating unit for generating a local clock from and/or in response to the central clock transmitted via the clock line.", "In a possible embodiment, clocking of the central clock preselecting unit may be set or influenced by a configuration.", "The local clock-generating unit is preferably implemented by using a frequency divider and/or a frequency multiplier, and the frequency divider ratio is preferably determined by the preselections of the clock preselecting means according to the clock determination based on the state.", "In a preferred variant, the logic cells or at least some of the logic cells include at least one ALU and/or are formed by such.", "It is possible and preferred if some of the logic cells contain at least one memory unit and/or register unit which may be assigned to the remaining logic cells.", "In particular, this unit may be provided for data to be processed and/or for configurations of the cell.", "It is possible that a plurality of logic cells are identical and are operated using different clocking corresponding to their particular configuration.", "It is possible in particular that all logic cells are identical.", "A patent is also claimed for a method for operating a field of clocked logic cells which may be set into different configuration states, a first state being determined, at least temporarily, for at least one first cell, a clock which is to be assigned to the first cell being determined dependent on the first state and the cell being operated using this clock; a second state is determined for at least one additional cell, a second clock which is to be assigned to the second cell being determined dependent on the second state and the second cell being operated using the second clock which differs from the first clock.", "As mentioned above, clocking may be preselected together with the configuration.", "The state is then the configuration state and/or is at least determined by it.", "In known and configurable logic cells, cells are typically combined in groups for executing complex operations.", "If individual cells execute suboperations which run in fewer clock cycles as is the case with those cells which are [engaged] in particularly drawn-out suboperations of the complex total operations executed by the group, it is preferred if these cells are operated at different clock rates, namely in such a way that the cells for less complex operations, thus operations which run in fewer clock cycles, are clocked slower than the other cells; it is preferred in particular if the cells of one group are clocked collectively in such a way that the number of blank cycles within the group is minimized.", "An alternative and/or an addition to this lies in the fact of temporarily changing the use of cells burdened with less complex tasks for a certain number of clock cycles, thus changing the use during a fixed number of clock cycles.", "In particular, the case may occur that the maximum clock cycle rate of PAEs and/or PAE groups is limited by their function and in particular by their interconnection.", "The propagation time of signals via bus systems plays an increasingly frequency-limiting role, in particular in advancing semiconductor technology.", "Henceforth, the method allows slower clocking of such PAEs and/or PAE groups, while other PAEs and/or PAE groups operate at a different and, if needed, higher frequency.", "It is suggested in a simplified embodiment to make the clock rate of the entire reconfigurable module (VPU) dependent on the maximum clock rate of the slowest PAE and/or PAE group.", "In other words, the central clock preselecting unit may be configured in such a way that the highest mutual operating clock of all PAEs and/or PAE groups (in other words the smallest common denominator of all maximum clock rates) is globally generated for all PAEs.", "The above-described method is particularly advantageous if the cells of the group process data sequentially, i.e., the result determined by one cell is passed on to one or multiple cells which are subsequently processing data.", "It should be noted that in addition to prioritizing tasks within the cell field for clock preselection, the condition of a power source may also be included in cell clocking determination.", "Clocking may be reduced overall in the case of a drop in supply voltage, in particular in mobile applications.", "Clocking-down for preventing an overtemperature by responding to a temperature sensor signal or the like is equally possible.", "It is also possible for the user to preset the clock preselection.", "Different parameters may jointly establish the clock-determining state.", "It was mentioned above that it is possible to perform time division multiplexing for carrying out multiple configurations on the same PAE.", "A preferred and enhanced design makes particularly resource-saving time division multiplexing for carrying out multiple configurations on the same PAE possible; the design may have advantages independently from the different clocking of individual cells, e.g., when latencies have to be taken into account which occur in the signal transmission of digital data via a bus, such as configuration data, data to be processed, or the like.", "These problems are particularly serious when reconfigurable modules, having reconfigurable units which are located in part comparatively far apart from one another, are to be operated at high clock frequencies.", "The problem arises here that due to the special configuration of VPUs a plurality of arbitrary PAEs is connected via buses and considerable data transmission traffic exists via the buses.", "The switching frequency of transistors is expected to further increase in modern and above all in future silicon technologies, while the signal transmission via buses is to increasingly become a performance-limiting factor.", "It is therefore suggested to decouple the data rate or frequency on the buses vis-a-vis the operating frequency of the data-processing PAEs.", "A particularly simple embodiment, preferred for simple implementations, operates in such a way that the clock rate of a VPU is only globally settable.", "In other words, a settable clock may be preselected for all PAEs or it may be configured by a higher-level configuration unit (CT).", "All parameters which have an effect on clocking determine this one global clock.", "Such parameters may be, for example, a temperature determination, a power reserve measurement of batteries, etc.", "A determining parameter may be in particular the maximum operating frequency of the slowest configuration which results as a function of a PAE configuration or a configuration of a group of PAEs.", "Since different configurations may include different numbers of PAEs over stretches of bus connections of different lengths, it was realized, in particular in bus signal transmission-limiting applications, that configurations may have different maximum frequencies.", "Configurations may have different maximum frequencies, as is known from FPGAs, for example, which depend on the particular function of the PAEs and in particular on the lengths of bus connections.", "The slowest configuration then ensures that the proper operation of this configuration is also ensured, and simultaneously reduces the power demand of all other configurations which is advantageous in particular when different portions of the data processing such as through the other configurations, which would possibly run at higher clock frequencies, are not needed prior to the slowest configuration.", "Also in cases where it must be absolutely ensured that proper operation takes place, the possibly only negligible performance loss occurring by clocking-down other configurations, which could run faster per se, is often acceptable.", "In an optimized embodiment, the frequency is only adapted to the configurations which are currently carried out on a VPU, in other words, the global frequency may be reset/reconfigured with each configuration.", "In an enhanced embodiment, the clock may then be configured globally, as well as, as described above, individually for each configurable element.", "It should be noted that different variants are possible, individually or in combination.", "In order to show a detailed example, it is assumed in the following, without this necessarily being the case, that the clock may be controlled individually in each PAE.", "This offers the following possibilities, for example: a) Controlled Enabling and Disabling of the Clock It is preferred that the processing clock of PAEs is disabled, i.e., the PAEs operate only in case of need; clock enabling, i.e., activating the PAE, may take place, for example, under at least one of the following conditions, namely when valid data is present; when the result of the previous computation is approved; due to one or more trigger signals; due to an expected or valid timing mark, compare DE 101 10 530.4 (PACT18).", "In order to cause clock enabling, each individual condition may be used either individually or in combination with other conditions, clock enabling being computed based on the logical combination of conditions.", "It should be noted that it is possible to put the PAEs into a power-saving operating mode while a clock is disabled, for example, through additionally partly switched-off or reduced power supply, or, should it be necessary because of other reasons, through extremely reduced sleeping clocks.", "b) Different Frequencies per PAE Technologies for controlling sequences in VPUs are known from PCT/DE 97/02949 (PACT02/PCT), PCT/DE 97/02998 (PACT04/PCT), and PCT/DE 00/01869 (PACT13/PCT).", "Special sequencers (SWTs) which control a large number of PAEs and which are responsible for their (re)configuration are configured in PCT/DE 97/02998 (PACT04/PCT).", "The (re)configuration is controlled by using status signals which are generated by the PAEs (triggers) and passed on to the SWTs, namely in that the SWT responds to the triggers, making the particular continuation of a sequence dependent on the triggers.", "A small memory for their configuration is assigned to each individual PAE in PCT/DE 97/02949 (PACT02/PCT).", "A sequencer passes through the memory and addresses the individual configurations.", "The sequencer is controlled by triggers and/or by the status of its PAE (into which it may be integrated, for example).", "During data processing, it is now possible that different sequencers in different PAEs have to carry out a different number of operations per transmitted data packet (compare DE 101 39 170.6 (PACT11), DE 101 42 903.7 (PACT11a), DE 101 44 732.9 (PACT11b), DE 101 45 792.8 (PACT11c), DE 101 54 260.7 (PACT11d), DE 102 07 225.6 (PACT11e), PCT/DE 00/01869 (PACT13/PCT)).", "This is described using a configuration as an example in which 3 sequencers are involved in processing a data packet, requiring a different number of operations for data packet processing.", "Example: Sequencer 1 (Seq1) requires 10 operations for processing a data packet, Sequencer 2 (Seq2) requires 5 operations for processing a data packet, Sequencer 3 (Seq3) requires 20 operations for processing a data packet.", "In order to obtain an optimum operation/power consumption ratio, the individual sequencers would have to be clocked as follows: Fmax=FSeq2/4=FSeq1/2=FSeq3 or at a maximum operating frequency of, for example, 100 MHz: FSeq1=50 MHz, FSeq2=25 MHz, FSeq3=100 MHz.", "It is suggested in particular to use different clock sources for each PAE and/or group of PAEs.", "For example, different techniques may be used for this purpose, either individually or jointly: 1) Clock dividers, individually programmable per PAE, which enable an individually configurable divider ratio based on one or more mutual base clocks.", "2) Clock multipliers (PLLs), individually programmable per PAE, which enable an individually configurable divider ratio based on one or more mutual base clocks.", "3) Deriving the particular PAE clock from the data stream of the particular data to be processed, e.g., by oversampling.", "An exemplary embodiment having different algorithms is illustrated in FIG.", "1.c) Configuration Clock Optimization of the power consumption is also favored in that the circuit components, necessary for executing a configuration, are clocked selectively, i.e., it is suggested to clock each PAE addressed and/or to completely disable the clock of those circuit components necessary for executing a configuration or a reconfiguration when no configuration or reconfiguration is being executed and/or to use static registers.", "In particular embodiments, the operating frequency of the PAEs or groups of PAEs may be made dependent on different and/or additional factors.", "The following is listed below as an example: 1.Temperature Measurement If the operating temperature reaches certain threshold values, the operating clock is reduced correspondingly.", "The reduction may take place selectively by initially operating those PAEs on a lower clock which represent the most irrelevant performance loss.", "In a particularly preferred embodiment, multiple temperature measurements may be performed in different regions and clocking may be adapted locally.", "2.Buffer Filling Levels IO-FIFOs (input-output-first-in-first-out-circuits) which decouple peripheral data transmissions from data processing within a VPU are known from DE 102 06 653.1 (PACT15), DE 102 07 224.8 (PACT15a), (PACT15b).", "One buffer for input data (input buffer) and/or one buffer for output data (output buffer) may be implemented, for example.", "A particularly efficient variable for determining the clock frequency may, for example, be determined from the filling level of the particular data buffers.", "The following effects and measures may occur, for example: a) An input buffer is largely full and/or the filling level rises abruptly: Clocking increase to accelerate processing.", "b) An input buffer is largely empty and/or the filling level drops abruptly: Clocking decrease to decelerate processing.", "c) An output buffer is largely full and/or the filling level rises abruptly: Clocking decrease to decelerate processing.", "d) An output buffer is largely empty and/or the filling level drops abruptly: Clocking increase to accelerate processing.", "Depending on the application and the system, suitable combinations may be implemented accordingly.", "It should be pointed out that such a clock frequency determination is implementable if a filling level determination means for a buffer, in particular an input and/or output buffer, alternatively also an intermediate buffer within a VPU array, is provided and if this filling level determination means is connected to a clock preselecting means for preselecting logic cell clocking so that this clock preselecting means is able to change the logic cell clocking in response to the buffer filling level.", "3.Battery Charge State It is imperative to be careful with the power supply, e.g., a battery, for mobile units.", "Depending on the power reserve, which may be determined based on the existing methods according to the related art, the frequency of PAEs and/or groups of PAEs is determined and is reduced in particular when the power reserve is low.", "Besides or in addition to optimizing data processing clocking it is also possible to accomplish an optimization of the data transmission with respect to the relationship between data transmission and data processing.", "In a particular embodiment, the clock controls of PAEs described may be enhanced in such a way that, by using a sequencer-like activation and a suitable register set, for example, multiple, preferably different, configuration words may be executed successively in multiple clocks.", "A sequencer, sequentially processing a number of configuration inputs, may be additionally assigned to the configuration registers and/or to a configuration memory which is possibly also decoupled and implemented separately (compare DE 102 06 653.1 (PACT15), DE 102 07 224.8 (PACT15a, PACT15b).", "The sequencer may be designed as a microcontroller.", "In particular, the sequencer may be programmable/configurable in its function such as Altera's module EPS448 (ALTERA Data Book 1993).", "Possible embodiments of such PAEs are described, for example, in the following patent applications which are included in their entirety for the purpose of disclosure: PCT/DE 97/02949 (PACT02/PCT), PCT/DE 97/02998 (PACT04/PCT), PCT/DE 00/01869 (PACT13/PCT), DE 101 10 530.4 (PACT18), DE 102 06 653.1 (PACT15), DE 102 07 224.8 (PACT15a, PACT 15b).", "For the following, it is initially assumed that multiple configuration words are combined into one configuration (PAKKEDCONF) and are configured on a PAE.", "The PACKEDCONF is processed in such a way that the individual configuration words are executed in chronological succession.", "The data exchange and/or status exchange between the individual timed configurations takes place via a suitable data feedback in the PAES; for example by using a suitable register set and/or another data exchange and/or status exchange means such as suitable memories and the like.", "This method allows a different timing for PAEs and bus systems.", "While PAEs process data at very high clock rates, for example, operands and/or results are transmitted via a bus at only a fraction of the clock rate of the PAES.", "The transmission time via a bus may be correspondingly longer.", "It is preferred if not only the PAEs or other logic units in a configurable and/or reconfigurable module are clockable at a different rate, but also if different clocking is provided for parts of a bus system.", "It is possible here to provide multiple buses in parallel whose speed is clocked differently, i.e., a bus which is clocked particularly high for providing a high-performance connection, parallel to a bus which is clocked lower for providing a power-saving connection.", "The connection clocked high may be used when longer signal paths have to be compensated, or when PAES, positioned close together, operate at a high frequency and therefore also have to exchange data at a high frequency in order to provide a good transmission here over short distances in which the latency plays a minor role at best.", "Therefore, it is suggested in a possible embodiment that a number of PAEs, positioned together locally and combined in a group, operate at a high frequency and possibly also sequentially and that local and correspondingly short bus systems are clocked high corresponding to the data processing rate of the group, while the bus systems, inputting the operands and outputting the results, have slower clock and data transmission rates.", "For the purpose of optimizing the power consumption, it would be alternatively possible to implement slow clocking and to supply data at a high speed, e.g., when a large quantity of inflowing data may be processed with only a minor operational effort, thus at low clock rates.", "In addition to the possibility of providing bus systems which are clocked using different frequencies it is also possible to provide multiple bus systems which are operable independently from one another and to then apply the PAEs in a multiplex-like manner as required.", "This alone makes it possible to operate reconfigurable modules particularly efficiently in resource multiplexing, independently from the still existing possibility of differently clocking different bus systems or different bus system parts.", "It is possible here to assign different configurations to different resources according to different multiplexing methods.", "According to PCT/DE 00/01869 (PACT13/PCT), a group of PAEs may be designed as a processor in particular.", "In the following embodiments, for example, different configurations are assigned to data-processing PAEs using time-division multiplexing, while bus systems are assigned to the different configurations using space-division multiplexing.", "In the assignment of resources, i.e., the assignment of tasks to PAEs or a group of PAEs to be carried out by the compiler or a similar unit, the given field may then be considered as a field of the n-fold variable and code sections may be transferred to this field of resources, which is virtually scaled up by the factor n, without the occurrence of problems, particularly when code sections are transferred in such a way that no interdependent code sections have to be configured into a PAE which is used in a multiplex-like manner.", "In the previous approach, a PACKEDCONF was composed of at least one configuration word or a bundle of configuration words for PAEs which belong to one single application.", "In other words, only configuration words which belong together were combined in the PACKEDCONF.", "In an enhanced embodiment, at least one or more configuration words per each different configuration are entered into a PACKEDCONF in such a way that the configuration word or words which belong together in a configuration are combined in a configuration group and the configuration groups thus created are combined in the PACKEDCONF.", "The individual configuration groups may be executed in chronological succession, thus in time-division multiplexing by a timeslice-like assignment.", "This results in time division multiplexing of different configuration groups on one PAE.", "As described above, the configuration word or the configuration words within a configuration group may also be executed in chronological succession.", "Multiplexers which select one of the configuration groups are assigned to the configuration registers and/or to a configuration memory, which is possibly also decoupled and implemented separately (compare DE 102 06 653.1 (PACT15), DE 102 07 224.8 (PACT15a, PACT 15b).", "In an enhanced embodiment, a sequencer (as described above) may be additionally assigned which makes the sequential processing of configuration words within configuration groups possible.", "Using the multiplexers and the optional sequencer, a resource (PAE) may be assigned to multiple different configurations in a time-division multiplex method.", "Among one another, different resources may synchronize the particular configuration group to be applied, for example by transmitting a configuration group number or a pointer.", "The execution of the configuration groups may take place linearly in succession and/or cyclically, with a priority being observed.", "It should be noted here in particular that different sequences may be processed in a single processor element and that different bus systems may be provided at the same time so that no time is wasted in establishing a bus connection which may take some time due to the long transmission paths.", "If a PAE assigns its first configuration to a first bus system and, on execution of the first configuration, couples the same to the bus system, then it may, in a second configuration, couple a different or partially different bus system to the former if spacial multiplexing for the bus system is possible.", "The execution of a configuration group, each configuration group being composed of one or more configuration words, may be made dependent on the reception of an execution release via data and/or triggers and/or an execution release condition.", "If the execute release (condition) for a configuration group is not given, the execute release (condition) may either be awaited, or the execution of a subsequent configuration group may be continued.", "The PAEs preferably go into a power-saving operating mode during the wait for an execute release (condition), for example with a disabled clock (gated clock) and/or partially disabled or reduced power supply.", "If a configuration group cannot be activated, then, as mentioned above, the PAEs preferably also go into a power-saving mode.", "The storage of the PACKEDCONF may take place by using a ring-type memory or other memory or register means, the use of a ring-type memory resulting in the fact that after the execution of the last input, the execution of the first input may be started again (compare PCT/DE 97/02998 (PACT04/PCT)).", "It should be noted that it is also possible to skip to a particular execution directly and/or indirectly and/or conditionally within the PACKEDCONF and/or a configuration group.", "In a preferred method, PAEs may be designed for processing of configurations in a corresponding time-division multiplexing method.", "The number of bus systems between the PAEs is increased such that sufficient resources are available for a sufficient number of configuration groups.", "In other words, the data-processing PAEs operate in a time-division multiplex method, while the data-transmitting and/or data-storing resources are adequately available.", "This represents a type of space division multiplexing, a first bus system being assigned to a first temporarily processed configuration, and a second bus system being assigned to an additional configuration; the second bus system runs or is routed spacially separated from the first bus system.", "It is possible at the same time and/or alternatively that the bus systems are also entirely or partially operated in time-division multiplexing and that multiple configuration groups share one bus system.", "It may be provided here that each configuration group transmits its data as a data packet, for example, a configuration group ID being assigned to the data packet (compare APID in DE 102 06 653.1 (PACT15), DE 102 07 224.8 (PACT 15a, PACT 15b)).", "Subsequently it may be provided to store and sort the particular data packets transmitted based on their assigned identification data, namely between different buses if required and for coordinating the IDs.", "In an enhanced method, memory sources may also be run in a time-division multiplex, e.g:, by implementing multiple segments and/or, at a change of the configuration group, by writing the particular memory/memories according to PCT/DE 97/02998 (PACT04/PCT) and/or PCT/DE 00/01869 (PACT13/PCT) into a different or even external memory or by loading from the same.", "In particular the methods according to DE 102 06 653.1 (PACT15), DE 102 07 224.8 (PACT15a, PACT 15b) may be used (e.g., MMU paging and/or APID).", "The adaptation of the operating voltage to the clock should be noted as a further possibility for conserving resources.", "Semiconductor processes typically allow higher clock frequencies when they are operated at higher operating voltages.", "However, this causes substantially higher power consumption and may also reduce the service life of a semiconductor.", "An optimum compromise may be achieved in that the voltage supply is made dependent on the clock frequency.", "Low clock frequencies may be operated at a low supply voltage, for example.", "With increasing clock frequencies, the supply voltage is also increased (preferably up to a defined maximum).", "The present invention, as an example, is explained in greater detail below with reference to the enclosed drawing.", "It should be noted that this exemplary description is not limiting and that in isolated cases and in different figures identical or similar units may be denoted using different reference numbers.", "As an example, FIG.", "1 shows a reconfigurable data processing unit (VPU) (0101).", "A configuration unit (CT, 0103) for the control and execution of the configuration and reconfiguration is superordinated to an array of PAEs (0102) which are configurable and reconfigurable independently from one another.", "In this connection, particular reference is made to the various applications of the applicant and the disclosure content of the patents and technologies mentioned in the introduction.", "In addition, a central clock generator (0104) is assigned to the data processing unit.", "In a possible embodiment, the clock rate of the central clock generator may be preselected by configuration unit 0103.In a possible embodiment, the clock rate of each PAE and/or groups of PAEs and their bus connections may also be preselected by configuration unit 0103.According to FIG.", "2, configuration unit 0103 feeds configuring data via a configuration line 0103a into respective cells 2 of which only one is illustrated as an example.", "Furthermore, the clock signal of central clock generator 0104 is fed to cell 0102 via a clock line 0104a.", "Via a data bus input 0205a and a data bus output 0205b, reconfigurable cell 0102 communicates with other cells and additionally has a data processing unit, e.g., an arithmetic logic unit ALU 0206, and preferably an internal data memory 0207 and a configuration memory 0208 into which configuring instructions from configuration unit 0103 are fed via a configuration instruction extractor 0209 in order to configure the data processing unit, e.g., ALU 0206, as a response.", "In addition, configuration [instruction] extractor 0209 is connected to a frequency divider/multiplier factor preselecting input 0210a of a frequency divider/frequency multiplier 0210 which is designed to divide or multiply the clock signal of central clock generator 0104 on clock line 0104a according to a clock ratio preselected via input 0210a and to feed the clock signal to the data processing unit, e.g., arithmetic logic unit ALU 0206, and possibly other units of reconfigurable cell 0102 via a line 0211.Using an optional data bus monitoring circuit 0212, 0210 may be activated in such a way that the frequency is controlled depending on the data reception or the data transmission.", "Furthermore, a multiplexer 0213 for selecting different configurations and/or configuration groups may optionally be integrated dependent on 0212.Furthermore, the multiplexer may optionally be activated by a sequencer 0214 in order to make sequential data processing possible.", "In particular, intermediate results may be managed in data memory 0207.While the general configuration of the cell was described in part in the applicant's applications mentioned in the introduction, the presently described clock dividing system, the associated circuit, and the optimization of its operation are at least novel and it should be pointed out that these facts may and shall be associated with the required hardware changes.", "The entire system and in particular configuration unit 0103 is designed in such a way that, together with a configuring signal with which a configuration word is fed via configuration line 0103a via configuration word extractor 0209 to data processing unit 0206 or upstream and/or downstream and/or associated memory 0208, a clock dividing/multiplying signal may also be transmitted which is extracted by configuration word extractor 0209 and transmitted to frequency divider/multiplier 0210, so that, as a response, 0210 may clock data processing unit 0206 and possibly also other units.", "It should be pointed out that, as a response to an input signal to the cell, there are also other possibilities instead of unit 0209 to vary clocking of an individual data processing unit 0206 with reference to a central clock unit 0104, via data bus monitoring circuit 0212, for example.", "Described only as an example with reference to FIGS.", "3 and 4, an entire field of all reconfigurable logic units 0102 may be operated using the above-described embodiment, but possibly also by implementing the units in a different way.", "For example, a 3×3 field of reconfigurable cells is configured in such a way, according to FIG.", "3a, that a first cell 0102a is used for analyzing an input/output signal.", "Cells 0102b, 0102c are presently not needed and are therefore denoted as not configured (n. c.).", "Cells 0102d through 0102i together form a group which executes a complex arithmetic operation; an addition takes place in cell 0102d, a subtraction takes place in cell 0102e, a multiplication takes place in cell 0102f, a loop is run in cell 0102g, a multiple addition being executed within the loop, a division takes place in cell 0102h, and an addition in turn takes place in cell 0102i.", "Cells 0102d through 0102i are connected to one another in group 0301, indicated by dot and dash lines, in such a way that data is sequentially and pipeline-like processed by the cells.", "As is indicated in the second row of the table in FIG.", "3b, the operations within cells 0102d and 0102e are executed in a different number of clock cycles.", "The number of clock cycles is denoted there and it is clear that an addition or a subtraction may be executed in one clock cycle; the division, however, requires 32 clock cycles.", "The third line of the table in FIG.", "3b denotes which value is assigned to the frequency divider of each cell in order to achieve optimum power usage at a constant data throughput through the cell.", "Only the cell in which the division takes place is operated at the highest clock; the clock ratio here is 1.This cell requires the longest time for the operation assigned to it.", "Since a new result has to be delivered only every 32 clock pulses to cell 0102h executing the division, cells 0102d and 0102e are clocked slower by the appropriate factor of 32; the frequency divider ratio for these cells is therefore 32, as can be seen in FIG.", "3b.", "Whereas, the multiplication running in two clock cycles has a frequency divider ratio of 16, and the more complex loop of cell 0102g running in 16 clock cycles is assigned a frequency divider ratio of only 2.These clock ratios are initially known at the configuration, in which the individual cells are compiled in groups and are assigned to each cell within the group since they were determined by the compiler at program compilation and may therefore be input into the cell at its configuration.", "It is denoted in the fourth row from the top which clock rate results from a central clock of 256 MHz.", "If the processor unit having the separately clockable reconfigurable logic cells is operated in an application where the voltage may drop, e.g., due to exhausting voltage supply capacities, it may be provided that, at a drop in the supply voltage, the entire frequency is reduced to a critical value U1; all cells are subsequently clocked slower by one half so that division cell 0102h too runs only at 128 MHz, while cell 0102d is clocked at 4 MHz.", "Cell 0102a, executing a query of the mouse pointer having a lower priority, is no longer clocked at 8 MHz as previously but rather at 2 MHz, i.e., depending on the prioritization, different slowdowns according to the importance of the task are assigned to the respective groups at a voltage drop or under other circumstances.", "If, for other reasons, the temperature still rises, the heat generation in the logic cell field may be further reduced by an additional clock rate reduction for the logic cells, as is indicated in the last row of FIG.", "3b.", "It is understood that, for example, a particular individual sensor for determining the condition such as the supply voltage and/or the temperature may be provided whose sensor signal is fed to the cells in a conditioned manner; a corresponding sensor system may be assigned to each cell and/or the central clock is possibly modifiable.", "This makes it possible to optimally operate a processor field energy-efficiently; the cooling capacity required is reduced and it is clear that, since as a rule not all cells may and/or must be permanently operated at the highest clock frequency, heat sinks and the like may be dimensioned appropriately smaller which in turn offers additional cost advantages.", "It should be noted that in addition to the query regarding a supply voltage, a temperature, the prioritization of computations, and the like, other conditions may determine the clock.", "For example, a hardware switch or a software switch may be provided with which the user indicates that only low clocking or higher clocking is desired.", "This makes an even more economical and targeted handling of the available power possible.", "It may be provided in particular that, at the user's request or at an external request, the central clock rate in total may be reduced; the clock divider ratios within the cell array, however, are not changed in order to avoid the requirement of reconfiguring all cells, e.g., at an extreme temperature rise.", "Moreover, it should be pointed out that a hysteresis characteristic may be provided in determining the clock rates, when a temperature-sensitive change of the clock frequencies is to be performed, for example.", "FIG.", "4 once more shows the data processing unit (VPU) according to FIG.", "1.Different groups within the VPU are operated using different frequencies f which are derived from a frequency normal n generated by 0104.It should be expressly noted that multiple frequency normals (n1 .", ".", ".", "nn) may be generated by multiple 0104 and may be used within one VPU.", "FIG.", "5 shows a simple exemplary embodiment for the operation of a PAE according to FIG.", "2.A data bus (0205a) delivers operands ia1 and ia2 to an ALU (0206) which in turn delivers the result of the computation oa to 0205b.", "The PAE is only activated, i.e., clocked and/or supplied with current, when data bus monitoring circuit 0212 recognizes the acceptance of the previous result oa by the receiver and the arrival of operands ia1 and ia2 necessary for the operation.", "In other words, the PAE is only activated when all working conditions and requirements are met.", "The clock release is carried out by 0210, the clock source is 0104a.", "FIG.", "6 corresponds to FIG.", "5 with the exception that a sequencer (0214) is additionally activated which controls a multicyclical configuration (e.g., a complex computation such as a matrix multiplication or the like).", "The sequencer extracts the operations from the configuration memory or from a section of the configuration memory.", "In the example shown, operations op1, op2, op3, op4, op5 are carried out sequentially.", "Result oa is conveyed after completion and the PAE has to be activated again.", "The data transmission occurring on data bus 0205a/b is illustrated in FIG.", "6a.", "It should be pointed out that the data routing via the bus may take place in a manner known per se as is known from other applications and/or publications of the present user, i.e., collision preventions and deadlock situations may be prevented for one configuration at a time in a manner known per se.", "In order to execute op1, operands ia must be available via 0205a (0601); the data transmissions for the remaining cycles may be undefined in principle.", "Thereafter, 0205a may preferably transmit the subsequent operands (0602) for which the execution time of op2, op3, op4, op5 is available, thus creating an essential temporal decoupling, allowing the use of slower and/or, in particular, longer bus systems.", "During the execution of op2, op3, op4, op5, data of other configurations may alternatively (0603) be transmitted via the same bus system 0205a using a time-division multiplex method.", "Following op5, result oa is applied to bus 0205b (0601); the data transmissions for the remaining cycles may be undefined in principle.", "The time prior to op5, i.e., during the execution of op1, op2, op3, op4, may be used for transmitting the previous result (0602).", "This again creates an essential temporal decoupling, allowing the use of slower and/or, in particular, longer bus systems.", "During the execution of op1, op2, op3, op4, data of other configurations may alternatively (0603) be transmitted via the same bus system 0205b using a time-division multiplex method.", "For clock multiplication, 0210 may use a PLL.", "A PLL may be used in particular in such a way that the operating clock of the PAE for executing op1, op2, op3, op4, op5 is five times that of the bus clock.", "In this case, the PAE may act as a PAE without a sequencer having only one (unicyclical) configuration and the same clock as the bus clock.", "FIG.", "7 corresponds to FIG.", "6 plus the addition that multiple configuration groups (ga, gb, gc) share the PAE in a time-division multiplexed manner and each group has connections to a separate (space-division multiplexed) bus system (ia/oa, ib/ob, ic/oc).", "A multiplexer in 0214 cyclically selects the groups ga, gb, gc.", "Provided the data monitoring circuit 0212 generates a valid execution release (condition) for a configuration group, the particular configuration group is executed; otherwise the execution release (condition) may be awaited or, preferably, a different subsequent configuration group may be selected.", "The configuration groups may be run through cyclically.", "One configuration group may contain multiple configuration words (ga={ka1, ka2}, gb={kb1}, gc={kc1, kc2, kc3}).", "The configuration words may be executed sequentially in 0214 using a sequencer.", "FIG.", "7a shows the bus transmissions according to the example in FIG.", "7.0701 corresponds to 0601, 0702 corresponds to 0602, 0703 corresponds to 0603; a separate bus system is used thereby for each group ga, gb, gc.", "In addition, a possible bus transmission using a time-division multiplex for the bus systems is illustrated in 0704.The input data of all groups is transmitted via an input bus system and the output data of all groups is transmitted via an output bus system.", "The undefined intermediate cycles are either unused or are free for other data transmissions." ] ]	Patent_10469909
[ [ "Method and Device for Treating and Processing Data", "Procedures and methods for managing and transmitting data within multidimensional systems of transmitters and receivers are described.", "Splitting a data stream into a plurality of independent branches and subsequent merging of the individual branches to form a data stream is to be performable in a simple manner, the individual data streams being recombined in the correct sequence.", "This method is of importance in particular for executing reentrant code.", "The method is well suited, in particular, for configurable architectures; particular attention is paid to the efficient control of configuration and reconfiguration." ], [ "1-31.", "(canceled) 32.A method for controlling a pipeline-type data processing system or a bus system, comprising: alternating different protocols to permit data processing in each cycle.", "33.The method as recited in claim 32, wherein one of the protocols confirms receipt of data by a receiver.", "34.The method as recited in claim 33, wherein one of the protocols confirms an expected receipt of data by a receiver.", "35.The method as recited in claim 34, further comprising: when the data confirmed for an expected receipt cannot be received by a receiver, writing the data into a buffer register and subsequently no further expected receipt of data by a receiver is confirmed until the buffer register is emptied.", "36.The method as recited in claim 35, wherein the buffer register is emptied as soon as the receiver resumes receiving data, before other additional data is sent to the receiver.", "37.A method for transmitting data of one transmitter to a plurality of receivers, comprising: logically gating acknowledgments of receipt of data by all receivers.", "38.A method for transmitting data of a plurality of transmitters to one receiver, comprising: storing a sequence of transmission requests of a plurality of transmitters; and enabling a transmission of data in the sequence.", "39.A method for transmitting data of a plurality of transmitters to one receiver, comprising: assigning to each transmitter upon a bus access request a transmitter number, which identifies the transmitter's position in the plurality of transmitters.", "40.The method as recited in claim 39, wherein all transmitter numbers are called in sequence by a call number generator by communicating a current call number to all transmitters, each transmitter comparing the communicated call number with its transmitter number and claiming the bus in the case of a match.", "41.The method as recited in claim 39, wherein the transmitter numbers are incremented in each time unit.", "42.The method as recited in claim 40, further comprising: arbitrating the bus when a plurality of transmitters has been assigned the same transmitter number.", "43.The method as recited in claim 41, wherein the call number generator does not increment until no transmitter has arbitrated the bus further.", "44.A method for managing data streams, comprising: assigning an identifier to data in the data stream.", "45.The method as recited in claim 44, wherein the identifier defines a chronological sequence.", "46.The method as recited in claim 44, wherein the identifier defines a source address or a target address.", "47.The method as recited in claim 45, wherein a merger of data in the original sequence is defined by a bus system, based on the identifier.", "48.The method as recited in claim 45, wherein a merger of data in an original sequence is defined by a memory, based on the identifier.", "49.The method as recited in claim 45, further comprising: transmitting the identifier via a peripheral interface.", "50.The method as recited in claim 44, wherein the identifier is written into memories together with the data.", "51.A method for partitioning a graph, comprising: introducing memories at the section edges of the graph.", "52.The method as recited in claim 51, wherein a memory is used at each edge of the graph.", "53.The method as recited in claim 51, wherein multiplexers merge a plurality of edges upstream from a memory.", "54.The method as recited in claim 51, further comprising: storing an identifier together with the data.", "55.A method for constructing sequencers from a plurality of programmable array elements, comprising: assigning an identifier assigned to data; and using the identifier for at least one of the addressing data sources and data targets.", "56.A method for constructing sequencers from a plurality of programmable array element, comprising: assigning an identifier to data, the identifier containing a data processing instruction.", "57.A method for pipeline-type data processing comprising: connecting FIFO buffers between data processing elements for chronological separation.", "58.The method as recited in claim 57, wherein the FIFO buffers have configurable latencies to balance the delay in the data paths.", "59.A FIFO memory method, comprising: resuming a readout procedure at a previously read data word.", "60.A FIFO memory method, comprising: resuming a write procedure at a previously written data word.", "61.The method as recited in claim 59, further comprising: saving in save register an address position of a data word at whose address a procedure may be repeated.", "62.The method as recited in claim 61, further comprising: testing an empty or full state of the FIFO by comparison with the save register.", "63.The method as recited in claim 61, wherein the save register may be set at any desired address." ], [ "The present invention describes procedures and methods for managing and transferring data within multidimensional systems of transmitters and receivers.", "Splitting a data stream into a plurality of independent branches and subsequent merging of the individual branches to form a data stream is to be performable in a simple manner, the individual data streams being recombined in the correct sequence.", "This method is of importance in particular for executing reentrant code.", "The described method is well suited, in particular, for configurable architectures; particular attention is paid to the efficient control of configuration and reconfiguration.", "The object of the present invention is to provide a novel method for commercial use.", "The achievement of the object is claimed independently.", "Preferred embodiments are found in the subclaims.", "Reconfigurable architecture is defined herein as modules (VPU) having a configurable function and/or interconnection, in particular integrated modules having a plurality of unidimensionally or multidimensionally positioned arithmetic and/or logic and/or analog and/or storage and/or internally/externally interconnecting modules, which are connected to one another directly or via a bus system.", "These generic modules include in particular systolic arrays, neural networks, multiprocessor systems, processors with a plurality of arithmetic units and/or logic cells and/or communication/peripheral cells (IO), interconnecting and networking modules such as crossbar switches, as well as known modules of the type FPGA, DPGA, Chameleon, XPUTER, etc.", "Reference is also made in particular in this context to the following patents and patent applications of the same applicant: P 44 16 881.0-53, DE 197 81 412.3, DE 197 81 483.2, DE 196 54 846.2-53, DE 196 54 593.5-53, DE 197 04 044.6-53, DE 198 80 129.7, DE 198 61 088.2-53, DE 199 80 312.9, PCT/DE 00/01869, DE 100 36 627.9-33, DE 100 28 397.7, DE 101 10 530.4, DE 101 11 014.6, PCT/EP 00/10516, EP 01 102 674.7, PACT02, PACT04, PACT05, PACT08, PACT10, PACT11, PACT13, PACT21, PACT13, PACT15b, PACT18(a), PACT25(a,b).", "The entire contents of these documents are hereby included for the purpose of disclosure.", "The above-mentioned architecture is used as an example to illustrate the invention and is referred to hereinafter as VPU.", "The architecture includes an arbitrary number of logic (including memory) and/or memory cells and/or networking cells and/or communication/peripheral (IO) cells (PAEs—Processing Array Elements) which may be positioned to form a unidimensional or multidimensional matrix (PA); the matrix may have different cells of any desired configuration.", "Bus systems are also understood here as cells.", "A configuration unit (CT) which affects the interconnection and function of the PA is assigned to the entire matrix or parts thereof.", "DESCRIPTION OF THE INVENTION The configurable cells of a VPU must be synchronized for the proper processing of data.", "Two different protocols are used for this purpose; one for the synchronization of the data traffic and another one for sequence control of the data processing.", "Data is preferably transmitted via a plurality of configurable bus systems.", "Configurable bus system means in particular that any PAEs transmit data and the connection to the receiving PAEs and the receiving PAEs themselves in particular are configurable in any desired manner.", "The data traffic is preferably synchronized using handshake protocols, which are transmitted with the data.", "In the following description, simple handshakes as well as complex procedures are described, whose preferred use depends on the particular application to be executed or the amount of applications.", "Sequence control takes place via signals (triggers) which indicate the status of a PAE.", "Triggers may be transmitted independently of the data via freely configurable bus systems, i.e., they may have different transmitters and/or receivers and preferably also have handshake protocols.", "Triggers are generated by a status of a transmitting PAE (e.g., zero flag, overflow flag, negative flag) by relaying individual states or combinations.", "Data processing cells (PAEs) within a VPU may assume different processing states, which depend on the configuration status of the cells and/or incoming or received triggers: “not configured”: no data processing “configured”: GO all incoming data is computed.", "STOP incoming data is not computed.", "STEP one computation is performed.", "GO, STOP, and STEP are triggered by the triggers described below: Handshake Synchronization A particularly simple yet powerful handshake protocol, which is preferably used when transmitting data and triggers, is described in the following.", "The control of the handshake protocol is preferably hard-wired in the hardware and may be an essential component of a VPU's data processing paradigm.", "The principles of this protocol have been described in PACT02.A RDY signal which indicates the validity of the information is also transmitted with each piece of information transmitted by a transmitter via any bus.", "The receiver only processes information that is provided with a RDY signal; all other information is ignored.", "As soon as the information has been processed by the receiver and the receiver is able to receive new information, it indicates, by sending an acknowledgment signal (ACK) to the transmitter, that the transmitter may transmit new information.", "The transmitter always waits for the arrival of ACK before it sends data again.", "A distinction is made between two operating modes: a) “dependent”: All inputs that receive information must have a valid RDY before the information is processed.", "Then ACK is generated.", "b) “independent”: as soon as an input that receives information has a valid RDY, an ACK is generated for this particular input if the input is able to receive data, i.e., the preceding data has been processed; otherwise it waits for the data to be processed.", "Data processing synchronization and control may be performed according to the related art via a hardwired state machine (see PACT02), a state machine having a fine-grained configuration (see PACT01, PACT04) or, preferably, via a programmable sequencer (PACT13).", "The programmable state machine is configured according to the sequence to be executed.", "Altera's EPS448 module (ALTERA Data Book 1993) implements such a programmable sequencer, for example.", "One particular function of handshake protocols for VPUs is the performance of pipeline-type data processing, in which in each cycle data may be processed in each PAE in particular.", "This requirement results in particular demands on the operation of the handshakes.", "The problem and the achievement of this object are shown using the example of a RDY/ACK protocol: FIG.", "1a shows a configuration of a pipeline within a VPU.", "The data is sent via (preferably configurable) bus systems (0107, 0108, 0109) to registers (0101, 0104), which have an optionally data processing logic (0102, 0105) connected downstream.", "The logic has an associated output stage (0103, 0106), which preferably also has a register for sending the results to a bus again.", "The RDY/ACK synchronization protocol is preferably transmitted both via the bus systems (0107, 0108, 0109) and via the data processing logic (0102, 0105).", "The two meanings of the terms of the RDY/ACK protocol are as follows: a) ACK means “receiver will receive data,” having the effect that the pipeline operates in each cycle.", "However, the problem arises that due to the hard-wiring, in the event of a pipeline stall, the ACK runs asynchronously through all the stopped stages of the pipeline.", "This results in considerable timing problems, in particular in the case of large VPUs and/or high clock frequencies.", "b) ACK means “receiver has received data,” having the effect that the ACK always runs only to the next stage where there is a register.", "The problem that arises here is that the pipeline only operates in every other cycle due to the delay of the register that is required in the hardwired implementation.", "The object is achieved by combining both meanings as shown in FIG.", "1b, which illustrates a section of stages 0101 through 0103.Protocol b) is used on bus systems (0107, 0108, 0109) in that a register (0110) delays the incoming RDY by one cycle by writing the transmitted data into an input register, and relays it again onto the bus as an ACK.", "This stage (0110) operates almost as a protocol converter between a bus protocol and the protocol within a data processing logic.", "The data processing logic uses protocol a), which is generated by a downstream protocol converter (0111).", "The 0111 unit has the distinguishing feature that a preliminary statement must be made about whether the incoming data from the data processing logic is actually also received by the bus system.", "This is accomplished by introducing an additional buffer register (0112) in the output stages (0103, 0106) for the data to be transmitted to the bus system.", "The data generated by the data processing logic is written to the bus system and into the buffer register at the same time.", "If the bus is unable to receive the data, i.e., no ACK is sent by the bus system, the data is stored in the buffer register and is sent to the bus system via a multiplexer (0113) as soon as the bus system is ready.", "If the bus system is immediately ready to receive the data, the data is relayed directly to the bus via the multiplexer (0113).", "The buffer register enables acknowledgment in the meaning a), because acknowledgment may be sent using “receiver will receive data” as long as the buffer register is empty, because writing into the buffer register ensures that the data is not lost.", "Triggers Triggers, whose operating principles are described in PACT08, are used in VPU modules for transmitting simple information.", "Triggers are transmitted using a unidimensional or multidimensional bus system divided into segments.", "The individual segments may be equipped with drivers for improving the signal quality.", "The particular trigger connections, which are implemented by the interconnection of various segments, are programmed by the user and configured via the CT. Triggers for example transmit mainly, but not exclusively, the following information or any possible combinations thereof: Status information of arithmetic units (ALUs), such as carry division by zero zero negative underflow/overflow Results of comparisons and/or loops n bit information (for small n) Interrupt requests generated internally or externally.", "Triggers are generated by any cells and are activated by any events in the individual cells.", "In particular, triggers may be generated by a CT or an external unit located outside the cell array or the module.", "Triggers are received by any cells and analyzed by any possible method.", "In particular, triggers may by analyzed by a CT or an external unit located outside the cell array or the module.", "Triggers are mainly used for sequence control within a VPU, for example, for comparisons and/or loops.", "Data paths and/or branchings may be enabled or disabled by triggers.", "Another important area of application of triggers is the synchronization and activation of sequences and their information exchange, as well as the control of data processing in the cells.", "Triggers may be managed and data processing may be controlled according to the related art by a hardwired state machine (see PACT02, PACT08), a state machine having a fine-grained configuration (see PACT01, PACT04, PACT08), (Chameleon), or preferably by a programmable state machine (PACT13).", "The programmable state machine is configured in accordance with the sequence to be executed.", "Altera's EPS448 module (ALTERA Data Book 1993) implements such a programmable sequencer, for example.", "Basic Method The simple synchronization method using RDY/ACK protocols makes the processing of complex data streams difficult, because observing the correct sequence ties up considerable resources.", "The correct implementation is the programmer's responsibility.", "Additional resources are also required for the implementation.", "In the following, a simple method for achieving this object is described.", "1:n Transmission This case is trivial: The transmitter writes the data onto the bus.", "The data is stable on the bus until the ACK is received as acknowledgment from all receivers (the data “resides”).", "RDY is pulsed, i.e., is applied for one cycle to prevent the data from being incorrectly read multiple times.", "Since RDY activates multiplexers and/or gates and/or other appropriate transmission elements which control the data transfer depending on the implementation, this activation is stored (RdyHold) for the time of the data transmission.", "This causes the position of gates and/or multiplexers and/or other appropriate transmission elements to remain valid even after the RDY pulse and thus valid data to remain on the bus.", "As soon as a receiver has received the data, it acknowledges using an ACK (see PACT02).", "It should be mentioned again that the correct data remains on the bus until it is received by the receiver(s).", "ACK is also preferably transmitted as a pulse.", "If an ACK passes through a multiplexer and/or gate, and/or another appropriate transmission element in which RDY was previously used for storing the activation (see RdyHold), this activation is now cleared.", "To transmit 1:n, it is advisable to hold ACK, i.e., to use no pulsed ACK, until a new RDY is received, i.e., ACK also “resides.” The ACKs received are AND-gated at each bus node representing a branching to a plurality of receivers.", "Since the ACKs “reside,” a “residing” ACK which represents the ACKs of all receivers remains at the transmitter.", "In order to keep the running time of the ACK chain through the AND gate as low as possible, it is recommended that a tree-shaped configuration be chosen or generated during the routing of the program to be executed.", "Residing ACKs may cause, depending on the implementation, the problem that RDY signals for which there was actually no ACK are ACK-ed because an old ACK resided for too long.", "One way of avoiding this problem is to basically pulse ACK and to store the incoming ACK of each branch at a branching.", "An ACK pulse is not relayed toward the transmitter and all stored ACKs (AckHold) and possibly the RdyHolds are not cleared until the ACKs of all branches have been received.", "FIG.", "1c shows the principle of the method.", "A transmitter 0120 transmits data via a bus system 0121 together with a RDY 0122.A plurality of receivers (0123, 0124, 0125, 0126) receive the data and the particular RDY (0122).", "Each receiver generates an ACK (0127, 0128, 0129, 0130), which are gated via an appropriate boolean logic (0131, 0132, 0133), for example a logical AND function, and sent to the transmitter (0134).", "FIG.", "1c shows one possible preferred embodiment having two receivers (a, b).", "An output stage (0103) transmits data and the associated (in this case pulsed) RDY (0131).", "RdyHold stages (0130) upstream from the target PAEs translate the pulsed RDY into a residing RDY.", "In this example, a residing RDY should have the boolean value b′1.The contents of all RdyHold stages are returned to 0103 via a chain of logical OR functions (0133).", "If a target PAE acknowledges the receipt of data, the corresponding RdyHold stage is only reset by the incoming ACK (0134).", "Thus, the meaning of the returned signal is b′1=“some PAE or other has not received the data.” As soon as all RdyHold stages have been reset, the information b′0=“all PAEs have received the data” is received by 0103 via the OR chain (0133), which is evaluated as ACK.", "The outputs (0132) of the RdyHold stages may also be used for activating bus switches as described previously.", "A logical b′0 is supplied to the last input of an OR chain to ensure proper operation of the chain.", "n:1-Transmission This case is relatively complex.", "(F1) On the one hand, a plurality of transmitters must be multiplexed onto one receiver; (F2) on the other hand, the time sequence of the transmissions must generally be observed.", "In the following, several methods are described to achieve this object.", "It should be pointed out that in principle no method is to be preferred.", "Rather, the most suitable method should be selected according to the system and the algorithms to be executed from the point of view of programmability, complexity, and cost.", "A simple n:1 transmission may be implemented by connecting a plurality of data paths to the inputs of each PAE.", "The PAEs are configured as multiplexer stages.", "Incoming triggers control the multiplexer and select one of the plurality of data paths.", "If necessary, tree structures may be constructed from PAEs configured as multiplexers to merge a plurality of data streams (large n).", "The method requires special attention on the programmer's part to ensure correct chronological sorting of the different data streams.", "In particular, all data paths should have the same length and/or delay to ensure the correct sequence of the data.", "More effective methods for merging are described below: Since F1 seems to be easily implementable using any arbiter and a downstream multiplexer, the discussion should begin with F2.The time sequence cannot be observed using simple arbiters.", "FIG.", "2 shows a first possible example of implementation.", "A FIFO (0206) is used to store on a bus system (0208) and execute the time sequences of transmission requests correctly.", "For this purpose, a unique number representing its address is assigned to each transmitter (0201, 0202, 0203, 0204).", "Each transmitter requests a data transmission to bus system 0208 by displaying its address on a bus (0209, 0210, 0211, 0212).", "The particular addresses are stored in a FIFO (0206) via a multiplexer (0205) according to the sequence of the transmission requests.", "The FIFO is executed step-by-step, and the address of the particular FIFO entry is displayed on another bus (0207).", "This bus addresses the transmitters and the transmitter having the corresponding address receives access to bus 0208.The internal memories of the VPU technology may be used, for example, as FIFO for such a procedure (see PACT04, PACT13).", "However, on closer examination, the following problem arises: as soon as a plurality of transmitters wish to access the bus, one transmitter must be selected whose address is then stored in the FIFO.", "In the next cycle, the next transmitter is then selected, and so forth.", "The selection may take place via an arbiter (0205).", "This eliminates the simultaneity, which however generally represents no problem.", "For real time applications, a prioritizing arbiter might be used.", "The method, however, fails because of the simple reason: At time t, three transmitters S1, S2, S3 request receiver E. S1 is stored at t, S2 is stored at t+1, and S3 is stored at t+2.However, at t+1 S4 and S5, at t+2 also S6 and again S1 request the receiver.", "Because the new requests overlap with the old ones, processing very quickly becomes extremely complex and requires considerable additional hardware resources.", "Thus the method described in FIG.", "2 is to be preferably used for simple n:1 transitions which, if possible, have no simultaneous bus requests.", "According to this discussion, it seems to be advisable not to store one transmitter per cycle, but the set of all transmitters that request the transmission in a given cycle.", "In the following cycle, the new set is then stored.", "If several transmitters request the transmission in the same cycle, these are arbitrated at the time the memory is processed.", "Storing a plurality of transmitter addresses at the same time is, however, very complicated.", "A simple implementation is achieved by the following embodiment in FIG.", "3: An additional counter (REQCNT, 0301) counts the number of cycles T. Each transmitter (0201, 0202, 0203, 0204) which requests the transmission at cycle t stores the value of REQCNT (REQCNT(t)) at cycle t as its address.", "Each transmitter which requests the transmission at cycle t+1 stores the value of REQCNT (REQCNT(t+1)) at cycle t+1 as its address.", ".", ".", ".", "Each transmitter which requests the transmission at cycle t+n stores the value of REQCNT (REQCNT(t+n)) at cycle t+n as its address.", "The FIFO (0206) stores the values of REQCNT(tb) at a given cycle tb.", "The FIFO displays a stored value of REQCNT as a transmission request on a separate bus (0207).", "Each transmitter compares this value with the one it has stored.", "If the values are identical, it transmits the data.", "If a plurality of transmitters have the same value, i.e., simultaneously wish to transmit data, the transmission is now arbitrated by a suitable arbiter (CHNARB, 0302b) and sent to the bus by a multiplexer (0302a) activated by the arbiter.", "A possible exemplary embodiment of the arbiter is described in the following.", "If no transmitter responds to a REQCNT value, i.e., the arbiter has no more bus requests for arbitration (0303), the FIFO switches to the next value.", "If the FIFO has no more valid entries (empty), the values are identified as invalid to prevent erroneous bus access.", "In a preferred embodiment, only those values of REQCNT are stored in the FIFO (0206) for which there was a bus request of a transmitter (0201, 0202, 0203, 0204).", "For this purpose, each transmitter signals its bus request (0310, 0311, 0312, 0313), which are logic gated (0314), e.g., by an OR function.", "The resulting transmission request of all transmitters (0315) is supplied to a gate (0316) which supplies only those REQCNT values to the FIFO (0206) at which there was an actual bus request.", "The above-described procedure may be further optimized according to a preferred embodiment corresponding to FIG.", "4 as follows: A linear sequence of values (REQCNT(tb)) is generated by REQCNT (0410) if, instead of all cycles t, only those cycles are counted in which there is a bus request by a transmitter (0315).", "The FIFO is now replaceable by a simple counter (SNDCNT, 0402), which now also counts linearly and whose value (0403) enables the particular transmitters according to 0207, due to the linear sequence of values, generated by REQCNT, which now has no gaps.", "SNDCNT continues to increment as long as no transmitter responds to the value from SNDCNT.", "As soon as the value of REQCNT is identical to the value of SNDCNT, SNDCNT stops counting, since the last value has been reached.", "It is true for all implementations that the maximum required width of REQCNT is equal to log2 (number_of_transmitters).", "When the largest possible value is exceeded, REQCNT and SNDCNT restart at the minimum value (usually 0).", "Arbiters A plurality of arbiters may be used as CHNARB according to the related art.", "Depending on the application, prioritized or unprioritized arbiters are better suited, prioritized arbiters having the advantage that they are able to give preference to certain tasks for real time tasks.", "A serial arbiter, which is implementable in the VPU technology in a particularly simple and resource-saving manner, is described in the following.", "In addition, the arbiter offers the advantage of working in a prioritizing mode, which permits preferred processing of certain transmissions.", "A possible basic configuration of a bus system is initially described in FIG.", "5.Modules of the generic VPU type have a network of parallel data bus systems (0502), each PAE having connection to at least one data bus for data transmission.", "A network is usually made up of a plurality of equivalent parallel data buses (0502); each data bus may be configured for one data transmission.", "The remaining data buses may be freely available for other data transmissions.", "It should be furthermore mentioned that the data buses may be segmented, i.e., using configuration (0521) a bus segment (0502) may be switched through to the adjacent bus segment (0522) via gates (G).", "The gates (G) may be made up of transmission gates and preferably have signal amplifiers and/or registers.", "A PAE (0501) preferably picks up, data from one of the buses (0502) via multiplexers (0503) or a comparable circuit.", "The enabling of the multiplex system is configurable (0504).", "The data (results) generated by a PAE are preferably supplied to a bus (0502) via a similar independently configurable (0505) multiplexer circuit.", "The circuit described in FIG.", "5 is labeled using bus nodes.", "A simple arbiter for a bus node may be implemented as illustrated in FIG.", "6 as follows: Basic element 0610 of a simple serial arbiter may be made up by two AND gates (0601, 0602), FIG.", "6a.", "The basic element has an input (RDY, 0603) through which an input bus shows that it is transmitting data and requesting an enable to the receiver bus.", "Another input (ACTIVATE, 0604) which in this example is showing, via a logical 1 level, that none of the preceding basic elements has currently arbitrated the bus and therefore arbitration by this basic element is allowed.", "Output RDY_OUT (0605) shows, for example, to a downstream bus node that the basic element has enabled the bus access (if there is a bus request (RDY)) and ACTIVATE_OUT (0606) shows that the basic element is not currently performing any (more) enabling because no bus request (RDY) exists (any longer) and/or no previous arbiter stage has occupied the receiver bus (ACTIVE).", "A serial prioritizing arbiter is obtained by the serial chaining of ACTIVATE and ACTIVATE_OUT via basic elements 0610, the first basic element according to FIG.", "6b, whose ACTIVATE input is always activated, having the highest priority.", "The above-described protocol ensures that within the same SNDCNT value each PAE only performs one data transmission, because a subsequent data transmission would have another SNDCNT value.", "This condition is required for proper operation of the serial arbiter, because this ensures the processing sequence of the enable requests (RDY) necessary for prioritization.", "In other words, an enable request (RDY) cannot appear later during an arbitration on the basic elements which already show, via ACTIVATE_OUT, that they enable no bus access.", "Locality and Running Time The method is applicable, in principle, over long paths.", "Beyond a length depending on the system frequency, transmission of the data and execution of the protocol are no longer possible in a single cycle.", "One approach is to design the data paths to be of exactly the same length and merge them at one point.", "This makes all control signals for the protocol local, which makes it possible to increase the system frequency.", "To balance the data paths, FIFO stages may be used, which operate as delay lines having configurable delays.", "They will be described in more detail below.", "A very advantageous approach in which data paths may also be merged in a tree shape may be constructed as follows: Modified Protocol, Time Stamp The prerequisite is that a data path be divided into a plurality of branches and re-merged later.", "This is usually accomplished at branching points such as programmer-constructed “IF” or “CASE” nodes; FIG.", "7a shows a CASE-like configuration as an example.", "A REQCNT (0702) is assigned to the last PAE upstream from a branching (0701), at the latest; REQCNT assigns a value (time stamp), which is then to be always transmitted together with the data word, to each data word.", "REGCNT increments linearly with each data word, so that the position of a data word within a data stream is determinable via a unique value.", "The data words subsequently branch off into different data paths (0703, 0704, 0705).", "The associated value (time stamp) is transmitted via the data paths with each data word.", "A multiplexer (0707) re-sorts the data words into the correct sequence upstream from the PAE(s) (0708) which further process the merged data path.", "For this purpose, a linearly counting SNDCNT (0706) is associated with the multiplexer.", "The value (time stamp) assigned to each data word is compared to the value of SNDCNT.", "The multiplexer selects the matching data word.", "If no matching data word is found at a certain point in time, no selection is made.", "SNDCNT only increments if a matching data word has been selected.", "To achieve maximum clock frequency, the data paths must be merged locally to the highest possible degree.", "This minimizes the conductor lengths and keeps the associated run times short.", "If necessary, the data path lengths are to be adjusted via register stages (pipelines) until it is possible to merge all data paths at a common point.", "Attention must be paid to making the lengths of the pipelines approximately the same to prevent excessive time shifts between the data words.", "Use of the Time Stamp for Multiplexing The output of a PAE (PAE-S) is connected to a plurality of PAEs (PAE-E).", "Only one of the PAEs should process the data in each cycle.", "Each PAE-E has a different hard-wired address, which is compared with the TimeStamp bus.", "The PAE-S selects the receiving PAE by outputting the address of the receiving PAE to the TimeStamp bus.", "In this way the PAE for which the data is intended is addressed.", "Predictive Design and Task Switch The problem of predictive design is known from conventional microprocessors.", "It occurs when the data processing depends on a result of the preceding data processing; however, processing of the dependent data is begun in advance—without the required results being available—for reasons of performance.", "If the result is different from what has been assumed, the data based on erroneous assumptions must be reprocessed (misprediction).", "This may also occur in VPUs in general.", "By re-sorting and similar procedures this problem may be minimized; however, its occurrence may never be ruled out.", "A similar problem occurs when the data processing is aborted, before it has been completed, due to a unit (such as the task scheduler of an operating system, real-time request, etc.)", "of a higher level than data processing within the PAs.", "In this case, the status of the pipeline must be saved so that the data processing resumes downstream from the point of the operands that resulted in the computation of the last finished result.", "Two relevant states occur in a pipeline: RD At the beginning of a pipeline, the reception or request of new data is displayed; DONE At the end of a pipeline, the correct processing of data for which no misprediction occurred is displayed.", "Furthermore, the MISS_PREDICT state may be used, which shows that a misprediction occurred.", "It may be helpful to generate this status by negating the DONE status at the appropriate point in time.", "Special FIFOs PACT04 and PACT13 disclose methods in which data is kept in memories from which it is read for processing and in which results are stored.", "For this purpose, a plurality of independent memories may be used, which may operate in different operating modes; in particular, direct access, stack mode, or FIFO operating mode may be used.", "Data is normally processed linearly in VPUs, so that the FIFO operating mode is often preferentially used.", "For example, a special extension of the memories should be considered for the FIFO operating mode, which directly supports prediction and enables reprocessing of mispredicted data in the event of misprediction.", "Furthermore, the FIFO supports task switches at any point in time.", "We shall initially discuss the extended FIFO operating modes using the example of a memory providing read access (read side) within a given data processing run.", "The exemplary FIFO is illustrated in FIG.", "8.The configuration of the write circuit having a conventional write pointer (WR_PTR, 0801) which advances with each write access (0810) corresponds to the related art.", "The read circuit has the conventional counter (RD_PTR, 0802), for example, which counts each read word according to a read signal (0811) and modifies the read address of the memory (0803) accordingly.", "Novel, with respect to the related art, is an additional circuit (DONE_PTR, 0804), which does not document the data which has been read out, but the data which has been read out and correctly processed; in other words, only the data where no error has occurred and whose result was output at the end of the computation and a signal (0812) was displayed as a sign of the correct end of the computation.", "Possible circuits are described in the following.", "The FULL flag (0805) (according to the related art), which shows that the FIFO is full and unable to store additional data, is now generated by a comparison (0806) of DONE_PTR with WR_WTR which ensures that data which may have to be reused due to a possible misprediction is not overwritten.", "The EMPTY flag (0807) is generated, according to the conventional configuration, by comparison (0808) of RD_PTR with the WR_PTR.", "If a misprediction (MISS_PREDICT, 0809) occurred, the read pointer is loaded with the value DONE_PTR+1.Data processing is thus restarted at the value that triggered the misprediction.", "Two possible exemplary configurations of DONE_PTR should be discussed in more detail.", "a) Implementation by a Counter DONE_PTR is implemented as a counter, which is set equal to RD_PTR when the circuit is reset or at the beginning of a data processing run.", "An incoming signal (DONE) indicates that the data has been processed successfully (i.e., without misprediction).", "DONE_PTR is then modified so that it points to the next data word being processed.", "b) Implementation by a Subtractor As long as the length of the data processing pipeline is always exactly known and it is assured that the length is constant (i.e., no branching into pipelines of different lengths occurs), a subtractor may be used.", "The length of the pipeline from when the memory is connected to the recognition of a possible misprediction is stored in an associated register.", "After a misprediction, data processing must therefore be reinitialized at the data word which may be computed via the difference.", "On the write side, in order to save the result of the data processing of a configuration, an appropriately configured memory is required, the function of DONE_PRT being implemented for the write pointer to overwrite (mis)computed results during a new data processing run.", "In other words, the functions of the read/write pointer are reversed according to the addresses in brackets in the drawing.", "If data processing is interrupted by another source (e.g., task switch of an operating system), it is sufficient to save DONE_PTR and to reinitialize the data processing at a later point in time at DONE_PTR+1.FIFOs for Input/Output Stages, e.g., 0101, 0103 In order to balance data paths and/or states of different edges of a graph or different branches of a data processing run (trigger, see PACT08, PACT13), it is useful to use configurable FIFOs at the outputs or inputs of the PAEs.", "The FIFOs have adjustable latencies, so that the delay of different edges/branches, i.e., the run times of data over different but usually parallel data paths, are adjustable to one another.", "As a pipeline may be held up within a VPU by pending data or a pending trigger, the FIFOs are also useful for compensating such delays.", "The FIFOs described in the following accomplish both functions: A FIFO stage may be configured, for example, as follows (see FIG.", "9): A multiplexer (0902) is connected downstream from a register (0901).", "The register stores the data (0903) and also its correct existence, i.e., the associated RDY (0904).", "Data is written into the register when the adjacent FIFO stage which is situated closer to the FIFO output (0920) indicates that it is full (0905) and a RDY (0904) exists for the data.", "The multiplexer relays the incoming data (0903) directly to the output (0906) until the data has been written into the register and thus the FIFO stage itself is full, which is indicated (0907) to the adjacent FIFO stage, which is situated closer to the input (0921) of the FIFO.", "Receipt of data in a FIFO stage is acknowledged with an input acknowledge (IACK, 0908).", "The output of data from a FIFO is acknowledged by an output acknowledge (OACK, 0909).", "OACK reaches all FIFO stages at the same time and causes the data to be shifted forward in the FIFO by one stage.", "Individual FIFO stages may be cascaded to form FIFOs of any desired length (FIG.", "9a).", "For this purpose, all IACK outputs are logically gated with one another, for example, by an OR function (0910).", "The mode of operation is elucidated using the example of FIG.", "10.a, b. Appending a Data Word A new data word is passed on via the multiplexers of the individual FIFO stages to the registers.", "The first full FIFO stage (1001) signals to the upstream stage (1002), using the stored RDY, that it cannot receive data.", "The upstream stage (1002) has no RDY stored, but is aware of the “full” status of the downstream stage (1001).", "Therefore the stage stores the data and the RDY (1003) and acknowledges the storage by an ACK to the transmitter.", "The multiplexer (1004) of the FIFO stage switches over in such a way that, instead of the data path, it relays the contents of the register to the downstream stage.", "Removing a Data Word If an ACK (1011) is received by the last FIFO stage, the data of each upstream stage is transmitted to the particular downstream stage (1010).", "This is accomplished by applying a global write cycle to each stage.", "Because all multiplexers are already set according to the register contents, all data slips one line downward in the FIFO.", "Removing and Simultaneously Appending a Data Word If the global write cycle has been applied, no data word is stored in the first free stage.", "Because the multiplexer of this stage still forwards the data to the downstream stage, the first full stage (1012) stores the data.", "Its data is stored by the downstream stage in the same cycle as described above.", "In other words: new data to be written automatically slips into the now first free FIFO stage (1012), i.e., the previously last full FIFO stage, which has been emptied by the arrival of ACK.", "Configurable Pipeline For certain applications it may be advantageous to switch, using a switch (0930), individual multiplexers of the FIFO in the FIFO stage shown in FIG.", "9 as an example in such a way that basically the corresponding register is switched on.", "A fixed settable latency or delay time is thus configurable via the switch for the data transmission.", "Merging Data Streams Three methods are available for merging data streams, each being best suited to particular applications: a) local merge, b) tree merge, c) memory merge.", "Local Merge Local merge is the simplest variant, where all data streams are preferably merged at a single point or relatively locally and immediately split again if appropriate.", "A local SNDCNT selects, via a multiplexer, the exact data word whose time stamp corresponds to the value of SNDCNT and therefore is now expected.", "Two options should be explained in more detail on the basis of FIGS.", "7a and 7b.", "a) A counter SNDCNT (0706) is incremented for each incoming data packet.", "A comparator which compares the particular count with the time stamp of the data path is connected downstream in each data path.", "If the values coincide, the current data packet is relayed to the downstream PAEs via the multiplexer.", "b) The approach of a) is extended by assigning a target data path to the currently active data path, preferably via a translation procedure, for example, a CT configurable lookup table (0710), after the selection of this data path as the source data path.", "The source data path is determined by comparing (0712) the time stamp arriving with the data according to method a) with a SNDCNT (0711), the coinciding data path is addressed (0714) and selected via a multiplexer (0713).", "Using the lookup table (0710), for example, the address (0714) is assigned to a target data path address (0715), which selects the target path via a demultiplexer (0716).", "If the above-described structure is implemented in bus nodes as in Figure, the data link of the PAE (0718) associated with the bus node may also be established via the exemplary lookup table (0710), for example, via a gate function (transmission gates) (0717) to the input of the PAE.", "A particularly effective exemplary circuit is illustrated in FIG.", "7c.", "A PAE (0720) has three data inputs (A, B, C) as in the XPU128ES, for example.", "The bus system (0733) connections to the data inputs, for example, may be configurable and/or multiplexable, and selectable for each clock cycle.", "Each bus system transmits data, handshakes, and the associated time stamp (0721).", "Inputs A and C of the PAE (0720) are used for relaying the time stamp of the data channels to the PAE (0722, 0723).", "The individual time stamps may be bundled by the SIMD bus system described in the following, for example.", "The bundled time stamps are unbundled again in the PAE and each time stamp (0725, 0726, 0727) is individually compared (0728) to an SNDCNT (0724) implemented/configured in the PAE.", "The results of the comparisons are used for activating the input multiplexers (0730) in such a way that the bus system is connected to a bus (0731) using the correct time stamp.", "The bus is preferably connected to input B to permit data to be relayed to the PAE according to 0717, 0718.The output demultiplexers (0732) for relaying the data to different bus systems are also activated by the results, the results being preferably re-sorted by a flexible translation, for example, by a lookup table (0729), to enable the results to be freely assigned to selecting bus systems via demultiplexers (0732).", "Tree Merge In many applications it is desirable to merge parts of a data stream at a plurality of points, which results in a tree-like structure.", "The problem is that it is impossible to make a central decision on the selection of a data word, but the decision is distributed over multiple nodes.", "Therefore, the particular value of SNDCNT must be transferred to all nodes.", "However, in the case of high clock frequencies, this is only accomplishable with a latency, which occurs, for example, due to a plurality of register stages during the transmission.", "Therefore, this approach initially yields no reasonable performance.", "A method for improving the performance is allowing local decisions to be made in each node, independently of the value of SNDCNT.", "A simple approach, for example, is to select the data word with the smallest time stamp at a node.", "This approach, however, becomes problematic if a data path delivers no data word to a node during a cycle.", "Then it is impossible to decide which data path is to be preferred.", "The following algorithm improves on this situation: a) Each node receives a standalone SNDCNT counter SNDCNTK.", "b) Each node should have n input data paths (P0, .", ".", ".", "Pn) c) Each node may have a plurality of output data paths, which are selected via a translation procedure, for example, a lookup table which is configurable by a higher-level configuration unit CT, depending on the input data path.", "d) The root node has a main SNDCNT to which all SNDCNTK are synchronized if appropriate.", "The following algorithm is used to select the correct data path: I.", "If data appears on all input data paths Pn: a) select the data path P(Ts) having the smallest time stamp Ts.", "b) assign K:=Ts+1; SNDCNT>Ts+1, then SNDCNTK:=SNDCNT.", "II.", "If data does not appear on all input data paths Pn: a) select a data path only if the time stamp Ts==SNDCNTK.", "b) SNDCNTK:=SNDCNT+1.c) SNDCNT:=SNDCNT+1.III.", "If no assignment takes place in a cycle, then: a) SNDCNTK:=SNDCNT.", "IV.", "The root node has the SNDCNT which is incremented for each selection of a valid data word and ensures the correct sequence of the data words at the root of the tree.", "All other nodes are synchronized to the value of SNDCNT if necessary (see 1-3).", "There is a latency which corresponds to the number of registers, which must be introduced for bridging the segment from SNDCNT to SNDCNTK.", "FIG.", "11 shows a possible tree, which is constructed, for example, of PAEs in a manner similar to those of the XPU128ES VPU.", "A root node (1101) has an integrated SNDCNT, whose value is available at output H (1102).", "The data words at inputs A and C are selected according to the above-described procedure and the particular data word is supplied to output L in the correct sequence.", "The PAEs of the next hierarchical level (1103) and on each additional higher hierarchical level (1104, 1105) work similarly, but with the following difference: The integrated SNDCNTK is local, and the particular value is not forwarded.", "SNDCNTK is synchronized with SNDCNT, whose value is applied to input B, according to the above-described procedure.", "SNDCNT may be pipelined between all nodes, however, in particular between the individual hierarchical levels, for example, via registers.", "Memory Merge In this procedure, memories are used for merging data streams.", "A memory location is assigned to each value of the time stamp.", "The data is then stored in the memory according to the value of its time stamp; in other words, the time stamp is used as the address of the memory location for the assigned data.", "This creates a data space which is linear to the time stamp, i.e., is sorted according to the time stamp.", "The memory is not enabled for further processing, i.e., read out linearly, until the data space is complete, i.e., all the data is stored.", "This is easily determinable, for example, by counting how many pieces of data have been written into a memory.", "If as many pieces of data have been written as the memory has data entries, it is full.", "The following problem arises during the execution of the basic principle: Before the memory is filled without any gap, a time stamp overrun may occur.", "An overrun is defined as follows: A time stamp is a number from a finite linear arithmetic space (TSR).", "The time stamp is specified strictly monotonously, whereby each specified time stamp is unique within the TSR arithmetic space.", "If the end of the arithmetic space is reached when a time stamp is specified, the specification is continued from the beginning of TSR; this results in a point of discontinuity.", "The time stamps specified now are no longer unique with respect to the preceding ones.", "It must always be ensured that these points of discontinuity are taken into account during processing.", "The arithmetic space (TSR) must therefore be selected to be sufficiently large for no ambiguity to be created in the most unfavorable case by two identical time stamps occurring within the data processing.", "In other words, the TSR must be sufficiently large for no identical time stamps to exist within the processing pipelines and/or memories in the most unfavorable case which may occur within the subsequent processing pipelines and/or memories.", "If a time stamp overrun occurs, the memories must always be able to respond to such overrun.", "It must therefore be assumed that, after an overrun, the memories will contain both data having the time stamp before the overrun (“old data”) and data having the time stamp after the overrun (“new data”).", "The new data cannot be written into the memory locations of the old data, since they have not yet been read out.", "Therefore several (at least two) independent memory blocks are provided, so that the old and new data may be written separately.", "Any method may be used to manage the memory blocks.", "Two options are discussed in more detail: a) If it is always ensured that the old data of a given time stamp value is received before the new data of this time stamp value, it is tested whether the memory location for the old data is still free.", "If this is the case, old data is present, and the data is written to the memory location; if not, new data is being applied, and the data is written to the memory location for the new data.", "b) If it is not ensured that the old data of a given time stamp value is received before the new data of this time stamp value, the time stamp may be provided with an identifier which differentiates the old time stamp from the new time stamp.", "This identifier may be one or more bits long.", "In the event of time stamp overrun, the identifier is linearly modified.", "In this way, old and new data is provided with unique time stamps.", "The data is assigned to one of the multiple data blocks according to the identifier.", "Identifiers whose maximum numerical value is considerably less than the maximum numerical value of the time stamps are preferably used.", "A preferred ratio may be given by the following formula: identifiermax<time stampmax/2.Use of Memories for Partitioning Wide Graphs As known from PACT13, large algorithms must be partitioned, i.e., divided into a plurality of partial algorithms so that they fit a given arrangement and number of PAEs of a VPU.", "The partitioning must be performed both efficiently with respect to performance and naturally, while preserving the correctness of the algorithm.", "One essential aspect is the management of data and states (triggers) of the particular data paths.", "In the following, we shall present methods for improved and simplified management.", "In many cases it is not possible to section a data flow graph at one edge only (see FIG.", "12a for example), because the graph is too wide, for example, or there are too many edges (1201, 1202, 1203) at the section point (1204).", "Partitioning may be performed according to the present invention by sectioning along all edges according to FIG.", "12b.", "The data of each edge of a first configuration (1213) is written into a separate memory (1211).", "It should be expressly pointed out that, together with (or possibly also separately from) the data, all relevant status information of the data processing also runs over the edges (for example, in FIG.", "12b) and may be written into the memories.", "The status information is represented in VPU technology by triggers (see PACT08), for example.", "After reconfiguration, the data and/or status information of a subsequent configuration (1214) is read out from the memories and processed further by this configuration.", "The memories work as data receivers of the first configuration (i.e., in a mainly write mode) and as data transmitters of the subsequent configuration (i.e., in a mainly read mode).", "The memories (1211) themselves are a part/resource of both configurations.", "To correctly process the data further, it is necessary to know the correct chronological sequence in which the data was written into the memories.", "Basically this may be ensured by a) sorting the data streams when writing into a memory, and/or b) sorting the data streams when reading out from a memory, and/or c) saving the sorting sequence with the data and making it available to the subsequent data processing.", "For this purpose, control units which are responsible for managing the data sequences and data relationships both when writing the data (1210) into the memories (1211) and when reading out the data from the memories (1212) are assigned to the memories.", "Depending on the configuration, different management modes and corresponding control mechanisms may be used.", "Two possible corresponding methods should be elucidated in more detail with reference to FIG.", "13.The memories are assigned to an array (1310, 1320) of PAEs, in a manner similar to the data processing method according to PACT04.a) In FIG.", "13a, the memories generate their addresses synchronously, for example, by common address generators, which are independent but synchronized.", "In other words, the write address (1301) is incremented in each cycle regardless of whether a memory actually has valid data to be stored.", "Thus, a plurality of memories (1303, 1304) have the same time base, i.e., write/read address.", "An additional flag (VOID, 1302) for each data memory position in the memory indicates whether valid data has been written into a memory address.", "The VOID flag may be generated by the RDY flag (1305) assigned to the data; accordingly, when reading out a memory, the data RDY flag (1306) is generated from the VOID flag.", "For reading out the data by the subsequent configuration, a common read address (1307), which is advanced in each cycle, is generated similarly to the writing of the data.", "b) In the example of FIG.", "13b it is more efficient to assign a time stamp to each data word according to the previously described method.", "The data (1317) is stored with the particular time stamp (1311) in the particular memory position.", "Thus no gaps are formed in the memories, which are more efficiently utilized.", "Each memory has independent write pointers (1313, 1314) for the data-writing configuration and read pointers (1315, 1316) for the subsequent data-reading configuration.", "According to the known method (e.g., according to FIG.", "7a or FIG.", "11), the chronologically correct data word is selected when reading on the basis of the associated time stamp stored (1312) with it.", "The data may also be sorted into the memories/from the memories according to different algorithmically suitable methods such as a) by assigning a memory location using the time stamp; b) by sorting into the data stream according to the time stamp; c) by storing in each cycle together with a VALID flag; d) by storing the time stamp and forwarding it to the subsequent algorithm when reading out the memory.", "Depending on the application, a plurality of (or all) data paths may also be merged upstream from the memories via the merge method according to the present invention.", "Whether this is done depends essentially on the available resources.", "If too few memories are available, merging upstream from the memories is necessary or desirable.", "If too few PAEs are available, preferably no additional PAEs are used for a merge.", "Extension of the Peripheral Interface (IO) Using Time Stamp In the following, a method of assigning time stamps to IO channels for peripheral modules and/or external memories will be described.", "The method may serve different purposes such as to allow proper sorting of data streams between transmitter and receiver and/or selecting unique data stream sources and/or targets.", "The following discussion will be illustrated using the example of the interface cells from PACT03.PACT03 describes a method of bundling buses internal to the VPU and of data exchange between different VPUs or VPUs and peripherals (IO).", "One disadvantage of this method is that the data source is no longer identifiable by the receiver, nor is the correct chronological sequence ensured.", "The following novel methods eliminate this problem; some or more of the methods described may be used and possibly combined according to the specific application.", "a) Identification of the Data Source FIG.", "14 as an example describes such an identification between arrays (PAs, 1408) made up of reconfigurable elements (PAEs) of two VPUs (1410, 1420).", "An arbiter (1401) selects on a data transmission module (VPU, 1410) one of the possible data sources (1405) to connect it to the IO via a multiplexer (1402).", "The address of the data source (1403), together with the data (1404), is sent to the IO.", "The data-receiving module (VPU, 1411) selects, according to the address (1403) of the data source, the particular receiver (1406) via a demultiplexer (1407).", "The address transmitted (1403) may be assigned to the receiver (1406) in a flexible manner via a translation procedure, for example, a lookup table which is configurable by a higher-level configuration unit (CT), for example.", "It should be expressly pointed out that interface modules connected upstream from the multiplexers (1402) and/or downstream from the demultiplexers (1407) according to PACT03 and/or PACT15 may be used for the configurable connection of bus systems.", "b) Compliance with the Chronological Sequence b1) The simplest procedure is to send the time stamp to the IO and to leave the evaluation to the receiver which receives the time stamp.", "b2) In another version, the time stamp is decoded by the arbiter which only selects the transmitter having the correct time stamp and sends to the IO.", "The receiver receives the data in the correct sequence.", "Methods a) and b) are usable together or separately depending on the requirements of the particular application.", "Furthermore, the method may be extended by specifying and identifying channel numbers.", "A channel number identifies a given transmitter area.", "For example, a channel number may be composed of a plurality of IDs, such as that of the bus within a module, the module, and/or the module group.", "This also makes identification easy even in applications with a large number of PAEs and/or a combination of several modules.", "In using channel numbers, instead of transmitting individual data words, a plurality of data words are preferably combined into a data packet and then transmitted with the specification of the channel number.", "The individual data words may be combined via a suitable memory such as described in PACT18 (BURST-FIFO), for example.", "It should be pointed out that the addresses and/or time stamps which have been transmitted may preferably be used as identifiers or parts of identifiers in bus systems according to PACT15.The method according to PACT07 is included in its entirety in the present patent, which may also be extended by the above-described identification method.", "Furthermore, the data transmission methods according to PACT18, for which the above-described method may also be applied, are included in their entirety.", "Sequencer Structure The use of time stamps or comparable methods makes a simpler structure of sequencers made up of PAE groups possible.", "The buses and basic functions of the circuit are configured, and the detail function and data addresses are flexibly set via an OpCode at run time.", "A plurality of these sequencers may also be constructed and operated within a PA (PAE arrays).", "The sequencers within a VPU may be constructed according to the algorithm.", "Examples have been given in multiple documents of the inventor which are incorporated in the present invention in their entirety.", "In particular, reference should be made to PACT13, where the construction of sequencers from a plurality of PAEs is described, which is to be also used as an exemplary basis for the description that follows.", "In detail, the following configurations of sequencers may be freely adapted, for example: type and number of IO/memories type and number of interrupts (e.g., via triggers) instruction set number and type of registers.", "A simple sequencer may be constructed from, for example, 1.an ALU for performing the arithmetic and logical functions; 2.a memory for storing data, similar to a register set; 3.a memory as a code source for the program (e.g., normal memory according to PACT22/24/13 and/or CT according to PACT10/PACT13 and/or special sequencers according to PACT04).", "If appropriate, the sequencer is extended by IO elements (PACT03, PACT22/24).", "In addition, additional PAEs may be added as data sources or data receivers.", "Depending on the code source used, the method according to PACT08 may be used, which allows OpCodes of a PAE to be directly set via data buses, as well as data sources/targets to be specified.", "The addresses of the data sources/targets may be transmitted by time stamp methods, for example.", "Furthermore, the bus may be used for transmitting the OpCodes.", "In an exemplary implementation according to FIG.", "15, a sequencer has a RAM for storing the program (1501), a PAE for computing the data (ALU) (1502), a PAE for computing the program pointer (1503), a memory as a register set (1504), and an IO for external devices (1505).", "The interconnection creates two bus systems: an input bus to ALU IBUS (1506) and an output bus from ALU OBUS (1507).", "A four-bit wide time stamp is assigned to each bus, which addresses the source IBUS-ADR (1508) and the target OBUS-ADR (1509), respectively.", "The program pointer (1510) is transmitted from 1504 to 1501.1501 returns the OpCode (1511).", "The OpCode is split into instructions for the ALU (1512) and the program pointer (1513), as well as the data addresses (1508, 1509).", "The SIMD procedures and bus systems described in the following may be used for splitting the bus.", "1502 is configured as an accumulator machine and supports the following functions, for example: ld <reg> load accumulator (1520) from register add_sub <reg> add/subtract register to/from accumulator sl_sr shift accumulator rl_rr rotate accumulator st <reg> write accumulator into register Three bits are needed for the instructions.", "A fourth bit specifies the type of operation: adding or subtracting, shifting right or left.", "1502 delivers the ALU status carry to trigger port 0 and 0 to trigger port 1.<reg> is coded as follows: 0-7 data register in 1504 8 input register (1521) program pointer computation 9 IO data 10 IO addresses Four bits are needed for the addresses.", "1503 supports the following operations via the program pointer: jmp jump to address in input register (2321) jt0 jump to address in input register given when trigger0 set jt1 jump to address in input register given when trigger1 set jt2 jump to address in input register given when trigger2 set jmpr jump to PP plus address in input register Three bits are needed for the instructions.", "A fourth bit specifies the type of operation: adding or subtracting.", "OpCode 1511 is also split into three groups having four bits each: (1508, 1509), 1512, 1513.1508 and 1509 may be identical for the given instruction set.", "1512, 1513 are sent to the C register of the PAEs (see PACT22/24), for example, and decoded as instruction within the PAEs (see PACT08).", "According to PACT13 and/or PACT11, the sequencer may be built into a more complex structure.", "For example, additional data sources, which may originate from other PAEs, are addressable via <reg>=11, 12, 13, 14, 15.Additional data receivers may also be addressed.", "Data sources and data receivers may have any structure, in particular PAEs.", "It should be noted that the circuit illustrated only needs 12 bits of OpCode 1511.Thus, for a 32-bit architecture, 20 bits are optionally available for extending the basic circuit.", "The multiplexer functions of the buses may be implemented according to the above-described time stamp method.", "Other designs are also possible; for example, PAEs may be used as multiplexer stages.", "SIMD Arithmetic Units and SIMD Bus Systems When using reconfigurable technologies for executing algorithms, an important paradox occurs: On the one hand, complex ALUs are needed to obtain maximum computing performance, while the complexity should be minimum for the reconfiguration; on the other hand, the ALUs should be as simple as possible to facilitate efficient bit level processing; also, the reconfiguration and data management should be accomplished intelligently and quickly in such a way that it is programmed in an efficient and simple manner.", "Previous technologies use a) very small ALUs having little reconfiguration support (FPGAs) and are efficient on the bit level; b) large ALUs (Chameleon) having little reconfiguration support, c) a mixture of large ALUs and small ALUs having reconfiguration support and data management (VPUs).", "Since the VPU technology represents the most powerful technique, an optimum method should be built on this technology.", "It should be expressly pointed out that this method may also be used for the other architectures.", "The surface needed for effective control of reconfiguration is relatively high with approx.", "10,000 to 40,000 gates per PAE.", "If fewer gates are used, only simple sequence control is possible, which considerably limits the programmability of VPUs and rules out their use as general purpose processors.", "Since the object is to achieve a particularly rapid reconfiguration, additional memories must be provided, which again considerably increases the number of required gates.", "Therefore, to obtain a reasonable compromise between reconfiguration complexity and computing performance, large ALUs (extensive functionality and/or large bit width) must be used.", "However, using excessively large ALUs decreases the usable parallel computing performance per chip.", "For excessively small ALUs (e.g., 4 bits), the complexity for configuring complex functions (e.g., 32-bit multiplication) is excessively high.", "In particular, the wiring complexity grows into ranges that are no longer commercially feasible.", "11.1 Use of SIMD Arithmetic Units To reach an ideal compromise between processing of small bit widths, wiring complexity, and the configuration of complex functions, the use of SIMD arithmetic units is proposed.", "Arithmetic units having bit width m are split so that n individual blocks having bit width b=m/n are obtained.", "For each arithmetic unit it is specified via configuration whether an arithmetic unit is to operate without being split or whether it should be split into one or more blocks of the same or different bit widths.", "In other words, an arithmetic unit may also be split in such a way that different word widths are configured simultaneously within an arithmetic unit (e.g., 32-bit width split into 1×16, 1×8, and 2×4 bits).", "The data is transmitted between the PAEs in such a way that the split data words (SIMD-WORD) are combined to data words having bit width m and transmitted over the network as a packet.", "The network always transmits a complete packet, i.e., all data words are valid within a packet and are transmitted according to the known handshake method.", "11.1.1 Re-Sorting the SIMD-WORD For efficient use of SIMD arithmetic units, a flexible and efficient re-sorting of the SIMD-WORD within a bus or between different buses is required.", "The bus switch according to FIGS.", "5, 7b, c may be modified so that the individual SIMD-WORDs are interconnected in a flexible manner.", "For this purpose, the multiplexers are designed to be splittable according to the arithmetic units in such a way that the split may be defined by the configuration.", "In other words, instead of using one multiplexer having a width m bits per bus, for example, n individual multiplexers having a width b=m/n bits are used.", "It is thus possible to configure the data buses for a data width of b bits.", "The matrix structure of the buses (FIG.", "5) permits the data to be re-sorted in a simple manner, as shown in FIG.", "16c.", "A first PAE sends data via two buses (1601, 1602), which are each divided into four partial buses.", "A bus system (1603) connects the individual partial buses to additional partial buses located on the bus.", "A second PAE contains partial buses sorted differently on its two input buses (1604, 1605).", "The handshakes of the buses between two PAEs having two arithmetic units (1614, 1615), for example, are logically gated in FIG.", "16a so that a common handshake (1610) is generated for the re-sorted bus (1611) from the handshakes of the original buses.", "For example, a RDY may be generated for a re-sorted bus from a logical AND gating of all RDYs of the data for buses delivering to this bus.", "The ACK of a bus which delivers data may also be generated from an AND gating of the ACKs of all buses which process the data further.", "The common handshake controls a control unit (1613) for managing the PAEs (1612).", "Bus 1611 is split into two arithmetic units (1614, 1615) within the PAE.", "In a first embodiment variant, the handshakes are gated within each individual bus node.", "This permits a bus system having width m, containing n partial buses having width b, to be assigned a single handshake protocol.", "In a further, particularly preferred embodiment, all bus systems are designed to have width b, which corresponds to the smallest implementable input/output data width b of a SIMD word.", "Corresponding to the width of the PAE data paths (m), an input/output bus is now composed of m/b—n partial buses of width b.", "For example, in the case of a smallest SIMD word width of 8 bits, a PAE having three 32-bit input buses and two 32-bit output buses actually has 3×4 eight-bit input buses and 2×4 eight-bit output buses.", "All handshake and control signals are assigned to each of the partial buses.", "The output of a PAE transmits them, using the same control signals, to all n partial buses.", "Incoming acknowledge signals of all partial buses are gated logically, for example, using an AND function.", "The bus systems are able to freely connect and independently route each partial bus.", "The bus system and, in particular, the bus nodes, do not process or gate the handshake signals of the individual buses independently of their routing, arrangement, and sorting.", "For data received by a PAE, the control signals of all n partial buses are gated in such a way that a control signal of overall validity, similar to a bus control signal, is generated for the data path.", "For example, in a “dependent” operating mode according to the definition, RdyHold stages may be used for each individual data path, and the data is not received by the PAE until all RdyHold stages signal the presence of data.", "In an “independent” operating mode according to the definition, the data of each partial bus is written individually into the input register of the PAE and acknowledged, which immediately frees the partial bus for a subsequent data transmission.", "The presence of all required data from all partial buses in the input registers is detected within the PAE by the appropriate logical gating of the RDY signals stored for each partial bus in the input register, whereupon the PAE starts the data processing.", "The important advantage of this method is that the SIMD property of PAEs has no specific influence on the bus system used.", "Only more buses (n) (1620) of a smaller width (b) and the associated handshakes (1621) are needed, as illustrated in FIG.", "16b.", "The interconnection itself remains unaffected.", "The PAEs link and manage the control lines locally.", "This makes additional hardware unnecessary in the bus systems for managing and/or linking the control lines." ] ]	Patent_10469910
[ [ "Tube clamp", "For obtaining a hose clamp which has a continuously smooth inner surface free of gaps and which requires an as short as possible length of band material, the inner band end portion is provided with a tongue the length of which is approximately half the amount by which the hose clamp is narrowed during tightening.", "During the first half of the tightening movement, the tongue is guided transversely to the band longitudinal direction by a projection which engages a slot provided in the outer band portion.", "The slot at its end is continued by a window which starts at a step, and the tongue, during the second half of the tightening movement, passes through the window which then takes over the guidance of the tongue." ], [ "1-10.", "(canceled) 11.A hose clamp made of an open band having an inner band portion with an inner band end, an outer band portion with an outer band end overlapping said inner band portion, the clamp being adapted to be transferred from a closed condition in which said outer band end is connected to said inner band portion, to a tightened condition in which the clamp has a reduced inner dimension, the clamp comprising a tongue formed at said inner band end, the tongue having a length which is substantially smaller than the difference between the inner dimension of the clamp in said closed condition and the reduced inner dimension of the clamp in said tightened condition, an aperture provided in said outer band portion for receiving said tongue in said tightened condition, a step projecting inwardly, as seen from said outer band end, extending transversely to the band longitudinal direction within an area of an end of the aperture nearer to said outer band end, and having a height corresponding to the thickness of the band material, and guide means provided in said outer band portion between said outer band end and said aperture for laterally guiding said tongue prior to its entry into said aperture: 12.The clamp of claim 11 wherein said aperture is a window for said tongue to pass in said tightened condition.", "13.The clamp of claim 11 wherein said guide means includes beads formed in said outer band portion on both sides of said tongue.", "14.The clamp of claim 13 wherein said inner band portion has two beads in an area adjacent said tongue in the band longitudinal direction, which beads are engaged, in said tightened condition, by said two beads formed in said outer band portion.", "15.The clamp of claim 13 wherein the width of each bead is ¼ to 1/10 of the band width.", "16.The clamp of claim 11, comprising a point-shaped embossment provided near the free end of said tongue, said embossment facing said outer band portion.", "17.The clamp of claim 11 wherein said guide means includes a slot continuous with said aperture in said outer band portion and a projection formed on an outer side of said tongue for engaging said slot.", "18.The clamp of claim 17 wherein said projection is a nose punched out of said tongue.", "19.The clamp of claim 11 comprising ear-type tightening means provided between said guide means and said outer band end.", "20.The clamp of claim 11 comprising an undulation provided between said guide means and said outer band end." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>U.S.", "Pat.", "No.", "4,299,012 discloses a hose clamp which consists of an open band with overlapping band ends and which is adapted to be transferred from a closed condition in which the outer band end is connected to the inner band portion, to a tightened condition having a reduced inner dimension, comprising a tongue formed at the inner band end, an aperture provided in the outer band portion for receiving the tongue in the tightened condition, and a step which projects inwardly as seen from the outer band end, which extends transversely to the band longitudinal direction within the area of the end of the aperture nearer to the outer band end, and which has a height corresponding to the thickness of the band material.", "This hose clamp has proved suitable for fixing even thin, hard hoses to pipe nipples due to the fact that, in the tightened condition, the inner surface of the clamp is free of gaps and steps and therefore supports the hose at all circumferential positions.", "For tightening the known hose clamp, a so-called “Oetiker ear” provided in the outer band section is narrowed by means of a pair of grippers.", "A tongue provided at the inner band end serves to bridge the gap remaining underneath the ear, the tongue cooperating with a tongue channel provided in the outer band portion.", "The channel is formed by two parallel cuts extending in the longitudinal direction of the band, wherein the two band portions outside these cuts are offset inwardly at their ends close to the outer band end, and the central portion situated between the cuts is offset inwardly at its end remote from the outer band end, each offset corresponding to the thickness of the band material.", "In the closed but not yet tightened condition of the hose clamp, hooks provided at the inner band end are inserted in openings near the outer band end.", "The tongue and the tongue channel are so arranged and dimensioned in the circumferential direction of the hose clamp in such a way that, in this closed condition, the free end of the tongue just reaches the channel and the portion of full band width adjacent the tongue bridges the spacing between the legs of the ear.", "In tightening, the tongue moves completely into the tongue channel until its end comes to lie close to the offset of the centre band portion between the two cuts, and the portion of full band width adjacent the tongue comes to lie close to the offset at the other end of the tongue channel.", "In this tightened condition, while gaps occur near the offsets, these gaps at no location extend across the full band width so that the hose is supported even in these regions by at least part of the band width, and tightness between the hose and the nipple is ensured over the entire circumference.", "In the hose clamp known from EP 0 570 742 A1, the tongue during tightening enters a space between two beads formed in the inner surface of the outer band section, which beads project inwardly by an amount corresponding to the thickness of the band material and terminate in a step of the same height.", "In order to make sure that the tongue is laterally guided throughout the tightening process, the tongue and beads have such a length in the circumferential direction of the hose clamp that the tongue reaches the space between the beads already in the closed condition of the hose clamp.", "U.S. Pat.", "No.", "4,315,348 describes a hose clamp having a separate insert portion bridging the ear, with two tongue-type ends extending through windows provided in the outer band section.", "The invention is based on the object to remove at least in part such disadvantages as occur with comparable hose clamps of the prior art.", "A more specific object may be seen in providing a hose clamp which requires an as small as possible length of band material at a given clamp diameter.", "The solution of this object in accordance with the invention which consists of an open band with overlapping band ends and which is adapted to be transferred from a closed condition in which the outer band end is connected to the inner band portion, to a tightened condition having a reduced inner dimension, comprising a tongue formed at the inner band end, an aperture provided in the outer band portion for receiving the tongue in the tightened condition, and a step which projects inwardly as seen from the outer band end, which extends transversely to the band longitudinal direction within the area of the end of the aperture nearer to the outer band end, and which has a height corresponding to the thickness of the band material, that the length of the tongue is substantially shorter than the difference between the inner dimension of the hose clamp in its closed condition and that of the hose clamp in its tightened condition, and that the outer band portion in the region between its outer band end and the aperture has means for laterally guiding the tongue prior to its entry into the aperture.", "The tongue provided at the inner band end is guided during tightening of the hose clamp consecutively by two different measures wherein, during the first part of the tightening movement, the tongue is remote from the aperture and only in the second portion enters the aperture and is guided by it.", "The tongue can thus be shorter by roughly one half than in the prior art, which results in a corresponding saving in band material length.", "The embodiment wherein the aperture is a window for the tongue to pass in the tightened condition of the hose clamp leads to a specifically safe guiding and retaining of the central band section transverse to the band.", "An easily obtained possibility of guiding the tongue in the first part of the tightening movement is obtainable wherein the guiding means includes beads formed in the outer band portion on both sides of the tongue clamp of claim 3 and wherein the inner band end portion has two beads in the area adjacent the tongue in the band longitudinal direction, which beads are engaged, in the tightened condition of the hose clamp, by the two beads in the outer band portion and wherein the width of each bead is ¼ to 1/10 of the band width.", "For reducing the friction which occurs between the tongue and the outer band portion during tightening of the hose clamp a point-shaped embossment facing the outer band portion is provided near the free end of the tongue.", "A specifically safe guidance of the tongue transverse to the band longitudinal direction in the first part of the tightening movement is provided when the guiding means includes a slot continuous with the aperture in the outer band portion and a projection on the outer side of the tongue for engaging the slot and wherein the projection is a nose punched out of the tongue.", "The invention is adapted for use with hose clamps with and without ear-like tightening means wherein an ear-type tightening means is provided between the guiding means and the outer band end and wherein an undulation is provided between the guiding means and the outer band end.", "Advantageous embodiments will now be explained in more detail with reference to the drawings.", "In the drawings," ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 shows a hose clamp according to a first embodiment in the open condition; FIG.", "2 is a somewhat enlarged cross-section through the tongue and the outer band portion of the hose clamp according to FIG.", "1 in the tightened condition; FIG.", "3 shows an earless hose clamp according to a second embodiment in the closed, not yet tightened condition; FIG.", "4 is a somewhat enlarged partial view of the hose clamp according to FIG.", "3 , viewed from the inside; FIG.", "5 represents a hose clamp having an Oetiker ear according to a third embodiment of the invention in the closed, not yet tightened condition; and FIG.", "6 is an enlarged partial view of the hose clamp according to FIG.", "5 , viewed from the inside.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "This application is the national stage of PCT Application PCT/EP02/03259 filed Mar.", "22, 2002, which claims benefit of EP Patent Application 01107515.7 filed Mar.", "26, 20001.BACKGROUND OF THE INVENTION U.S. Pat.", "No.", "4,299,012 discloses a hose clamp which consists of an open band with overlapping band ends and which is adapted to be transferred from a closed condition in which the outer band end is connected to the inner band portion, to a tightened condition having a reduced inner dimension, comprising a tongue formed at the inner band end, an aperture provided in the outer band portion for receiving the tongue in the tightened condition, and a step which projects inwardly as seen from the outer band end, which extends transversely to the band longitudinal direction within the area of the end of the aperture nearer to the outer band end, and which has a height corresponding to the thickness of the band material.", "This hose clamp has proved suitable for fixing even thin, hard hoses to pipe nipples due to the fact that, in the tightened condition, the inner surface of the clamp is free of gaps and steps and therefore supports the hose at all circumferential positions.", "For tightening the known hose clamp, a so-called “Oetiker ear” provided in the outer band section is narrowed by means of a pair of grippers.", "A tongue provided at the inner band end serves to bridge the gap remaining underneath the ear, the tongue cooperating with a tongue channel provided in the outer band portion.", "The channel is formed by two parallel cuts extending in the longitudinal direction of the band, wherein the two band portions outside these cuts are offset inwardly at their ends close to the outer band end, and the central portion situated between the cuts is offset inwardly at its end remote from the outer band end, each offset corresponding to the thickness of the band material.", "In the closed but not yet tightened condition of the hose clamp, hooks provided at the inner band end are inserted in openings near the outer band end.", "The tongue and the tongue channel are so arranged and dimensioned in the circumferential direction of the hose clamp in such a way that, in this closed condition, the free end of the tongue just reaches the channel and the portion of full band width adjacent the tongue bridges the spacing between the legs of the ear.", "In tightening, the tongue moves completely into the tongue channel until its end comes to lie close to the offset of the centre band portion between the two cuts, and the portion of full band width adjacent the tongue comes to lie close to the offset at the other end of the tongue channel.", "In this tightened condition, while gaps occur near the offsets, these gaps at no location extend across the full band width so that the hose is supported even in these regions by at least part of the band width, and tightness between the hose and the nipple is ensured over the entire circumference.", "In the hose clamp known from EP 0 570 742 A1, the tongue during tightening enters a space between two beads formed in the inner surface of the outer band section, which beads project inwardly by an amount corresponding to the thickness of the band material and terminate in a step of the same height.", "In order to make sure that the tongue is laterally guided throughout the tightening process, the tongue and beads have such a length in the circumferential direction of the hose clamp that the tongue reaches the space between the beads already in the closed condition of the hose clamp.", "U.S. Pat.", "No.", "4,315,348 describes a hose clamp having a separate insert portion bridging the ear, with two tongue-type ends extending through windows provided in the outer band section.", "The invention is based on the object to remove at least in part such disadvantages as occur with comparable hose clamps of the prior art.", "A more specific object may be seen in providing a hose clamp which requires an as small as possible length of band material at a given clamp diameter.", "The solution of this object in accordance with the invention which consists of an open band with overlapping band ends and which is adapted to be transferred from a closed condition in which the outer band end is connected to the inner band portion, to a tightened condition having a reduced inner dimension, comprising a tongue formed at the inner band end, an aperture provided in the outer band portion for receiving the tongue in the tightened condition, and a step which projects inwardly as seen from the outer band end, which extends transversely to the band longitudinal direction within the area of the end of the aperture nearer to the outer band end, and which has a height corresponding to the thickness of the band material, that the length of the tongue is substantially shorter than the difference between the inner dimension of the hose clamp in its closed condition and that of the hose clamp in its tightened condition, and that the outer band portion in the region between its outer band end and the aperture has means for laterally guiding the tongue prior to its entry into the aperture.", "The tongue provided at the inner band end is guided during tightening of the hose clamp consecutively by two different measures wherein, during the first part of the tightening movement, the tongue is remote from the aperture and only in the second portion enters the aperture and is guided by it.", "The tongue can thus be shorter by roughly one half than in the prior art, which results in a corresponding saving in band material length.", "The embodiment wherein the aperture is a window for the tongue to pass in the tightened condition of the hose clamp leads to a specifically safe guiding and retaining of the central band section transverse to the band.", "An easily obtained possibility of guiding the tongue in the first part of the tightening movement is obtainable wherein the guiding means includes beads formed in the outer band portion on both sides of the tongue clamp of claim 3 and wherein the inner band end portion has two beads in the area adjacent the tongue in the band longitudinal direction, which beads are engaged, in the tightened condition of the hose clamp, by the two beads in the outer band portion and wherein the width of each bead is ¼ to 1/10 of the band width.", "For reducing the friction which occurs between the tongue and the outer band portion during tightening of the hose clamp a point-shaped embossment facing the outer band portion is provided near the free end of the tongue.", "A specifically safe guidance of the tongue transverse to the band longitudinal direction in the first part of the tightening movement is provided when the guiding means includes a slot continuous with the aperture in the outer band portion and a projection on the outer side of the tongue for engaging the slot and wherein the projection is a nose punched out of the tongue.", "The invention is adapted for use with hose clamps with and without ear-like tightening means wherein an ear-type tightening means is provided between the guiding means and the outer band end and wherein an undulation is provided between the guiding means and the outer band end.", "Advantageous embodiments will now be explained in more detail with reference to the drawings.", "In the drawings, BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 shows a hose clamp according to a first embodiment in the open condition; FIG.", "2 is a somewhat enlarged cross-section through the tongue and the outer band portion of the hose clamp according to FIG.", "1 in the tightened condition; FIG.", "3 shows an earless hose clamp according to a second embodiment in the closed, not yet tightened condition; FIG.", "4 is a somewhat enlarged partial view of the hose clamp according to FIG.", "3, viewed from the inside; FIG.", "5 represents a hose clamp having an Oetiker ear according to a third embodiment of the invention in the closed, not yet tightened condition; and FIG.", "6 is an enlarged partial view of the hose clamp according to FIG.", "5, viewed from the inside.", "DETAILED DESCRIPTION OF THE DRAWINGS The open hose clamp shown in FIGS.", "1 and 2 is manufactured from a steel band 10 having a width of, e.g., 10 mm and a thickness of, e.g., 1 mm and is pre-bent to the configuration shown, with mutually overlapping band ends.", "In this condition, the inner band end portion 11 is opposite an outer band portion referenced 12.In order to seal, e.g.", "a hose (not shown) with respect to a nipple (not shown), the hose clamp is applied to the hose portion to be sealed.", "Upon engagement of a hook 13, provided at the inner band portion into openings 14 located near the outer band end, an overall approximately U-shaped “Oetiker ear” 15 provided in the inner band portion 12 is compressed by means of grippers so that the hose clamp is narrowed and tightened around the hose.", "A tongue 17 is integrally formed at the inner band end portion 11, the tongue having a width of, e.g., 4 mm at the assumed band width of 10 mm.", "The length of the tongue is somewhat greater than half the amount by which the ear 15 is narrowed and the circumference of the hose clamp is reduced during tightening.", "Two lateral beads 18 extending in the longitudinal direction of the band and projecting radially inwardly are formed in the opposite outer band portion 12.The beads 18 are located hear the outer band edges, each having a width of approximately 1/10 to at most ¼ of the band width.", "The length of the beads 18 in the longitudinal direction of the band is essentially the same as the length of the tongue 17.The beads 18 terminate at a step 19 which projects inwardly as seen from the outer band end, extends transversely to the band longitudinal direction and has a height corresponding to the thickness of the band material.", "At the start of step 19, thus at the end of the beads 18 remote from the outer band end, an overall rectangular window 20 starts, which has a width slightly larger than the width of the tongue 17 and a length that extends beyond the step 19.Two inwardly projecting beads 21 are formed in the inner band end portion 11 on both sides of the tongue 17 and starting at the end of the tongue, the beads 18 having a length corresponding at least to the length of the beads 18.Further, the free end of the tongue 17 is provided with an outwardly projecting point-shaped embossment 22.In the closed, not yet tightened condition, in which the hooks 13 provided on the inner band portion engage the openings 14 provided in the outer band portion 12, the free end of the tongue 17 is just between the beads 18 provided in the outer band portion.", "When the ear 15 is then narrowed for tightening the hose clamp, the tongue 17 at first moves between the beads 18 and is guided by them, thereby preventing the inner band end from moving relative to the outer band portion 12 transversely to the band longitudinal direction.", "During this movement, the embossment 22 reduces the friction between the tongue 17 and the outer band portion 12.In the second half of this tightening movement, the tongue 17 reaches the window 20 and passes therethrough while the beads 18 provided in the outer band portion 12 engage the beads 21 formed in the inner band end portion 11.In the completely tightened condition, in which the ear 15 is closed down to a small gap, the shoulders which exist at the inner band end portion 11 on both sides of the tongue 17 are at the step 19 with a small gap remaining at these two lateral positions.", "Since the height of the step 19 corresponds to the thickness of the hose clamp material and since the tongue 17 at this location extends further and finally through the window 20, the result in the tightened condition is an inner surface of the hose clamp which is continuously smooth and free of gaps.", "The alternative embodiments of the hose clamp shown in FIGS.", "3 to 6 differ from that of FIG.", "1 in that the tongue 17 in the first half of the tightening movement is guided not by lateral beads provided in the outer band portion but by an outward projection 23 formed at the free end of the tongue, which projection 23 engages a narrower slot 24 continuous with the window 20.The projection may be formed as a nose or, alternatively, as a rearwardly bent hook.", "A hook has the advantage that it may engage at the closed end of the slot 24, thereby rendering an inadvertent opening of the hose clamp difficult.", "The hose clamp of FIGS.", "3 and 4 is an earless hose clamp, wherein the resiliency required for sealing a hose to a nipple is achieved by an undulation 25.On the other hand, the hose clamp of FIGS.", "5 and 6 is a hose clamp which, just as that of FIG.", "1, has an Oetiker ear 15, with an undulation 25 being provided as an additional elasticity reserve." ] ]	Patent_10469912

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